Humanized anti-PVR antibody drug conjugate

Abstract

The present invention provides humanized anti-PVR (CD155) antibody drug conjugates (ADCs) and their use in the treatment of disease, particularly cancer.

Description

[Technical Field]

[0001] The present invention is in the field of immunotherapy and relates to humanized anti-PVR (CD155) antibody-drug conjugates (ADCs) and their use in the treatment of diseases, particularly cancer. [Background technology]

[0002] Antibodies are widely used as carriers of various diagnostic and therapeutic agents due to their specificity for antigens on the surface of target cells and molecules. For example, antibodies conjugated with labels and reporter groups, such as fluorescent dyes, radioisotopes, and enzymes, are used for labeling and imaging applications. Conjugation with cytotoxic and chemotherapeutic agents allows targeted delivery of drugs to specific tissues or structures, such as specific cell types or growth factors, thereby minimizing the impact on normal, healthy tissues and significantly reducing the side effects associated with chemotherapy treatment. Antibody-drug conjugates (ADCs) have broad therapeutic potential in several disease areas, particularly cancer, and represent a novel class of targeted drugs for disease treatment. A typical ADC contains a targeting antibody, a connector or linker for drug attachment, and a highly active payload (e.g., a drug) as an effector. Since the US FDA approval of Adcetris in 2011 and Kadcyla in 2013, ADC-based drug development has become widespread for cancer treatment. Currently, more than 80 ADCs are in clinical development, and 11 ADCs (9 containing small molecule payloads and 2 containing biological toxins) have been approved for use by the FDA (Baah et al., 2021 May 15;26(10):2943).

[0003] Drug compounds belong to various chemical families. For example, auristatins are microtubule-disrupting agents derived from the marine shellless mollusk Dolabella auricularia, known as dolastatins. Various derivatives of auristatins have been synthesized, including monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). MMAE and MMAF were developed by Seattle Genetics and are used as payloads in ADCs. MMAF and MMAE have advantages and disadvantages. MMAE has higher membrane permeability and a lower IC50 than MMAF. However, MMAF is more hydrophilic, has a lower tendency to aggregate, and exhibits lower systemic toxicity than MMAE (Park et al., Molecules 2019, 24, 2754). Ravtansine (DM4) is another example of a toxin that may be used in ADCs. Ravtansine is a maytansinoid (a chemical derivative of maytansine) with the ability to disrupt microtubule function. Another maytansinoid, emtansine (DM1), binds to the ends of microtubules and inhibits their dynamic instability.

[0004] The poliovirus receptor (PVR), also known as CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. PVR has previously been described as a tumor antigen and a potential target for therapeutic intervention, due to its upregulated expression in neuroectodermal cancers such as glioblastoma multiforme and medulloblastoma, colorectal cancer (Solecki et al., J. Biol. Chem. 2002, 277:25697-700), and pancreatic cancer (Nishiwada et al., Anticancer Res. 2015, 35(4):2287-97). PVR is known to enhance serum-induced activation of the Ras-Raf-MEK-ERK signaling pathway, upregulate cyclin D2 and E, and downregulate p27Kip1, ultimately shortening the G0 / G1 phase of the cell cycle (Kakunaga 2004, J. Biological Chemistry, 279, 36419-36425). Therefore, inhibition of PVR in tumor cells is expected to reduce their viability. PVR also plays an important role in angiogenesis, regulating VEGF-induced angiogenesis by regulating the interaction between vascular endothelial growth factor receptor 2 (VEGFR2) and integrin α(v)β(3) and the VEGFR2-mediated Rap1-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012, 110(5), 716-26). In addition, PVR forms a complex with IGF1R and is involved in tyrosine protein kinase Met (cMet) signaling, and inhibiting this complex reduced cell viability and angiogenesis (Lee et al., Scientific Reports 2014,20,4,7139).

[0005] In recent years, PVR has emerged as an important immune checkpoint ligand (Brilc PK et al., 2019 Cell Mol Immunology). PVR expression is upregulated on both malignant cells and tumor-infiltrating myeloid cells in both humans and mice. PVR- / - mice exhibit reduced tumor growth and metastasis via upregulation of DNAM-1 (CD226) and enhanced effector function of CD8+ T cells and NK cells, respectively. Blockade of programmed cell death protein 1 (PD-1) or both PD-1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is more effective in settings where PVR is limited, suggesting the clinical potential of combination therapy using PD-1 / PD-L1 and PVR blockade. Furthermore, because PD-L1 and PVR expression are independently regulated in clinical settings, it was possible to stratify patients treated with anti-PD-1 antibodies into four groups according to their PD-L1 and PVR expression levels. High expression of PVR in patients with low PD-L1 expression led to an increase in non-responders. This result was further validated using a genetically engineered cancer model. These findings reinforce the importance of PVR as a critical immune checkpoint in tumor immunotherapy (Lee BR et al., JCI.Insight 2020). The involvement of PVR in metastasis was demonstrated by injecting cancer cells into the tail of mice and measuring metastasis to the lung. Upregulated PVR in cancer cells has been shown to trans-interact with platelet counter-receptors, and this trans-interaction promotes cancer cell metastasis to the lung (Morimoto et al., Oncogene (2008) 27, 264-273).

[0006] WO2017 / 149538, by some of the inventors of the present invention, discloses murine antibodies and fragments thereof that bind to PVR, encoding polynucleotide sequences, and hybridoma cells that produce these antibodies.

[0007] U.S. Patent Application Publication No. 2007 / 0041985 discloses molecules that specifically bind to at least one intracellular or extracellular domain of PVR, which molecules have the ability to modulate the receptor-mediated adhesion, trafficking and / or invasive behavior of cells expressing PVR or its derivatives.

[0008] U.S. Patent Application Publication No. 2009 / 0215175 provides molecules (e.g., small molecules, oligonucleotides, polypeptides, antibodies, and antibody fragments) that modulate PVR functions necessary for cell adhesion, trafficking, invasion, and / or metastatic potential, which can be used to treat metastatic cells, metastasis, and cancer.

[0009] WO2021 / 070181, by some of the inventors of the present invention, discloses humanized anti-PVR antibodies that can restore immune activity and upregulate the surface expression of DNAM1 (CD226) on CD8 and NK cells. The humanized antibodies are particularly useful for treating PVR-expressing tumors. One of the disclosed antibodies is NB1088 (referred to herein as NTX1088).

[0010] U.S. Patent Application Publication No. 2022 / 0056146 discloses that humanized antibodies or antigen-binding fragments that bind to the poliovirus receptor (PVR) can be used in the preparation of antibody-drug conjugates (ADCs) that target nucleic acids, peptides and proteins, drugs and radiopharmaceuticals to cancer cells.

[0011] WO2019 / 102456 discloses immunotoxins for treating cancer, and describes anti-PVR antibodies and their immunotoxins for treating glioblastoma.

[0012] Although several ADCs have been approved for clinical use and have shown robust efficacy in several indications, they are associated with significant toxicities that limit their use. Such toxicities may be caused by antibody target recognition on healthy tissues or by drug release into the circulation. Therefore, there is an unmet need to provide novel ADCs that are tumor-specific, safe, and potent and can be used as new cancer therapeutics. Summary of the Invention

[0013] The present invention provides antibody-drug conjugates (ADCs) comprising an antibody, or an antigen-binding portion thereof, specific for human PVR (CD155) and a cytotoxin. According to some embodiments, the antibody is a humanized antibody. In some embodiments, the ADCs provided herein are useful for treating cancers expressing PVR, particularly refractory tumors.

[0014] An ADC designated NTX1088, which contains a specific payload conjugated to a specific humanized anti-PVR antibody, has been disclosed to be highly effective in targeting therapeutic and diagnostic agents to cancer cells, including resistant types of cancer. This ADC was found to be highly specific and to have minimal effects on non-tumor cells (PVR-negative target cells or non-dividing PVR-positive normal cells). The ADC described herein was found to be more effective and safer than previously published anti-PVR ADCs and is believed to be useful in the treatment and diagnosis of various cancers.

[0015] Selecting an appropriate linker and toxin to achieve maximum therapeutic efficacy without excessive toxicity is nontrivial and requires extensive experimentation. The antibody-linker-toxin combination must release the toxin in target cells, effectively killing these cells while minimizing damage to normal cells. Not just any antibody exhibiting high specificity is suitable for ADCs; it must also enable rapid internalization, which is essential for both efficacy and safety, to reduce the chance of off-target release of the ADC. Furthermore, ADC candidate compounds must be designed, synthesized, and tested in vitro and in vivo to determine their efficacy and toxicity. The specific humanized anti-PVR antibodies described herein, particularly NTX1088, have been disclosed to be highly effective and suitable for use in ADCs.

[0016] The ADCs described herein were found to be effective against highly resistant cancers such as glioblastoma, as demonstrated in U87, a common GBM cell model. These results contrast with previous attempts to use other anti-PVR ADCs against highly resistant tumors.

[0017] In one aspect, the invention provides an antibody-drug conjugate (ADC) comprising a humanized anti-PVR antibody or antigen-binding portion thereof conjugated to an active or detectable moiety (payload), wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO:1, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO:2.

[0018] According to some embodiments, the humanized antibody comprises a heavy chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO: 3 (NYWIE), SEQ ID NO: 4 (EIFPGSGRINFNEKFKG), and SEQ ID NO: 5 (TKIYGNSFDY), and a light chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO: 6 (KASQDVGTAVV), SEQ ID NO: 7 (WASSRHE), and SEQ ID NO: 8 (QQYSRYPLT).

[0019] According to some embodiments, the humanized antibody comprises a heavy chain comprising a variable region having an amino acid sequence at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identical to SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the humanized antibody comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 1. According to some embodiments, the humanized antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1.

[0020] According to some embodiments, the humanized antibody comprises a light chain comprising a variable region having an amino acid sequence at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identical to SEQ ID NO: 2. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the humanized antibody comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 2. According to some embodiments, the humanized antibody comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2.

[0021] According to some embodiments, the humanized antibody comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 2 (referred to herein as NTX1088).

[0022] According to some embodiments, the humanized antibody is an IgG antibody. According to some embodiments, the humanized antibody has a heavy chain constant region selected from IgG4 and IgG1. In certain embodiments, the humanized antibody, or antigen-binding portion thereof, is of the IgG4 subclass. In certain embodiments, the humanized antibody, or antigen-binding portion thereof, is of the IgG1 subclass.

[0023] According to some embodiments, the humanized antibody or fragment thereof comprises a human IgG4 constant region with a S228P (also referred to as S241P) substitution in the hinge region.

[0024] According to some embodiments, the humanized antibody comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:18 and a light chain having the amino acid sequence set forth in SEQ ID NO:19.

[0025] According to some embodiments, the humanized antibody, or antigen-binding portion thereof, is a whole antibody, a Fab, a F(ab)2, a single domain antibody, or a single chain variable fragment (scFv).

[0026] According to some embodiments, the active moiety (payload) is a toxin.

[0027] In other embodiments, the payload is a detectable moiety, such as a radioactive or fluorescent moiety.

[0028] Any chemical or biological entity capable of killing or inhibiting the growth of tumor cells in vivo can be used as a toxin in conjunction with the ADCs of the invention. According to some embodiments, the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, and RNA polymerase inhibitors.

[0029] 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).

[0030] According to some embodiments, the toxin is saporin.

[0031] According to some embodiments, the toxin is a maytansine derivative. In particular embodiments, the maytansine derivative is DM4 or DM1.

[0032] According to some embodiments, the toxin is a quinoline alkaloid. According to particular embodiments, the quinoline alkaloid is SN-38.

[0033] According to some embodiments, the toxin is selected from the group consisting of DM4, MMAE, and SN-38. In certain embodiments, the toxin is DM4 or MMAE.

[0034] According to some embodiments, the toxin is a topoisomerase I inhibitor. According to some embodiments, the toxin is a camptothecin derivative. According to an exemplary embodiment, the toxin is exatecan.

[0035] According to certain exemplary embodiments, the ADC comprises a toxin selected from the group consisting of saporin, MMAE, DM1, DM4, SN-38, and exatecan, each possibility representing a separate embodiment of the present invention.

[0036] In some embodiments, the toxin is directly attached to the antibody. In other embodiments, the antibody and the toxin are attached via a linker. In some embodiments, the toxin is covalently attached to the humanized antibody, either directly or via a linker.

[0037] According to some embodiments, the linker is cleavable. According to other embodiments, the linker is not cleavable. According to some embodiments, the cleavable linker is selected from the group consisting of an enzymatically cleavable linker, a pH-sensitive linker, and a reducible linker. According to some embodiments, the linker is an enzymatically cleavable linker. According to certain embodiments, the linker is a pH-sensitive linker. According to some embodiments, the linker is a reducible linker.

[0038] According to some embodiments, the linker is selected from the group consisting of maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-amino-benzyloxycarbonyl (MC-VC-PAB), maleimidomethylcyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)butanoate (sulfo-SPDB), and Lys-PAB-CO (lysine-ρ-aminobenzyl-C═O).

[0039] According to yet other embodiments, the linker comprises a stretch of 1 to 30 amino acid residues. According to some embodiments, the linker comprises two cysteine ​​residues used for cysteine ​​conjugation. According to certain exemplary embodiments, the linker comprises valine (Val) and alanine (Ala) residues. According to some embodiments, the linker is a valine-alanine (VA) linker, i.e., a stretch of 2 to 20 amino acids consisting of Ala and Val residues. In certain embodiments, the linker consists of the amino acids valine-alanine.

[0040] According to some embodiments, the ADC comprises the antibody NTX1088.

[0041] According to some embodiments, the ADC comprises the antibody NTX1088 and a toxin selected from the group consisting of MMAE, MMAF, DM1, DM4, SN-38, and exatecan.

[0042] According to some embodiments, the ADC comprises the antibody NTX1088, the toxin MMAE, and the linker MC-VC-PAB (referred to herein as NTX1088-MMAE). According to some embodiments, the ADC comprises the toxin MMAF and the linker MC (referred to herein as NTX1088-MMAF). According to some embodiments, the conjugate comprises the toxin DM1 and the linker SMCC (referred to herein as NTX1088-DM1). According to some embodiments, the conjugate comprises the toxin DM4 and the linker SPDB (referred to herein as NTX1088-DM4). According to some embodiments, the conjugate comprises the toxin SN38 and the linker Lys-PAB-CO (referred to herein as NTX1088-SN38). According to some embodiments, the conjugate comprises the toxin exatecan and the valine-alanine linker (referred to herein as NTX1088-exatecan).

[0043] According to some embodiments, the ADC comprises an anti-PVR antibody that competes with an antibody described herein for specific binding to a PVR molecule. According to particular embodiments, the ADC comprises an anti-PVR antibody that specifically binds to a PVR molecule in competition with an antibody comprising a heavy chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:3 (NYWIE), SEQ ID NO:4 (EIFPGSGRINFNEKFKG), and SEQ ID NO:5 (TKIYGNSFDY), and a light chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:6 (KASQDVGTAVV), SEQ ID NO:7 (WASSRHE), and SEQ ID NO:8 (QQYSRYPLT).

[0044] According to another aspect, the present invention provides a pharmaceutical composition comprising a conjugate as described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0045] Any mode of administration can be used to deliver the compositions of the present invention to a subject in need thereof, including parenteral and enteral modes of administration.

[0046] According to some embodiments, the pharmaceutical composition is formulated for injection or infusion. According to some embodiments, the pharmaceutical composition is formulated for intravenous (IV) administration. In certain embodiments, the pharmaceutical composition is formulated for intratumoral (IT) administration.

[0047] According to some embodiments, the conjugate or pharmaceutical composition is for use in treating cancer in an individual.

[0048] According to some embodiments, the cancer is a resistant type of cancer. According to some embodiments, the cancer is characterized by expression of PVR. According to some embodiments, the cancer is characterized by high expression of PVR. According to some embodiments, the cancer is characterized by overexpression of PVR.

[0049] In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of liver cancer, lung cancer, colon cancer, glioblastoma, adrenal cancer, uterine cancer, testicular cancer, head and neck cancer, pancreatic cancer, and breast cancer. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the cancer is glioblastoma. According to some embodiments, the cancer is colorectal adenocarcinoma. According to certain embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0050] According to additional embodiments, the cancer is selected from the group consisting of bile duct cancer, colon adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, kidney papillary cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, skin cutaneous melanoma, stomach adenocarcinoma, testicular germ cell tumor, uterine endometrial cancer, bladder urothelial carcinoma, pheochromocytoma and paraganglioma, lung squamous cell carcinoma, mesothelioma, cervical squamous cell carcinoma, cervical adenocarcinoma, kidney chromophobe, stomach cancer, gastroesophageal junction cancer, small cell lung cancer, adrenal cancer, gallbladder cancer, small intestine cancer, and anal cancer, with each possibility representing a separate embodiment of the present invention.

[0051] According to some embodiments, the cancer is selected from the group consisting of lung cancer, cervical cancer, urothelial cancer, pancreatic cancer, ovarian cancer, colon cancer, hepatocellular carcinoma, esophageal cancer, and brain cancer.

[0052] According to some embodiments, the cancer is a hematological cancer.

[0053] According to some embodiments, the hematological cancer is a leukemia, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL); a lymphoma, including Hodgkin's disease and non-Hodgkin's lymphoma; and multiple myeloma.

[0054] According to some embodiments, the cancer is a resistant and / or refractory cancer.

[0055] According to some embodiments, the individual is a human.

[0056] According to some embodiments, the use further comprises an agent that downregulates the activity or expression of an immune co-inhibitory receptor.

[0057] According to some embodiments, the immune co-inhibitory receptor is selected from the group consisting of PD-1, PD-L1, TIGIT, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55 (CD160), LAIR1, SIGLEC10, CD112R, CD112, ILT-4, and 2B4, with each possibility representing a separate embodiment of the present invention.

[0058] According to some embodiments of the invention, the use further comprises use in combination with an additional ADC.

[0059] According to a further aspect, the present invention provides an antibody-drug conjugate (ADC) comprising a humanized anti-PVR antibody conjugated to a toxin for use in treating resistant or refractory cancer, wherein the antibody comprises a heavy chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:3 (NYWIE), SEQ ID NO:4 (EIFPGSGRINFNEKFKG) and SEQ ID NO:5 (TKIYGNSFDY), and a light chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:6 (KASQDVGTAVV), SEQ ID NO:7 (WASSRHE) and SEQ ID NO:8 (QQYSRYPLT).

[0060] According to some embodiments, the antibody comprises a heavy chain comprising a variable region having the sequence set forth in SEQ ID NO:1 and a light chain comprising a variable region having the sequence set forth in SEQ ID NO:2.

[0061] According to an additional aspect, the present invention provides an antibody-drug conjugate (ADC) comprising a humanized anti-PVR antibody conjugated to a toxin for use in treating resistant or refractory cancer, wherein the antibody is NTX1088.

[0062] According to another aspect, the present invention provides a method of treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a conjugate or pharmaceutical composition described herein. In certain embodiments, the cancer is a solid tumor. According to further embodiments, the cancer is a non-solid tumor. In certain embodiments, the cancer is selected from the group consisting of glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, and lung cancer. In certain embodiments, the method of treating cancer comprises preventing or reducing the formation, growth, or spread of metastases in a subject.

[0063] According to some embodiments, the cancer is a resistant or refractory cancer.

[0064] According to some embodiments, the cancer is resistant to chemotherapy or radiation.

[0065] According to some embodiments, the individual has become resistant to prior immunotherapy, chemotherapy, or radiation therapy.

[0066] In other embodiments, the cancer is a blood cancer. According to some embodiments, the hematological cancer is selected from leukemia, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphoma, including Hodgkin's disease and non-Hodgkin's lymphoma; and multiple myeloma.

[0067] According to some embodiments, the individual is a human.

[0068] According to some embodiments, the method of treating cancer comprises administering or administering at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, or immunotherapy.

[0069] According to some embodiments, a method of treating cancer comprises administering a conjugate described herein and an additional anti-cancer agent, wherein the additional anti-cancer agent is selected from the group consisting of an immunomodulatory agent, an activated lymphocyte cell, a kinase inhibitor, and a chemotherapeutic agent.

[0070] 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 known as CD134), killer cell immunoglobulin-like receptor (KIR), TIGIT, Nectin-2, CTLA-4, NKG2A, GITR, and any other checkpoint molecule, or a combination thereof. Each possibility represents a separate embodiment of the present 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.

[0071] According to some embodiments, the method of treating cancer comprises preventing or reducing the formation, growth or spread of metastases in a subject.

[0072] Also described herein are methods of manufacturing a composition for treating cancer in an individual afflicted with cancer, the method comprising the step of combining a conjugate described herein with a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, and lung cancer.

[0073] According to another aspect, the invention provides an antibody-drug conjugate (ADC) comprising a humanized anti-PVR antibody or antigen-binding portion thereof conjugated to a radioactive moiety or labeling tag, wherein the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO:1, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO:2.

[0074] The humanized antibody is as described above.

[0075] According to another aspect, the invention provides a method for delivering an antibody-drug conjugate (ADC) to a cell, the method comprising contacting a cell with an ADC, wherein the ADC comprises a humanized anti-PVR antibody, or an antigen-binding portion thereof, conjugated to a toxin, wherein the antibody, or antigen-binding portion thereof, comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO:1, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO:2.

[0076] Any of the ADCs described above may be used in the methods of the present invention. According to certain embodiments, the ADC comprises a humanized antibody, which comprises a heavy chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:3 (NYWIE), SEQ ID NO:4 (EIFPGSGRINFNEKFKG), and SEQ ID NO:5 (TKIYGNSFDY), and a light chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:6 (KASQDVGTAVV), SEQ ID NO:7 (WASSRHE), and SEQ ID NO:8 (QQYSRYPLT).

[0077] According to some embodiments, the cell is a tumor cell.

[0078] According to some embodiments, the method includes administering the ADC to cells of a subject. In certain embodiments, the subject is a human subject.

[0079] According to some embodiments, the cell is a cell of a cancer that expresses or overexpresses PVR. In certain embodiments, the cell is a cell of a cancer selected from the group consisting of melanoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, colon cancer, cervical cancer, renal cancer, lung cancer, thyroid cancer, prostate cancer, brain cancer, kidney cancer, pharyngeal cancer, laryngeal cancer, bladder cancer, liver cancer, fibrosarcoma, endometrial cancer, glioblastoma, sarcoma, myeloma, leukemia, and lymphoma.

[0080] According to some embodiments, the method further comprises the step of additionally administering to the subject an immunomodulatory agent, activated lymphocyte cells, a kinase inhibitor, a chemotherapeutic agent, or other anti-cancer agent.

[0081] According to another aspect, the invention provides a method of delivering an ADC described herein to cells of a subject, said method comprising the step of administering the conjugate to the subject.

[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 PVR in a biological sample of said subject using at least one antibody conjugate as described herein.

[0083] The diagnosed cancer may be any of the cancer types described herein above, particularly cancers that express PVR.

[0084] According to another aspect, the present invention further provides a method for determining or quantifying the expression of PVR, comprising the steps of contacting a biological sample with an antibody conjugate described herein and measuring the level of complex formation.

[0085] According to some embodiments, the method for detecting or quantifying expression of PVR comprises: i. incubating the sample with an antibody conjugate described herein; ii. using the complex to detect bound PVR.

[0086] According to some embodiments, the method comprises: iii. Comparing the amount of (ii) with a standard curve obtained from reference samples containing known amounts of PVR; iv. calculating the amount of PVR in the sample from the standard curve; Further includes:

[0087] According to some particular embodiments, the sample is a body fluid or a solid tissue. In some embodiments, the method is carried out in vitro or ex vivo.

[0088] Kits for measuring PVR expression in a biological sample are also provided, comprising at least one conjugate described herein and a means for measuring PVR expression. In some embodiments, the kit further comprises instructional materials directing use of the kit.

[0089] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description set forth below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given for purposes 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 the detailed description. [Brief explanation of the drawings]

[0090] [Figure 1] Figures 1A-1C show the correlation between PVR expression levels (high and low mRNA levels indicated by thick and thin curves, respectively) and survival probability over time. The correlations were significant (p<0.001) for cervical cancer (Figure 1A), urothelial carcinoma (Figure 1B), and lung cancer (Figure 1C). Data sets were obtained from TCGA and analyzed with Protein Atlas. [Figure 2] FIG. 2 shows widespread and robust expression of human PVR in biopsies of the most prevalent solid tumor types, as measured by immunohistochemistry and assessed by H-score. [Figure 3] Figures 3A-3B demonstrate the enhanced ability of humanized anti-PVR NTX1088-based ADCs to induce killing of the PVR+EGFR+ glioblastoma multiforme (GBM) cell lines U251 (Figure 3A) and U87 (Figure 3B). Biotinylated NTX1088 (black bars) or Erbitux (trademark, anti-EGFR mAb, gray bars) were the catalysts for generating the toxin saporin (ZAP)-based ADCs. [Figure 4] Figure 4 depicts killing induced by the NTX1088-ADC in cells positive and negative for PVR expression. The ADC is based on saporin (ZAP), as in Figure 3. A549 (lung adenocarcinoma) and MDA-MB-231 (triple-negative breast cancer, TNBC) target cells were confirmed to express PVR. Jeg-3 (choriocarcinoma) cells, which do not express PVR, were used to assess the specificity of the ADC. [Figure 5] Figures 5A-5D depict the ability of NTX1088-ADCs to robustly and specifically induce tumor cell killing in vitro. TNBC target cells MDA-MB-231 (Figure 5A) and MDA-MB-468 (Figure 5B), colon cancer cells RKO (Figure 5C), and human PVR-null CHO cells (Figure 5D) were incubated with the presented NTX1088-based ADCs. [Figure 6] Figures 6A-6B depict the ability of NTX1088-based ADCs to induce robust killing of tumor cells representative of intractable cancer types. SKOV-3 cells, a model of ovarian cancer, and U87 cells, a model of GBM (Figures 6A and 6B, respectively), were incubated with NTX1088-ADCs at concentrations ranging from 12 to 0.01 μg / ml. Here, the most potent versions of the ADCs, as depicted in Figure 5, were tested. [Figure 7] Figures 7A-7B depict the in vivo efficacy of select NTX1088-based ADCs against the aggressive GBM model, U87. Female nude mice were subcutaneously implanted with 5x106 U87 cells. Treatment with the primary NTX1088-based ADC was initiated after tumors reached an average size of approximately 160 mm3. A dose of 5 mg / kg was administered, as indicated by the arrow in Figure 7A. The effect of the MMAE-based ADC is shown for individual mice in Figure 7B (excluding other treatments). [Figure 8]Figure 8 shows a representative image of a tissue micrograph from the liver of a female PVRTg21 mouse. This sample demonstrates robust membrane expression of human PVR throughout the tissue, supporting the validity of this strain for ADC toxicity assessment. [Figure 9] Figure 9 depicts the in vivo efficacy of NTX1088-based ADCs against H322M cells, a non-small cell lung cancer (NSCLC) model. Female nude mice were implanted with 5x106 H322M cells subcutaneously (SC). Treatment with the primary NTX1088-based ADC was initiated after tumors exceeded 130 mm3. MMAE-conjugated NTX1088 (black squares) at 5 mg / kg or exatecan-conjugated NTX1088 (vehicle, gray circles) at 10 mg / kg was administered intravenously, as indicated by the arrows. PBS was administered as an experimental control (vehicle, gray filled circles). DETAILED DESCRIPTION OF THE INVENTION

[0091] The present invention provides antibody-drug conjugates, or ADCs, that comprise the humanized anti-PVR antibodies described herein and that are useful for treating cancer. Advantageously, the ADCs described herein comprise nearly fully humanized antibodies, thereby avoiding the risk of adverse immune responses to the antibodies and therefore likely to be safe for use in humans. Furthermore, the ADCs described herein are highly active and suitable for use in treating highly resistant cancers.

[0092] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, one of ordinary skill in the art will understand that the provided embodiments may be practiced without these details. Throughout this specification and the following claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," are to be construed in an open, inclusive sense, i.e., "including, but not limited to," unless the context dictates otherwise. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise. Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments. As used herein, the term "about" refers to an amount that is closer to the specified amount, up to 10% or less.

[0093] According to one aspect, the invention includes an antibody-drug conjugate (ADC), which comprises a humanized anti-PVR antibody, or antigen-binding portion thereof, conjugated to a toxin, wherein the antibody, or antigen-binding portion thereof, comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO:1, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to SEQ ID NO:2.

[0094] According to some embodiments, the humanized antibody is NTX1088, which comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:1 and a light chain variable region having the amino acid sequence of SEQ ID NO:2.

[0095] According to another aspect, the present invention includes an antibody-drug conjugate (ADC), which comprises a humanized anti-PVR antibody, or an antigen-binding portion thereof, conjugated to a toxin selected from the group consisting of SN-38, DM1, DM4, MMAE, and MMAF, wherein the humanized antibody, or antigen-binding portion thereof, comprises: (1) a heavy chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:3 (NYWIE), SEQ ID NO:4 (EIFPGSGRINFNEKFKG), and SEQ ID NO:5 (TKIYGNSFDY); and (2) a light chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:6 (KASQDVGTAVV), SEQ ID NO:7 (WASSRHE), and SEQ ID NO:8 (QQYSRYPLT).

[0096] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence QVQLVQSGAE(L / V)KKPGASVK(I / V)SCKATGYTFSNYWIEW(I / V)(K / R)QAPGQGLEW(I / M)GEIFPGSGRINFNEKFKGR(A / V)TFTADTSI(D / S)T(T / A)YM(Q / E)LS(S / R)L(T / R)SDD(S / T)AVYYCARTKIYGNSFDYWGQGT(T / L)VTVSS (SEQ ID NO: 9), and a heavy chain variable region comprising the amino acid sequence DI(M / Q)MTQSPS(F / S)LSASVGDRVTITC(K / R)ASQDVGTAV(V / A)WYQQKPGKAPK(L / S)LIYWASSRHEGVP(D / S)RF(T / S)GSGSGTDFTLTISS and a light chain variable region comprising: LQ(S / P)EDFA(D / T)YFCQQYSRYPLTFGQGT KLEIK (SEQ ID NO: 10).

[0097] According to some embodiments, the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, 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:15, SEQ ID NO:16, and SEQ ID NO:17. SEQ ID NOs:11-17 are humanized variants of NTX1088 selected based on improved productivity and can be assembled into complete V-region sequences lacking important T-cell epitopes, as described in WO2021070181. This variant comprises five heavy chains (VH1-VH5) and four light chains (Vκ1-Vκ4) containing an N56E substitution.

[0098] According to some embodiments, the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, and the light chain comprises a variable region having an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0099] According to some embodiments, the humanized antibody comprises a combination of a heavy chain variable region and a light chain variable region, the combination comprising: i. a heavy chain variable region sequence set forth in SEQ ID NO: 1 and a light chain variable region sequence set forth in SEQ ID NO: 2; ii. a heavy chain variable region sequence set forth in SEQ ID NO: 12 and a light chain variable region sequence set forth in SEQ ID NO: 16; iii. a heavy chain variable region sequence set forth in SEQ ID NO: 13 and a light chain variable region sequence set forth in SEQ ID NO: 2; iv. A heavy chain variable region sequence set forth in SEQ ID NO: 13 and a light chain variable region sequence set forth in SEQ ID NO: 16; v. a heavy chain variable region sequence set forth in SEQ ID NO: 12 and a light chain variable region sequence set forth in SEQ ID NO: 2; vi. a heavy chain variable region sequence set forth in SEQ ID NO: 1 and a light chain variable region sequence set forth in SEQ ID NO: 16; vii. a heavy chain variable region sequence set forth in SEQ ID NO: 14 and a light chain sequence set forth in SEQ ID NO: 2; viii. The heavy chain variable region sequence set forth in SEQ ID NO: 14 and the light chain variable region sequence set forth in SEQ ID NO: 16 are selected from the group consisting of:

[0100] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises the amino acid sequence identical to that set forth in SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence identical to that set forth in SEQ ID NO:2.

[0101] Conjugates of the present invention include humanized antibodies as described herein. Antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Thus, antibodies include, but are not limited to, full-length antibodies, fragments and portions thereof that retain their binding specificity, including any specific binding portion, including those having any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including, but not limited to, Fab, F(ab'), Fv, and scFv (single-chain or related entities). Monoclonal antibodies are typically one of a substantially homogeneous antibody composition, such that individual antibodies within a monoclonal antibody composition are identical except for naturally occurring mutations that may be present in minor amounts. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0102] The term "antibody" is used herein 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. This term also encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies, multispecific antibodies, e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, and includes 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.

[0103] Although several methods are known in the art for determining the CDR sequences of a given antibody molecule, there is no standard, unambiguous method. Determination of CDR sequences from antibody heavy and light chain variable regions can be performed according to any method known in the art, including, but not limited to, the KABAT, Chothia, and IMGT methods. The selected set of CDRs may include sequences identified by one or more methods; for example, some CDR sequences may be determined using KABAT and some CDR sequences may be determined using IMGT. In some embodiments, the CDR sequences of mAb variable regions are determined using the IMGT method. For example, CDR determination is performed according to Kabat (Wu TT and Kabat EA, J Exp Med, 1970;132:211-50) and IMGT (Lefranc MP et al., Dev Comp Immunol, 2003,27:55-77).

[0104] When "CDR having a sequence" or similar terminology is used, options in which the CDR comprises the sequence specified are included as well as options in which the CDR consists of the sequence specified.

[0105] The provided antibody may be an antibody fragment. The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the same antigen as the intact antibody. 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, and the single-chain antibody fragment contains a variable heavy chain region and / or a variable light chain region, such as an scFv.

[0106] A "humanized" antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the framework region (FR) amino acid residues are derived from human FRs. A humanized antibody may also comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody generally 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 of the FR residues of a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0107] Amino acid residues in the Fc domain can be substituted to render them ineffective, meaning that the Fc domain does not bind to an Fc receptor or can bind with such low affinity and / or avidity that binding does not result in Fc receptor signaling. The Fc domain can be ineffective for binding to an Fcγ receptor. Examples of Fcγ receptors to which the Fc domain can be ineffective include, but are not limited to, FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a), FcγRIIIA (CD16a) F158 variant, FcγRIIIA (CD16a) V158 variant, or FcγRIIIB (CD16b). The Fc domain can have one or more, two or more, three or more, or four or more amino acid substitutions that reduce binding of the Fc domain to an Fc receptor.

[0108] According to some embodiments, the complex comprises a humanized antibody having a mutated Fc domain that prevents FcγR-mediated internalization. According to some embodiments, the humanized antibody comprises an Fc-null domain. According to certain embodiments, the Fc domain is nullified for binding to Fcγ receptors.

[0109] As used herein, "Fc null" refers to a domain that binds weakly or not at all to one or more Fcγ receptors.

[0110] antibody-drug conjugates The present invention provides a conjugate comprising a humanized antibody disclosed herein and a toxin.

[0111] According to some embodiments, the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, and RNA polymerase inhibitors, with each possibility representing a separate embodiment of the present invention.

[0112] In certain embodiments, the toxin is a microtubule-disrupting agent. In an exemplary embodiment, the toxin is an auristatin or a derivative thereof. In certain embodiments, the auristatin derivative is monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF).

[0113] According to some embodiments, the toxin is a saponin.

[0114] According to some embodiments, the toxin is a maytansine derivative. According to particular embodiments, the maytansine derivative is DM4 or DM1.

[0115] According to some embodiments, the toxin is a quinoline alkaloid. According to particular embodiments, the quinoline alkaloid is SN-38.

[0116] According to some embodiments, the toxin is a topoisomerase I inhibitor. According to some embodiments, the toxin is a camptothecin derivative. According to certain exemplary embodiments, the toxin is exatecan.

[0117] According to additional embodiments, the toxin is selected from the group consisting of MMAE, MMAF, saporin, DM4, DM1, SN-38, calicheamicin, DXd, exatecan, PBD, duocarmycin, sandramycin, α-amanitin, chaetocin, CYT997, daunorubicin, 17-AAG, agrochelin A, doxorubicin, methotrexate, colchicine, cordycepin, epothilone B, hygrolysine, herboxidiene, ferulenol, curubrin, paclitaxel, englerin A, taltoburin, triptolide, cryptophycin, and nemorubicin. Each possibility represents a separate embodiment of the present invention.

[0118] According to some embodiments, the toxin is SN-38. According to some embodiments, the toxin is DM1. According to some embodiments, the toxin is DM4. According to some embodiments, the toxin is MMAE. According to some embodiments, the toxin is MMAF.

[0119] In some embodiments, the antibody is directly conjugated to the toxin. In other embodiments, the antibody and the toxin are conjugated via a linker. In some embodiments, the humanized antibodies described herein are covalently conjugated to the toxin.

[0120] According to some embodiments, the linker is cleavable. According to additional embodiments, the linker is not cleavable.

[0121] According to some embodiments, the linker is cleaved in response to a change in pH or redox potential. According to some embodiments, the linker is cleaved upon contact with a lysosomal enzyme.

[0122] According to some embodiments, the linker is 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)valerate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl)aminobenzoate (SLAB), 6-maleimidocaproyl-valine-citrulline ...MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline (MP), 6-maleimidocaproyl-valine-citrulline ( and a moiety selected from the group consisting of phosphorus-p-aminobenzyloxycarbonyl (MC-vc-PAB), Val-Cit-PABC, N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 3-(pyridin-2-yldithio)propionate (SPDP), Phe-Lys(Fmoc)-PAB, Aloc-D-Ala-Phe-Lys(Aloc)-PAB-PNP, Boc-Phe-(Alloc)Lys-PAB-PNP, and perfluorophenyl 3-(pyridin-2-yldisulfanyl)propanoate. Each possibility represents a separate embodiment of the present invention.

[0123] According to another aspect, the present invention provides a pharmaceutical composition comprising a conjugate as described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0124] According to some embodiments, the pharmaceutical composition according to the present invention is for use in treating cancers characterized by expression of PVR. According to other embodiments, the pharmaceutical composition according to the present invention is for use in treating cancers characterized by overexpression of PVR. Cancer types associated with PVR expression and overexpression can be identified using known databases, such as The Cancer Genome Atlas (TCGA). According to certain embodiments, cancers treatable with the composition according to the present invention include glioblastoma multiforme (GBM), adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), liver hepatocellular carcinoma (LIHC), colon and rectal adenocarcinoma (COAD, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), papillary renal cell carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), and endometrioid carcinoma (Uterine Endometrioid). Cancer (UCEC), 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), breast cancer (BRCA), low-grade glioma (LGG), diffuse large B-cell lymphoma (DLBC). Each possibility represents a separate embodiment of the present invention.

[0125] In some embodiments, the ADCs provided herein are useful for treating refractory tumors. As used herein, the term "refractory" refers to cancers associated with poor clinical outcomes, e.g., insufficient effectiveness of known therapies. Non-limiting examples of "refractory" tumors include lung cancer, pancreatic cancer, ovarian cancer, colon cancer, esophageal cancer, and brain cancer.

[0126] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of having, or at risk of having at least one disease for which the described compositions and methods are useful for treating. According to some embodiments, the individual is a mammal. According to some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​According to some embodiments, the individual is a human.

[0127] As used herein, the term "effective amount" refers to the amount of a therapeutic agent that, when administered to a mammal, produces a biological effect. Biological effects include, but are not limited to, a decrease in tumor growth, a reduction in tumor metastasis, or an increase in the survival time of an animal bearing a tumor. A "therapeutic amount" is a concentration of a drug calculated to produce a therapeutic effect. A therapeutic amount encompasses a range of dosages capable of inducing a therapeutic response in a population of individuals. The mammal may be a human individual. The human individual may be afflicted with a tumor or suspected of being afflicted with a tumor.

[0128] As used herein, the term "combination" or "combination therapy" can refer to either simultaneous administration of the combined items or sequential administration of the combined items. As described herein, when combination refers to sequential administration of the items, the items can be administered in any temporal order.

[0129] As used herein, the term "checkpoint inhibitor" refers to an agent that inhibits biological molecules ("checkpoint molecules") that are produced by an organism and negatively regulate the anti-tumor / cancer activity of T cells in that organism. Checkpoint molecules include, but are not limited to, PD-1, PD-L-1, PD-L-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, TIGIT, IDO, KIR, A2AR, B7-H3, B7H4, CEACAM1, and CD112R.

[0130] The molecules of the present invention as active ingredients are dissolved, dispersed, or mixed in a pharmaceutically acceptable excipient that is compatible with the active ingredient, as is well known. Suitable excipients include, for example, water, saline, phosphate-buffered saline (PBS), glucose, 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.

[0131] As used herein, the term "treatment" refers to both therapeutic and prophylactic or preventative treatment. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0132] The terms "cancer" and "tumor" refer to a physiological condition in mammals characterized by unregulated cell growth. Cancer is a type of disease in which a group of cells exhibits uncontrolled or unwanted growth. Cancer cells can also spread to other locations, leading to the formation of metastases. The spread of cancer cells within the body occurs, for example, via lymph or blood. Uncontrolled growth, invasion, and metastasis formation are also referred to as malignant characteristics of cancer. These malignant characteristics distinguish cancer from benign tumors, which usually do not undergo invasion or metastasis.

[0133] According to some embodiments, the ADCs described herein are used to treat refractory or resistant cancers.

[0134] As used herein, the term "resistant cancer" refers to a cancer that is so insensitive to treatment with abundant anti-cancer drugs (e.g., chemotherapy) or radiation that it does not affect cancer growth and its symptoms are not improved, alleviated, or relieved by drug or radiation therapy, or to treatment with immunomodulatory agents, including, but not limited to, antibody-based molecules, immune cells, CAR cells, cytokines, etc. The term also refers to cases where certain treatments, such as radiation, are not feasible. As used herein, the term "resistant cancer" is used interchangeably with "refractory" cancer.

[0135] According to some embodiments, the ADCs are used to treat subjects with refractory cancer. According to particular embodiments, the subjects have already been treated with chemotherapy and / or radiation.

[0136] In some embodiments, the resistant cancer is selected from the group consisting of glioblastoma, pancreatic cancer, colon cancer, liver cancer, lung cancer, skin cancer, ovarian cancer, esophageal cancer, and endometrial cancer. In some embodiments, the resistant cancer is glioblastoma (GBM).

[0137] According to some embodiments, the cancer is a resistant type of cancer. According to some embodiments, the cancer is characterized by expression of PVR. According to some embodiments, the cancer is characterized by high expression of PVR. According to some embodiments, the cancer is characterized by overexpression of PVR, with cancer cells containing, on average, 2, 3, 4, or 5 times more PVR molecules than corresponding non-cancerous cells. According to some embodiments, the cancer is characterized by having a tumor proportion score (TPS) of greater than 50% as assessed by immunohistochemistry (IHC). In certain embodiments, the cancer is characterized by having an H-score (histochemical score) of at least 1.

[0138] According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen that includes administering at least one additional anti-cancer agent.

[0139] According to some embodiments, the anti-cancer agent is selected from the group consisting of antimetabolites, antimitotic agents, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginase, alkylating agents, antitumor antibiotics, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0140] In some embodiments, the antimetabolite is selected from the group consisting of cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. In some embodiments, the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine. In some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irinotecan. In some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, and procarbazine. In some embodiments, the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin. According to some embodiments, the topoisomerase II inhibitor is selected from the group consisting of etoposide and teniposide. Each possibility represents a separate embodiment of the present invention.

[0141] According to another aspect, the present invention provides a method for treating cancer in an individual suffering from cancer, the method comprising the step of administering to the individual a therapeutically effective amount of a conjugate or 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 selected from the group consisting of pembrolizumab, nivolumab, AMP-514, tislelizumab, spartalizumab, or and a PD-1-binding fragment. In certain embodiments, the PD-1 signaling inhibitor is an antibody that specifically binds to PD-L-1 or PD-L-2. In certain embodiments, the antibody that specifically binds to 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 PD-1 signaling inhibitor comprises an Fc fusion protein that binds to 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 PD-1 signaling inhibitor 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 (BMS202); (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] glycinamide, N-(2-mercaptoacetyl)-L-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.

[0142] Also described herein are methods of preparing a composition for treating cancer in an individual afflicted with cancer, the method comprising admixing the conjugate with a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of glioblastoma, colon cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, prostate cancer, and lung cancer.

[0143] In 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, with each possibility representing a separate embodiment of the present invention. [Example]

[0144] The present invention will now be described in a non-limiting manner with reference to the following examples, which, together with the above descriptions,

[0145] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, immunological, and recombinant DNA techniques. Such techniques are well known in the art. Other general references to well-known procedures are provided throughout the specification for the convenience of the reader.

[0146] (Example 1) High PVR mRNA expression correlates with poor survival in various cancers Based on the FPKM value of PVR, patients were classified into two expression groups, and the correlation between PVR expression level and patient survival was examined. The prognosis of patients in each group was examined using Kaplan-Meier survival rates. Figure 1 shows the plot for conditions in which PVR expression is defined as an unfavorable prognostic gene (meaning that the risk of death in the high expression group was significantly higher than in the low expression group, p<0.001), and in which survival rates were significantly worse when compared using the log-rank test.

[0147] (Example 2) PVR is expressed in the majority of solid tumors We evaluated the expression levels of PVR in different human malignancies. PVR expression was detected by standard immunohistochemistry using a commercially available rabbit monoclonal antibody clone D3G7H and cancer tissue microarrays. Staining was digitized, intensity quantified, and H-scores were calculated within and between conditions. Figure 2 shows that PVR expression levels were elevated in most conditions analyzed, with varying frequency. These data support the therapeutic potential of targeting PVR via NTX1088-conjugated ADCs across multiple conditions. Elevated PVR expression was observed in liver cancer, colon cancer, adrenal cancer, uterine cancer, testicular cancer, squamous cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer, bladder cancer, prostate cancer, bile duct cancer, skin cancer, HNSCC cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, and melanoma.

[0148] (Example 3) NTX1088 acts as an ADC promoter and inhibits EGFR + PVR + Superior to Erbitux in inducing cell killing To assess the potential of the mAbs for use as ADCs, streptavidin-saporin (ZAP, IT-27-250 (ATS)) was used. The mAbs shown in Figure 3 were biotinylated at a 1:1 ratio using a biotinylation kit (Ab207195, Abcam). Figure 3 shows the EGFR + PVR + 3A and 3B depict the results of an initial screen involving NTX1088 and Erbitux targeting GBM cell lines U251 and U87 (respectively). 3Cells were seeded and allowed to adhere for 4-6 hours. ADCs were added, and cells were incubated with the ADCs for 96 hours. Tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega G9242) standard protocol. NTX1088 demonstrated significantly greater killing of both target cells compared to Erbitux.

[0149] (Example 4) NTX1088-ADC specifically kills targets only when PVR is expressed To assess the specificity of NTX1088-based ADCs, streptavidin-saporin was used as described in Figure 3. Figure 4 depicts the efficacy of a single dose (10 nM) against A549 (lung adenocarcinoma) and MDA-MB-231 (TNBC) target cells, which have been confirmed to express PVR. Jeg-3 (choriocarcinoma) cells, which do not express PVR, were used to assess the specificity of the ADCs. 2x10 per well 3 Cells were seeded and allowed to adhere for 4-6 hours. The ADC was added, and the cells were incubated with the ADC for 96 hours. After 96 hours, assay samples were collected and tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega G9242) standard protocol. As shown in Figure 4, specific killing of PVR-expressing positive targets was more than 20-fold higher than killing of PVR-negative target cells, confirming the high specific activity and demonstrating the potential safety of the NTX1088-based ADC.

[0150] (Example 5) Evaluation of NTX1088-based ADCs by linker-payload matrix The proposed linker-payload combinations were selected according to the desired release mechanism in combination with different payloads. These ADCs were produced according to standard protocols from Abzena. Briefly, the mAb was reduced and incubated with an excess of linker-payload to achieve a desired drug-to-antibody ratio (DAR) of 4 for linker-payloads other than SN-38, which had a DAR of 8. The final products were then purified, and the DAR was determined by LC / MS analysis. A summary of the ADC properties is shown in Table 1. [Table 1]

[0151] (Example 6) Efficacy evaluation of NTX1088-based ADCs in a linker-payload matrix Selected tumor cell lines representing a variety of solid tumors were used to evaluate the in vitro efficacy of various linker-payload combinations listed in Table 1. 2x10 target cells were presented per well. 3Cells were seeded and allowed to adhere for 4–6 hours. ADCs were added at 4-fold dilutions to concentrations ranging from 12 to 0.75 μg / ml, and cells were incubated with the ADCs for an additional 72 hours. Tumor cell killing was then assessed using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega G9242) according to standard protocols. Robust killing of MDA-MB-231 cells (triple-negative breast cancer), MDA-MB-468 cells (triple-negative breast cancer), and RKO cells (colorectal adenocarcinoma) is depicted in Figures 5A–5C, respectively. CHO cells not expressing human PVR were not killed by most linker-payload combinations, except for carbonate-linked SN-38, which is released into the medium in a nonspecific manner. Cytotoxicity was significant and specific for all payload types, with DM4 having the most potent effect. Of note, SN-38 at 0.75 μg / ml caused minimal killing of CHO cells but over 70% killing of RKO and MDA-MB-468 cells, again suggesting the presence of a significant PVR-specific killing component in this ADC.

[0152] Although the type of target clearly influenced activity, the most promising candidates were NTX1088-MMAE, NTX1088-DM4, and NTX1088-SN38.

[0153] (Example 7) NTX1088-based ADCs are potent against cell lines representative of refractory solid tumors The in vitro efficacy of selected lead compounds, linker-payload NTX1088-ADCs, was evaluated using selected tumor cell lines representing difficult-to-treat solid cancers. Killing assays were performed as described in Figure 5 using 4-fold dilutions over the ADC range of 12 to 0.01 μg / ml. Robust killing of SKOV-3 cells (ovarian cancer) and U87 cells (GBM) is shown in Figures 6A-6B, respectively. Significant killing (p<0.01) was observed for all linker payloads at all doses up to 0.75 μg / ml. Surprisingly, killing of U87 cells was significantly lower with both NTX1088-DM4 and NTX1088-MMAE, and the highest ADC concentrations did not reach the EC-50, suggesting this is a highly resistant cancer model.

[0154] (Example 8) Select NTX1088-based ADCs result in tumor regression in an in vivo model of invasive GBM Female nude mice were treated with 5x10 cells in 1:1 Matrigel. 6 U87 cells were injected subcutaneously (SC). The average tumor volume was 160 mm 3 Once the mice reached 100 mg / kg / day, they were randomly assigned to four groups (n = 7 per group) and blinded to receive intravenous injections of either PBS (vehicle), NTX1088-MMAE, NTX1088-DM4, or NTX1088-SN38 at a dose of 5 mg / kg every four days for three consecutive doses, followed by a single dose of 5 mg / kg 25 days after the last dose.

[0155] As shown in Figure 7, despite the high efficacy of NTX1088-SN38 against the U87 target observed in vitro (Figure 6B), there was no effect on tumor growth in the group treated with this ADC compared to the experimental control group. NTX1088-DM4 resulted in nearly complete tumor regression, consistent with the high efficacy of this linker payload observed in vitro. Finally, although not anticipated by the in vitro results, NTX1088-MMAE was the most robust ADC, completely regressing tumors. These results contrast with previous attempts to use other anti-PVR ADCs against highly resistant tumor cells such as U87. For example, in WO2019 / 102456, the described anti-PVR ADC did not affect tumor growth of U87 cells (Figure 6B of WO2019 / 102456). Unexpectedly, the tested NTX1088-DM4 and NTX1088-MMAE (but not NTX1088-SN38) were able to induce tumor regression. These results confirm that NTX1088-ADC is a novel and unobvious therapeutic agent.

[0156] These combined findings support the clinical development of a new class of agents, NTX1088-based ADCs, for the treatment of solid tumors.

[0157] These in vivo findings were unexpected given the very different activity profiles seen in the in vitro assays (Figures 5 and 6).

[0158] (Example 9) The NTX1088 ADC demonstrated a clear safety profile in vivo when tested in mice expressing human PVR TgPVR21 mice are a strain that expresses full-length human PVR (doi:10.1073 / pnas.88.3.951) and are recognized as a mouse model for testing the pathogenicity of poliovirus vaccines. We used these animals to evaluate the safety of the most potent NTX1088-ADC. Based on the above results, we selected NTX1088-MMAE. TgPVR21 mice have been reported to express human PVR in multiple tissues. Because the human liver expresses the highest levels of PVR among all normal tissues (The Human Protein Atlas (proteinatlas.org)), we considered the liver to be the most relevant target organ. To examine human PVR expression on mouse hepatocytes, we used the IHC described above (Example 3) and the commercially available anti-PVR antibody clone D37GH, which does not cross-react with mouse PVR. Figure 8 shows representative histological data obtained from the liver of a female PVRTg21 mouse. The tested samples demonstrated robust membrane expression of PVR throughout the tissues. Given the nature of the MMAE toxin, its toxicity should be evident upon acute exposure. Next, three PVRTg21 female mice (13 weeks old) per group received a single intravenous injection of PBS or 7.5 or 10 mg / kg of NTX1088-MMAE. Toxicity was assessed by collecting blood samples from the treated mice on days 1 and 10 post-injection (Tables 2 and 3, respectively). Liver enzyme levels were measured and normalized to those of PBS-treated animals. No significant changes in liver enzymes were observed at any time point in either treatment group. No weight loss was observed during this period (data not shown). These findings suggest that NTX1088 is safe and well-tolerated, even when PVR is widely expressed in normal tissues. The observed safety profile (no toxic effects) is a result of the combination of an antibody and a specific payload that selectively targets dividing cells. This result is highly unexpected, especially considering the robust antitumor effect seen with a fraction of this dose (Example 8). Taken together, these results demonstrate that NTX1088 can be used as a safe and potent ADC in the treatment of PVR-positive cancers. [Table 2] [Table 3]

[0159] (Example 10) NTX1088-based ADC demonstrates high therapeutic efficacy in the NSCLC model H322M Female nude mice were treated with 5x10 cells in 1:1 Matrigel. 6 H322M cells were injected subcutaneously (SC). The tumors were 130 mm 3 At this time, mice were randomized into three groups (n = 6 per group) and intravenously injected with either PBS, 5 mg / kg NTX1088-MMAE, or 10 mg / kg NTX1088-exatecan in a blinded manner on days 0, 4, and 9. As shown in Figure 9, all ADC-treated groups showed initial tumor growth, followed by robust tumor inhibition compared to the control (PBS) group and tumor regression compared to tumor volume at the start of treatment. These results demonstrate that NTX1088-based ADCs exert robust therapeutic effects when conjugated to tubulin- or TopoI-targeting agents. The kinetics of efficacy differed between the two NTX1088 ADCs, demonstrating potentially unpredictable differences in the impact of specific Ab-drug combinations. array

[0160] [Table 4]

Claims

1. An antibody-drug conjugate (ADC) comprising a humanized anti-PVR antibody or an antigen-binding portion thereof, wherein the antibody or the antigen-binding portion comprises a heavy chain and a light chain, the heavy chain comprising a variable region having an amino acid sequence identical to at least about 90% of SEQ ID NO: 1, and the light chain comprising a variable region having an amino acid sequence identical to at least about 90% of SEQ ID NO:

2.

2. The antibody-drug conjugate according to claim 1, wherein the heavy chain variable region includes the CDR sequence described in SEQ ID NO: 3 (NYWIE), SEQ ID NO: 4 (EIFPPGSGRINFNEKFKG), and SEQ ID NO: 5 (TKIYGNSFDY), and the light chain variable region includes the CDR sequence described in SEQ ID NO: 6 (KASQDVGTAVV), SEQ ID NO: 7 (WASSRHE), and SEQ ID NO: 8 (QQYSRYPLT).

3. The antibody-drug conjugate according to claim 1, wherein the humanized antibody comprises a heavy chain having a variable region having the amino acid sequence described in SEQ ID NO: 1 and a light chain having a variable region having the amino acid sequence described in SEQ ID NO: 2 (referred to as NTX1088).

4. The humanized antibody or its antigen-binding portion is a monoclonal antibody, Fab, F(ab) ２ The antibody-drug conjugate according to claim 1, wherein the conjugate is a single-domain antibody or a single-chain variable fragment (scFv).

5. The antibody-drug conjugate according to claim 1, wherein the toxin is selected from the group consisting of topoisomerase inhibitors, microtubule inhibitors, DNA synthesis inhibitors, and RNA polymerase inhibitors.

6. The antibody-drug conjugate according to claim 1, wherein the toxin is selected from the group consisting of auristatin or its derivatives, mytansin derivatives, and quinoline alkaloids.

7. The antibody-drug conjugate according to claim 1, wherein the toxin is selected from the group consisting of exatecan, DM4, MMAE, and SN-38.

8. The antibody-drug conjugate according to claim 1, wherein the antibody and the toxin are linked via a linker.

9. The antibody-drug conjugate according to claim 8, wherein the linker is cleavable.

10. The antibody-drug conjugate according to claim 9, wherein the cleavable linker is selected from the group consisting of an enzymatically cleavable linker, a pH-sensitive linker, and a reductible linker.

11. The antibody-drug conjugate according to claim 8, wherein the linker comprises a portion selected from the group consisting of valine-alanine (VA), maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-VC-PAB), maleimidomethylcyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), and Lys-PAB-CO (lysine-ρ-aminobenzyl-C=O).

12. The antibody-drug conjugate according to claim 3, wherein the conjugate comprises (1) the toxin exatecan and the linker valine-alanine (VA) (referred to as NTX1088-exatecan), (2) the toxin MMAE and the linker MC-VC-PAB (referred to as NTX1088-MMAE), (3) the toxin MMAF and the linker MC (referred to as NTX1088-MMAF), (4) the toxin DM1 and the linker SMCC (referred to as NTX1088-DM1), (5) the toxin DM4 and the linker SPDB (referred to as NTX1088-DM4), or (6) the toxin SN38 and the linker Lys-PAB-CO (referred to as NTX1088-SN38).

13. The antibody-drug conjugate according to claim 1, wherein the antibody comprises a heavy chain variable region having the sequence described in SEQ ID NO: 1 and a light chain variable region having the sequence described in SEQ ID NO: 2, the linker comprises valine-alanine (VA), and the toxin is exatecan.

14. A pharmaceutical composition comprising the antibody-drug conjugate described in claim 1 and a pharmaceutically acceptable excipient, carrier, or diluent.

15. A pharmaceutical composition according to claim 14 for use in the treatment of cancer.

16. The pharmaceutical composition according to claim 15, wherein the cancer is an intractable cancer.

17. The pharmaceutical composition according to claim 15, wherein the cancer is selected from the group consisting of prostate cancer, ovarian cancer, colorectal cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, glioblastoma, adrenal cancer, uterine cancer, skin cancer, testicular cancer, and head and neck cancer.

18. A complex comprising a humanized anti-PVR antibody or an antigen-binding portion thereof, compounded with a detectable portion, a radioactive portion, or a labeled tag, wherein the antibody or the antigen-binding portion comprises a heavy chain and a light chain, the heavy chain comprising a variable region having at least about 90% the same amino acid sequence as SEQ ID NO: 1, and the light chain comprising a variable region having at least about 90% the same amino acid sequence as SEQ ID NO:

2.

19. An in vitro method for diagnosing or predicting cancer, comprising the step of determining the expression level of PVR in a biological sample using at least one complex described in claim 18.

20. An antibody-drug conjugate (ADC) comprising a toxin-bound humanized anti-PVR antibody for use in the treatment of drug-resistant or refractory cancer, wherein the antibody comprises a heavy chain having a variable region having the sequence described in SEQ ID NO: 1 and a light chain having a variable region having the sequence described in SEQ ID NO: 2.

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