BR112018067522B1 - Isolated monoclonal antibody and its variant, polynucleotide sequence, plasmid, pharmaceutical composition and use thereof. - Google Patents

Abstract

The present invention provides monoclonal antibodies that recognize the poliovirus receptor (PVR) and inhibit its binding to the T-cell immunoreceptor with Ig and ITIM domains (TIGIT). The present invention further provides pharmaceutical compositions comprising the antibodies and methods for their use in cancer immunotherapy, infection treatment, and diagnosis.

Description

FIELD OF THE INVENTION

[0001] The invention is in the field of immunotherapy and relates to antibodies and fragments thereof that bind to the poliovirus protein receptor, to polynucleotide sequences encoding these antibodies, and to hybridoma cells that produce these antibodies. The invention further relates to diagnostic and therapeutic compositions comprising these antibodies and to methods of treating and diagnosing diseases, particularly cancer, using these monoclonal antibodies.

BACKGROUND OF THE INVENTION

[0002] Cancer immunotherapy is used to generate and enhance an antitumor immune response, for example, through treatment with antibodies specific to antigens in tumor cells, with fusions of antigen-presenting cells with tumor cells, or through specific activation of antitumor T cells. The ability to recruit immune cells (e.g., T cells) against tumor cells in a patient provides a therapeutic modality to combat types of cancer and metastasis that, until now, have been considered incurable.

[0003] The T cell-mediated immune response includes multiple sequential steps regulated by a balance between co-stimulatory and co-inhibitory signals that control the magnitude of the immune response. Inhibitory signals, called immune checkpoints, are crucial for maintaining self-tolerance and also for limiting immune-mediated collateral damage to tissue. These signals are altered as an infection or immune provocation clears, worsens, or persists, and these alterations affect the T cell response and reshape the immune response.

[0004] The expression of immune checkpoint proteins can be regulated by tumors. For example, upregulation of programmed death ligand-1 (PD-L1) on the surface of cancer cells allows them to escape the host immune system by inhibiting T cells through PD-1 binding that might otherwise attack these tumor cells. Therefore, immune checkpoints represent significant barriers to the activation of functional cellular immunity against cancer. Consequently, antagonistic antibodies specific to inhibitory ligands on immune cells are considered viable anticancer agents and are being evaluated in clinics (e.g., Nivolumab and Pembrolizumab). Another example of an immune checkpoint molecule is the T cell immunoreceptor with Ig and ITIM domains (TIGIT). TIGIT is a co-inhibitory molecule expressed on various immune cells, including T cells and Natural Killer cells (NK cells). TIGIT binds with high affinity to the poliovirus receptor (PVR).

[0005] The poliovirus receptor (PVR), also called CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. It has been previously described as a tumor antigen and as a potential target for therapeutic intervention, since its expression is upregulated in neuroectodermal cancers, including glioblastoma multiforme, medulloblastoma and colorectal carcinoma (Solecki et al., J. Biol. Chem. 2002, 277; 25697-700), as well as in pancreatic cancer (Nishiwada et al., Anticancer Res. 2015, 35(4), 2287-97). PVR is also known to enhance serum-induced activation of Ras-Raf-MEK-ERK signaling, the upregulated D2 and downregulated p27Kip1 cyclins, eventually shortening the G0/G1 phase of the cell cycle (Kakunaga 2004, J. Biological Chemistry, 279, 36419-36425). For this reason, it is contemplated that blocking PVR in tumor cells reduces tumor cell viability. PVR also plays a critical role in angiogenesis and is suggested to regulate VEGF-induced angiogenesis by controlling the interaction of VEGFR2 with integrin α(v)β(3), and the VEGFR2-mediated Rap1-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012, 110(5), 716-26). Additionally, PVR forms a complex with IGF1R and participates in Met signaling, and blocking complex formation reduced angiogenesis and cell viability (Lee et al., Scientific Reports 2014, 20, 4, 7139).

[0006] The involvement of PVR in metastasis was demonstrated by injecting cancer cells into the tails of mice and measuring metastasis to the lungs. It was shown that upregulated PVR in cancer cells transinteracts with its counterreceptor on platelets, and that this transinteraction enhances metastasis of cancer cells to the lungs (Morimoto et al., Oncogene (2008) 27, 264-273).

[0007] US Patent Application No. 20070041985 discloses molecules that specifically bind to at least one intracellular or extracellular domain of PVR or any derivative thereof, wherein the molecule has the ability to modulate receptor-mediated adhesion, trafficking and/or invasion behavior of a cell expressing PVR or any derivative thereof.

[0008] US Patent Application No. 20090215175 provides molecules (e.g., small chemical compounds, oligonucleotides, polypeptides, antibodies, and antibody fragments) that modulate PVR functions necessary for cell adhesion, trafficking, invasion, and/or metastatic potential. The molecules can be used for the treatment of cells that have metastatic potential, metastasis, and cancer.

[0009] PCT Application Publication No. WO 2006/124667 discloses the modulation of the zB7R1 protein (TIGIT) via monoclonal antibodies that block the binding of TIGIT to its ligand PVR.

[0010] There is an unmet need to provide additional and more effective, specific, safe and/or stable agents that, alone or in combination with other agents, enhance immune system cells to attack virus- or tumor-infected cells by inhibiting PVR binding to TIGIT.

SUMMARY OF THE INVENTION

[0011] The present invention provides antibodies and fragments thereof that recognize the poliovirus receptor (PVR), prevent its binding to the T-cell immunoreceptor with Ig and ITIM domains (TIGIT), and inhibit suppressive activity on lymphocytes such as T cells and natural killer (NK) cells. The anti-PVR antibodies disclosed herein are capable of binding to PVR present on cancer cells. These antibodies and fragments thereof are characterized by having unique sets of complementarity-determining regions (CDR) sequences, high affinity and high specificity to PVR, and are useful in cancer immunotherapy to combat tumor immune evasion, as stand-alone therapy and in combination with other anticancer agents. The antibodies are also useful in the treatment of viral infections.

[0012] It is now revealed that the high-affinity anti-PVR antibodies disclosed in this document block the TIGIT-PVR interaction and restore NK and T cell activity. The antibodies showed high specificity to human PVR. These properties make the monoclonal antibodies (mAbs) of the present invention valuable candidates for use in cancer immunotherapy, enabling the administration of lower doses with fewer side effects.

[0013] Advantageously, the anti-PVR mAbs according to the invention can induce T cell proliferation better than anti-PD-1 and CTLA-4 mAbs in an in vitro PD-L1 model (A549). The induction effect was shown in peripheral blood mononuclear cells (PMBCs) and purified CD4 and CD8 T cells. Additionally, the PVR mAbs were also able to induce NK cell activation in most of the target cells tested. Furthermore, some of the anti-PVR antibodies described herein have in vitro anticancer activity comparable to that of a known agent, Erbitux®, currently used in therapy. Additionally, some of the anti-PVR antibodies described herein showed a synergistic effect when combined with additional anticancer agents, such as anti-PD-1 and CTLA-1 antibodies and epidermal growth factor receptor (EGFR). Additionally, it was found that some of the anti-PVR antibodies induce antibody-dependent cell-mediated cytotoxicity (ADCC). It is further revealed that some anti-PVR antibodies according to the invention did not exhibit any blocking effect on DNAM1 co-stimulatory signaling, therefore, they have no deletion effect on other immune induction signals.

[0014] Interestingly, despite the high sequence similarity between human and rodent PVR sequences, the antibodies of the present invention are highly specific for human PVR.

[0015] It is further revealed that, unexpectedly, some chimeric monoclonal antibodies comprising human constant chain showed an enhanced effect on immune cell activation compared to their equivalent murine monoclonal antibodies.

[0016] Some of the anti-PVR mAbs described in this document were also able to reduce tumor cell viability in an immunologically independent manner by blocking PVR in tumor cells. Without focusing on any specific mechanisms of action, it is suggested that this activity results from PVR's ability to shorten the G0/G1 phase of the cell cycle.

[0017] According to one aspect, the present invention provides an isolated monoclonal antibody that binds to the poliovirus receptor (PVR), or an antibody fragment thereof comprising at least the antigen-binding portion, wherein the isolated antibody or antibody fragment is selected from the group consisting of: i. three CDRs of a variable heavy chain (HC) region comprising SEQ ID NO: 77 and three CDRs of a variable light chain (LC) region comprising SEQ ID NO: 79, or an analog or derivative thereof having at least 90% sequence identity with said fragment or antibody sequence; ii. three complementarity-determining regions (CDRs) of a variable heavy chain region comprising SEQ ID NO: 69 and three CDRs of a variable light chain region comprising SEQ ID NO: 71, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence; and iii. three CDRs of a variable heavy chain region comprising SEQ ID NO: 73 and three CDRs of a variable light chain region comprising SEQ ID NO: 75, or an analog or derivative thereof having at least 90% sequence identity with said fragment or antibody sequence.

[0018] Antibodies comprising CDR sequences contained in heavy and light chain homologs for SEQ ID Nos: 2, 10, 18, 26, 34 or 42 are also covered by the scope of the present invention. According to some modes, SEQ ID NO: 2 is interchangeable with SEQ ID NO: 69. According to some modes, SEQ ID NO: 10 is interchangeable with SEQ ID NO: 71. According to some modes, SEQ ID NO: 18 is interchangeable with SEQ ID NO: 73. According to some modes, SEQ ID NO: 26 is interchangeable with SEQ ID NO: 75. According to some modes, SEQ ID NO: 34 is interchangeable with SEQ ID NO: 77. According to some modes, SEQ ID NO: 42 is interchangeable with SEQ ID NO: 79.

[0019] There are several known methods in the art for determining the CDR sequences of a given antibody molecule; however, there is no unequivocal standard method. The determination of CDR sequences from the variable regions of antibody heavy and light chains can be performed according to any method known in the art, including, but not limited to, the methods known as KABAT, Chothia, and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using KABAT and some using IMGT, for example.

[0020] According to some embodiments, the isolated monoclonal antibody or fragment comprises the CDR sequences of a monoclonal antibody denoted Anti-PVR 4E5 (or hPVR.07), namely, the three CDR sequences contained in the variable heavy chain region presented in SEQ ID NO: 69 and the three CDR sequences contained in the variable light chain region presented in SEQ ID NO: 71.

[0021] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR1, comprising the sequence GFDFSRYW (SEQ ID NO: 4). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2, comprising the sequence EIHPDSSKINYTPSQ (SEQ ID NO: 6). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR3, comprising the sequence PDGNYNALDYW (SEQ ID NO: 8).

[0022] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR1 heavy chain, which comprises the sequence RYW. According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR1 heavy chain, which comprises the sequence RYWMT (SEQ ID NO: 80). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR3 heavy chain, which comprises the sequence PDGNYNALDY (SEQ ID NO: 82).

[0023] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) the HC CDR1 comprising the sequence GFDFSRYW (SEQ ID NO: 4); (ii) the HC CDR2 comprising the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6); and (iii) the HC CDR3 comprising the sequence: PDGNYNALDYW (SEQ ID NO: 8).

[0024] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) the HC CDR1 comprising the sequence RYW; (ii) the HC CDR2 comprising the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6); and (iii) the HC CDR3 comprising the sequence: PDGNYNALDY (SEQ ID NO: 82).

[0025] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR1 light chain comprising the sequence KASQDVGTAVT (SEQ ID NO: 12). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR2 light chain comprising the sequence WASTRHT (SEQ ID NO: 14). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR3 light chain comprising the sequence QQYSRYPYT (SEQ ID NO: 16). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) the CDR1 LC comprising the sequence KASQDVGTAVT (SEQ ID NO: 12); (ii) the CDR2 LC comprising the sequence: WASTRHT (SEQ ID NO: 14); and (iii) the CDR3 of HC comprises the sequence: QQYSRYPYT (SEQ ID NO: 16).

[0026] According to some specific embodiments, the isolated monoclonal antibody or fragment comprises the CDR1 heavy chain sequence comprising the sequence: GFDFSRYW (SEQ ID NO: 4), the CDR2 heavy chain comprising the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), the CDR3 heavy chain comprising the sequence: PDGNYNALDYW (SEQ ID NO: 8), the CDR1 light chain comprising the sequence: KASQDVGTAVT (SEQ ID NO: 12), the CDR2 light chain comprising the sequence: WASTRHT (SEQ ID NO: 14) and the CDR3 light chain comprising the sequence: QQYSRYPYT (SEQ ID NO: 16), or analogues thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the hypervariable region (HVR) sequence.

[0027] According to some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: iv. the CDR1 heavy chain having a sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 80v. . the CDR2 heavy chain having a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 81vi. . the CDR3 heavy chain having a sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 82vii. the CDR1 light chain having a sequence presented in SEQ ID NO: 12viii. the CDR2 light chain having a sequence presented in SEQ ID NO: 14eix. the CDR3 light chain having a sequence presented in SEQ ID NO: 16.

[0028] According to some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

[0029] According to other specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

[0030] According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises the variable heavy chain region presented in SEQ ID NO: 69), or an analog or derivative thereof, which has at least 90% sequence identity with the variable heavy chain region sequence.

[0031] According to some embodiments, the analogue of SEQ ID NO: 2 is the variable heavy chain region that has a sequence presented in SEQ ID NO: 69.

[0032] According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises the variable light chain region presented in SEQ ID NO: 71), or an analog thereof, which has at least 90% sequence identity with the variable light chain region sequence.

[0033] According to some embodiments, the analog of SEQ ID NO: 10 is the variable light chain region that has a sequence presented in SEQ ID NO: 71.

[0034] According to a specific embodiment, the isolated monoclonal antibody or fragment thereof comprises a variable heavy chain region having a sequence presented in SEQ ID NO: 2 or SEQ ID NO: 69 and a variable light chain region having the sequence presented in SEQ ID NO: 10 or SEQ ID NO: 71, or an analog thereof, having at least 90% sequence identity with the light and/or heavy chain sequence.

[0035] The invention also encompasses the antibody or antibody fragment capable of high-affinity binding to an epitope within the human PVR protein to which the 4E5 monoclonal antibody (mAb) binds.

[0036] According to other embodiments, the isolated monoclonal antibody comprises the CDR sequences of a monoclonal antibody denoted 7D4 (or hPVR.01), namely, the three CDR sequences contained in the variable region of the heavy chain presented in SEQ ID NO: 73 and the three CDR sequences contained in the variable region of the light chain presented in SEQ ID NO: 75.

[0037] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR1 comprising the sequence GYTFTEYTMH (SEQ ID NO: 20). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR2 comprising the sequence GIDPNNGGTNYNQNFKG (SEQ ID NO: 22). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a heavy chain CDR3 comprising the sequence VIPLEY (SEQ ID NO: 24). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) the HC CDR1 comprising the sequence GYTFTEYTMH (SEQ ID NO: 20); (ii) the HC CDR2 comprising the sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); and (iii) the CDR3 of HC comprises the sequence: VIPLEY (SEQ ID NO: 24).

[0038] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises CDR1 heavy chain, which comprises the sequence EYTMH (SEQ ID NO: 83).

[0039] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR1 light chain comprising the sequence KASQNVYTNVA (SEQ ID NO: 28). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR2 light chain comprising the sequence SASYRYR (SEQ ID NO: 30). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR3 light chain comprising the sequence QQYNSYPLA (SEQ ID NO: 32). According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) the CDR1 LC comprising the sequence KASQNVYTNVA (SEQ ID NO: 28); (ii) the CDR2 LC comprising the sequence: SASYRYR (SEQ ID NO: 30); and (iii) the CDR3 of HC comprises the sequence: QQYNSYPLA (SEQ ID NO: 32).

[0040] According to some specific embodiments, the isolated monoclonal antibody comprises the heavy chain CDR1 comprising the sequence GYTFTEYTMH (SEQ ID NO: 20), the heavy chain CDR2 comprising the sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), the heavy chain CDR3 comprising the sequence: VIPLEY (SEQ ID NO: 24), the light chain CDR1 comprising the sequence: KASQNVYTNVA (SEQ ID NO: 28), the light chain CDR2 comprising the sequence: SASYRYR (SEQ ID NO: 30) and the light chain CDR3 comprising the sequence: QQYNSYPLA (SEQ ID NO: 32), or analogues thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the HVR sequence.

[0041] According to some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: the heavy chain CDR1 having a sequence selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 83, the heavy chain CDR2 having a sequence presented in SEQ ID NO: 22, the heavy chain CDR3 having a sequence presented in SEQ ID NO: 24, the light chain CDR1 having a sequence presented in SEQ ID NO: 28, the light chain CDR2 having a sequence presented in SEQ ID NO: 30 and the light chain CDR3 having a sequence presented in SEQ ID NO: 32.

[0042] According to some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32.

[0043] According to other specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDR sequences consisting of: SEQ ID NO: 83, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30 and SEQ ID NO: 32.

[0044] According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises the variable heavy chain region presented in SEQ ID NO: 73, or an analog or derivative thereof, which has at least 90% sequence identity with the variable heavy chain region sequence.

[0045] According to some embodiments, the analogue of SEQ ID NO: 18 is the variable heavy chain region that has a sequence presented in SEQ ID NO: 73.

[0046] According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises the variable light chain region presented in SEQ ID NO: 75, or an analog thereof, which has at least 90% sequence identity with the variable light chain region sequence.

[0047] According to some embodiments, the analog of SEQ ID NO: 26 is the variable light chain region that has a sequence presented in SEQ ID NO: 75

[0048] According to a specific embodiment, the isolated monoclonal antibody or fragment thereof comprises a variable heavy chain region having a sequence presented in SEQ ID NO: 18 or SEQ ID NO: 73 and a variable light chain region having the sequence presented in SEQ ID NO: 26 or SEQ ID NO: 75, or an analog thereof, having at least 90% sequence identity with the light and/or heavy chain sequence.

[0049] The invention also includes an antibody or antibody fragment capable of binding with high affinity to an epitope within the human PVR protein to which mAb 7D4 binds.

[0050] According to other embodiments, the isolated monoclonal antibody comprises the CDR sequences of a monoclonal antibody denoted 5B9 (or hPVR.09), namely, the three CDR sequences contained in the variable region of the heavy chain presented in SEQ ID NO: 77 and the three CDR sequences contained in the variable region of the light chain presented in SEQ ID NO: 79.

[0051] According to some embodiments, the isolated monoclonal antibody comprises the complementarity-determining region (CDR) sequences contained in the variable heavy chain region presented in SEQ ID NO: 34 and the three CDR sequences contained in the variable light chain region sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54. Each possibility represents a separate embodiment of the invention.

[0052] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR1 heavy chain comprising the sequence GYTFSNYWIE (SEQ ID NO: 36). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR2 heavy chain comprising the sequence EIFPGSGRINFNEKFKG (SEQ ID NO: 38). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR3 heavy chain comprising the sequence TKIYGNSFDY (SEQ ID NO: 40). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR1 heavy chain comprising the sequence SNYWIE (SEQ ID NO: 84).

[0053] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) the HC CDR1 comprises the sequence SNYWIE (SEQ ID NO: 84); (ii) the HC CDR2 comprises the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38); and (iii) the HC CDR3 comprises the sequence: TKIYGNSFDY (SEQ ID NO: 40).

[0054] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR1 light chain comprising the sequence KASQDVGTAVV (SEQ ID NO: 44). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR2 light chain comprising the sequence WASSRHN (SEQ ID NO: 46). According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR3 light chain comprising the sequence QQYSRYPLT (SEQ ID NO: 48).

[0055] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises the CDR1 light chain comprising the sequence KASQDVGTAV (SEQ ID NO: 85).

[0056] According to certain embodiments, the isolated monoclonal antibody or antibody fragment comprises: (i) the LC CDR1 comprises the sequence KASQDVGTAV (SEQ ID NO: 85); (ii) the LC CDR2 comprises the sequence: WASSRHN (SEQ ID NO: 46); and (iii) the HC CDR3 comprises the sequence: QQYSRYPLT (SEQ ID NO: 48).

[0057] According to additional embodiments, the CDR2 of LC comprises the sequence presented in SEQ ID Nos: 46, 56, 57, 58, 59, 60 or 61. Each possibility represents a separate embodiment of the invention.

[0058] According to some specific embodiments, the isolated monoclonal antibody or fragment comprises the CDR1 heavy chain sequence comprising the sequence: GYTFSNYWIE (SEQ ID NO: 36), the CDR2 heavy chain comprising the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), the CDR3 heavy chain comprising the sequence: TKIYGNSFDY (SEQ ID NO: 40), the CDR1 light chain comprising the sequence: KASQDVGTAVV (SEQ ID NO: 44), the CDR2 light chain comprising the sequence: WASSRHN (SEQ ID NO: 46) and the CDR3 light chain comprising the sequence: QQYSRYPLT (SEQ ID NO: 48), or analogues thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the HVR sequence.

[0059] According to some specific embodiments, the isolated monoclonal antibody or fragment consisting of: CDR1 heavy chain having a sequence selected from the group consisting of SEQ ID NO: 36 and SEQ ID NO: 84, CDR2 heavy chain having a sequence presented in SEQ ID NO: 38, CDR3 heavy chain having a sequence presented in SEQ ID NO: 40, CDR1 light chain having a sequence selected from the group consisting of SEQ ID NO: 44 and SEQ ID NO: 85, CDR2 light chain having a sequence presented in SEQ ID NO: 46 and CDR3 light chain having a sequence presented in SEQ ID NO: 48.

[0060] According to some specific embodiments, the isolated monoclonal antibody or fragment consisting of: CDR1 heavy chain having a sequence presented in SEQ ID NO: 36 or SEQ ID NO: 84, CDR2 heavy chain having a sequence presented in SEQ ID NO: 38, CDR3 heavy chain having a sequence presented in SEQ ID NO: 40, CDR1 light chain having a sequence presented in SEQ ID NO: 44 or SEQ ID NO: 85, CDR2 light chain having a sequence selected from the group consisting of: SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61; and the CDR3 light chain which has a sequence presented in SEQ ID NO: 48. Each possibility represents a separate embodiment of the invention.

[0061] According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises the variable heavy chain region presented in SEQ ID NO: 77, or an analog or derivative thereof, which has at least 90% sequence identity with the variable heavy chain region sequence.

[0062] According to some embodiments, the analogue of SEQ ID NO: 34 is the variable heavy chain region that has a sequence presented in SEQ ID NO: 77.

[0063] According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises the variable light chain region presented in SEQ ID NO: 79, or an analog thereof, which has at least 90% sequence identity with the variable light chain region sequence.

[0064] According to some embodiments, the analog of SEQ ID NO: 42 is the variable light chain region that has a sequence presented in SEQ ID NO: 79.

[0065] According to a specific embodiment, the isolated monoclonal antibody or fragment thereof comprises a variable heavy chain region having a sequence selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 77 and a variable light chain region having a sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO: 79, or an analog thereof, having at least 90% sequence identity with the light and/or heavy chain sequence.

[0066] According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises a variable heavy chain region having the sequence shown in SEQ ID NO: 34 and a variable light chain region having the sequence shown in SEQ ID NO: 42, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 54, or an analogue thereof, having at least 90% sequence identity with the light and/or heavy chain sequence. Each embodiment represents a separate embodiment of the invention.

[0067] The invention also includes an antibody or antibody fragment capable of binding with high affinity to an epitope within the human PVR protein to which mAb 5B9 binds.

[0068] According to some embodiments, the isolated antibody or fragment thereof recognizes human PVR with an affinity of at least 10⁻⁸ M. According to other embodiments, an antibody or antibody fragment binds with an affinity of 10⁻⁸ M, 5x10⁻⁸ M, 10⁻⁸ M, 5x10⁻⁹ M, 10⁻¹⁰ M, 5x10⁻¹¹ M, or even higher, to human PVR. Each possibility represents a separate embodiment of the invention.

[0069] Analogs and derivatives of isolated mAb antibodies, and antibody fragments described above, are also covered by the scope of the invention. In some embodiments, particular mAb analogs or isolates or fragments thereof comprising at least one variable region presented and a sequence selected from the group consisting of: SEQ ID NOs: 2, 10, 18, 26, 34 and 42 are also covered by the scope of the present invention. Isolated mAbs or fragments thereof comprising at least one variable region presented in a sequence selected from the group consisting of: SEQ ID NOs: 69, 71, 73, 75, 77 and 79 are also covered by the scope of the present invention.

[0070] According to some embodiments, the antibody or antibody fragment analog has at least 90% sequence identity with the hypervariable region of the reference antibody sequence.

[0071] According to certain embodiments, the analogue or derivative of the isolated antibody or fragment thereof has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with a variable region of the reference antibody sequence. Each possibility represents a separate embodiment of the invention.

[0072] According to some embodiments, the antibody or antibody fragment according to the invention comprises a variable heavy chain region presented in SEQ ID NO: 2 or SEQ ID NO: 69 or an analog having at least 95% sequence similarity to said sequence. According to other embodiments, the antibody or antibody fragment according to the invention comprises a variable heavy chain region presented in SEQ ID NO: 18 or SEQ ID NO: 73 or an analog having at least 95% sequence similarity to said sequence. According to other embodiments, the antibody or antibody fragment according to the invention comprises a variable heavy chain region presented in SEQ ID NO: 34 or SEQ ID NO: 77 or an analog having at least 95% sequence similarity to said sequence.

[0073] According to some embodiments, the antibody or antibody fragment comprises a variable light chain region presented at SEQ ID NO: 10 or SEQ ID NO: 71 or an analog that has at least 95% sequence similarity to said sequence. According to other embodiments, the antibody or antibody fragment comprises a variable light chain region presented at SEQ ID NO: 26 or SEQ ID NO: 75 or an analog that has at least 95% sequence similarity to said sequence. According to other embodiments, the antibody or antibody fragment comprises a variable light chain region presented at SEQ ID NO: 42 or SEQ ID NO: 79 or an analog that has at least 95% sequence similarity to said sequence.

[0074] According to some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, wherein: (i) the heavy chain comprises SEQ ID NO: 2 and the light chain comprises SEQ ID NO: 10; (ii) the heavy chain comprises SEQ ID NO: 18 and the light chain comprises SEQ ID NO: 26; or (iii) the heavy chain comprises SEQ ID NO: 34 and the light chain comprises SEQ ID NO: 42. Analogs of the antibodies or fragments, which have at least 95% sequence similarity to said heavy or light chains, are also included.

[0075] According to other embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, wherein: (i) the heavy chain comprises SEQ ID NO: 69 and the light chain comprises SEQ ID NO: 71; (ii) the heavy chain comprises SEQ ID NO: 73 and the light chain comprises SEQ ID NO: 75; or (iii) the heavy chain comprises SEQ ID NO: 77 and the light chain comprises SEQ ID NO: 79. Analogs of the antibodies or fragments, which have at least 95% sequence similarity to said heavy or light chains, are also included.

[0076] According to some embodiments, the analog has at least 96, 97, 98, or 99% sequence identity with variable regions of antibody light or heavy chains described above. According to some embodiments, the analog comprises no more than an amino acid substitution, deletion, or addition to one or more CDR sequences of the hypervariable region, namely, any of the CDR sequences presented in SEQ ID NOs: 4, 6, 8, 12, 14, 16, 20, 22, 24, 28, 30, 32, 36, 38, 40, 44, 46, 48, 80, 81, 82, 83, 84, and 85. Each embodiment represents a separate embodiment of the present invention. According to some embodiments, the amino acid substitution is a conservative substitution.

[0077] According to some embodiments, the antibody or antibody fragment comprises a hypervariable region (HVR) having the light and heavy chain regions defined above, in which 1, 2, 3, 4 or 5 amino acids have been substituted, deleted and/or added. Each possibility represents a separate embodiment of the invention.

[0078] According to some embodiments, the antibody or antibody fragment comprises an HVR having the light and heavy chain regions defined above, in which an amino acid has been substituted. According to specific embodiments, the antibody or antibody fragment comprises a CDR as defined above, in which an amino acid has been substituted. According to some specific embodiments, the antibody or antibody fragment comprises a CDR2 light chain as defined above, in which an amino acid has been substituted.

[0079] According to some specific embodiments, the antibody or antibody fragment comprises a CDR2 light chain having a sequence presented in SEQ ID NO: 55 (WASSRHX), wherein X is selected from the group consisting of A, R, D, E, P and T. Each possibility represents a separate embodiment of the invention.

[0080] According to some embodiments, the antibody or antibody fragment comprises a CDR2 light chain having a sequence selected from the group consisting of SEQ ID NO: 56 to 61.

[0081] According to some embodiments, the isolated monoclonal antibody or antibody fragment comprises a set of CDRs selected from the group consisting of: i. a set of six CDRs wherein: HC CDR1 is selected from GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); HC CDR2 is EIFPGSGRINFNEKFKG (SEQ ID NO: 38); HC CDR3 is TKIYGNSFDY (SEQ ID NO: 40); LC CDR1 is selected from KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); LC's CDR2 is selected from the group consisting of: WASSRHN (SEQ ID NO: 46), WASSRHA (SEQ ID NO: 56), WASSRHR (SEQ ID NO: 57), WASSRHD (SEQ ID NO: 58), WASSRHE (SEQ ID NO: 59), WASSRHP (SEQ ID NO: 60) and WASSRHT (SEQ ID NO: 61); and LC's CDR3 is QQYSRYPLT (SEQ ID NO: 48). ii. a set of six CDRs wherein: HC's CDR1 sequence is selected from GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); HC's CDR2 is selected from EIHPDSSKINYTPSQ (SEQ ID NO: 6) and EIHPDSSKINYTPSQKD (SEQ ID NO: 81); HC's CDR3 is selected from PDGNYNALDYW (SEQ ID NO: 8) and PDGNYNALDY (SEQ ID NO: 82); LC's CDR1 is KASQDVGTAVT (SEQ ID NO: 12); LC's CDR2 is WASTRHT (SEQ ID NO: 14); and LC's CDR3 is QQYSRYPYT (SEQ ID NO: 16).iii. a set of six CDRs wherein: HC's CDR1 sequence is selected from GYTFTEYTMH (SEQ ID NO: 20) and EYTMH (SEQ ID NO: 83); HC's CDR2 is GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); HC's CDR3 is VIPLEY (SEQ ID NO: 24); LC's CDR1 is KASQNVYTNVA (SEQ ID NO: 28); LC's CDR2 is SASYRYR (SEQ ID NO: 30); and LC's CDR3 is QQYNSYPLA (SEQ ID NO: 32).

[0082] The present invention, therefore, provides a monoclonal antibody that specifically binds to the human protein PVR, or a binding fragment thereof, wherein said monoclonal antibody or fragment comprises a set of six CDR sequences, the set being selected from the group consisting of: i. SEQ ID NOs. 4, 6, 8, 12, 14 and 16; ii. SEQ ID NOs. 20, 22, 24, 28, 30 and 32; iii. SEQ ID NOs. 36, 38, 40, 44, 46 and 48; iv. SEQ ID NOs. 36, 38, 40, 44, 55 and 48; v. SEQ ID NOs. 80, 81, 82, 12, 14 and 16; vi. SEQ ID NOs. 83, 22, 24, 28, 30 and 32;vii. SEQ ID NOs. 84, 38, 40, 85, 46 and 48; eviii. SEQ ID NOs. 84, 38, 40, 85, 55 and 48.

[0083] The present invention also provides monoclonal antibodies and binding fragments thereof, comprising a heavy chain and a light chain, wherein said chains comprise a set of variable region heavy chain sequences and variable region light chain sequences, wherein said set is selected from the group consisting of: i. SEQ ID NOs: 2 and 10; ii. SEQ ID NOs: 69 and 71; iii. SEQ ID NOs: 18 and 26; iv. SEQ ID NOs: 73 and 75; v. SEQ ID NOs: 34 and 42; and vi. SEQ ID NOs: 77 and 79.

[0084] According to some embodiments, the antibody or antibody fragment is able to inhibit the binding of human PVR to TIGIT expressed on T cells or NK cells.

[0085] According to a specific embodiment, the mAb is selected from the group consisting of: a chimeric antibody and an antibody fragment comprising at least the antigen-binding portion of an antibody. According to specific embodiments, the antibody is a chimeric antibody. According to still other embodiments, the chimeric antibody comprises the human constant region. According to still other embodiments, the chimeric monoclonal antibody comprises the human IgG1 constant region. According to a specific embodiment, the antibody fragment is selected from the group consisting of: Fab, Fab’, F(ab’)2, Fd, Fd’, Fv, dAb, isolated CDR region, single-chain antibody (scab), “diabodies” and “linear antibodies”. Each possibility represents a separate embodiment of the present invention.

[0086] According to some embodiments, the antibody or antibody fragment comprises a scaffold sequence selected from the group consisting of: mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, human IgG2, human IgG3, and human IgG4. Each embodiment represents a separate embodiment of the present invention.

[0087] According to some embodiments, a conjugate comprising the antibody or a fragment thereof as described above is provided.

[0088] According to some embodiments, the conjugate comprises a carrier protein.

[0089] Polynucleotide sequences encoding monoclonal antibodies, which have high affinity and specificity for PVR, as well as vectors and host cells carrying these polynucleotide sequences, are provided according to another aspect of the present invention.

[0090] According to some embodiments, the polynucleotide sequences that encode the amino acid sequences of the HC variable region and the light LC variable region described above are provided.

[0091] According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain capable of binding to an epitope within the human PVR protein to which it binds: (i) a monoclonal antibody (in the present document, identified as 4E5), which has a variable heavy chain region of SEQ ID NO: 2 and a variable light chain region of SEQ ID NO: 10; (ii) a monoclonal antibody (in the present document, identified as 7D4), which has a variable heavy chain region of SEQ ID NO: 2 and a variable light chain region of SEQ ID NO: 10; (iii) a monoclonal antibody (in the present document, identified as 7D4), which has a variable heavy chain region of SEQ ID NO: 18 and a variable light chain region of SEQ ID NO: 26; or (iii) a monoclonal antibody (identified herein as 5B9) that has a variable heavy chain region with SEQ ID NO: 34 and a variable light chain region with SEQ ID NO: 42.

[0092] According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 69. According to some embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 71.

[0093] According to other embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 73. According to further embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence shown in SEQ ID NO: 26 or SEQ ID NO: 75.

[0094] According to other embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 77. According to further embodiments, the polynucleotide sequence encodes an antibody or antibody fragment or chain comprising the sequence shown in SEQ ID NO: 42 or SEQ ID NO: 79.

[0095] According to some embodiments, the polynucleotide sequence according to the invention encodes an antibody or antibody fragment or chain comprising the six CDR sequences: (i) the CDR1 heavy chain having the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), the CDR2 heavy chain having the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), the CDR3 heavy chain having the sequence: PDGNYNALDY (SEQ ID NO: 82), the CDR1 light chain having the sequence: KASQDVGTAVT (SEQ ID NO: 12), the CDR2 light chain having the sequence: WASTRHT (SEQ ID NO: 14) and the CDR3 light chain having the sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) the heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), the heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), the heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), the light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), the light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30) and the light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) the heavy chain CDR1 having the sequence SNYWIE (SEQ ID NO: 84), the heavy chain CDR2 having the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), the heavy chain CDR3 having the sequence: TKIYGNSFDY (SEQ ID NO: 40), the light chain CDR1 having the sequence: KASQDVGTAV (SEQ ID NO: 85), the light chain CDR2 having the sequence: WASSRHN (SEQ ID NO: 46) and the light chain CDR3 having the sequence: QQYSRYPLT (SEQ ID NO: 48).

[0096] According to some embodiments, the polynucleotide sequences defined above encode a molecule selected from the group consisting of: an antibody, an antibody fragment comprising at least one antigen-binding moiety, and an antibody conjugate comprising said antibody or antibody fragment. Each embodiment represents a separate embodiment of the present invention.

[0097] According to some embodiments, the polynucleotide sequence, which encodes a variable region of monoclonal antibody heavy chain, comprises a sequence presented in SEQ ID NO: 1 or SEQ ID NO: 68 or a variant thereof, which has at least 90% sequence identity.

[0098] According to some embodiments, the polynucleotide sequence, which encodes a variable region of monoclonal antibody heavy chain, comprises a sequence presented in SEQ ID NO: 17 or SEQ ID NO: 72 or a variant thereof, which has at least 90% sequence identity.

[0099] According to some embodiments, the polynucleotide sequence, which encodes a variable region of monoclonal antibody heavy chain, comprises a sequence presented in SEQ ID NO: 33 or SEQ ID NO: 76 or a variant thereof, which has at least 90% sequence identity.

[0100] According to some embodiments, the polynucleotide sequence, which encodes a variable region of monoclonal antibody light chain, comprises a sequence presented in SEQ ID NO: 9 or SEQ ID NO: 70 or a variant thereof, which has at least 90% sequence identity.

[0101] According to some embodiments, the polynucleotide sequence, which encodes a variable region of monoclonal antibody light chain, comprises a sequence presented in SEQ ID NO: 25 or SEQ ID NO: 74 or a variant thereof, which has at least 90% sequence identity.

[0102] According to some embodiments, the polynucleotide sequence, which encodes a variable region of monoclonal antibody light chain, comprises a sequence presented in SEQ ID NO: 41 or SEQ ID NO: 78 or a variant thereof, which has at least 90% sequence identity.

[0103] The present invention provides, according to some embodiments, a polypeptide comprising at least one sequence encoded by at least one polynucleotide sequence disclosed above.

[0104] In a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one antibody chain or fragment thereof according to the present invention. According to some embodiments, the nucleic acid construct is a plasmid.

[0105] According to some embodiments, the plasmid comprises a polynucleotide sequence presented in SEQ ID NO: 1, SEQ ID NO: 17 or SEQ ID NO: 33.

[0106] According to some embodiments, the plasmid comprises a polynucleotide sequence presented in SEQ ID NO: 68, SEQ ID NO: 72 or SEQ ID NO: 76.

[0107] According to other embodiments, the plasmid comprises a polynucleotide sequence presented in SEQ ID NO: 9, SEQ ID NO: 25 or SEQ ID NO: 41.

[0108] According to other embodiments, the plasmid comprises a polynucleotide sequence presented in SEQ ID NO: 70, SEQ ID NO: 74 or SEQ ID NO: 78.

[0109] In yet another aspect, the present invention provides a hybridoma cell capable of producing an antibody or an antibody fragment comprising specific CDR sequences and/or variable regions of specific heavy and light chains defined above.

[0110] According to some embodiments, a hybridoma cell is provided comprising at least one polynucleotide sequence disclosed above.

[0111] According to some embodiments, the hybridoma is capable of producing a monoclonal antibody comprising the six complementarity-determining regions (CDRs) sequences: (i) the CDR1 heavy chain with the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), the CDR2 heavy chain with the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), the CDR3 heavy chain with the sequence: PDGNYNALDY (SEQ ID NO: 82), the CDR1 light chain with the sequence: KASQDVGTAVT (SEQ ID NO: 12), the CDR2 light chain with the sequence: WASTRHT (SEQ ID NO: 14) and the CDR3 light chain with the sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) the heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), the heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), the heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), the light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), the light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30) and the light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) the heavy chain CDR1 having the sequence SNYWIE (SEQ ID NO: 84), the heavy chain CDR2 having the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), the heavy chain CDR3 having the sequence: TKIYGNSFDY (SEQ ID NO: 40), the light chain CDR1 having the sequence: KASQDVGTAV (SEQ ID NO: 85), the light chain CDR2 having the sequence: WASSRHN (SEQ ID NO: 46) and the light chain CDR3 having the sequence: QQYSRYPLT (SEQ ID NO: 48).

[0112] The antibodies or fragments thereof according to the present invention may be attached to a cytotoxic chemical moiety, a radioactive chemical moiety or an identifiable chemical moiety.

[0113] The present invention provides, according to another aspect, a pharmaceutical composition comprising, as an active ingredient, at least one antibody, antibody fragment or conjugates thereof, which recognize PVR with high affinity and specificity and optionally at least one acceptable pharmaceutical vehicle, salt, diluent or excipient.

[0114] According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or a fragment thereof that is capable of binding to an epitope within the human PVR protein, to which a monoclonal antibody selected from the group consisting of: (i) an antibody herein identified as 4E5 (also denoted PVR.07), which has a variable heavy chain region of SEQ ID NO: 2 and a variable light chain region of SEQ ID NO: 10; (ii) an antibody herein identified as 7D4 (also denoted PVR.01), which has a variable heavy chain region of SEQ ID NO: 18 and a variable light chain region of SEQ ID NO: 26; and (iii) an antibody herein identified as 5B9 (also denoted PVR.09), which has a variable heavy chain region of SEQ ID NO: 34 and a variable light chain region of SEQ ID NO: 42.

[0115] According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or antibody fragment thereof comprising the six CDRs: (i) the CDR1 heavy chain having the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), the CDR2 heavy chain having the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), the CDR3 heavy chain having the sequence: PDGNYNALDY (SEQ ID NO: 82), the CDR1 light chain having the sequence: KASQDVGTAVT (SEQ ID NO: 12), the CDR2 light chain having the sequence: WASTRHT (SEQ ID NO: 14) and the CDR3 light chain having the sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) the CDR1 heavy chain having the sequence EYTMH (SEQ ID NO: 83), a heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), a heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), a light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), a light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30), and a light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) a heavy chain CDR1 with sequence: SNYWIE (SEQ ID NO: 84), a heavy chain CDR2 with sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), a heavy chain CDR3 with sequence: TKIYGNSFDY (SEQ ID NO: 40), a CDR1 a light chain with the sequence: KASQDVGTAV (SEQ ID NO: 85), a CDR2 light chain with the sequence: WASSRHN (SEQ ID NO: 46), and a CDR3 light chain with the sequence: QQYSRYPLT (SEQ ID NO: 48).

[0116] According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a variable heavy chain region having a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 69, SEQ ID NO: 18, SEQ ID NO: 73, SEQ ID NO: 34 and SEQ ID NO: 77. Each embodiment represents a separate embodiment of the invention.

[0117] According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a variable light chain region having a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 71, SEQ ID NO: 26, SEQ ID NO: 75, SEQ ID NO: 42 and SEQ ID NO: 79. Each embodiment represents a separate embodiment of the invention.

[0118] According to the specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a variable heavy chain region having the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 69 and a variable light chain region having the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 71.

[0119] According to the specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a variable heavy chain region having the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 73 and a variable light chain region having the sequence shown in SEQ ID NO: 26 or SEQ ID NO: 75.

[0120] According to the specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a variable heavy chain region having the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 77 and a variable light chain region having the sequence shown in SEQ ID NO: 42 or SEQ ID NO: 79.

[0121] According to some embodiments, the pharmaceutical composition comprises a combination of at least two antibodies, or antibody fragments, that recognize human PVR.

[0122] According to still other embodiments, the pharmaceutical composition comprises an mAb or fragment that specifically binds to PVR according to the invention, and an mAb or fragment that specifically binds to a different antigen, such as a tumor antigen, cell receptor, or immunomodulatory protein.

[0123] Pharmaceutical compositions are also provided, comprising at least one antibody, antibody fragment or antibody conjugate according to the invention, for use in restoring NK cytotoxicity by inhibiting PVR binding to TIGIT expressed on NK cells.

[0124] According to some embodiments, the antibody, antibody fragment or antibody conjugate is able to inhibit the binding of human PVR to TIGIT expressed on T cells.

[0125] According to some embodiments, the pharmaceutical composition according to the present invention is for use in cancer immunotherapy or in enhancing the immune response.

[0126] Cancer can be any cancer that expresses PVR. According to some modalities, cancer overexpresses PVR.

[0127] According to some embodiments of the invention, the cancer is metastatic cancer. According to some embodiments, the pharmaceutical composition according to the present invention is for use in inhibiting the formation or distribution of metastases or reducing the total number of metastases in an individual.

[0128] According to some embodiments of the invention, the cancer is selected from the group consisting of melanoma, breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, colon cancer, cervical cancer, kidney cancer, lung cancer, thyroid cancer, prostate cancer, brain cancer, renal cancer, throat cancer, laryngeal carcinoma, bladder carcinoma, liver carcinoma, fibrosarcoma, endometrial cell carcinoma, glioblastoma, sarcoma, myeloid, leukemia, and lymphoma. Each possibility represents a separate embodiment of the invention.

[0129] According to some modalities, the cancer is a solid cancer. According to some specific modalities, the solid cancer is selected from the group consisting of melanoma (skin), lung, colon, breast, uterine, and renal cancer. According to specific modalities, the cancer is selected from the group consisting of breast cancer, lung cancer, and liposarcoma.

[0130] According to other embodiments, the cancer is hematological cancer. According to some embodiments, the hematological cancer is myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, or myelodysplastic syndrome. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the cancer is leukemia. According to specific embodiments, the cancer is acute myeloid leukemia (AML).

[0131] According to some embodiments, the pharmaceutical composition according to the present invention is for use in the treatment of a viral infection.

[0132] According to some embodiments, viral infection is caused by a virus that binds to a target cell via a PVR on the surface of the infected cell.

[0133] According to some embodiments, the pharmaceutical composition according to the present invention is for use in the treatment of a disease or disorder related to angiogenesis. According to certain embodiments, the disease or disorder related to angiogenesis is selected from the group consisting of: cancer, proliferative cell eye diseases (ocular diseases), retinal disorders, and inflammatory disease. Each possibility represents a separate embodiment of the invention.

[0134] According to yet another aspect, the present invention provides a method of inhibiting the binding of human PVR to at least one ligand using a monoclonal antibody or antibody fragment defined above.

[0135] According to a further aspect, the present invention provides a method for enhancing the immune response in an individual who needs it, comprising administering to said individual a therapeutically effective amount of a monoclonal antibody, antibody fragment or antibody conjugate defined above.

[0136] According to yet another aspect, the present invention provides a method for treating cancer comprising administering to an individual in need thereof a therapeutically effective amount of a pharmaceutical composition comprising at least one antibody, antibody fragment or conjugate thereof, which recognizes human PVR with high affinity and specificity and is capable of inhibiting its binding to its ligand.

[0137] According to some embodiments, the antibody in the administered pharmaceutical composition is selected from the group consisting of: (i) a monoclonal antibody comprising the CDR sequences contained in the variable heavy chain region presented in SEQ ID NO: 2 and the CDR sequences contained in the variable light chain region presented in SEQ ID NO: 10; (ii) a monoclonal antibody comprising the CDR sequences contained in the variable heavy chain region presented in SEQ ID NO: 18 and the CDR sequences contained in the variable light chain region presented in SEQ ID NO: 26; or (iii) a monoclonal antibody comprising the CDR sequences contained in the variable heavy chain region presented in SEQ ID NO: 34 and the CDR sequences contained in the variable light chain region presented in SEQ ID NO: 42.

[0138] According to some specific embodiments, the monoclonal antibody in the administered pharmaceutical composition comprises: the heavy chain CDR1 comprises the sequence selected from the group consisting of GFDFSRYW (SEQ ID NO: 4) and RYWMT (SEQ ID NO: 80); the heavy chain CDR2 comprises the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6); the heavy chain CDR3 comprises the sequence: PDGNYNALDY (SEQ ID NO: 82); the light chain CDR1 comprises the sequence: KASQDVGTAVT (SEQ ID NO: 12); the light chain CDR2 comprises the sequence: WASTRHT (SEQ ID NO: 14) and the light chain CDR3 comprises the sequence: QQYSRYPYT (SEQ ID NO: 16). Each possibility represents a separate embodiment of the invention.

[0139] According to other specific embodiments, the monoclonal antibody in the administered pharmaceutical composition comprises: the CDR1 heavy chain comprises the sequence EYTMH (SEQ ID NO: 83); the CDR2 heavy chain comprises the sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22); the CDR3 heavy chain comprises the sequence: VIPLEY (SEQ ID NO: 24); the CDR1 light chain comprises the sequence: KASQNVYTNVA (SEQ ID NO: 28); the CDR2 light chain comprises the sequence: SASYRYR (SEQ ID NO: 30) and the CDR3 light chain comprises the sequence: QQYNSYPLA (SEQ ID NO: 32).

[0140] According to other specific embodiments, the monoclonal antibody in the administered pharmaceutical composition comprises: the CDR1 heavy chain comprises the sequence SNYWIE (SEQ ID NO: 84); the CDR2 heavy chain comprises the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38); the CDR3 heavy chain comprises the sequence: TKIYGNSFDY (SEQ ID NO: 40); the CDR1 light chain comprises the sequence: KASQDVGTAV (SEQ ID NO: 85); the CDR2 light chain comprises the sequence: WASSRHN (SEQ ID NO: 46) and the CDR3 light chain comprises the sequence: QQYSRYPLT (SEQ ID NO: 48).

[0141] According to some embodiments of the invention, the therapeutically effective amount results in a decrease in tumor size or in the number of metastases in the individual.

[0142] According to some modalities, the cancer treatment method comprises administering or performing at least one additional anticancer therapy. According to certain modalities, the additional anticancer therapy is surgery, chemotherapy, radiotherapy, or immunotherapy.

[0143] According to some embodiments, the method for cancer treatment comprises the administration of a monoclonal antibody that recognizes human PVR with high affinity and specificity and an additional anticancer agent. According to some embodiments, the additional anticancer agent is selected from the group consisting of: immunomodulator, activated lymphocyte cell, kinase inhibitor, and chemotherapeutic agent.

[0144] According to other embodiments, the additional immunomodulator is an antibody, antibody fragment, or antibody conjugate that binds to an antigen other than the human PVR.

[0145] According to some embodiments, the traditional immunomodulator is an antibody against an immune checkpoint molecule. According to some embodiments, the traditional immunomodulator 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 receptors (KIR), TIGIT, and any combination thereof. Each embodiment represents a separate embodiment of the invention.

[0146] According to some embodiments, the anticancer agent is selected from the group consisting of: Erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide and any combination thereof. Each possibility represents a separate embodiment of the invention.

[0147] According to some embodiments, the anticancer agent is an epidermal growth factor receptor (EGFR) inhibitor. According to some embodiments, the EGFR inhibitor is selected from the group consisting of: Cetuximab (Erbitux®), Panitumumab (Vectibix®) and necitumumab (Portrazza®). According to some embodiments, the EGFR inhibitor is Cetuximab (Erbitux®).

[0148] According to some embodiments of the invention, the individual is a human individual.

[0149] According to some embodiments of the invention, the use further comprises the use of an agent that downregulates the activity or expression of an immune co-inhibitory receptor.

[0150] According to some embodiments of the invention, the immune cell is a T cell.

[0151] According to some embodiments of the invention, the immune co-inhibitory receptor is selected from the group consisting of PD-1, TIGIT, DNAM-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55, LAIR1, SIGLEC10 and 2B4. Each possibility represents a separate embodiment of the invention.

[0152] According to one aspect, the present invention provides a method for modulating the function and/or activity of the immune system comprising modulating the binding of PVR to TIGIT using an antibody according to the invention.

[0153] According to one aspect, the present invention provides a method for preventing or treating a viral infection of a virus that uses CD155 as an entry receptor, in an individual who needs it, wherein the method comprises administering to the individual a therapeutically effective amount of an anti-PVR monoclonal antibody described in this document. According to some embodiments, the virus is selected from the group consisting of: poliovirus, coxsackievirus, adenovirus and human deficiency virus (HIV).

[0154] According to yet another aspect, the present invention provides a method for treating a disease or disorder related to angiogenesis. According to certain embodiments, the disease or disorder related to angiogenesis is selected from the group consisting of: cancer, proliferative cell eye diseases (ocular diseases), retinal disorders, and inflammatory disease. Each possibility represents a separate embodiment of the invention.

[0155] According to some modalities, the method of cancer treatment involves preventing or reducing the formation, growth, or spread of metastases in an individual through the inhibition of angiogenesis.

[0156] According to one aspect, the present invention provides a method for diagnosing or prognosticating cancer or infectious disease in an individual, the method comprising determining the level of PVR expression in a biological sample from said individual using at least one antibody as described in this document.

[0157] The present invention further comprises, according to another aspect, a method for determining or quantifying PVR expression, wherein the method comprises placing a biological sample in contact with an antibody or antibody fragment and measuring the level of complex formation, wherein the antibody or antibody fragment comprises the complementarity-determining regions (CDRs) selected from the group consisting of: (i) the heavy chain CDR1 having the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), the heavy chain CDR2 having the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), the heavy chain CDR3 having the sequence: PDGNYNALDY (SEQ ID NO: 82), the light chain CDR1 having the sequence: KASQDVGTAVT (SEQ ID NO: 12), the light chain CDR2 having the sequence: WASTRHT (SEQ ID NO: (i) the CDR3 light chain with sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) the CDR1 heavy chain with sequence: EYTMH (SEQ ID NO: 83), the CDR2 heavy chain with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), the CDR3 heavy chain with sequence: VIPLEY (SEQ ID NO: 24), the CDR1 light chain with sequence: KASQNVYTNVA (SEQ ID NO: 28), the CDR2 light chain with sequence: SASYRYR (SEQ ID NO: 30) and the CDR3 light chain with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) the heavy chain CDR1 having the sequence SNYWIE (SEQ ID NO: 84), the heavy chain CDR2 having the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), the heavy chain CDR3 having the sequence: TKIYGNSFDY (SEQ ID NO: 40), the light chain CDR1 having the sequence: KASQDVGTAV (SEQ ID NO: 85), the light chain CDR2 having the sequence: WASSRHN (SEQ ID NO: 46) and the light chain CDR3 having the sequence: QQYSRYPLT (SEQ ID NO: 48).

[0158] The determination and quantification of the methods can be performed in vitro or ex vivo according to some modalities or can be used in the diagnosis of conditions associated with PVR expression. The antibodies according to the present invention can also be used to set up examination methods. For example, an enzyme-linked immunosorbent assay (ELISA), or a radioimmunoassay (RIA) can be constructed to measure levels of secreted or cell-associated polypeptide using the antibodies and methods known in the art.

[0159] According to some embodiments, the method for detecting or quantifying the presence of PVR comprises the steps of: 1) incubating a sample with a specific antibody for PVR or an antibody fragment thereof comprising at least one antigen-binding portion; 11) detecting the bound PVR using a detectable probe.

[0160] According to some embodiments, the method further comprises the steps of:111) comparing the amount of (ii) with a standard curve obtained from a reference sample containing a known amount of PVR; and112) calculating the amount of PVR in the sample from the standard curve.

[0161] According to some particular modalities, the sample is a body fluid.

[0162] Depending on the modality, the method is performed in vitro or ex vivo.

[0163] A kit for measuring PVR expression in the biological sample is also provided comprising at least one antibody or antibody fragment comprising complementarity-determining regions (CDRs) selected from the group consisting of: (i) the heavy chain CDR1 having the sequence: GFDFSRYW (SEQ ID NO: 4) or RYWMT (SEQ ID NO: 80), the heavy chain CDR2 having the sequence: EIHPDSSKINYTPSQ (SEQ ID NO: 6), the heavy chain CDR3 having the sequence: PDGNYNALDY (SEQ ID NO: 82), the light chain CDR1 having the sequence: KASQDVGTAVT (SEQ ID NO: 12), the light chain CDR2 having the sequence: WASTRHT (SEQ ID NO: 14) and the light chain CDR3 having the sequence: QQYSRYPYT (SEQ ID NO: 16); (ii) the heavy chain CDR1 with sequence EYTMH (SEQ ID NO: 83), the heavy chain CDR2 with sequence: GIDPNNGGTNYNQNFKG (SEQ ID NO: 22), the heavy chain CDR3 with sequence: VIPLEY (SEQ ID NO: 24), the light chain CDR1 with sequence: KASQNVYTNVA (SEQ ID NO: 28), the light chain CDR2 with sequence: SASYRYR (SEQ ID NO: 30) and the light chain CDR3 with sequence: QQYNSYPLA (SEQ ID NO: 32); or (iii) the heavy chain CDR1 having the sequence SNYWIE (SEQ ID NO: 84), the heavy chain CDR2 having the sequence: EIFPGSGRINFNEKFKG (SEQ ID NO: 38), the heavy chain CDR3 having the sequence: TKIYGNSFDY (SEQ ID NO: 40), the light chain CDR1 having the sequence: KASQDVGTAV (SEQ ID NO: 85), the light chain CDR2 having the sequence: WASSRHN (SEQ ID NO: 46) and the light chain CDR3 having the sequence: QQYSRYPLT (SEQ ID NO: 48).

[0164] According to one aspect, the present invention provides a kit for detecting cancer, wherein the diagnostic kit comprises a fragment antibody thereof as disclosed herein.

[0165] According to some embodiments, the invention provides a method for diagnosing, assessing the severity of, or analyzing the stage of an immunological disease or a proliferative disease comprising determining the expression or activity of PVR in a sample from an individual using an antibody according to the present invention or a fragment or conjugate thereof, and comparing the expression or activity of PVR with a reference quantity of PVR expression or activity. Said reference quantity may be obtained from a sample taken from a normal individual, from the same individual while at a different stage of the disease, or determined from clinical data from a large population of individuals.

[0166] Additional embodiments and the full scope of applicability of the present invention will become apparent from the detailed description provided hereafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided for illustrative purposes only, as various alterations and modifications encompassed by the spirit and scope of the invention will become apparent to those skilled in the art from the present detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0167] Figures 1A to 1C are graphs representing the correlation of PVR mRNA expression levels (high or low as indicated) with survival probability. The correlation was measured for lung cancer (Figure 1A), breast cancer (Figure 1B), and liposarcoma (Figure 1C). mRNA expression datasets were obtained from the GEO site and analyzed using the bioprofiling.de site, as follows: for lung cancer, GEO dataset ID: GSE31210; for breast cancer, GEO dataset ID: GSE25055; and for liposarcoma, GEO dataset ID: GSE30929.

[0168] Figure 2 is a schematic illustration of receptor expression in immune and tumor cells. TIGIT refers to a co-inhibitory receptor in many immune cells (e.g., T cells); DNAM-1 (also called CD226) refers to an activating receptor in many immune cells (e.g., T cells); Fc receptor refers to the strongly activating receptor expressed primarily in NK cells, but also in myeloid cells including neutrophils and macrophages; PVR refers to an inhibitory ligand for TIGIT (weaker binding to DNAM-1), expressed by many tumor cells; Nectin-2 refers to an activating ligand for DNAM-1 (marginal recognition by TIGIT), expressed by many tumor cells. Anti-PVR binding according to the present invention shows a dual effect: 1) enhancement of tumor cell extermination via Fc receptors; and 2) increased activation of immune cells by blocking interaction with TIGIT.

[0169] Figures 3A to 3D are graphs representing FACS analysis of the four generated anti-PVR antibodies. The effectiveness of the antibodies in blocking the direct binding of TIGIT-Fc to a tumor cell line is shown. Figure 3A illustrates a non-blocking anti-PVR antibody (anti-PVR mAb antibody, 2G3, also called hPVR.17). Figures 3B to 3D show that three of the generated antibodies, namely 5B9 (also called hPVR.09), 7D4 (also called hPVR.01), and 4E5 (also called hPVR.07), respectively, are blocking anti-PVR mAbs as shown by the blockade of TIGIT-Ig binding. Hybridoma broths (5 μl/well) were added to HepG2 cells. TIGIT-Fc was used at 2 μg/well to a final concentration of 20 μg/ml, and the levels of bound TIGIT per cell were measured by FACS after the addition of fluorescently identified anti-Fc Ab. The filled histograms represent the background staining by the anti-Fc reagent.

[0170] Figure 4 is a graph showing how blocking PVR-TIGIT interactions with the anti-PVR mAb antibody, 7D4 (also called hPVR.01) enhances NK cell kill of the human cell line MDA-MB-231 (Breast Adenocarcinoma). Specific kill is calculated based on [35S]--Methionine secretion from target cells. The control (Ctrl) is unrelated mouse IgG. P-value = 0.0056.

[0171] Figure 5 is a graph representing the fact that such blocking of PVR-TIGIT interactions with the use of the anti-PVR 4E5 mAb (also called hPVR.07) intensifies the NK cell extermination of the human cancer cell line, HepG2 (hepatocellular carcinoma). No - HepG2 extermination without mAb; anti-PVR 4E5 - HepG2 extermination with mouse anti-PVR 4E5mIgG1 (without human Fc receptor activation); anti-PVR 4E5hIgG1 - HepG2 extermination with anti-PVR 4E5-hIgG1 (human Fc receptor activation), Erbitux (P.C) - a positive control mAb Erbitux (anti-EGFR). All mAbs were used at 10 μg/ml. P values: anti-PVR 4E5 - 0.04, anti-PVR 4e5hIgG1 - 0.000746 and Erbitux (positive control) - 0.003219.

[0172] Figures 6A to 6O are graphs representing the fact that human tumor cell lines express PVR and Nectin-2. Melanoma cells (Figures 6A to 6E), breast cancer cells (Figures 6F to 6H), colorectal cells (Figure 6I), kidney cells (Figure 6J), lung cancer cells (Figure 6K), prostate cancer cells (Figure 6L), brain tumor cells (Figure 6M), and hepatocellular carcinoma cells (Figures 6N to 6O) all express PVR and Nectin-2. mAbs were used at 0.2 μg/well: a commercial anti-Nectin-2 mAb and the anti-PVR mAb 4E5 (also called hPVR.07).

[0173] Figures 7A to 7C are FACS analysis plots representing the fact that PVR is the main TIGIT ligand. Figure 7A illustrates the fact that HepG2 cells (human hepatocellular carcinoma cells) express PVR and Nectin-2. Figure 7B illustrates the fact that the purified anti-PVR mAb 4E5 (also called hPVR.07) (0.15 μg) almost completely blocks (above 97%) the binding of TIGIT-Ig (2 μg/ml), despite the fact that these cells also express Nectin-2. Figure 7C illustrates the fact that CHO cells express high levels of hNectin-2. Figure 7D shows a lack of staining of the same CHO cells as Figure 7C by all PVR mAbs, meaning that there is no direct recognition of Nectin-2 by anti-PVR mAbs and therefore the TIGIT binding blockade seen in Figure 7B cannot be explained by Nectin-2 blockade, but instead is the result of a direct PVR blockade.

[0174] Figures 8A to 8C represent similar binding efficacy of all anti-PVR mAbs to human PVR. All three blocking clones generate similar binding (less than 10% difference) to both endogenous PVR HepG2 cells (Figure 8A) and overexpressed hPVR B16-hPVR cells (Figure 8B). Binding was also examined using the African Green Monkey Vero cell line (Figure 8C), which expresses a PVR protein with 93% similarity to human PVR. In this case, the different Abs showed differential staining intensities. 0.2 μg of each mAb was used in all cases.

[0175] Figure 9 is a graph representing the fact that the anti-PVR antibodies of the invention do not recognize canine PVR. Human TIGIT-Fc (10 μg/ml) has strong cross-reactivity and binds to the canine MDCK cell line. None of the PVR mAbs could bind to these cells, suggesting that they do not recognize canine PVR.

[0176] Figures 10A to 10D represent the fact that Nectin-2 is preferentially bound by DNAM-1 and not by TIGIT. Cells overexpressing both PVR and Nectin-2 were stained using the indicated antibody concentrations. Figure 10A illustrates the binding of TIGIT-Fc to cells expressing Nectin-2; Figure 10B illustrates the binding of DNAM-FC to cells expressing Nectin-2. Figure 10C illustrates the binding of TIGIT to cells expressing PVR. Figure 10D illustrates the binding of DNAM-1 to cells expressing PVR.

[0177] Figure 11 shows the effect of anti-PVR antibodies on T cell proliferation. Human PBMCs were identified with CFSE and incubated with target cells in the presence of the indicated antibodies. The results are presented as increased proliferation by multiplication compared to the control. The results are from 7 pooled experiments, with a total of 10 healthy donors. The P values: for mAb 4E5 - 0.000241, for mAb 5B9 - 1.96E-05, for anti-PD-1 - 0.016303, for anti-CTLA4 - 0.000171 and for 4E5hIgG1 - 0.008176.

[0178] Figure 12 shows the combined effect of anti-PVR antibodies and other antibodies on T cell proliferation. Human PBMCs were identified with CFSE and incubated with target cells in the presence of the indicated antibody combination. Results are presented as increased proliferation by multiplication compared to the control. Results are from 7 independent experiments using 12 healthy donors. P values: anti-PD1+anti-CTLA - 47.54E-03, anti-PD-1+4E5 - 7.02E-02, anti-PD-1+5B9 - 1.11E-04, anti-CTLA4+4E5 - 1.37E-03, anti-CTLA4+5B9 - 5.47E-06.

[0179] Figure 13 depicts the specific effect of anti-PVR antibodies on CD8 T cell proliferation. Human PBMCs were identified with CFSE and incubated with target cells (A549) in the presence of the indicated antibodies. CD8-positive cells were counted using FACS after 9 to 12 days in culture. Results are presented as increased proliferation by multiplication compared to the control. The results are from 2 independent experiments using healthy PBMC donors.

[0180] Figure 14 represents the ratio of CD8 to CD4 cells after induction with different antibodies. Human PBMCs were identified with CFSE and incubated with target cells (A549) in the presence of the indicated antibodies. Cells were counted after 9 to 12 days in culture. The results are from 2 pooled experiments, with a total of 2 healthy donors.

[0181] Figure 15 shows the effect of anti-PVR antibodies on NK degranulation. Human NK cells were incubated with MDA-MB-231 cells (triple-negative breast cancer cell line) in the presence of the indicated antibodies. The results are representative of 7 independent experiments performed with 5 different healthy NK cell donors. P-values: PVR4E5 - 0.005063, PVR5B9 - 0.00374, PVR7D4 - 0.019448, PVR4E5-hIgG1 - 2.03E-05, PVR5B9-hIgG1 - 1.45E-05, PVR7D4-hIgG1 5.8E-05.

[0182] Figures 16A to 16D show the effect of anti-PVR antibodies on tumor cell survival in the absence of immune cells. The survival of A549 cells (Figure 16A), U373 cells (Figure 16B), HCT116 cells (Figure 16C) and Mel-624 cells (Figure 16D) was examined using the MTT cell survival assay in the presence of 50 micrograms/ml of the indicated mAb for 24 hours. Significance is calculated using a single-tailed Student's T-test, * < 0.05, ** < 0.03 and *** < 0.02.

DETAILED DESCRIPTION OF THE INVENTION

[0183] The present invention provides monoclonal antibodies specific for the human poliovirus receptor (PVR). The invention also provides the production and use of mAbs as therapeutic agents. In particular, the mAbs of the present invention can be used, alone or in combination with other agents, to restore and enhance the antitumor killing activity of immune cells and as diagnostic reagents.

[0184] The term “antigen,” as used in this document, refers to a molecule or a portion of a molecule capable of eliciting antibody formation and which is specifically bound by an antibody. An antigen may have one or more epitopes. The specific binding mentioned above is intended to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multiple other antibodies that may be evoked by other antigens. An antigen according to some embodiments of the present invention is a PVR protein.

[0185] The term “PVR”, as used in this document, refers to the poliovirus receptor, also known as CD155 (cluster of differentiation 155). The PVR is a transmembrane glycoprotein with an N-terminal signal sequence, three extracellular immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic tail. It has a molecular size of approximately 80 kDa and a structure composed of three Ig-like domains, specifically an outermost V-type domain followed by two C2-type domains. An exemplary PVR according to the invention is presented in the GenBank accession numbers: NP_001129240.1, NP_001129241.1, NP_001129242.2 and NP_006496.4. These poliovirus receptors share the extracellular domain sequence and therefore can be targeted by the affinity-binding chemical portion of the invention.

[0186] The term “antigenic determinant” or “epitope” as used herein refers to the region of an antigen molecule that reacts specifically with a particular antibody. Peptide sequences derived from an epitope can be used, alone or in conjunction with a chemical carrier moiety, by applying methods known in the art, to immunize animals and to produce additional polyclonal or monoclonal antibodies. Isolated peptides derived from an epitope can be used in diagnostic methods to detect antibodies.

[0187] It should be noted that affinity can be quantified using methods known as Surface Plasmon Resonance (SPR) (described in Scarano S, Mascini M, Turner AP, Minunni M. "Surface plasmon resonance imaging for affinity-based biosensors". Biosens Bioelectron. 2010, 25; 957-66), and can be calculated using, for example, a dissociation constant, Kd, so that a lower Kd reflects a higher affinity.

[0188] The antibodies or a fragment thereof according to the invention bind to an epitope in hPVR. Specifically, the antibodies bind to an epitope within amino acids 1 to 343 of PVR as presented in NP_006496.4.

[0189] Antibodies, or immunoglobulins, comprise two heavy chains linked to each other by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds in a “Y” configuration. Proteolytic digestion of an antibody produces Fv (variable fragment) and Fc (crystallizable fragment) domains. Antigen-binding domains, Fab, include regions where the polypeptide sequence varies. The term F(ab’)2 represents two Fab’ arms linked to each other by disulfide bonds. The central geometric axis of the antibody is called the Fc fragment. Each heavy chain has, at one end, a variable domain (VH) followed by numerous constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at the other end, where the variable domain of the light chain is aligned with the variable domain of the heavy chain and the constant domain of the light chain is aligned with the first constant domain of the heavy chain (CH1). The variable domains of each pair of light and heavy chains form the antigen-binding site. The domains in the light and heavy chains have the same general structure, and each domain comprises four scaffold regions, whose sequences are relatively conserved, joined by three hypervariable domains known as complementarity-determining regions (CDRs 1 to 3). These domains contribute to the specificity and affinity of the antigen-binding site.

[0190] The identification or determination of CDR from a given variable sequence of heavy or light chains is typically performed using one of several methods known in the art. For example, such determination is performed according to Kabat (Wu T.T and Kabat E.A., J Exp Med, 1970; 132:211-50) and IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77).

[0191] When the term “CDR that has a sequence”, or a similar term is used, it includes options where the CDR comprises the specified sequences and also options where the CDR consists of the specified sequence.

[0192] The antigen specificity of an antibody is based on the hypervariable region (HVR), namely, the unique CDR sequences of both the light and heavy chains that together form the antigen-binding site.

[0193] The heavy chain isotype (gamma, alpha, delta, epsilon, or mu) determines the immunoglobulin class (IgG, IgA, IgD, IgE, or IgM, respectively). The light chain consists of either of two isotypes (kappa, K, or lambda, α). Both isotopes are found in all antibody classes.

[0194] The term “antibody” is used in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies and antibody fragments long enough to exhibit the desired biological activity, namely binding to human PVR.

[0195] The antibody or antibodies according to the invention include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof, such as Fab or F(ab’)2 fragments. Single-chain antibodies are also within the scope of the present invention.

Antibody Fragments

[0196] “Antibody fragments” comprise only a portion of an intact antibody, generally including an antigen-binding site of the intact antibody and thus retaining the ability to bind to antigen. Examples of antibody fragments encompassed by this definition include: (i) the Fab fragment, which has VL, CL, VH, and CH1 domains; (ii) the Fab’ fragment, which is a Fab fragment that has one or more cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd fragment, which has VH and CH1 domains; (iv) the Fd’ fragment, which has VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (v) the Fv fragment, which has the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) consisting of a VH domain; (vii) isolated CDR regions; (viii) F(ab’)2 fragments, a bivalent fragment that includes two Fab’ fragments linked by a disulfide bridge in the junction region; (ix) single-chain antibody molecules (e.g., single-chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Huston et al., Proc. Natl. Acad. Sci. (USA) 1988, 85, 5879-5883); (x) “diabodies” with two antigen-binding sites, comprising a variable heavy chain (VH) domain connected to a variable light chain (VL) domain on the same polypeptide chain (see, for example, documents EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444 to 6448); (xi) “linear antibodies” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al. Protein Eng., 1995, 8, 1057 to 1062; and US Patent No. 5,641,870).

[0197] Several techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, Fab’-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab’)2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will become apparent to skilled technicians. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv).

[0198] Single-chain antibodies may be single-chain composite polypeptides that have antigen-binding capabilities and comprise amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain, i.e., VH-VL linked or single-chain Fv (scFv). Techniques for the production of single-chain antibodies (US Patent No. 4,946,778) can be adapted to produce single-chain antibodies for PVR.

[0199] The term “monoclonal antibody” (mAb), as used in this document, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minute quantities. Monoclonal antibodies are highly specific, being directed against a single antigen. Additionally, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The “monoclonal” modifier should not be constructed in such a way as to require antibody production by any particular method. mAbs can be obtained through methods known to those skilled in the art. For example, the monoclonal antibodies to be used according to the present invention can be produced from the hybridoma method, first described by Kohler et al., Nature 1975, 256, 495, or they can be produced by recombinant DNA methods (see, for example, US Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 1991, 352, 624 to 628 or Marks et al., J. Mol. Biol., 1991, 222:581 to 597.

[0200] The design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of functional heavy variable (VH) and light variable (VL) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria are described in Fields et al. 2013, 8(6), 1125-48.

[0201] The mAbs of the present invention can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD. A hybridoma that produces an mAb can be cultured in vitro or in vivo. High titers of mAbs can be obtained by in vivo production, wherein cells from individual hybridomas are injected intraperitoneally into pristine Balb/c mice to produce ascitic fluid containing high concentrations of the desired mAbs. The mAbs can be purified from such ascitic fluids, or from culture supernatants, using methods well known to those skilled in the art.

[0202] Anti-idiotype antibodies specifically immunoreactive with the hypervariable regions of an antibody of the invention are also understood.

[0203] The invention provides a monoclonal antibody or an antibody fragment comprising an antigen-binding domain (ABD) comprising three CDRs of a light chain and three CDRs of a heavy chain, wherein said ABD has at least 90% sequence identity or similarity to an ABD of a mouse monoclonal antibody comprising: (i) a variable heavy chain comprising amino acid sequence SEQ ID NO: 69 and a variable light chain comprising amino acid sequence SEQ ID NO: 71 (identified herein as 4E5 or hPVR.07); (ii) a variable heavy chain comprising amino acid sequence SEQ ID NO: 73 and a variable light chain comprising amino acid sequence SEQ ID NO: 75 (identified herein as 7D4 or hPVR.01); or (iii) a heavy variable chain comprising amino acid sequence SEQ ID NO: 77 and a light variable chain comprising amino acid sequence SEQ ID NO: 79 (identified herein as 5B9 or hPVR.09). Such an antibody may have an ABD domain that has at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or similarity, or 100% sequence identity with the corresponding ABD of 4E5, 7D4, or 5B9.

[0204] Sequence identity is the number of amino acids or nucleotides that exactly match between two different sequences. Sequence similarity allows conservative amino acid substitution to be determined as identical amino acids.

[0205] The invention also provides conservative amino acid variants of the antibody molecules according to the invention. Variants according to the invention that conserve the overall molecular structure of the encoded proteins can also be produced. Considering the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., “conservative substitutions,” can be made, for example, based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. The term “antibody analog,” as used herein, refers to an antibody derived from another antibody through one or more conservative amino acid substitutions.

[0206] The term “antibody variant,” as used herein, refers to any molecule comprising the antibody of the present invention. For example, fusion proteins, in which the antibody or an antigen-binding fragment thereof is linked to another chemical entity, are considered an antibody variant.

[0207] Analogs and variants of antibody sequences are also covered by the scope of the present application. These include, but are not limited to, conservative and non-conservative substitution, insertion and deletion of amino acids within the sequence. Such modification and the resulting antibody analog or variant are covered by the scope of the present invention, provided that they confer, or even enhance, the binding of the antibody to human PVR.

[0208] Conservative amino acid substitutions, as known to those skilled in the art, are within the scope of the present invention. Conservative amino acid substitutions include the substitution of one amino acid for another that has the same type of functional group or side chain, for example, aliphatic, aromatic, positively charged, negatively charged. These substitutions can improve oral bioavailability, penetration and targeting to specific cell populations, immunogenicity, and the like. A person skilled in the art will recognize that individual substitutions, deletions, or additions to a sequence of peptides, polypeptides, or proteins that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” in which the alteration results in the substitution of one amino acid for a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. For example, according to a table known in the art, each of the following six contains amino acids that are conservative substitutions for one another:113) Alanine (A), Serine (S), Threonine (T);114) Aspartic acid (D), Glutamic acid (E);115) Asparagine (N), Glutamine (Q);116) Arginine (R), Lysine (K);117) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and118) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

[0209] It should be emphasized that variant strand sequences are determined by sequencing methods using specific primers. Different sequencing methods employed on the same sequence may result in slightly different sequences due to technical issues and different primers, particularly at the sequence terminals. Therefore, different variants of the anti-PVR variable strand sequences are specified throughout the application.

[0210] The terms “molecule having the antigen-binding portion of an antibody” and “antigen-binding fragments,” as used herein, are intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab’ fragment, the F(ab’)2 fragment, the variable portion of the heavy and/or light chains thereof, Fab mini-antibodies (see, for example, WO 93/15210, U.S. Patent Application No. 08/256,790, WO 96/13583, U.S. Patent Application No. 08/817,788, WO 96/37621, U.S. Patent Application No. 08/999,554) and single-chain antibodies incorporating such a fraction. reactive, as well as any other type of molecule into which such a reactive antibody fraction has been physically inserted. Such molecules may be supplied by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis, or recombinant techniques.

[0211] The monoclonal antibodies in this document specifically include “chimeric” antibodies, in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species, or belonging to a particular antibody class or subclass, while the remainder of the chain (or chains) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 to 6855 (1984)). Additionally, grafting of the complementarity-determining region (CDR) can be performed to alter certain properties of the antibody molecule, including affinity or specificity. A non-limiting example of CDR grafting is disclosed in US patent no. 5,225,539.

[0212] Chimeric antibodies are molecules whose different portions are derived from different animal species, such as those that have a variable region derived from a murine mAb and a constant region from human immunoglobulin. Antibodies that have variable region framework residues substantially from human antibody (called an acceptor antibody) and CDRs substantially from a mouse antibody (called a donor antibody) are also called humanized antibodies. Chimeric antibodies are mainly used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but superior immunogenicity in humans, so human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (e.g., PCT patent applications WO 86/01533, WO 97/02671, WO 90/07861, WO 92/22653 and US patents 5,693,762, 5,693,761, 5,585,089, 5,530,101 and 5,225,539).

[0213] According to some specific embodiments, the monoclonal antibody is a chimeric monoclonal antibody.

[0214] According to some embodiments, the chimeric antibody comprises constant regions derived from human beings.

[0215] According to some embodiments, the human constant regions of the chimeric antibody are selected from the group consisting of: human IgG1, human IgG2, human IgG3 and human IgG4.

[0216] According to a specific embodiment, the chimeric monoclonal antibody or fragment thereof comprises a constant region subclass of human IgG1 subtype.

[0217] According to a particular embodiment, a chimeric monoclonal antibody that recognizes PVR is provided so as to comprise a set of six CDRs selected from the group consisting of: (i) SEQ ID Nos: 4 or 80, 6 or 81, 8 or 82, 12, 14, and 16; (ii) SEQ ID Nos: 20 or 83, 22, 24, 28, 30, and 32; and (iii) SEQ ID Nos: 36 or 84, 38, 40, 44 or 85, 46, and 48.

Pharmacology

[0218] In medicinal and pharmaceutical formulations, the active agent is preferably used together with one or more pharmaceutically acceptable vehicles and optionally any other therapeutic ingredients. The vehicle (or vehicles) must be pharmaceutically acceptable, in the sense of being compatible with the other ingredients of the formulation, and not unduly harmful to the recipient thereof. The active agent is supplied in an amount effective to achieve the desired pharmacological effect, as described above, and in an amount adequate to achieve the desired exposure.

[0219] Typically, the antibodies and fragments, and conjugates thereof, of the present invention comprising the antigen-binding portion of an antibody or comprising another polypeptide that includes a mimetic peptide will be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control the release of the active ingredient (molecule comprising the antigen-binding portion of an antibody) or to prolong its presence in a patient's system. Numerous suitable drug delivery systems are known and include, for example, implantable drug delivery systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres and the like. Controlled-release preparations may be prepared by using polymers to form complexes or adsorb the molecule according to the present invention. For example, biocompatible polymers include poly(ethylene-co-vinyl) acetate matrices and matrices of a polyanhydride copolymer of a stearic acid and sebaric acid dimer. The release rate of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends on the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of the dispersed particles.

[0220] The pharmaceutical composition of the present invention can be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intra-articularly, intralesionally, intratumorally or parenterally. In general, intravenous (i.v.) administration is used to deliver antibodies.

[0221] It will be evident to those of ordinary skill in the art that the therapeutically effective amount of the molecule according to the present invention will depend, among other things, on the administration schedule, the unit dose of the molecule administered, the possibility of the molecule being administered in combination with other therapeutic agents, the immunological status and health of the patient, the therapeutic activity of the molecule administered, its persistence in the bloodstream, and the decision of the responsible physician.

[0222] As used in this document, the term “therapeutically effective amount” refers to an amount of a drug effective in treating a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., delay, to some extent, and preferably stop) the infiltration of cancer cells into peripheral organs; inhibit (i.e., delay, to some extent, and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or alleviate, to some extent, one or more of the symptoms associated with the disorder. Since the drug can prevent the growth and/or destroy existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy, for example, can be measured by assessing survival duration, time to disease progression (TTP), response rates (RR), duration of response, and/or quality of life.

[0223] Cancers susceptible to treatment by the present invention include, but are not limited to: carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancies. The most specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder carcinoma, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade/follicular non-Hodgkin lymphoma (NHL); small lymphocytic NHL (SL); intermediate-grade/follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL); The invention relates to the treatment of: voluminous NHL disease; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); capillary lymphocyte leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. Cancerous conditions treatable by the invention include metastatic cancers.

[0224] According to other embodiments, the pharmaceutical composition according to the invention is for use in the treatment of cancer characterized by PVR overexpression. The types of cancer related to PVR overexpression can be identified using databases known as The Cancer Genome Atlas (TCGA). According to certain modalities, cancer is selected from a group consisting of adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), hepatocellular carcinoma of the liver (LIHC), colon and rectal adenocarcinoma (COAD, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), papillary kidney carcinoma (KIRP), lung adenocarcinoma (LUAD), squamous cell carcinoma of the head and neck (HNSC), prostate adenocarcinoma (PRAD), endometrial carcinoma of the uterine body (UCEC), cervical cancer (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladder carcinoma (BLCA), clear cell kidney carcinoma (KIRC), squamous cell carcinoma of the lung (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarian cystadenocarcinoma (OV), carcinoma of 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 invention.

[0225] The molecules of the present invention, as active ingredients, are dissolved, dispersed or mixed by addition to an excipient that is pharmaceutically acceptable and compatible with the active ingredient, as is a well-known fact. Suitable excipients are, for example, water, saline solution, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol or similar substances and combinations thereof. Other suitable vehicles are well known to those skilled in the art. Additionally, if desired, the composition may contain minimal amounts of auxiliary substances such as wetting and emulsifying agents, pH buffering agents.

[0226] The pharmaceutical composition according to the present invention can be administered together with an antineoplastic composition.

[0227] The term “treatment,” as used in this document, refers to both therapeutic treatment and prophylactic or preventive measures. Those who need treatment include those who already have the disorder, as well as those in whom the disorder should be prevented.

[0228] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include melanoma, lung, thyroid, breast, colon, prostate, liver, bladder, kidney, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterine, sarcoma, biliary, or endometrial cancer.

[0229] According to some embodiments, the cancer treatment method comprises administering the pharmaceutical composition as part of a treatment regimen that includes the administration of at least one additional anticancer agent.

[0230] According to some embodiments, the anticancer agent is selected from the group consisting of an antimetabolite, a mitotic inhibitor, a taxane, a topoisomerase inhibitor, a topoisomerase II inhibitor, an asparaginase, an alkylating agent, an antitumor antibiotic, and combinations thereof. Each possibility represents a separate embodiment of the invention.

[0231] According to some embodiments, the antimetabolite is selected from the group consisting of cytarabine, gludarabine, 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 irenotecan. According to some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosamide, mechlorethamine, melphalan, thiotepa, dacarbazine, and procarbazine. According to some embodiments, the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, 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.

[0232] According to some particular embodiments, the additional anticancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, daunorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0233] Monoclonal antibodies according to the present invention can be used as part of combination therapy with at least one anticancer agent. According to some embodiments, the additional anticancer agent is an immunomodulator, an activated lymphocyte cell, a kinase inhibitor, or a chemotherapeutic agent.

[0234] According to some embodiments, the anticancer agent is an immunomodulator, regardless of whether it is an agonist or antagonist, such as an antibody against an immune checkpoint molecule.

[0235] Checkpoint immunotherapy blockade has proven to be an exciting new avenue for cancer treatment. Immune checkpoint pathways consist of a range of co-stimulatory and inhibitory molecules that work together to maintain self-tolerance and protect tissues from damage by the immune system in response to physiological afflictions. Tumors exploit certain checkpoint pathways to evade the immune system. Therefore, inhibiting such pathways has emerged as a promising anticancer treatment strategy.

[0236] The anti-cytotoxic T lymphocyte antibody 4 (CTLA-4), ipilimumab (approved in 2011), was the first immunotherapeutic agent to show a benefit for the treatment of cancer patients. The antibody interferes with inhibitory signals during antigen presentation to T cells. The anti-programmed cell death antibody 1 (PD-1), pembrolizumab (approved in 2014), blocks negative immune regulatory signaling of the PD-1 receptor expressed by T cells. An additional anti-PD-1 agent was filed for regulatory approval in 2014 for the treatment of non-small cell lung cancer (NSCLC). Active research is currently exploring many other immunological checkpoints, including: CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activation gene 3 (LAG3), CD137, OX40 (also called CD134), and killer cell immunoglobulin-like receptors (KIR).

[0237] According to some specific embodiments, the immunomodulator is selected from the group consisting of: an antibody that inhibits CTLA-4, a human programmed cell death antiprotein 1 (PD-1), PD-L1 and PD-L2 antibodies, an activated cytotoxic lymphocyte cell, a lymphocyte activation agent, an antibody against CEACAM, an antibody against TIGIT, and an RAF/MEK pathway inhibitor. Each possibility represents a separate embodiment of the present invention. According to some specific embodiments, the additional immunomodulator is selected from mAb to PD-1, from mAb to PD-L1, from mAb to PD-L2, from mAb to CEACAM1, from mAb to CTLA-4, from mAb to TIGIT, Interleukin 2 (IL-2), or lymphokine-activated killer cell (LAK).

[0238] According to other embodiments, the additional anticancer agent is a chemotherapeutic agent. The chemotherapeutic agent, which could be administered together with the antibody according to the present invention, or separately, may comprise any of such agents known in the art, which exhibit anticancer activity, including, but not limited to: mitoxantrone, topoisomerase inhibitors, spindle venom inhibitors: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabine; podophyllotoxins: etoposide, irinotecan, topotecan, dacarbazine; antibiotics: daunorubicin (adriamycin), bleomycin, mitomycin; Nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferon, asparaginase; hormones: tamoxifen, leuprolide, flutamide, and megestrol acetate.

[0239] According to some embodiments, the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substituted urea, methylhydrazine derivatives, adrenocortical suppressant, adrenocorticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens and gonadotropin-releasing hormone analogs. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel and doxetaxel. One or more chemotherapeutic agents may be used.

[0240] In some embodiments, the pharmaceutical composition according to the present invention is for use in the treatment of cancer or for use in enhancing the immune response.

[0241] The term “intensify the immune response” refers to increasing the responsiveness of the immune system and inducing or prolonging its memory. The pharmaceutical composition according to the present invention can be used to stimulate the immune system through vaccination. Therefore, in one embodiment, the pharmaceutical composition can be used to enhance vaccination.

[0242] In certain embodiments, the cancer is selected from lung, thyroid, breast, colon, melanoma, prostate, liver, bladder, kidney, cervical, pancreatic, leukemia, lymphoma, myeloid, ovarian, uterine, sarcoma, biliary and endometrial cell cancers. Each possibility represents a separate embodiment of the invention.

[0243] According to some embodiments, a pharmaceutical composition comprising at least one antibody or fragment thereof according to the present invention, and a pharmaceutical composition comprising an additional immunomodulator or kinase inhibitor, are used in the treatment of cancer by separate administration.

[0244] According to yet another aspect, the present invention provides a method of treating cancer in an individual who needs it, comprising administering to said individual a therapeutically effective amount of a monoclonal antibody or antibody fragment according to the present invention.

[0245] The term “effective amount,” as used in this document, refers to a sufficient quantity of the monoclonal antibody or antibody fragment that, when administered to an individual, will have the intended therapeutic effect. The effective amount required to achieve the final therapeutic outcome may depend on numerous factors, including, for example, the specific type of tumor and the severity of the patient's condition, and the possibility of the combination being additionally co-administered with radiation. The effective amount (dose) of the active agents, in the context of the present invention, must be sufficient to effect a beneficial therapeutic response in the individual over time, including, but not limited to, inhibition of tumor growth, reduction in tumor growth rate, prevention of tumor and metastasis growth, and improved survival.

[0246] The toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the IC50 (the concentration that provides 50% inhibition) and the maximum tolerated dose for a compound of the material. Data obtained from these cell culture assays and animal studies can be used in formulating a dosage range for use in humans. The dosage may vary depending on, among other things, the dosage form employed, the dosage regimen chosen, the composition of the agents used for treatment, and the route of administration used, among other relevant factors. The exact formulation, route of administration, and dosage may be chosen by the individual physician in view of the patient's condition. Depending on the severity and responsiveness of the condition to be treated, the dosage may also be a single administration of a slow-release composition, where the course of treatment lasts from several days to several weeks or until a cure is achieved or a reduction in the disease state is attained. The amount of a compound to be administered will obviously depend on the individual being treated, the severity of the ailment, the method of administration, the decision of the attending physician, and all other relevant factors.

[0247] The term “administering” or “administration of” a substance, compound, or agent to an individual can be performed using a variety of methods known to those skilled in the art. For example, a compound or agent may be administered enterally or parenterally. Enterally refers to administration via the gastrointestinal tract, which includes per os, sublingual, or rectal. Parenteral administration includes intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, ocular, sublingual, intranasal, inhalation, intraspinal, intracerebral, and transdermal administration (by absorption, for example, through a skin duct). A compound or agent may also be suitably delivered by biodegradable or refillable polymeric devices or other devices, for example, patches and pumps, or formulations, that provide extended, slow, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and/or over one or more extended periods. In some modalities, administration includes both direct administration, which includes self-administration, and indirect administration, which includes the act of prescribing a drug. For example, as used in this document, a physician who instructs a patient to self-administer a drug, or to receive a drug administered by a third party, and/or who provides a patient with a prescription for a drug, administers the drug to the patient.

[0248] Antibodies are generally administered in the range of about 0.1 to about 20 mg/kg of patient weight, commonly about 0.5 to about 10 mg/kg, and frequently about 1 to about 5 mg/kg. In this regard, it is preferable to use antibodies that have a circulating half-life of at least 12 hours, preferably at least 4 days, more preferably up to 21 days. Chimeric antibodies are expected to have circulating half-lives of up to 14 to 21 days. In some cases, it may be advantageous to administer a large loading dose followed by periodic maintenance doses (e.g., weekly) throughout the treatment period. Antibodies can also be delivered by slow-release delivery systems, pumps, and other known delivery systems for continuous infusion.

[0249] The term “about” means that an acceptable error range, for example, up to 5% or 10%, for the particular value should be assumed.

Angiogenesis

[0250] According to one aspect, the present invention provides a pharmaceutical composition according to the present invention for use in the treatment of a disease or disorder related to angiogenesis.

[0251] Angiogenesis is an important cellular event in which vascular endothelial cells proliferate, separate, and reorganize to form new vessels from pre-existing vascular networks. There is strong evidence that the development of a vascular supply is essential for inflammation and pathological and normal proliferative processes. The vascular compartment is necessary not only for organ development and differentiation during embryogenesis but also for wound healing, tissue repair, and reproductive functions in the adult.

[0252] Angiogenesis is also implicated in the pathogenesis of a variety of disorders, including but not limited to, tumors, proliferative retinopathies, age-related macular degeneration, rheumatoid arthritis, and psoriasis. Angiogenesis is essential for the growth of most primary tumors and their subsequent metastasis. Tumors can absorb sufficient nutrients and oxygen through simple diffusion up to a size of 1 to 2 mm, at which point further growth requires the development of a vascular supply. This process is believed to involve the recruitment of adjacent mature host vasculature to begin sprouting new blood vessel capillaries, which grow toward, and subsequently infiltrate, the tumor mass. Additionally, tumor angiogenesis involves the recruitment of circulating endothelial precursor cells from the bone marrow to promote neovascularization.

Diagnosis

[0253] The present invention further discloses methods for cancer diagnosis and prognosis.

[0254] According to one aspect, the present invention provides a method for diagnosing and/or prognosticating cancer or infectious disease in an individual, wherein the method comprises the step of determining the level of PVR expression in a biological sample from said individual using at least one antibody as described in this document.

[0255] The term “biological sample” encompasses a variety of sample types obtained from an organism that can be used in a diagnostic or monitoring assay. The term includes blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and their progeny. Additionally, the term may include tumor or other circulating cells. The term specifically encompasses a clinical sample and additionally includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, urine, amniotic fluid, biological fluids including aqueous humor and vitreous humor for ocular samples and tissue samples. The term also encompasses samples that have been manipulated in any way after obtaining them, such as treatment with reagents, solubilization or enrichment for certain components.

[0256] The determination of PVR expression levels can be performed using an anti-PVR antibody identified as described in this document. Expression determination can be performed, for example, by ELISA.

[0257] The method of the invention may further comprise the step of comparing said expression level with a control level.

[0258] The following examples are presented to illustrate more fully some embodiments of the invention. They should in no way be interpreted as limiting the scope of the invention.

EXAMPLES Experimental Procedures

[0259] Reference is now made to the following examples, which, together with the descriptions above, illustrate the invention in a non-limiting way.

[0260] In general, the nomenclature used in this document and the laboratory procedures used in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are well known in the art. Other general references to well-known procedures are provided throughout this document for the convenience of the reader.

Example 1. High PVR expression correlates with poor prognosis.

[0261] PVR and Nectin-2 are ligands for the TIGIT receptor inhibitor (Figure 2). The results illustrate that high levels of PVR expression are correlated with poor cancer prognosis for lung cancer, breast cancer, and liposarcoma (Figures 1A to 1C, respectively). PVR GEO expression was correlated with survival using bioprofiling.de, whose relevant datasets are ID: GSE31210, GSE25055, GSE30929. Additionally, using the same analysis, Nectin-2 expression was primarily a positive marker for survival.

Example 2. Generation and purification of anti-PVR mAbs

[0262] To generate anti-PVR antibodies, a recombinant protein was produced and purified, hPVR-Fc, which combines the extracellular part of human PVR and the human Fc region of an immunoglobulin G vehicle as an immunogen.

[0263] BALB/c mice were injected with 50 μg of the immunogen in complete Freund's adjuvant and, after 2 weeks, in incomplete Freund's adjuvant. After 2 weeks, the sera were examined for antibody titers. Those showing the best responses (serum was monitored by ELISA assay for anti-hPVR-Fc antibody titers) were intensified with the immunogen in PBS. After three days, spleen cells were collected, and after red blood cell lysis, fused with SP2/0 cells. The cells were seeded in 20% RPMI 1640 medium, which contains hypoxanthine, aminopterin, and thymidine, for hybridoma selection and examined for mAbs using ELISA. Stable hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenic cells from an immunized mouse.

[0264] Positive results (cell lines secreting antibodies that recognize hPVR-Fc) were further selected to develop a product that will have several differentiating characteristics: a) high yield to reduce antibody production costs; b) lack of cross-reactivity between mouse and human PVR and several other ligands of immune cell receptors; c) a strong binding capacity with mature human native PVR molecules expressed on the surface of living cells (the antibodies were chosen from a comprehensive anti-hPVR monoclonal antibody cluster with proven ability to recognize hPVR in different techniques, e.g., flow cytometry, western blot, ELISA). In fact, human and mouse PVRs have a high level of homology, and it is not easy to generate a mouse monoclonal antibody that recognizes a human homolog. More importantly, human PVR is extensively glycosylated in its extracellular region. For these reasons, it is not easy to generate an antibody that recognizes a native protein using common antigens (such as E. coli derivatives).

[0265] The present inventors used hPVR-Fc immunogen, a molecule produced in human embryonic kidney 293 (HEK 293T) mammalian cells and purified under native conditions by immunoaffinity chromatography, to approximately simulate the native protein, human PVR. In conclusion, from a cluster of generated anti-hPVR mAbs, representatives that recognize a native human PVR, which form in living cells, were identified, which is a prerequisite for developing a derivative that would influence the human immune cell response during treatment.

[0266] Four anti-PVR antibodies were generated. Among these, three antibodies were blocking anti-PVR mAbs, namely, antibody 5B9 (also called hPVR.09), antibody 7D4 (also called hPVR.01), and antibody 4E5 (also called hPVR.07). All of these antibodies block TIGIT-Ig binding (as illustrated in Figures 3B to 3D, respectively). A fourth antibody was generated, which does not block TIGIT-Ig binding and was named 2G3 (also called hPVR.17) (Figure 3A).

[0267] The Biacore™ T100 surface plasmon resonance (SPR) biosensor (GE Healthcare) was used to determine Koff, Kon, and KD between antibodies and hPVR (Table 1). Table 1. Antibody affinity measurement using Biacore™

[0268] The chimeric monoclonal antibodies, comprising the human heavy chain constant IgG1 region presented in SEQ ID NO: 86 (corresponding to GenBank: AAA02914.1), were produced from the three antibodies above using methods known in the art.

Example 3. Blocking of PVR-TIGIT interactions with enhanced NK cell kill by anti-PVR mAbs from human cell lines.

[0269] Target cells were identified with [35S]-Methionine 12 hours prior to the assay. The indicated antibodies were added to a final concentration of 5 μg/ml and incubated with the identified targets (5000 cells/well) for 30 minutes on ice. Assays were performed in RPMI medium in 96 U-shaped plates at 37 °C for 5 hours. The identified targets were incubated with effector NK cells at an E:T ratio of 10:1. After incubation, the plates were centrifuged (1600 rpm, 5 min, 4 °C) and the supernatants (50 μl) were collected and transferred to Opti-opaque plates (Packard). 150 μl of scintillation fluid (Perkin Elmer) were added and analyzed by a micro beta, β-counter (Perkin Elmer). Maximum identification was determined by adding 100 μl of 0.1 N NaOH to an equal amount of targets (5000/well). Spontaneous release was determined in wells containing only target cells. Final specific kill was calculated as follows: ((radioactive readout - spontaneous release) / (maximum identification - spontaneous release)) * 100 = specific kill. As shown in Figure 4, NK cell culture with the anti-PVR 7D4 mAb (also called hPVR.01) intensified (doubled) NK cell kill of the human breast adenocarcinoma cell line, MDA-MB-231.

Example 4. Blocking PVR-TIGIT interactions with enhanced NK cell extermination by anti-PVR mAbs from human cancer cell lines

[0270] Target cells were identified with [35S]-Methionine 12 hours before the assay. The indicated antibodies were added to a final concentration of 5 μg/ml and incubated with the identified targets (5000 cells/well) for 30 minutes on ice. The cells were incubated with effector NK cells at an E:T ratio of 10:1. Assays were performed in RPMI medium in 96 U-shaped plates at 37 °C for 5 hours. After incubation, the plates were centrifuged (1600 rpm, 5 min, 4 °C) and the supernatants (50 μl) were collected and transferred to Opti-opaque plates (Packard). 150 μl of scintillation fluid (Perkin Elmer) were added and analyzed by a micro beta, β-counter (Perkin Elmer). Maximum identification was determined by adding 100 μl of 0.1 N NaOH to an equal amount of targets (5000/well). Spontaneous release was determined in wells containing only target cells. The final specific kill was calculated as follows: ((radioactive readout - spontaneous release) / (maximum identification - spontaneous release)) * 100 = specific kill.

[0271] As shown in Figure 5, blocking PVR-TIGIT interactions with the anti-PVR mAb 4E5 (also called hPVR.07) intensifies the NK cell kill of the human cancer cell line, HepG2 (hepatocellular carcinoma). Therefore, it is clear that blocking PVR leads to intensified kills of target cells. Kills are further intensified when the human IgG counterpart of 4E5 is used in a chimeric version of the mAb. Kills are equivalent to the positive control (Erbitux©).

Example 5. Human tumor cell lines express PVR and Nectin-2.

[0272] To examine the expression of PVR and Nectin-2 in tumor cells, the expression levels of these proteins in different tumor cell lines were examined by FACS analysis using the anti-PVR-4E5 antibody and the anti-Nectin-2 antibody (clone TX-31), both at 2 μg/ml. As shown in Figures 6A to 6O, several human tumor cell lines express PVR and Nectin-2. Specifically, melanoma cells (Figures 6A to 6E), breast cancer cells (Figures 6F to 6H), colorectal cells (Figure 6I), kidney cells (Figure 6J), lung cancer cells (Figure 6K), prostate cancer cells (Figure 6L), brain tumor cells (Figure 6M), and hepatocellular carcinoma cells (Figures 6N to 6O) are all shown to express PVR and Nectin-2.

Example 6. PVR is the main TIGIT binder.

[0273] Figures 7A to 7C demonstrate that PVR is the main TIGIT ligand. Specifically, it has been shown that HepG2 cells (human hepatocellular carcinoma cells) express both PVR and Nectin-2 (Figure 7A). HepG2 cell culture with purified anti-PVR mAb 4E5, also called hPVR.07 (0.15 μg/well), almost completely blocked TIGIT-Ig binding (2 μg/ml) (Figure 7B), despite the fact that these cells also express Nectin-2. As shown in Figures 7C and 7D, it is evident that there is no direct recognition of Nectin-2 by anti-PVR mAbs.

Example 7. Linking mAb clones to human and primate PVR.

[0274] As shown in Figures 8A to 8C, all tested anti-PVR antibody clones bind to human PVR (hPVR) using FACS analysis. Briefly, cells were trypsinized and transferred to staining at 2*10⁵ cells per well. The indicated antibodies were added for 30 min on ice. All antibodies were used at a final concentration of 2 μg/ml. Detection of bound anti-PVR antibodies was performed using mouse IgG-647. Mouse IgG1 kappa was used as a negative control.

[0275] Figure 8A illustrates the fact that endogenous hPVR was detected by FACS staining on the surface of HepG2 hepatocellular carcinoma cells. In Figure 8B, the murine cell line B16 was used. The murine PVR sequence is different from the human species and is not recognized by anti-human PVR antibodies, as can be seen by the lack of signal (left panel). When the full-length hPVR protein (1418 amino acids of NP_006496.4) was overexpressed in these cells, all three clones resulted in identical signal (right panel), thus further supporting the claim that this staining is the result of specific binding of these Abs to the human PVR protein.

[0276] The amino acid sequence of PVR is conserved across some species. The amino acid conservation of human PVR was compared with that of African green monkeys in silico and was found to be 93% similar to human PVR. FACS staining of African green monkey Vero cells demonstrated that monkey PVR is effectively recognized by all three human mAbs (Figure 8C). It was further found that these anti-human PVR antibodies do not recognize PVR from canines and rodents such as crickets or mice. Figure 9 shows the FACS analysis for MDCK cells expressing canine PVR. As can be seen, none of the anti-human Abs resulted in a positive signal, while PVR expression itself is suggested by the strong TIGIT-Ig signal.

Example 8. Nectin-2 is preferentially bound by DNAM-1

[0277] TIGIT-Fc and DNAM-1-Fc were used at the concentrations indicated in Figures 10 to stain cells overexpressing hNectin-2: the RPMI-8866 cell line (Figures 10A and 10B), or the B16-hPVR cell line (Figures 10C and 10D). Detection of bound fusion protein was performed using anti-human IgG APCs and analyzed by FACS.

[0278] For PVR binding, the signal by TIGIT-Fc was 2 to 4 times greater than the signal by DNAM-1-Fc, similarly to a previous report (Yu. X et al., 2009, Nat Immunol., 10(1):48-57). At the same time, hNectin-2 binding to DNAM-1-Fc was 2 to 10 times stronger than its binding to TIGIT-Fc, which is contradictory to a previous report (Yu. X et al. 2009), but corroborated by another study (Zhu Y et al., 2016, J Exp Med., 8;213(2):167-76). Taken together, the results illustrate that DNAM-1 is the preferred receptor for Nectin-2 binding. Consequently, blocking PVR will prevent inhibitory TIGIT signaling while allowing co-stimulatory signaling via DNAM-1. DNAM-1 mediates cell adhesion to other cells carrying its ligands.

Example 9. Anti-PVR antibodies enhance T cell proliferation.

[0279] To test the effect of anti-PVR mAbs on T cell proliferation, human peripheral blood mononuclear cells (PBMCs) were stained with carboxyfluorescein succinimidyl ester (CFSE) and incubated with target cells in the presence of antibodies at a concentration of 4 μg/ml. The CFSE dilution was measured in CD45-positive cells after 5 to 9 days in culture. As shown in Figure 11, the activity of 5B9 anti-PVR exceeds the activity of PD-1 and CTLA4 antibodies as a single agent. Furthermore, the chimeric anti-PVR clone 4E5hIgG1, which has a human IgG1 constant region, was superior to its mouse counterpart. Next, the combined effect of anti-PVR mAbs and other antibodies, which were found to enhance T cell proliferation, was examined. Human PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and incubated with target cells in the presence of antibodies at a concentration of 4 μg/ml. The CFSE dilution was measured in CD45-positive cells after 5 to 9 days in culture. The results show that the proliferation activity of 5B9 anti-PVR, when combined with PD-1 or CTLA-4, exceeds the activity of a combination of PD-1 and CTLA4 (Figure 12). Furthermore, the activity of a combination of anti-PVR 4E5 and CTLA-4 is equal to the combination of PD-1 and CTLA4. Next, specific CD8 induction was examined. As shown in Figure 13, the CD8 T cell proliferation activity of anti-PVR antibodies exceeds the activity of PD1. In addition, the induction activity of 5B9 anti-PVR antibody exceeds that of CTLA1. The CD8/CD4 proliferation ratio was evaluated for different antibodies (Figure 14). 5B9 anti-PVR showed the highest CD8/CD4 ratio. The DNA sequence of the variable heavy and light chains was used to construct chimeric antibodies, comprising the constant domains of human IgG1 isotype and the constant light human IgG kappa domain (CL). T cell proliferation induced by chimeric mouse and human counterpart antibodies was measured by the CFSE assay. The numbers shown in Table 2 represent the relative level of proliferation compared to the control. Table 2. A summary of the effect of anti-PVR antibodies on T cell proliferation.

[0280] Next, the effect of PVR antibodies on NK degranulation was examined. During degranulation, cytolytic granules in NK cells are released and lysosome-associated membrane protein-1 (LAMP-1, CD107a), which is present on the surface of cytolytic granules, is transported to the cell surface and becomes accessible for antibody binding. This marker allows the identification of activated NK cells. NK cells were incubated with 5 different target cells along with anti-PVR antibodies (mouse and their chimeric human counterpart antibodies). Degranulation was assessed using anti-CD107a antibodies. Table 3. Effect of anti-PVR antibodies on NK degranulation activity. * hPVR expression in Mel-624 is low, therefore the relative effect is low.

[0281] As shown in Table 3 and Figure 15, all antibodies showed NK degranulation activity, with the chimeric antibodies exhibiting significantly higher activity compared to their corresponding mouse antibody.

Example 10. Anti-PVR antibodies reduce the survival of tumor cells in the absence of immune cells.

[0282] The survival of A549, U373, HCT116, and Mel-624 cells was examined using the MTT cell survival assay in the presence of 50 micrograms/ml of different anti-PVR mAbs for 24 hours. As shown in Figures 16A to 16D and Table 4, PVR blockade by 5B9 mAb significantly reduced viability by 20 to 40% compared with mIgG. Table 4. Effect of anti-PVR antibodies on tumor cell survival.

[0283] Percentage of dead cells in 24 h across various target cell lines in relation to cells treated with mIgG.

Example 11. In vivo effect of anti-PVR antibodies on human tumor in a humanized mouse model - short-term humanization

[0284] The antitumor efficacy of antibodies is studied in vivo. To estimate the efficacy of the antibodies described in this document in inhibiting human cancer, the antibody is studied in a model that combines both tumors and lymphocytes of human origin. NOD scid gamma (NSG) type mice are grafted with hPBMCs to restore immune competence and challenged with human cancer cells. At a predetermined time point/tumor size, the mice are treated with human anti-PVR antibody according to the invention, receiving multiple doses at different time points after tumor challenge. The same experiments are performed with chimeric anti-PVR antibodies. Tumor growth curves and body weight are measured 3x/week and, upon sacrifice of the mice, extensive phenotypic analysis of TILs and immune populations in different organs is performed.

[0285] A similar model, with tumor cell lines in huNSG-like PBMC mice, is performed according to Gupta P., Oncoimmunology. March 6, 2015; 4(2): e981449.

Example 12. In vivo effect of anti-PVR antibodies on human tumor in a humanized mouse model - long-term humanization

[0286] To estimate the efficacy of anti-PVR antibodies in inhibiting human cancer, the antibody is studied in a model that combines both tumors and lymphocytes of human origin. Newborn NOD scid gamma (NSG) type pups are irradiated and grafted with CD34+ HSC to restore immune competence. 1 to 2 weeks after determining immune cell reconstitution, mice are challenged with human cancer cells and, at a predetermined time point/tumor size, treated with human anti-PVR antibody according to the invention, receiving multiple doses at different time points. The same experiments are performed with humanized anti-PVR antibodies. Tumor growth curves and body weight are measured 3x/week. Upon sacrifice of the mice, extensive phenotypic analysis of TILs and immune populations in different organs is performed.

[0287] A similar model used to study the tumor-inhibiting activity of the antibodies of the present invention was established by The Jackson Laboratory (http://immune-checkpoint.com/wp-content/uploads/sites/24/2015/01/Day-1-15.45-Rick-Huntress.pdf).

Example 13. Induction of IFNY secretion

[0288] To test the effect of anti-PVR mAbs on cytokine secretion, human peripheral blood mononuclear cells (PBMCs) from 2 healthy donors were incubated with target cells in the presence of antibodies (mIgG, 5B9mIgG, anti-CTL-4 antibody, named Ipilimumab), at concentrations of 1 and 0.1 μg/ml. IFNY levels after 6 days in culture were measured. A significant induction of IFNY by the 5B9 anti-PVR antibody was observed (P = 7.34548E11 for 1 μg/ml and 2.73179E08 for 0.1 μg/ml).

[0289] IFNγ secretion is a key component of antitumor immunity, the induction of IFNγ secretion by anti-PVR mAbs indicates the potential additional antitumor effect of these compounds in cancer treatment.

[0290] The aforementioned description of specific embodiments will reveal the general nature of the invention so completely that third parties may, through the application of current knowledge, readily modify and/or adapt such specific embodiments for various applications without undue experimentation and without departing from the generic concept, and therefore such adaptations and modifications must be and are intended to be understood within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the phraseology or terminology employed herein is for descriptive purposes and not for limitation.

Claims (11)

1. Isolated monoclonal antibody that binds to the human poliovirus receptor (PVR) or an antibody fragment thereof, characterized by comprising at least the antigen-binding portion, wherein the isolated antibody or antibody fragment comprises: a set of six CDRs wherein: HC CDR1 is selected from GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); HC CDR2 is EIFPGSGRINFNEKFKG (SEQ ID NO: 38); HC CDR3 is TKIYGNSFDY (SEQ ID NO: 40); LC CDR1 is selected from KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); The CDR2 of LC is selected from the group consisting of: WASSRHN (SEQ ID NO: 46), WASSRHA (SEQ ID NO: 56), WASSRHR (SEQ ID NO: 57), WASSRHD (SEQ ID NO: 58), WASSRHE (SEQ ID NO: 59), WASSRHP (SEQ ID NO: 60) and WASSRHT (SEQ ID NO: 61); and the CDR3 of LC is QQYSRYPLT (SEQ ID NO: 48), in which the isolated monoclonal antibody is capable of inhibiting the binding of PVR to the T cell immunoreceptor with Ig and ITIM domains (TIGIT). 2. Isolated monoclonal antibody or antibody fragment according to claim 1, characterized in that the CDR1 sequence of HC is selected from the group consisting of GYTFSNYWIE (SEQ ID NO: 36) and SNYWIE (SEQ ID NO: 84); the CDR2 sequence of HC consists of the sequence EIFPGSGRINFNEKFKG (SEQ ID NO: 38); the CDR3 sequence of HC consists of the sequence: TKIYGNSFDY (SEQ ID NO: 40); the CDR1 sequence of LC is selected from the group consisting of KASQDVGTAVV (SEQ ID NO: 44) and KASQDVGTAV (SEQ ID NO: 85); the CDR2 sequence of LC consists of the sequence: WASSRHE (SEQ ID NO: 59); and the CDR3 sequence of LC consists of the sequence: QQYSRYPLT (SEQ ID NO: 48). 3. Polynucleotide sequence, characterized by encoding the isolated monoclonal antibody or antibody fragment, as defined in claim 1 or 2, wherein the polynucleotide sequence comprises SEQ ID NO: 33 and SEQ ID NO: 41. 4. Plasmid, characterized by comprising the polynucleotide sequence as defined in claim 3. 5. Monoclonal antibody according to claim 1 or 2, characterized by being attached to a cytotoxic chemical moiety, a radioactive chemical moiety, or an identifiable chemical moiety. 6. Pharmaceutical composition, characterized by comprising, as an active ingredient, at least one isolated antibody or fragment thereof, as defined in claim 1 or 2, and an acceptable pharmaceutical vehicle, salt, diluent or excipient. 7. Use of the pharmaceutical composition as defined in claim 6, characterized by being in the manufacture of a medicament for the treatment of cancer. 8. Use of the pharmaceutical composition according to claim 7, characterized by further comprising the use, by said individual, of an additional immunomodulator, activated lymphocyte cell, kinase inhibitor, chemotherapeutic agent or any other anticancer agent. 9. Use of the pharmaceutical composition according to claim 8, characterized in that the additional immunomodulator is an antibody against an immune checkpoint molecule selected from the group consisting of PD-1, CTLA-4, PDL-1, CEACAM1, NKG2A, B7-H3, B7-H4, VISTA, CD112R, lymphocyte activating gene 3 (LAG3), CD137, OX40 (also called CD134), killer cell immunoglobulin-like receptors (KIR), TIGIT and any combination thereof. 10. Use of the pharmaceutical composition according to claim 7, characterized in that the cancer is a solid cancer. 11. Use of the pharmaceutical composition according to claim 7, characterized in that the cancer is a hematological cancer.