Antibodies against poliovirus receptor (PVR) and uses thereof

Abstract

SOLUTION: To provide humanized antibodies and antigen binding fragments thereof that bind to human poliovirus (PVR), the antibodies being useful in the treatment of tumors or cancers.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to humanized antibodies that specifically bind to the human poliovirus receptor (PVR) and contain specific sets of CDR and framework sequences. The present invention also relates to pharmaceutical compositions containing these humanized antibodies and uses thereof. [Background technology]

[0002] The poliovirus receptor (PVR), also known as CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. This receptor was previously described as a tumor antigen and a potential target for therapeutic intervention, due to its upregulated expression in neuroectodermal cancers, including glioblastoma multiforme, medulloblastoma, and colorectal cancer (Solecki et al., J. Biol. Chem. 2002, 277:25697-700), as well as 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, upregulation of cyclins D2 and E, and downregulation of p27Kip1, ultimately shortening the G0 / G1 phase of the cell cycle (Kakunaga 2004, J. Biological Chemistry, 279, 36419-36425). Therefore, blocking PVR in tumor cells is predicted to reduce their viability. PVR also plays an important role in angiogenesis, regulating the interaction between vascular endothelial growth factor receptor 2 (VEGFR2) and integrin α(v)β(3) and the VEGFR2-mediated Rap1-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012, 110(5), 716-26). In addition, PVR forms a complex with IGF1R, which is involved in tyrosine-protein kinase Met (cMet) signaling, and blocking the complex formation reduces cell viability and angiogenesis (Lee et al., Scientific Reports 2014, 20, 4, 7139).

[0003] In recent years, PVR has emerged as an important immune checkpoint ligand (Brilc PK et al. 2019 Cell Mol Immunology). PVR expression is upregulated in both malignant cells and tumor-infiltrating myeloid cells in humans and mice. PVR- / - mice exhibit reduced tumor growth and metastasis via DNAM-1 (CD226) upregulation, as well as enhanced effector function of CD8+ T and NK cells, respectively. Blockade of programmed cell death protein 1 (PD-1) or both PD-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA4) is more effective in settings where PVR is limited, suggesting the clinical potential of combination therapy using PD-1 / PD-L1 and PVR blockade (Li XY et al. JCI 2018). Furthermore, in clinical settings, PD-L1 and PVR expression are independently regulated, allowing patients treated with anti-PD-1 antibodies to be stratified into four groups according to PD-L1 and PVR expression levels. High PVR expression in patients with low PD-L1 expression enriched non-responders. This was further validated using genetically engineered cancer models. These findings support the significance of PVR as an important immune checkpoint in tumor immunotherapy (Lee BR et al. JCI.Insight 2020). The involvement of PVR in metastasis was demonstrated by injecting cancer cells into the tail of mice and measuring metastasis to the lung. It has been shown that upregulated PVR in cancer cells interacts with its counterreceptor in platelets, and this interaction enhances cancer cell metastasis to the lung (Morimoto et al., Oncogene (2008) 27, 264-273).

[0004] WO2017149538 to one of the present inventors discloses murine antibodies and fragments thereof that bind to PVR as well as encoding polynucleotide sequences and hybridoma cells that produce these antibodies.

[0005] U.S. Patent Application No. 20070041985 discloses molecules that specifically bind to at least one intracellular or extracellular domain of PVR, which molecules have the ability to modulate receptor-mediated adhesion, trafficking, and / or invasive behavior of cells expressing PVR or any derivative thereof.

[0006] U.S. Patent Application No. 20090215175 provides molecules (e.g., small molecule compounds, oligonucleotides, polypeptides, antibodies, and antibody fragments) that modulate PVR functions necessary for cell adhesion, trafficking, invasion, and / or metastasis. These molecules can be used to treat cells with metastatic potential, metastasis, and cancer.

[0007] There is an unmet need to provide humanized antibodies that recognize human PVR that are safer and more potent and can be used diagnostically and therapeutically in diseases involving PVR expression. Summary of the Invention

[0008] Described herein, in some embodiments, are humanized antibodies that specifically bind to the human poliovirus receptor (PVR, CD155) and interfere with PVR binding to at least one of the T cell immunoreceptor with ligand, Ig, and ITIM domains (TIGIT), CD96, and CD226 (DNAM-1). The humanized antibodies of the present invention, selected from a larger collection of antibody clones, have improved properties compared to other known anti-PVR antibodies. These improved properties include, but are not limited to, reduced immunogenic potential, improved binding affinity and activity, improved biophysical properties, and improved expression. Because PVR binding to CD226 downregulates surface expression of CD226 on T cells and NK cells, stimulating their killing, the antibodies of the present invention can restore the expression and / or activity of CD266 on these cells. Proper expression and function of CD226 allows for enhanced tumor killing by immune cells, particularly CD8+ T cells and NK cells.

[0009] A large collection of humanized antibodies has been produced by combining specific sets of CDR sequences and human framework sequences and introducing specific mutations into these sequences to produce improved antibodies with modified variable regions. The newly designed humanized variable regions minimize the risk of adverse immune reactions to the antibody by minimizing the occurrence of potential T-cell epitopes while preserving residues important for maintaining the antibody's conformation and binding affinity. The antibodies disclosed herein were designed based on factors including homology, T-cell epitopes, critical residues, and predicted structure.

[0010] Unexpectedly, a mutant with a glutamic acid to asparagine point mutation in the last residue of CDR2 of the light chain variable region (position 56 according to Kabat numbering) combined with a specific human framework showed strong affinity for human PVR and improved immunoreactivity.

[0011] Due to their low affinity, various humanized antibody variants according to the present invention have been found to be particularly suitable for use in chimeric antigen receptors (CARs), which may be useful for targeting highly expressed PVR tumor cells without targeting normal tissues.

[0012] Advantageously, the various humanized antibody variants according to the present invention have improved manufacturability and can be produced in exceptionally high yields compared to other variants.

[0013] It is disclosed herein that the humanized antibodies described herein exhibit high efficacy in cytotoxic T cell and NK cell stimulation and in treating cancer in humanized mouse models, including in vivo models of pancreatic and lung cancer.

[0014] Thus, the present invention provides, in some embodiments, highly specific, non-immunogenic, humanized antibodies against human PVR with improved affinity, activity, and / or reproducibility.

[0015] In one aspect, the present invention provides a humanized antibody that specifically binds to human polyviral receptor (PVR, CD155), or a fragment thereof comprising at least an antigen-binding site, wherein the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0016] In some embodiments, the antibody comprises a heavy chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, and a light chain variable region amino acid sequence comprising the CDR sequences set forth in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0017] In some embodiments, the humanized antibody or fragment thereof is an IgG monoclonal antibody. In some embodiments, the humanized monoclonal antibody has a heavy chain constant region selected from IgG4 and IgG1. In certain embodiments, the humanized antibody or fragment thereof is of the IgG4 subclass. In certain embodiments, the humanized antibody or antigen-binding fragment thereof is of the IgG1 subclass.

[0018] According to some embodiments, the humanized antibody or fragment thereof comprises a human IgG4 constant region with a S228P (also called S241P) substitution in the hinge region.

[0019] According to some embodiments, the humanized antibody or fragment thereof is a monoclonal antibody, a Fab, a F(ab)2, a single domain antibody, or a single chain variable fragment (scFv).

[0020] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence QVQLVQSGAE(L / V)KKPGASVK(I / V)SCKATGYTFSNYWIEW(I / V)(K / R)QAPGQGLEW(I / M)GEIFPGSGRINFNEKFKGR(A / V)TFTADTSI(D / S)T(T / A)YM(Q / E)LS(S / R)L(T / R)SDD(S / T)AVYYCARTKIYGNSFDYWGQGT(T / L)VTVSS (SEQ ID NO: 47), and a heavy chain variable region comprising the amino acid sequence DI(M / Q)MTQSPS(F / S)LSASVGDRVTITC(K / R)ASQDVGTAV(V / A)WYQQKPGKAPK(L / S)LIYWASSRHEGVP(D / S)RF(T / S)GSGSGTDFTLTISS and a light chain variable region comprising: LQ(S / P)EDFA(D / T)YFCQQYSRYPLTFGQGT KLEIK (SEQ ID NO: 48).

[0021] According to some embodiments, the humanized antibody or fragment thereof comprises: i. A set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 10 to 12; ii. a set of four heavy chain framework (FR) sequences, namely: (A) FR-H1 selected from the group consisting of SEQ ID NOs: 18, 22, and 26; (B) FR-H2 selected from the group consisting of SEQ ID NOs: 19, 23, and 28; (C) FR-H3 selected from the group consisting of SEQ ID NOs: 20, 24, 27, and 29; and (D) FR-H4 selected from the group consisting of SEQ ID NOs: 21 and 25. The chain variable region comprises:

[0022] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises: i. A set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 13 to 15; ii. a set of four light chain framework sequences: (A) FR-L1 selected from the group consisting of SEQ ID NOs: 30 and 34; (B) FR-L2 selected from the group consisting of SEQ ID NOs: 31 and 37; (C) FR-L3 selected from the group consisting of SEQ ID NOs: 32, 35, and 36; and (D) FR-L4, which is SEQ ID NO: 33. The light chain variable region comprises:

[0023] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: i. A set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 10 to 12; ii. a set of four heavy chain (HC) framework (FR) sequences, namely: (A) FR-H1 selected from the group consisting of SEQ ID NOs: 18, 22, and 26; (B) FR-H2 selected from the group consisting of SEQ ID NOs: 19, 23, and 28; (C) FR-H3 selected from the group consisting of SEQ ID NOs: 20, 24, 27, and 29; and (D) FR-H4 selected from the group consisting of SEQ ID NOs: 21 and 25. and the light chain variable region comprises: i. A set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 13 to 15; ii. a set of four light chain (LC) framework sequences (FR), namely: (A) FR-L1 selected from the group consisting of SEQ ID NOs: 30 and 34; (B) FR-L2 selected from the group consisting of SEQ ID NOs: 31 and 37; (C) FR-L3 selected from the group consisting of SEQ ID NOs: 32, 35, and 36; and (D) FR-L4, which is SEQ ID NO: 33. Includes:

[0024] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. In certain embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. In one embodiment, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0025] According to some embodiments, the humanized antibody comprises a combination of a heavy chain variable region and a light chain variable region, the combination comprising: i. a heavy chain variable region sequence set forth in SEQ ID NO: 1 and a light chain variable region sequence set forth in SEQ ID NO: 2; ii. a heavy chain variable region sequence set forth in SEQ ID NO: 4 and a light chain variable region sequence set forth in SEQ ID NO: 8; iii. A heavy chain variable region sequence set forth in SEQ ID NO: 5 and a light chain variable region sequence set forth in SEQ ID NO: 2; iv. A heavy chain variable region sequence set forth in SEQ ID NO: 5 and a light chain variable region sequence set forth in SEQ ID NO: 8; v. a heavy chain variable region sequence set forth in SEQ ID NO:4 and a light chain variable region sequence set forth in SEQ ID NO:2; vi. a heavy chain variable region sequence set forth in SEQ ID NO: 1 and a light chain variable region sequence set forth in SEQ ID NO: 8; vii. A heavy chain variable region sequence set forth in SEQ ID NO: 6 and a light chain sequence set forth in SEQ ID NO: 2, and viii. The heavy chain variable region sequence set forth in SEQ ID NO: 6 and the light chain variable region sequence set forth in SEQ ID NO: 8 is selected from the group consisting of:

[0026] In some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:1, and the light chain variable region comprises an amino acid sequence at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:2.

[0027] In some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:1, and the light chain variable region comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:2.

[0028] According to some embodiments, the humanized antibody inhibits binding of PVR to at least one of TIGIT, CD96, and CD226.

[0029] According to some embodiments, the antibody inhibits binding of PVR to TIGIT, CD96, and CD226.

[0030] According to some embodiments, the humanized antibody is an IgG4 antibody comprising the heavy chain sequence set forth in SEQ ID NO: 49, or a sequence with at least 90% identity. According to some embodiments, the humanized antibody is an IgG1 antibody comprising the heavy chain sequence set forth in SEQ ID NO: 50, or a sequence with at least 90% identity.

[0031] According to some embodiments, the humanized antibody comprises the light chain sequence set forth in SEQ ID NO:49.

[0032] According to some embodiments, humanized antibodies exhibit improved antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) compared to other known antibodies.

[0033] Polynucleotide sequences encoding the humanized antibody or antigen-binding fragment thereof are provided according to another aspect of the invention.

[0034] Some embodiments provide polynucleotide sequences that encode the amino acid sequences of the heavy chain variable region, the light chain variable region, or both, as described above.

[0035] Some embodiments provide a polynucleotide encoding a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0036] Some embodiments provide a polynucleotide encoding a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0037] According to some embodiments, the polynucleotide encodes a humanized antibody or antibody fragment thereof comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. Each combination of heavy chain variable region and light chain variable region represents a separate embodiment of the present invention.

[0038] According to some embodiments, the polynucleotide sequence encoding the humanized antibody heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 38-42, or a variant thereof having at least 90% sequence identity, with each possibility representing a separate embodiment of the present invention.

[0039] According to some embodiments, the polynucleotide sequence encoding the humanized antibody light chain variable region comprises a sequence selected from the group consisting of SEQ ID NOs: 43-46, or a variant thereof having at least 90% sequence identity, with each possibility representing a separate embodiment of the present invention.

[0040] In a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one humanized antibody chain or fragment thereof as described herein. According to some embodiments, the nucleic acid construct is a plasmid.

[0041] Also described are cell lines containing nucleic acids encoding the antibodies of the invention. The cell lines are for expression of the humanized antibodies or fragments thereof described herein. In some embodiments, the cell lines are mammalian cell lines, such as Chinese hamster ovary (CHO) cell lines.

[0042] According to some embodiments, the cell line is bacterial, plant, murine (e.g., NS0 and Sp2 / 0), rat (e.g., YB2 / 0), hamster (e.g., BHK and CHO), or human (e.g., PER.C6).

[0043] In one aspect, the present invention provides chimeric antigen receptors (CARs) comprising an extracellular portion (binding domain) containing any of the humanized antibodies or fragments thereof described herein. In some embodiments, CARs are provided that comprise combinations of the heavy and light chain variable region sequences described above, with unique combinations of CDR and framework sequences, resulting in improved binding and other properties.

[0044] According to some embodiments, the CAR comprises a combination of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0045] According to some embodiments, the CAR comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0046] According to some embodiments, the CAR comprises a combination of a humanized antibody heavy chain variable region and a light chain variable region, the combination comprising: i. a heavy chain variable region sequence set forth in SEQ ID NO: 1 and a light chain variable region sequence set forth in SEQ ID NO: 9; ii. a heavy chain variable region sequence set forth in SEQ ID NO: 3 and a light chain variable region sequence set forth in SEQ ID NO: 9; iii. A heavy chain variable region sequence set forth in SEQ ID NO: 4 and a light chain variable region sequence set forth in SEQ ID NO: 9; iv. a heavy chain variable region sequence set forth in SEQ ID NO: 5 and a light chain variable region sequence set forth in SEQ ID NO: 9, and v. The heavy chain variable region sequence set forth in SEQ ID NO: 6 and the light chain variable region sequence set forth in SEQ ID NO: 9 is selected from the group consisting of:

[0047] According to some embodiments, the CAR comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 1, 3, 4, 5, and 6, a light chain variable region sequence set forth in SEQ ID NO: 9, a transmembrane domain, and an intracellular T cell signaling domain.

[0048] Also provided by the present invention are single chain variable domains (scFv) comprising the heavy and light chain variable domains of the antibodies described herein. In some embodiments, a hinge region is present between the variable domains.

[0049] According to some embodiments, the amino acid sequence of the scFv is set forth in a sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, and analogs thereof having at least 90% sequence similarity to any of these sequences.

[0050] According to some embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NO:56 or SEQ ID NO:57.

[0051] In some embodiments, the CAR comprises an scFv sequence and at least one protein domain selected from the group consisting of a CD8 stalk domain, a CD28TM domain, a 41BB domain, and a CD3ζ (CD3Z, Zetta) domain. In some embodiments, the CAR comprises a CD8 stalk domain. In some embodiments, the CAR comprises a CD28TM domain. In some embodiments, the CAR comprises a CD3Z domain. In some embodiments, the CAR comprises a 41BB domain. In certain embodiments, the CAR comprises a CD8 stalk domain, a CD28TM domain, a 41BB domain, and a CD3Z domain.

[0052] According to some embodiments, the CAR comprises an scFv sequence comprising the PVR-binding site of any antibody disclosed above and at least one domain selected from the group consisting of a CD8 stalk domain, a CD28TM domain, a 41BB domain, and a CD3Z domain. According to certain embodiments, the CAR comprises an scFv sequence comprising the PVR-binding site of any antibody disclosed above and a CD8 stalk domain, a CD28TM domain, a 41BB domain, and a CD3Z domain.

[0053] Some embodiments provide lymphocytes engineered to express a CAR described herein. Some embodiments provide T cells engineered to express a CAR described herein. Further embodiments provide NK cells engineered to express a CAR described herein.

[0054] Certain embodiments provide engineered T cells that express an scFv sequence selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57, or an analog thereof having at least 90% sequence similarity to either of these sequences, a CD8 stalk domain, a CD28TM domain, a 41BB domain, and a CD3Z domain.

[0055] According to one aspect, the present invention provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one lymphocyte comprising a CAR described herein.

[0056] According to another aspect, the present invention provides a pharmaceutical composition comprising a humanized antibody or antigen-binding fragment described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0057] Any dosage form can be used to deliver the compositions of the present invention to a subject in need thereof, including parenteral and enteral dosage forms.

[0058] In some embodiments, the pharmaceutical composition is formulated for injection or infusion. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intratumoral administration.

[0059] According to some embodiments, the humanized antibody or antigen-binding fragment thereof, or pharmaceutical composition is for use in increasing surface expression and / or signaling of CD226 on CD8+ and CD4+ T cells.

[0060] According to multiple embodiments, the humanized antibody or antigen-binding fragment thereof, or pharmaceutical composition is for use in treating cancer in an individual. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, colon cancer, glioblastoma, adrenal cancer, uterine cancer, head and neck cancer, pancreatic cancer, and breast cancer. Each possibility represents a separate embodiment of the present invention.

[0061] According to some embodiments, the cancer is a hematological cancer.

[0062] According to some embodiments, the hematological cancer is selected from leukemias, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL), lymphomas, including Hodgkin's disease and non-Hodgkin's lymphoma, and multiple myeloma.

[0063] According to some embodiments, the individual is a human.

[0064] 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.

[0065] According to some embodiments, the immune co-inhibitory receptor is selected from the group consisting of PD-1, PD-L1, TIGIT, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55 (CD160), LAIR1, SIGLEC10, CD112R, CD112, ILT-4, and 2B4, with each possibility representing a separate embodiment of the present invention.

[0066] According to some embodiments of the invention, the use further comprises combination with an anti-endothelial growth factor receptor (EGFR) antibody.

[0067] According to another aspect, the present invention provides a method for increasing surface expression and / or signaling of CD226 on CD8+ and CD4+ T cells in an individual, the method comprising administering to the individual a therapeutically effective amount of a humanized antibody or antigen-binding fragment thereof, or a pharmaceutical composition described herein. In one embodiment, the CD8+ T cells are tumor-infiltrating CD8+ T cells.

[0068] In another aspect, the present invention provides a method of treating cancer in an individual in need thereof, the method comprising administering a therapeutically effective amount of a humanized antibody or antigen-binding fragment thereof, or a pharmaceutical composition to the individual. In some embodiments, the cancer comprises a solid tumor. In further embodiments, the cancer is a non-solid tumor. In some embodiments, the cancer is selected from the group consisting of glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, and lung cancer. In some embodiments, the method of treating cancer comprises preventing or reducing the formation, growth, or spread of metastases in a subject.

[0069] In another aspect, the present invention provides a method for treating cancer in an individual suffering from cancer, the method comprising administering to the individual a therapeutically effective amount of a humanized antibody or antigen-binding fragment thereof, or a pharmaceutical composition, and an inhibitor of PD-1, PD-L1, CTLA-4, or CD112R signaling. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is selected from the group consisting of glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, or lung cancer. In some embodiments, the inhibitor of PD-1 signaling is an antibody or fragment thereof that binds to PD-1. In some embodiments, the antibody or fragment thereof that binds to PD-1 is pembrolizumab, nivolumab, AMP-514, tislelizumab, spartalizumab, or a PD-1-binding fragment thereof. In some embodiments, the inhibitor of PD-1 signaling is an antibody that specifically binds to PD-L-1 or PD-L-2. In some embodiments, the antibody that specifically binds to PD-L1 or PD-L2 comprises durvalumab, atezolizumab, avelumab, BMS-936559, or FAZ053, or a PD-L1- or PD-L2-binding fragment thereof. In some embodiments, the inhibitor of PD-1 signaling comprises an Fc fusion protein that binds to PD-1, PD-L1, or PD-L2. In some embodiments, the Fc fusion protein comprises AMP-224 or a PD-1-binding fragment thereof. In some embodiments, the inhibitor of PD-1 signaling comprises a small molecule inhibitor of PD-1, PD-L1, or PD-L2.In certain embodiments, the small molecule inhibitor of PD-1, PD-L1, or PD-L2 signaling is selected from the group consisting of N-{2-[({2-methoxy-6-[(2-methyl[1,1′-biphenyl]-3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS202), (2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5-methylbenzyl)-D-serine hydrochloride , (2R,4R)-1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)benzyl)-4-hydroxypyrrolidine-2-carboxylic acid, 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenylindole, 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenyl-1h-indole, L-α-glutamine, N2,N6-bis( L-Seryl-L-asparaginyl-L-threonyl-L-seryl-L-α-glutamyl-L-seryl-L-phenylalanyl)-L-lysyl-L-phenylalanyl-L-arginyl-L-valyl-L-threonyl-L-glutaminyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutaminyl-L-isoleucyl-L-lysyl, (2S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1'-biphenyl]-3-yl)methoxy]phenyl]methyl ]-2-piperidinecarboxylic acid, glycinamide, N-(2-mercaptoacetyl)-L-phenylalanyl-N-methyl-L-alanyl-L-asparaginyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophyl-L-seryl-L-tryptophyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1→14)-thioether, or one or more of their derivatives or analogs.

[0070] Also described herein is a method for producing a composition for treating cancer in an individual suffering from cancer, the method comprising mixing a humanized antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is selected from the group consisting of glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, and lung cancer. Also described herein is a method for producing a humanized antibody or antigen-binding fragment thereof, the method comprising incubating a cell line described herein in a cell culture medium under conditions sufficient to allow expression and secretion of the humanized antibody or antigen-binding fragment thereof.

[0071] According to one aspect, the present invention further provides a method for diagnosing or prognosing cancer in a subject, the method comprising determining the expression level of PVR in a biological sample of the subject using at least one humanized antibody, fragment, or scFv described herein.

[0072] According to another aspect, the present invention further provides a method for determining or quantifying the expression of PVR, the method comprising contacting a biological sample with an antibody or antibody fragment described herein and measuring the level of complex formation.

[0073] According to some embodiments, the method for detecting or quantifying expression of PVR comprises: i. incubating the sample with an antibody or antibody fragment thereof specific for PVR, comprising at least an antigen-binding portion; ii. detecting bound PVR using a detectable probe; Includes:

[0074] According to some embodiments, the method comprises: iii. comparing the amount of (ii) with a standard curve obtained from reference samples containing known amounts of PVR; iv. Calculating the amount of PVR in the sample from the standard curve; Further includes:

[0075] According to some embodiments, the method includes indicating that the subject has a PVR-positive cancer if the amount of PVR is higher than a control or given standard.

[0076] According to some particular embodiments, the sample is a body fluid or a solid tissue. In some embodiments, the method is performed in vitro or ex vivo.

[0077] Kits for measuring PVR expression in a biological sample are also provided, comprising at least one antibody or antibody fragment described herein and a means for measuring PVR expression. In some embodiments, the kit further comprises instructional materials directing use of the kit.

[0078] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description set forth below. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0079] The novel features described herein are set forth with particularity in the appended claims. The nature and advantages of the features described herein will be better understood by reference to the following detailed description that sets forth illustrative examples, which utilize the principles of the features described herein, and the accompanying drawings. [Figure 1A]Figure 1A shows the affinity improvement for PVR binding by the N56E and N56D mutants. Biacore assays show the relative and absolute affinity of N56 substitutions (hIgG4 mutants) relative to PVR-HIS tagged versions (Sino Cat. no. 10109-H08H). Affinity shifts of >25% were considered significant. (*) The relative KD for each mutant was established by dividing the KD of the substitution by the KD of the parent N56 mutant (VH0VK0). [Figure 1B] Affinity and competition assays of N56 substitutions. In Figure 1B, the potency of a chimeric antibody (5B9 wild-type-WT with IgG4(S241P)HC) is compared to a variable region carrying the LC CDR2 sequence with a single amino acid substitution (to remove the deamidation site). Potency was measured in a competition assay using parental 5B9. The relative IC50 of each variant was determined by dividing its IC50 by the IC50 of the chimeric WT antibody tested in parallel. [Figure 2A] Figure 2 shows the improvement of cross-reactivity for monkey PVR binding by the N56E and N56D mutants. To assess maximum binding, a saturating dose (10 μg / ml) of the N56 mutant monoclonal antibody (shown on the X-axis) was used to stain PVR-expressing cells, and mean fluorescence intensity (MFI) values were determined by FACS analysis. Fold changes were calculated by dividing the MFI of each mutant by the MFI of the parent antibody (K0) (Figure 2A, NCI-H1975 (human lung) cells). Affinity shifts of more than 25% were considered significant. [Figure 2B] Figure 2B shows improved cross-reactivity of monkey PVR binding by N56E and N56D mutants. To assess maximum binding, a saturating dose (10 μg / ml) of N56 mutant monoclonal antibody (shown on the X-axis) was used to stain PVR-expressing cells, and mean fluorescence intensity (MFI) values were determined by FACS analysis. Fold changes were calculated by dividing the MFI of each mutant by the MFI of the parent antibody (K0) ((Figure 2B) Vero (African green monkey kidney) cells). Affinity shifts of more than 25% were considered significant. [Figure 3]Figure 1 shows the improvement of NK activation by N56E and N56T mutants. To characterize the N56 substitution mutants, a CD107a induction assay was performed using human NK cells from healthy donors and MDA-MB-231 as target cells at a 2:1 ratio. Abs were added at 600 pM. K0 was the parent clone (N56). All mutants resulted in significant CD107a induction (>240% of isotype IgG1). Furthermore, N56 substitutions N56E and N56T significantly improved CD107a induction compared to K0 (*p<0.04, **p<0.01). [Figure 4] Figure 1 shows the improvement of CD8+ T cell proliferation by N56E and N56T mutants. To characterize the N56 mutants, a T cell proliferation assay was performed. A549 cancer cells were used at an effector-to-target ratio of 4:1 using fresh CFSE-labeled human PBMCs in the presence of 2.5 μl / ml of PHAL. All N56 mutant monoclonal antibodies (X-axis) were added at 4 μg / ml, and the cocultures were incubated for 96 hours. Results of FACS analysis gated on CD8+ T cells are presented. The relative MFI of CFSE labeling was calculated by dividing the MFI of the IgG-treated group by the MFI of each mutant. Because increased proliferation resulted in a decrease in CFSE signal, the Y-axis represents the reciprocal of this ratio. The N56E and N56T mutants significantly increased CD8+ T cell proliferation compared to the parental clone (K0), marked with a # (*p<0.05, #<0.04, **p<0.01). [Figure 5A] Figure 1 illustrates the affinity of humanized variants for human PVR as measured by surface plasmon resonance (SPR). Absolute and relative results are shown, using the chimeric N56E mutant parent antibody (N56E VH0 / Vk0) as a baseline. [Figure 5B] Figure 1 illustrates their surface binding to PVR-expressing HEK293 cells as measured by flow cytometry. Absolute and relative results are shown, using the chimeric N56E mutant parent antibody (N56E VH0 / Vk0) as a baseline. [Figure 6A]Figure 1 shows the expression levels of humanized variants.Titers of individual variants after transient expression in HEK293EBNA cells are shown. [Figure 6B] Similarity of humanized variants to human variable domain germline sequences (FIG. 6B): Variable domain sequence identity of humanized heavy and light chain variants to human germline sequences. [Figure 7A] Figure 7 compares the biophysical properties of the humanized lead mutant NB1088 (right column) with those of the humanized mutant NB941 (left column) carrying the LC CDR2 5B9 WT sequence. Figure 7A shows the decrease in the generation of acidic species in NB1088 over time after stress testing at low pH. [Figure 7B] Figure 7B compares the biophysical properties of the humanized lead mutant NB1088 (right column) with those of the humanized mutant NB941 (left column) carrying the LC CDR2 5B9 WT sequence. Figure 7B shows the decrease in the generation of acidic species in NB1088 over time after stress testing at high concentrations and 40°C. [Figure 8] Figure 8 illustrates the EC50 of NB1088 binding to PVR (Figure 8A), the IC50 of NB1088 inhibition of PVR-TIGIT binding (Figure 8B), the IC50 of NB1088 inhibition of PVR-CD96 binding (Figure 8C), and the IC50 of NB1088 inhibition of PVR-CD226 binding (Figure 8D). [Figure 9] Figures 9A and 9B illustrate that NB1088 alone (Figures 9A and 9B) and in combination with PD1 blockade with pembrolizumab (Figure 9B) increases interferon-γ release in tumor / T cell co-culture systems using an antigen-specific T cell assay (human papillomavirus, HPV) (Figure 9A) or a non-specific allogeneic T cell assay (Figure 9B). **p<0.01, one-way ANOVA. [Figure 10]Figure 10 illustrates that NB1088 increases antibody-dependent cellular cytotoxicity when combined with an EGFR-binding antibody in the endothelial growth factor receptor (EGFR)-expressing breast cancer cell line A549 (Figure 10A), and that this coincides with increased interferon-γ release (Figure 10B). *p<0.05, **p<0.01, one-way ANOVA. [Figure 11] Figure 11 illustrates that PVR-expressing tumor cell lines (A549 or CaSki) can induce downregulation of CD226 expressed on CD8+ T cells (Figure 11A) and NK cells (Figure 11B), which can be restored by NB1088 but not by anti-TIGIT (Figures 11A and 11B). Figure 11A shows results obtained in a co-culture system using an antigen-specific T cell assay (human papillomavirus, HPV) or a non-specific allogeneic T cell assay. [Figure 12] Figure 12 illustrates that the NB1088-dependent increase in interferon-γ release by allogeneic or antigen-responsive CD8+ T cells (Figure 12A) or induction of antibody-dependent cellular cytotoxicity (ADCC)-responsive NK cells in the presence of EGFR blockade (Figure 12B) is dependent on CD226 activity, and in both cases, NB1088 exhibits superior activity to TIGIT inhibition (both Abs at 10 μg / ml). [Figure 13] Figure 13 illustrates that NB1088 has efficacy as monotherapy in a humanized mouse model of pancreatic cancer (Figure 13A) and a humanized mouse model of lung adenocarcinoma (Figure 13C, humanized mice) that is comparable to pembrolizumab (anti-PD-1, administered at the standard dose established in these models) (Figure 13B), where efficacy is dependent on the presence of human immune cells (Figure 13D, non-humanized mice) and correlates with increased CD226 expression on tumor-infiltrating CD8+ T cells (Figure 13E) (*p<0.05, ***p<0.001 by two-way ANOVA). [Figure 14]Figure 14A is related to the model in Figures 13C and 13E and illustrates that NB1088 increases the frequency of interferon-γ-positive (Figure 14A) and interferon-γ / CD107a double-positive (Figure 14B) CD8 TIL cells, and that NB1088 increases the frequency of interferon-γ / CD226+ double-positive CD8 T cells (Figure 14D), but not interferon-γ / CD226- single-positive CD8 T cells (Figure 14C). *p=0.0210, ***<0.0001, independent TEST. [Figure 15] Figure 15 illustrates the pharmacokinetics of NB1088 on circulating CD4 T cells from cynomolgus monkeys administered various doses of antibody once (at doses of 20 mg / kg and 50 mg / kg) or four times at weekly intervals (at a dose of 200 mg / kg) (Figure 15A), and the corresponding pharmacodynamic changes in CD226 surface expression (Figure 15B). [Figure 16] FIG. 1 shows the expression of human PVR in biopsies of various types of cancer as measured by immunohistochemistry and assessed by H-score. [Figure 17] Overall schematic diagram of the CAR-T construct. The scFv comprises the heavy and light chains (VH and VL, respectively) of a humanized antibody according to the invention. [Figure 18] Figure 1 shows robust interleukin 2 (IL2) secretion in Jurkat cells overexpressing the αPVR CAR-T construct over parental Jurkat cells. Parental Jurkat cells or Jurkat cells overexpressing the αPVR CAR-T constructs H4K2-NTX1088C or H3K4-NTX1034C (40K cells / well) were incubated with A549 or MDA-231 cells at a 1:1 E:T ratio for 24 hours, and IL2 secretion was quantified using Biolegend hIL-2 (cat431804). Both CAR-T drivers increased IL2 secretion 100-fold over parental Jurkat cells in the presence of the indicated targets. [Figure 19A]Figure 19 shows increased target cell killing by anti-PVR (αPVR) CAR-T. A549 cells (Figure 19A) or MDA-231 cells (Figure 19B) (both 200K cells) were plated in 12-well plates with CAR-T-PVR mutants NTX-1088C and NTX-1034C at E:T ratios of 0.4 and 0.8 to 1 (based on GFP positivity) in NK medium for 72 hours. Tumor cell killing was assessed using a standard CTG protocol (Promega G9241). [Figure 19B] Figure 19 shows increased target cell killing by anti-PVR (αPVR) CAR-T. A549 cells (Figure 19A) or MDA-231 cells (Figure 19B) (both 200K cells) were plated in 12-well plates with CAR-T-PVR mutants NTX-1088C and NTX-1034C at E:T ratios of 0.4 and 0.8 to 1 (based on GFP positivity) in NK medium for 72 hours. Tumor cell killing was assessed using a standard CTG protocol (Promega G9241). [Figure 19C] Figure 19 shows increased target cell killing by anti-PVR (αPVR) CAR-T. A549 cells (Figure 19A) or MDA-231 cells (Figure 19B) (both 200K cells) were plated in 12-well plates with CAR-T-PVR mutants NTX-1088C and NTX-1034C at E:T ratios of 0.4 and 0.8 to 1 (based on GFP positivity) in NK medium for 72 hours. Tumor cell killing was assessed using a standard CTG protocol (Promega G9241). [Figure 20] Figure 1 shows efficient hematological target cell killing by αPVR CAR-T. K562 (AML model) cells were incubated with αPVR CAR-T mutants NTX-1088C and NTX-1034C in RPMI+IL-2 medium for 18 hours at the E:T ratios indicated on the X-axis. Tumor cell killing was assessed using flow cytometry. Significant target cell killing was observed with both CAR-T drivers. DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention provides humanized monoclonal antibodies that recognize the poliovirus receptor (PVR). Advantageously, the antibodies of the present invention are almost completely humanized, thereby avoiding the risk of adverse immune responses to the antibodies and making them safe for in vivo use in humans. The antibodies of the present invention are characterized by having unique CDR sequences and novel humanized framework sequences and designs. More specifically, the monoclonal antibodies provided by the present invention have a specific combination of CDRs and non-fully humanized framework sequences, and have unique properties and improved safety and efficacy compared to known anti-PVR antibodies.

[0081] Some of the variants described herein have increased productivity and are expressed at higher levels compared to other humanized PVR antibodies containing the same CDR regions. Also disclosed herein are methods of using these antibodies to treat cancer in individuals.

[0082] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, one of ordinary skill in the art will understand that the provided embodiments may be practiced without these details. Unless the context requires otherwise, throughout the following specification and claims, terms such as "comprise" and variations such as "comprises" and "comprising" should be construed in their open and inclusive sense, i.e., "including, but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise. It should also be noted that the term "or" is used throughout in its sense to include "and / or" unless expressly indicated otherwise. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0083] As used herein, the term "PVR" refers, in some embodiments, to the poliovirus receptor, also known as CD155 (cluster of differentiation 155), protein ID: Q92692. PVR is a transmembrane glycoprotein with an N-terminal signal sequence, three extracellular immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic tail. PVR has a molecular size of approximately 80 kDa and a structure composed of three Ig-like domains, specifically an outermost V-like domain followed by two C2-like domains. The humanized antibodies described herein have affinity for human PVR (hPVR). In some embodiments, the antibodies have some affinity for PVR proteins from other animals, particularly primates. Advantageously, affinity for other primates, such as African green monkeys, allows for further testing of humanized antibodies for safety and efficacy in nonclinical trials. Affinity for PVRs from evolutionarily more distant animals, such as rodents, has not been observed.

[0084] As used herein, the term "about" refers to an amount that is closer to 10% or less of the stated amount.

[0085] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of having, or at risk of developing at least one disease for which the described compositions and methods are useful for treating. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In some embodiments, the individual is a human.

[0086] As used herein, the term "combination" or "combined treatment" can refer to simultaneous administration of the substances to be combined, or sequential administration of the substances to be combined. As described herein, when the combination refers to sequential administration of the substances, the substances can be administered in any temporal order.

[0087] The terms "cancer" and "tumor" refer to a physiological condition in mammals characterized by deregulated cell proliferation. Cancer is a class of diseases in which a group of cells exhibits unregulated or unwanted growth. Cancer cells can also spread to other locations, potentially resulting in the formation of metastases. The spread of cancer cells within the body can occur, for example, via lymph or blood. Unregulated growth, invasion, and metastasis formation are also referred to as malignant properties of cancer. These malignant properties distinguish cancers from benign tumor-derived cancers, which generally do not invade or metastasize.

[0088] As used herein, the term "effective amount" refers to the amount of a therapeutic agent that, when administered to a mammal, produces a biological effect. Biological effects include, but are not limited to, inhibiting or blocking receptor-ligand interactions (e.g., PVR-TIGIT, PD-1-PD-L1 / PD-L-2), inhibiting signal transduction pathways, reducing tumor growth, reducing tumor metastasis, or prolonging the survival of tumor-bearing animals. A "therapeutic amount" is the concerted amount of a drug calculated to produce a therapeutic effect. A therapeutic amount encompasses a range of dosages capable of inducing a therapeutic response in a population of individuals. The mammal may be a human individual. The human individual may be afflicted with or suspected of being afflicted with a tumor.

[0089] As used herein, "checkpoint inhibitors" refers to drugs that inhibit biological molecules ("checkpoint molecules") produced by an organism that negatively regulate the anti-tumor / anti-cancer activity of T cells in the organism. Checkpoint molecules include, but are not limited to, PD-1, PD-L-1, PD-L-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, PVR, TIGIT, IDO, KIR, A2AR, B7-H3, B7H4, CEACAM1, NOX2, CD112R, and CD112.

[0090] Among the antibodies provided are monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Antibodies include antibody-containing molecules such as antibody conjugates and chimeric molecules. Thus, antibodies include, but are not limited to, full-length antibodies as well as fragments and portions that retain their binding specificity, such as, but not limited to, biologically relevant (antigen-binding) fragments or specific binding portions thereof, including, but not limited to, any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), as well as Fab, F(ab')2, Fv, and scFv (single-chain or related entities). Monoclonal antibodies are generally found within a substantially homogeneous antibody composition. Thus, the individual antibodies contained within a monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. Polyclonal antibodies are preparations containing different antibodies of various sequences, typically directed against two or more different determinants (epitopes). Monoclonal antibodies may contain a human IgG1 constant region. Monoclonal antibodies may contain a human IgG4 constant region.

[0091] The term "antibody" is used broadly herein and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments including single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term further encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0092] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each full-length heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).The precise amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the “Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (the “Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745 (the “Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme), Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70, ("Aho" numbering scheme), and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modeling antibodies on the WEB," Protein Eng. 2000 Dec;13(12):819-24 ("AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0093] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia numbering schemes are based on the most common antibody region sequence lengths, with insertions accommodated by an insertion letter, e.g., "30a," and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0094] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is necessary for binding the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (V H and V L ) have a similar overall structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). H or V L The V domains may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen each contain a complementary V domain. L or V H V from an antibody that binds to an antigen to screen a library of domains H or V Ldomains can be used to isolate them (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0095] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv or sFv), and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment containing the variable heavy chain region and / or the variable light chain region, such as an scFv.

[0096] The term "antigen" as used herein refers to a molecule or a portion thereof that can induce antibody formation and specifically bind to an antibody. An antigen may have one or more epitopes. Specific binding, as referred to above, indicates that an antigen reacts in a highly selective manner with its corresponding antibody, rather than with the numerous other antibodies that may be induced by other antigens. The antigen according to some embodiments of the present invention is human PVR.

[0097] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as well as production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment containing a non-naturally occurring configuration, one with two or more antibody regions or chains joined by a synthetic linker, e.g., a polypeptide linker, and / or one not produced by enzymatic digestion of a naturally occurring intact antibody. In some embodiments, the antibody fragment is an scFv.

[0098] A "humanized" antibody is one in which all or nearly all CDR amino acid residues are derived from non-human CDRs and all or nearly all FR amino acid residues are derived from human FRs. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has been humanized, generally to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0099] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or a non-human source utilizing a human antibody repertoire, including a human antibody library, or other human antibody-encoding sequence. The term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as those in which all or nearly all CDRs are non-human.

[0100] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains, as well as other peptides, such as linkers and connecting peptides, may contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, a polypeptide may include a native sequence or modifications to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or accidental, as through mutation of the host producing the protein or errors due to PCR amplification.

[0101] The percent (%) sequence identity to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity; however, any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of known ways using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is authored by Genentech, Inc., and the source code has been filed with the U.S. Copyright Office, Washington, DC 20559, along with user documentation, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0102] In situations where ALIGN-2 is utilized to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a particular % of the given amino acid sequence A to the given amino acid sequence B) is calculated as 100 × the fraction X / Y, where X is the number of amino acid residues scored as a perfect match by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and Y is the total number of amino acid residues. It is recognized that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity between A and B will not equal the % amino acid sequence identity between B and A. Unless otherwise specified, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0103] The terms "homology," "homology," or "percent homology," as used herein to describe an amino acid or nucleic acid sequence relative to a reference sequence, can be determined using the formula set forth by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, as revised in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula is incorporated into the Basic Local Alignment Search Tool (BLAST) of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent sequence identity can be determined using the most recent version of BLAST available as of the filing date of this application.

[0104] In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Variants typically differ from the polypeptides specifically disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants may occur naturally or may be produced synthetically, e.g., by modifying one or more of the above polypeptide sequences of the present invention, assessing one or more biological activities of the polypeptides described herein, and / or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody; amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0105] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDRs and FRs. Amino acid substitutions can be introduced into the antibody of interest and screened for a desired activity, such as retention or improvement of antigen binding, reduction of immunogenicity, or improvement of antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0106] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, where the substitutions, insertions, or deletions do not substantially reduce antibody binding to the antigen. For example, conservative substitutions that do not substantially reduce binding affinity may be made within a CDR. Such modifications may occur outside of CDR "hot spots." Mutant V H and V L In some embodiments of the sequences, each CDR is unmodified.

[0107] Modifications (e.g., substitutions) may be made in CDRs, for example, to improve antibody affinity. Such modifications are made in CDRs encoding codons with high mutation rates during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting mutants can be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, CDR randomization, or oligonucleotide-directed mutagenesis) can be used to improve antibody affinity (see, e.g., Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2001)). CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, e.g., Cunningham and Wells Science, 244:1081-1085 (1989)). CDR-H3 and CDR-L3, in particular, are often targeted. Alternatively or additionally, a crystal structure of the antigen-antibody complex is targeted to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain the desired properties.

[0108] Amino acid sequence insertions and deletions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions and deletions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., in ADEPT) or a polypeptide that extends the serum half-life of the antibody. An example of an intrasequence insertional variant of an antibody molecule is an insertion of three amino acids in the light chain. An example of a terminal deletion is an antibody with a deletion of seven or fewer amino acids at the end of the light chain.

[0109] In some embodiments, the dissociation constant (K D ) for the antibody target, human poliovirus receptor (CD155), is about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 M). D can be measured by any suitable assay. In one embodiment, KD can be measured using a surface plasmon resonance (SPR) assay (e.g., using a BIACORE®-2000 or BIACORE®-3000).

[0110] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region herein refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. Fc regions include native-sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., the Fc region of a human IgG1, IgG2, IgG3, or IgG4) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0111] In some embodiments, the antibodies of the present disclosure are variants that retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assays may be utilized (e.g., ACTI™ and CytoTox96® non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC), monocytes, macrophages, and natural killer (NK) cells.

[0112] The antibodies may have increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US 2005 / 0014934). Such antibodies may comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn, and may comprise an Fc region with a substitution at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 according to the EU numbering system (see, e.g., U.S. Patent No. 7,371,826). Other exemplary Fc region variants are also contemplated (see, e.g., Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260 and 5,624,821, and WO 94 / 29351).

[0113] In some embodiments, it may be desirable to generate cysteine-engineered antibodies, e.g., "ThioMAbs," in which one or more residues of an antibody are substituted with a cysteine residue. According to some embodiments, the substituted residues occur at accessible sites of the antibody. The reactive thiol group can be positioned at the site for conjugation to other moieties, such as a drug moiety or a linker-drug moiety, to generate an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region.

[0114] In some embodiments, the antibodies provided herein can be further modified to contain additional nonproteinaceous moieties that are known and available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamic acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, these can be the same or different molecules.

[0115] The antibodies described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide containing two or more nucleotide bases. In some embodiments, a nucleic acid is a component of a vector that can be used to transcribe a polypeptide encoded by a polynucleotide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a genome-integrating vector, or "integrating vector," that can integrate into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements used to control transcription, such as promoters, enhancers, and polyadenylation signals, can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in the host, usually conferred by an origin of replication, and a selection gene can also be incorporated to facilitate recognition of transformants. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses may be used. Plasmid vectors may be linearized for integration into chromosomal locations. Vectors may contain sequences that induce site-specific integration (e.g., AttP-AttB recombination) into a defined location or a restricted set of sites in the genome. Additionally, vectors may contain sequences derived from transposable elements.

[0116] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise transfect appropriate cells with the nucleic acid, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell culture are known in the art. In some embodiments, the cells are eukaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. In some embodiments, the mammalian cells are cell lines useful for producing antibodies, such as Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or PER.C6® cells. In some embodiments, the nucleic acids encoding the antibodies are integrated into a genomic locus of a cell useful for producing the antibody. In some embodiments, methods of making antibodies are described herein, comprising culturing cells containing nucleic acids encoding the antibodies under in vitro conditions sufficient to allow the production and secretion of the antibody.

[0117] In one embodiment, described herein is a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location; and (b) a cryoprotectant. In one embodiment, the cryoprotectant comprises glycerol. In one embodiment, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antibody having (i) a heavy chain amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 4, 5, or 6 and (ii) a light chain amino acid sequence set forth in any one of SEQ ID NOs: 2, 7, 8, or 9; and (b) a cryoprotectant. In one embodiment, the cryoprotectant comprises glycerol. In one embodiment, the master cell bank is placed in a suitable vial or container that can withstand freezing with liquid nitrogen.

[0118] Also described herein are methods of producing the antibodies described herein. Such methods include incubating cells or cell lines containing nucleic acid encoding the antibody in cell culture medium under conditions sufficient to allow expression and secretion of the antibody, and further harvesting the antibody from the cell culture medium. The harvesting step may further include one or more purification steps to remove viable cells, cell debris, non-antibody proteins or polypeptides, unwanted salts, buffers, and medium components. In certain embodiments, additional purification steps include centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification, and / or ion exchange chromatography.

[0119] Antibodies Described Herein In some aspects, the present specification describes an anti-human PVR (anti-hPVR) antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof binds to human PVR at the PVR-TIGIT interface. In some embodiments, the anti-hPVR antibody or antigen-binding fragment thereof can compete with any one or more of TIGIT, CD96, and CD226.

[0120] In one embodiment, the EC of the humanized antibody or antigen-binding fragment thereof 50 In certain embodiments, the EC of the hPVR antibody for binding to PVR is less than about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, or 0.01 nM. 50 is about 5 nM to 1 nM, about 5 nM to about 2 nM, about 4 nM to about 2 nM, about 4 nM to about 3 nM, or about 3 nM to about 2 nM.

[0121] Median effective concentration (EC 50 ) refers to the concentration of antibody that induces a response halfway between baseline and maximum after a specified exposure time.

[0122] According to some embodiments, the antibody is a recombinant antibody. According to certain embodiments, the antibody is a recombinant humanized monoclonal antibody.

[0123] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0124] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0125] According to some embodiments, the humanized antibody or antigen-binding fragment thereof is NB1088 (SEQ ID NO: 1 and SEQ ID NO: 2).

[0126] In one aspect herein, described herein is a humanized antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and wherein the humanized antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0127] In another aspect herein, described is a humanized antibody or antigen-binding fragment thereof comprising a heavy chain and an immunoglobulin light chain, wherein the heavy chain comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:1, and the light chain comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:2, and wherein the humanized antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0128] In one embodiment, the present specification describes a humanized antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and wherein the humanized antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0129] In certain embodiments, described herein is a humanized antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, and wherein the humanized antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155). Each possibility represents a separate embodiment of the present invention.

[0130] In one embodiment, the present specification describes a humanized antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least about 98% identical to the amino acid sequence set forth in SEQ ID NO:1, and the light chain comprises an amino acid sequence at least about 98% identical to the amino acid sequence set forth in SEQ ID NO:2, and wherein the humanized antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0131] In one embodiment, the present specification describes a humanized antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:1, and the light chain comprises an amino acid sequence at least about 99% identical to the amino acid sequence set forth in SEQ ID NO:2, and wherein the humanized antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0132] In one embodiment, the present specification describes a humanized antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:1 and the light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:2, and wherein the humanized antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0133] According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 7. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 8. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 9. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 3 and the light chain sequence set forth in SEQ ID NO: 2. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 3 and the light chain sequence set forth in SEQ ID NO: 7. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 3 and the light chain sequence set forth in SEQ ID NO: 8. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 3 and the light chain sequence set forth in SEQ ID NO: 9. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 4 and the light chain sequence set forth in SEQ ID NO: 2. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO:4 and the light chain sequence set forth in SEQ ID NO:7. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO:4 and the light chain sequence set forth in SEQ ID NO:8. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO:4 and the light chain sequence set forth in SEQ ID NO:9. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO:5 and the light chain sequence set forth in SEQ ID NO:2. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO:5 and the light chain sequence set forth in SEQ ID NO:7. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO:5 and the light chain sequence set forth in SEQ ID NO:8. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO:5 and the light chain sequence set forth in SEQ ID NO:9. According to some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO:6 and the light chain sequence set forth in SEQ ID NO:2.According to some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 6 and a light chain sequence set forth in SEQ ID NO: 7. According to some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 6 and a light chain sequence set forth in SEQ ID NO: 8. According to some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 6 and a light chain sequence set forth in SEQ ID NO: 9.

[0134] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence QVQLVQSGAE(L / V)KKPGASVK(I / V)SCKATGYTFSNYWIEW(I / V)(K / R)QAPGQGLEW(I / M)GEIFPGSGRINFNEKFKGR(A / V)TFTADTSI(D / S)T(T / A)YM(Q / E)LS(S / R)L(T / R)SDD(S / T)AVYYCARTKIYGNSFDYWGQGT(T / L)VTVSS (SEQ ID NO: 47), and a heavy chain comprising the amino acid sequence DI(M / Q)MTQSPS(F / S)LSASVGDRVTITC(K / R)ASQDVGTAV(V / A)WYQQKPGKAPK(L / S)LIYWASSRHEGVP(D / S)RF(T / S)GSGSGTDFTLTISS and a light chain region comprising: LQ(S / P)EDFA(D / T)YFCQQYSRYPLTFGQGT KLEIK (SEQ ID NO: 48).

[0135] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, where position 11 is L, position 20 is I, position 37 is I, position 38 is K, position 48 is I, position 68 is A, position 77 is D, position 79 is T, position 82 is Q, position 85 is S, position 87 is T, position 91 is S, or position 114 is T, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0136] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, position 20 is I, position 37 is I, position 38 is K, position 48 is I, position 68 is A, position 77 is D, position 79 is T, position 82 is E, position 85 is R, position 87 is R, position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0137] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, position 20 is V, position 37 is V, position 38 is R, position 48 is M, position 68 is V, position 77 is S, position 79 is A, position 82 is E, position 85 is R, position 87 is R, position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0138] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, position 20 is V, position 37 is I, position 38 is K, position 48 is I, position 68 is V, position 77 is S, position 79 is T, position 82 is E, position 85 is R, position 87 is R, position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0139] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, position 20 is V, position 37 is V, position 38 is R, position 48 is I, position 68 is V, position 77 is S, position 79 is T, position 82 is E, position 85 is R, position 87 is R, position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0140] According to some embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO:48, where position 3 is M, position 10 is F, position 24 is K, position 34 is V, position 46 is L, position 60 is D, position 63 is T, position 80 is S, or position 85 is D, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0141] According to some embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO:48, where position 3 is Q, position 10 is S, position 24 is K, position 34 is V, position 46 is L, position 60 is D, position 63 is S, position 80 is P, or position 85 is D, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0142] According to some embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO:48, where position 3 is Q, position 10 is S, position 24 is R, position 34 is V, position 46 is L, position 60 is S, position 63 is S, position 80 is P, or position 85 is T, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0143] According to some embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO:48, where position 3 is Q, position 10 is S, position 24 is R, position 34 is A, position 46 is L, position 60 is S, position 63 is S, position 80 is P, or position 85 is T, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0144] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, position 20 is V, position 37 is V, position 38 is R, position 48 is I, position 68 is V, position 77 is S, position 79 is T, position 82 is E, position 85 is R, position 87 is R, position 91 is T, or position 114 is L, or any combination thereof; and the light chain comprises the amino acid sequence set forth in SEQ ID NO:48, wherein position 3 is Q, position 10 is S, position 24 is K, position 34 is V, position 46 is L, position 60 is D, position 63 is S, position 80 is P, or position 85 is D, or any combination thereof.

[0145] According to a further embodiment, the heavy chain CDR1 sequence is GYTFSNYWIE (SEQ ID NO: 58).

[0146] According to some embodiments, the human constant region of the antibody is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

[0147] According to some embodiments, the human constant region of the antibody is selected from the group consisting of human IgG1 and human IgG4.

[0148] According to some embodiments, the humanized antibody is an IgG4 antibody comprising the heavy chain sequence set forth in SEQ ID NO: 49, or a sequence with at least 90% identity. According to some embodiments, the humanized antibody is an IgG1 antibody comprising the heavy chain sequence set forth in SEQ ID NO: 50, or a sequence with at least 90% identity.

[0149] According to some embodiments, the humanized antibody comprises the light chain sequence set forth in SEQ ID NO:49.

[0150] Treatment method In some embodiments, the anti-hPVR antibodies disclosed herein are useful for treating cancer or tumors. Treatment refers to a method that seeks to improve or ameliorate the disease being treated. With respect to cancer, treatment includes, but is not limited to, a decrease in tumor volume, a decrease in tumor volume growth, an increase in progression-free survival, or overall life expectancy. In some embodiments, treatment affects the remission of the cancer being treated. In some embodiments, treatment encompasses use as a prophylactic or maintenance dose intended to prevent the recurrence or progression of a previously treated cancer or tumor. Those skilled in the art will understand that not all individuals will respond equally or at all to a given treatment, yet these individuals are still considered to be treated.

[0151] In certain embodiments, the anti-hPVR antibodies and antigen-binding fragments described herein are for use in the manufacture of a medicament for, or in a method of treating, a PVR-positive cancer.

[0152] In some embodiments, the anti-hPVR antibodies or antigen-binding fragments described herein are used as monotherapy to treat cancers or tumors that are refractory to treatment with checkpoint inhibitors. Refractory cancer refers to cancers / tumors that develop progressive disease despite treatment with a checkpoint inhibitor alone. In some embodiments, the checkpoint inhibitor is a PD-1, PD-L1, or PD-L2 inhibitor. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is an antibody or antigen-binding fragment that specifically binds to PD-1 (CD279), including pembrolizumab, nivolumab, AMP-514, spartalizumab, tislelizumab (BGB-A317), or PD-1 (CD279)-binding fragments thereof. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is a PD-L2 Fc fusion protein (e.g., AMP-224). In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor comprises an antibody or PD-L1-binding fragment that specifically binds to PD-L1 (CD274). In some embodiments, the antibody or antigen-binding fragment that specifically binds to PD-L1 (CD274) comprises durvalumab (MEDI4376), atezolizumab, avelumab, BMS-936559, or FAZ053, or a PD-L1 (CD274)-binding fragment thereof. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor comprises an antibody or PD-L2-binding fragment that specifically binds to PD-L2 (CD273).In certain embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is selected from the group consisting of N-{2-[({2-methoxy-6-[(2-methyl[1,1′-biphenyl]-3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS202), (2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5-methylbenzyl)-D-serine hydrochloride, (2R,4R)- 1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)benzyl)-4-hydroxypyrrolidine-2-carboxylic acid, 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenylindole, 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenyl-1h-indole, L-α-glutamine, N2,N6-bis(L-seryl-L-α) (2S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1'-biphenyl]-3-yl)methoxy]phenyl]methyl]-2-piperidinyl, ... and one or more small molecule inhibitors such as a carboxylic acid, glycinamide, N-(2-mercaptoacetyl)-L-phenylalanyl-N-methyl-L-alanyl-L-asparaginyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophyl-L-seryl-L-tryptophyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1→14)-thioether, or a derivative or analog thereof.

[0153] In some embodiments, the anti-hPVR antibody or antigen-binding fragment thereof is for use in combination with a PD-1, PD-L1, or PD-L2 inhibitor. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is an antibody or antigen-binding fragment that specifically binds to PD-1 (CD279), including pembrolizumab, nivolumab, AMP-514, spartalizumab, tislelizumab (BGB-A317), or a PD-1 (CD279)-binding fragment thereof. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is a PD-L2 Fc fusion protein (e.g., AMP-224). In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is an antibody or PD-L-1-binding fragment that specifically binds to PD-L-1 (CD274). In some embodiments, the antibody or antigen-binding fragment that specifically binds to PD-L-1 (CD274) comprises durvalumab (MEDI4376), atezolizumab, avelumab, BMS-936559, or FAZ053, or a PD-L-1 (CD274)-binding fragment thereof. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor comprises an antibody or PD-L2-binding fragment that specifically binds to PD-L2 (CD273).In certain embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is selected from the group consisting of N-{2-[({2-methoxy-6-[(2-methyl[1,1′-biphenyl]-3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS202), (2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5-methylbenzyl)-D-serine hydrochloride, (2R,4R)- 1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)benzyl)-4-hydroxypyrrolidine-2-carboxylic acid, 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenylindole, 3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenyl-1h-indole, L-α-glutamine, N2,N6-bis(L-seryl-L-α) (2S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1'-biphenyl]-3-yl)methoxy]phenyl]methyl]-2-piperidinyl, ... and one or more small molecule inhibitors such as a carboxylic acid, glycinamide, N-(2-mercaptoacetyl)-L-phenylalanyl-N-methyl-L-alanyl-L-asparaginyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophyl-L-seryl-L-tryptophyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1→14)-thioether, or a derivative or analog thereof.

[0154] In certain embodiments, the anti-hPVR antibody or antigen-binding fragment thereof is for use in combination with an EGFR inhibitor or an EGFR-binding antibody.

[0155] In some embodiments, the anti-hPVR antibody or antigen-binding fragment thereof is for use in treating cancer or tumor. In some embodiments, the cancer or tumor is a solid cancer or tumor. In some embodiments, the cancer or tumor is a blood cancer or tumor. In some embodiments, the cancer or tumor is a tumor of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, and / or liver. In some embodiments, tumors treatable with the antibodies of the invention include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma. In certain embodiments, the tumor / cancer is selected from the group consisting of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytoma, Bartholin's adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrial adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, and thyroid cancer. sarcoma), focal nodular hyperplasia, gastric adenoma, germinal line tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatocellular adenomatosis, hepatocellular carcinoma, insulinite, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung carcinoma, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, schwannoma, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid Selected from the group consisting of leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, warty carcinoma, vaginal / vulvar carcinoma, VIPpoma, and Wilms' tumor.In certain embodiments, tumors / cancers treated with one or more antibodies of the present disclosure include brain tumors, head and neck cancer, colorectal cancer, acute myeloid leukemia, pre-B-cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III, or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell carcinoma, and non-small cell carcinoma, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma, and breast cancer, preferably ductal carcinoma and / or breast carcinoma. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include glioblastoma. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include pancreatic cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include ovarian cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include lung cancer. In certain embodiments, cancers treated with one or more antibodies of the present disclosure include prostate cancer. In some embodiments, the cancer treated with one or more antibodies of the present disclosure comprises colon cancer. In some embodiments, the cancer treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In some embodiments, the cancer is refractory to other treatments. In some embodiments, the cancer treated is recurrent. In some embodiments, the cancer is recurrent / refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.

[0156] It will be apparent to those skilled in the art that a therapeutically effective amount of a molecule according to the invention will depend, among other factors, on the administration schedule, the unit dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, the patient's immune status and health status, the therapeutic activity of the administered molecule, its persistence in the circulation, and the judgment of the treating physician.

[0157] In some embodiments, the antibody can be administered to a subject in need thereof by any route suitable for administering an antibody-containing pharmaceutical composition, such as subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral. In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered subcutaneously. In some embodiments, the antibody is administered intratumorally. In some embodiments, the antibody is administered according to a suitable dosing schedule, for example, weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once monthly. In some embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In some embodiments, a therapeutically acceptable amount is from about 0.1 mg / kg to about 50 mg / kg. In some embodiments, a therapeutically acceptable amount is from about 1 mg / kg to about 40 mg / kg. In some embodiments, a therapeutically acceptable amount is from about 5 mg / kg to about 30 mg / kg. A therapeutically effective amount includes an amount sufficient to alleviate one or more symptoms associated with the disease or affliction being treated.

[0158] The antibodies of the present invention can be used in CAR-based adoptive immunotherapy, which utilizes engineered lymphocytes containing CARs to treat cancer. The CAR-T system is described herein as a non-limiting example.

[0159] T cell therapy utilizes chimeric antigen receptors (CARs) in the treatment of cancer or tumors (i.e., CAR-T cell therapy). CAR-T cell therapy is a cellular immunotherapy that involves administering genetically engineered T cells to cancer patients, which act on tumor cells and induce their apoptosis. Genetically engineered T cells are prepared by expressing CARs, which contain the variable regions of an antibody (VL and VH) combined with an intracellular domain, such as a fragment of the CD3 ζ chain sequence, on T cells using gene transfer technology. CARs are a collective term for chimeric proteins in which the light and heavy chains of the variable regions of a monoclonal antibody specific for a tumor antigen are linked to each other and then to a T cell receptor (TCR) chain at the C-terminus.

[0160] In some embodiments, the CAR comprises at least one protein domain selected from the group consisting of a CD8 stalk domain, a CD28TM domain, a 41BB domain, and a CD3ζ domain. In some embodiments, the CAR comprises a CD8 stalk domain. In some embodiments, the CAR comprises a CD28TM domain. In some embodiments, the CAR comprises a CD3ζ signaling domain. In some embodiments, the CAR comprises a 41BB domain. In certain embodiments, the CAR comprises a CD8 stalk domain, a CD28TM domain, a 41BB domain, and a CD3ζ domain.

[0161] Some embodiments provide chimeric antigen receptors (CARs) comprising a heavy chain variable region (VH) and a light chain variable region (VL) according to the present invention. Some embodiments provide genetically modified lymphocytes expressing a CAR on their surface. Some specific embodiments provide genetically modified T cells (CAR-T cells) expressing a CAR on their surface.

[0162] According to some embodiments, the CAR comprises a combination of a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence with at least 90%, 92%, 94%, 96%, or 98% sequence identity to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence with at least 90%, 92%, 94%, 96%, or 98% sequence identity to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0163] According to some embodiments, the CAR comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0164] According to some embodiments, the CAR comprises a combination of a humanized antibody heavy chain variable region and a light chain variable region, the combination comprising: i. a heavy chain variable region sequence set forth in SEQ ID NO: 1 and a light chain variable region sequence set forth in SEQ ID NO: 9; ii. a heavy chain variable region sequence set forth in SEQ ID NO: 3 and a light chain variable region sequence set forth in SEQ ID NO: 9; iii. A heavy chain variable region sequence set forth in SEQ ID NO: 4 and a light chain variable region sequence set forth in SEQ ID NO: 9; iv. a heavy chain variable region sequence set forth in SEQ ID NO: 5 and a light chain variable region sequence set forth in SEQ ID NO: 9, and v. The heavy chain variable region sequence set forth in SEQ ID NO: 6 and the light chain variable region sequence set forth in SEQ ID NO: 9 is selected from the group consisting of:

[0165] According to some embodiments, the CAR comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 1, 3, 4, 5, and 6, a light chain variable region sequence set forth in SEQ ID NO: 9, a transmembrane domain, and an intracellular T cell signaling domain.

[0166] In some embodiments, a CAR is provided comprising an scFv sequence set forth in SEQ ID NO: 56 or SEQ ID NO: 57, or an analog thereof having at least 90%, 92%, 94%, 96%, or 98% sequence similarity to either of these sequences. In certain aspects, the invention provides cells comprising a CAR described herein. In some embodiments, the cell expresses or is capable of expressing a CAR of the invention. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is selected from a T cell and a natural killer (NK) cell.

[0167] Some embodiments provide lymphocytes engineered to express a CAR described herein. Some embodiments provide T cells engineered to express a CAR described herein.

[0168] Further embodiments provide NK cells engineered to express a CAR described herein.

[0169] The present invention further discloses methods for diagnosing and prognosing cancer.

[0170] According to one aspect, the present invention provides a method for the diagnosis and / or prognosis of cancer or an infectious disease in a subject, the method comprising determining the expression level of PVR in a biological sample of the subject using at least one antibody described herein.

[0171] Pharmaceutically Acceptable Excipients, Carriers, and Diluents In some embodiments, the anti-PVR antibodies of the present disclosure are included in pharmaceutical compositions containing one or more pharmaceutically acceptable excipients, carriers, and diluents. The carriers must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not overly harmful to the recipient. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in an amount appropriate to achieve the desired exposure.

[0172] In some embodiments, the antibody of the present disclosure is administered suspended in a sterile solution. In some embodiments, the solution contains about 0.9% NaCl. In some embodiments, the solution contains about 5.0% dextrose. In some embodiments, the solution further contains one or more of a buffer such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid such as glycine or arginine; an antioxidant such as ascorbic acid or methionine; or a chelating agent such as EDTA or EGTA.

[0173] Typically, antibodies of the present invention, as well as fragments and conjugates thereof, containing the antigen-binding portion of an antibody or another polypeptide, including a peptidomimetic, are suspended in a sterile saline solution for therapeutic use. Alternatively, pharmaceutical compositions may be formulated to control the release of the active ingredient (a molecule containing the antigen-binding portion of an antibody) or to prolong its presence in the patient's system. Many suitable drug delivery systems are known, including implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled-release formulations can be prepared by using polymers to complex or adsorb the molecules of the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of polyanhydride copolymers of stearic acid dimer and sebaceous acid. The rate at which the molecules of the present invention, i.e., antibodies or antibody fragments, are released from such matrices depends on the molecular weight of the molecule, the amount of molecule in the matrix, and the size of the dispersed particles.

[0174] In some embodiments, antibodies of the present disclosure are lyophilized for transport / storage and reconstituted prior to administration. In some embodiments, lyophilized antibody formulations include bulking agents such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or combinations thereof. The lyophilized formulations can be packaged in vials made of glass or other suitable non-reactive materials. Once formulated, the antibodies, with or without reconstitution, can be buffered at a specific pH, generally below 7.0. In some embodiments, the pH can be 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0.

[0175] In some embodiments, lymphocytes carrying the CAR described herein are transported / stored before use. Cells are typically cryopreserved when not being used immediately. Cryopreservation methods and storage media suitable for CAR-carrying cells are known in the art (see, for example, Wang, et al. 2019 May;21(5):566-578).

[0176] Also described herein are kits comprising one or more of the antibodies described herein in a suitable container and one or more additional components selected from instructions for use, diluents, excipients, carriers, and administration devices. In some embodiments, the kits comprise a means for measuring expression of human PVR.

[0177] In some embodiments, described herein are methods for preparing a cancer treatment, the methods comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure. In some embodiments, described herein are methods for preparing a cancer treatment for storage or transport, the methods comprising lyophilizing one or more antibodies of the present disclosure. [Example]

[0178] The following illustrative examples represent embodiments of the compositions and methods described herein and are not intended to be limiting in any way.

[0179] Example 1 - Improved affinity of PVR binding by N56E and N56D mutants The variable region of the chimeric anti-PVR antibody 5B9, disclosed in WO2017 / 149538, carries a deamidated sequence (asparagine-glycine) in CDR2 of the light chain (WASSRHNG, SEQ ID NO: 17). Seven chimeric mutants were generated by introducing point mutations at residue asparagine N56. To evaluate the binding affinity of the N56 substitution mutants, wild-type (WT) and substitution mutant IgG4 (S241P) monoclonal antibodies were immobilized on a protein A capture chip. Binding was tested with the analyte PVR conjugated to a histidine tag (PVR-HIS, Sino Cat. no. 10109-H08H). Dilution range: 5-point 2-fold dilutions from 50 nM to 3.125 nM. Instrument: Biacore T200 (serial number 1909913) running Biacore T200 Evaluation Software V2.0.1. Running buffer: HBS-P+, 300 mM NaCl, 1 mg / ml BSA. Flow rate: 30 μl / min. Association: 350 s, Dissociation: 800 s. Renaturation: 10 mM glycine pH 1.5. Analysis: 1:1 binding. K of displacement D K is the K of the parent N56 mutant (VH0VK0) D The relative K for each substitution is calculated by dividing D A significant (>25%) improvement in affinity was noted for the N56E (Asp) and N56D (Glu) mutants (Figure 1A). Binding of the chimeric mutants to human PVR expressed on HEK293 EBNA cells was assessed by flow cytometry in a competition assay with the parental 5B9 antibody (WT, Figure 1B).

[0180] Example 2 - Improved cross-reactivity for monkey PVR binding by N56E and N56D mutants The binding of N56-mutant antibodies to cell-bound human (protein id: Q92692) and green monkey (African green monkey, protein id: UniProtkB-P32506) PVR was examined. Figure 2A shows the relative binding of all mutants added at saturating concentrations (10 μg / ml) to NCI-H1975 cells expressing human PVR. Figure 2B shows the relative binding of all mutants added at saturating concentrations (10 μg / ml) to Vero cells expressing green monkey PVR. For detection, goat anti-human 647 antibody (Jackson immunoresearch 109-606-088) was used at a 1:250 dilution. Cell binding of Abs was analyzed by FACS. Fold change was calculated by dividing the MFI of each mutant by the MFI of the parent antibody (K0). A significant (>25%) increase in cross-reactivity was observed for the N56E and N56D mutants.

[0181] Example 3 - Improved NK activation by N56E and N56T mutants NK cells from healthy donors were incubated in the presence of selected N5 6-substitution mutants and the target breast cancer cell line (MDA-MB-231) at a 2:1 E:T ratio for 2 hours at 37°C. NK cell activation was measured by induction of CD107a surface expression, and the fold change over control IgG was calculated for each mutant (Y-axis). All monoclonal antibodies were used at 600 pM (0.09 μg / ml) (as determined by two-tailed Student's t-test). * p<0.04, ** As shown in Figure 3, the N56E and N56T mutants exhibited improved NK activation compared to K0, as evidenced by increased CD107a expression.

[0182] Example 4 - Improvement of CD8 T cell proliferation by N56E and N56T mutants Human PBMCs were fluorescently labeled with CFSE (C34554 ThermoFischer) and incubated with A549 target breast cancer cells in the presence of 2.5µl / ml PHA-L (Roche) and the indicated antibody variants at 4µg / ml. After 96 hours of culture, immune cells were harvested, stained with anti-human CD8, and analyzed by FACS. CD8+ T cell proliferation was assessed by CFSE signal intensity. CFSE levels in IgG-treated cells were set at 1. Results are presented as the fold increase in proliferation compared to the control. Because increased proliferation resulted in a decrease in CFSE signal, the Y-axis represents the reciprocal of this ratio. Experiments were performed in quadruplicate. Results are shown for a single PBMC donor. The data show that variants N56E and N56T have a significantly stronger effect on CD8+ T cell proliferation in the presence of tumor cells compared to the parental antibody (Figure 4, two-tailed Student's t-test). * p<0.05, ** p<0.01).

[0183] Example 5 - Identification of humanized 5B9 variants with improved productivity The N56E antibody variant performed best in competitive assays and was selected as the lead variant for humanization. Based on structural analysis, a large set of preliminary sequence segments was identified that were used to generate the 5B9 humanized variants. These segments were selected and analyzed using iTope™ technology (Perry et al., 2008) for in silico analysis of peptide binding to human MHC class II alleles and TCED™ for known antibody sequence-associated T cell epitopes (Bryson et al., 2010). Sequence segments identified as significant non-human germline binders to human MHC class II or that scored significant hits against TCED™ were discarded. This resulted in a reduced set of segments, and these combinations were further analyzed, as described above, to confirm that the junctions between the segments did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V-region sequences lacking significant T cell epitopes. Subsequently, five heavy chain (VH1 to VH5) and four light chain (containing the N56E substitution) (Vκ1 to Vκ4) sequences were selected.

[0184] [Table 1]

[0185] [Table 2]

[0186] [Table 3]

[0187] All mutants were tested for binding by SPR (Figure 5A), and mutants with twice the affinity of the parent antibody were tested for cell surface PVR binding by flow cytometry (Figure 5B). All mutants, except for the Vk4-containing mutant, showed very similar binding compared to the parent mouse / human chimeric molecule carrying the N56E substitution (IgG4(S241P)N56E_VH0 / Vκ0). Note that humanization removed N-linked glycosylation in the light chain at position N20FR1.

[0188] To select lead candidates, we examined their expression levels after transient expression in HEK293EBNA cells and their similarity to human germline sequences (Figure 6). Figure 6A summarizes the titers of all mutants after transient transfection. The mutant VH4 / Vk2 exhibited the highest expression titer and shared a high percentage of sequence identity with the human germline gene (Figure 6B). Finally, we evaluated the productivity of the VH4 / Vk2 mutant (NB1088) compared with an identical mutant, NB1088, but containing the original deamidation-competent LC CDR2 of 5B9 (WASSRHNG), designated NB0941. We then determined the biophysical properties of NB0941 and NB1088. As shown in Figures 7A and 7B, high pH stress and incubation at 40°C revealed changes in capillary isoelectric focusing (cIEF), particularly an increase in the proportion of acid species, likely due to deamidation. These changes were more pronounced in NB0941 than in NB1088. Therefore, NB1088, which has optimized immunogenicity, expression, and binding profiles as well as desirable biophysical properties, was selected as the lead humanized variant for functional analysis.

[0189] The decreased affinity observed for some of the mutants is particularly advantageous for designing CAR drivers, given the fact that normal tissues express minimal levels of PVR. Results (Figure 16) show that PVR is overexpressed in a variety of tumors, enabling efficient targeting of these tumors by PVR-driven CAR-T. Potential safety concerns can be easily addressed by "down-tuned" affinity anti-PVR mutants, as described by Liu et al. (Cancer research, 2015; Volume 75, Issue 17).

[0190] Example 6 - NB1088 inhibits binding of PVR to TIGIT, CD96, and CD226 NB1088 was tested for its ability to block the binding of TIGIT, CD96, and CD226 to PVR. Dissociated CHO (Chinese Hamster Ovary) cells stably expressing human PVR were incubated with NB1088 at the indicated concentrations for 20 minutes on ice, followed by the addition of biotinylated recombinant TIGIT, CD96, or CD226-Fc at 10 μg / ml for an additional 120 minutes on ice. After washing, surface-bound NB1088 was detected with an anti-human Alexa-488-conjugated secondary antibody, and biotinylated proteins were detected with Alexa647-conjugated streptavidin and analyzed by flow cytometry. Figure 8A shows that NB1088 had an EC of approximately 3.3 nanomolar. 50 The IC of NB1088 to compete with TIGIT, CD96, or CD226 for PVR binding was 50 are 1.1, 1.1, and 1.9, respectively, as shown in Figures 8B to 8D.

[0191] Example 7 – NB1088 stimulates cytotoxic T and NK cells The ability of NB1088 to stimulate T cell and NK cell activity in vitro was determined. Using an antigen-specific human papillomavirus (HPV) assay, 30,000 HPV+ human cervical epidermoid carcinoma cell line (CaSki cells) and 30,000 HPV-specific CD8+ T cells were co-cultured overnight with control IgG or NB1088 at 10 μg / ml. Interferon-γ release into the supernatant was detected using a human interferon-γ-specific MSD system. As shown in Figure 9A, NB1088 increased interferon-γ release from human HPV-specific CD8+ (cytotoxic) T cells when incubated with HPV+ CaSki cells. To test CD8+ T cell activity in an allogeneic system, PBMCs were pre-activated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) for 3 days, allowed to rest overnight in the absence of PHA / IL-2, and then CD8+ T cells were isolated using a magnetic negative isolation procedure. 10,000 A549 tumor cells and 100,000 healthy donor CD8+ T cells were co-cultured overnight in the presence of 100 U / ml IL2 and 1 μg / ml anti-CD28 antibody. As shown in Figure 9B, NB1088 increased interferon-γ release from CD8+ T cells to a greater extent than anti-PD-1 (pembrolizumab), and this release was further increased when NB1088 was combined with anti-PD-1 antibody.

[0192] The effect of NB1088 on antibody-dependent cellular cytotoxicity (ADCC) was also determined. NK cells from healthy donors were isolated from PBMCs and allowed to settle overnight using a magnetic negative isolation procedure. 10,000 PVR+ and EGFR+ A549 tumor cells and 50,000 NK cells were incubated with either control IgG, control IgG and the anti-EGFR antibody cetuximab (5 μg / ml), or cetuximab and NB1088. NK cell activity in mediating antibody-dependent cellular cytotoxicity or interferon-γ release was determined by analyzing the viability of adherent A549 cells after co-culture and removal of NK cells using Cell Titer Glow, or by MSD analysis of the supernatants as described above. As shown in Figures 10A and 10B, NB1088 was able to increase NK cell-mediated killing of A549 cells as well as interferon-γ release when incubated with cetuximab.

[0193] Example 8 - NB1088 restores CD226 expression and activity on CD8 T and NK cells We determined the ability of NB1088 to affect CD226 function. CD226 (DNAM-1) is a cell surface glycoprotein receptor expressed by NK cells and T cells. It functions as a ligand for PVR, supporting tumor killing by CD8+ T and NK cells. In this function, CD226 opposes TIGIT and CD96, which are inhibitory molecules expressed on T and NK cells. Therefore, the increase in CD226 function caused by NB1088 suggests that NB1088 enhances the activity of T and NK cells and has broad antitumor activity. The effect of NB1088 on CD226 expression and function was tested in antigen-specific and allogeneic coculture systems as described above. As shown in Figures 11A and 11B, coculture of CD8 T and NK cells with PVR+ target cells significantly reduced CD226 surface expression on CD8 T and NK cells. NB1088, but not anti-TIGIT, restored cell surface expression of CD226 on CD8 T cells or NK cells regardless of the coculture system (Figures 11A and 11B). The functional consequences of increased CD226 expression on T cells and NK cells after NB1088 treatment were assessed using the antigen-specific and allogeneic coculture systems described above with minor modifications (Figures 12A and 12B). The increase in CD226 expression correlated with significantly higher levels of interferon-γ release after NB1088 treatment compared with control IgG or anti-TIGIT treatment (Figures 12A and 12B). The superior T and NK cell activity with NB1088 treatment was mediated, at least in part, through CD226 activity. Anti-CD226 (DX11, 20 μg / ml) reduced NB1088-dependent interferon-γ release by both allogeneic and antigen-stimulated CD8 T cells (FIG. 12A) and NK cells after A549 coculture (FIG. 12B) to levels observed with anti-TIGIT. These data demonstrate that NB1088 improves T and NK cell activity more than TIGIT blockade by increasing CD226 expression and / or function.

[0194] Example 9 – Efficacy of NB1088 Monotherapy in a Humanized Mouse Tumor Xenograft Model The ability of NB1088 to kill either tumors A549 (lung adenocarcinoma) or HPAF (pancreatic) in humanized mouse models was determined. Briefly, 5 × 10 6 Tumor cells (A549 or HPAF) were mixed with activated human peripheral blood mononuclear cells (HBMCs) at a 1:1 ratio in Matrigel and implanted subcutaneously into the flanks of immunodeficient NOD / SCID mice (12 animals per condition). As shown in Figures 13A and 13B, NB1088 was able to reduce tumor volume at least as well as the anti-PD-1 antibody pembrolizumab. NB1088 was also able to reduce tumor volume in the A549 / PBMC model (Figure 13C), but not A549 cells alone (Figure 13D). In the A549 / PBMC model, tumor volume reduction correlated with increased CD226 expression on CD8 T cells isolated from NB1088-treated tumors (Figure 13E). The effect of NB1088 on CD8 T cell effector function ex vivo was also assessed (Figures 14A and 14B). Single-cell suspensions digested from tumors were stimulated with anti-CD28 / anti-CD3 in the presence of brefeldin A and anti-CD107a for 5 hours at 37°C. After stimulation, cells were stained and interferon-γ production by CD8+ T cells was detected using flow cytometry with standard surface and intracellular staining methods. As shown in Figures 14A and 14B, NB1088 increased the frequency of tumor-derived CD8+ T cells that were overall interferon-γ positive (Figure 14A) and polyfunctional interferon-γ / CD107a double positive (Figure 14B). Furthermore, the increased frequency of interferon-γ-positive CD8+ T cells in NB1088-treated tumors was derived exclusively from CD226-positive CD8+ T cells (Figures 14C and 14D), suggesting an important in vivo contribution of CD226 function to the antitumor activity of NB1088.

[0195] Example 10 – Pharmacokinetic and pharmacodynamic changes of NB1088 on CD226 expression on CD4 T cells in cynomolgus monkeys The pharmacokinetic properties of NB1088 were measured in cynomolgus monkeys (two females per dose group) after a single or 4 × 1-week IV bolus injections at doses of 2, 50, or 200 mg / kg. Additionally, changes in CD226 expression on circulating peripheral CD4 T cells were assessed. Figure 15 shows the plasma concentration (µg / ml) of NB1088 as a function of time (hours) and dose. Based on an in vitro potency assay using a cynomolgus monkey PBMC assay, the IC90 and 10×IC90 were calculated. NB1088 exhibited a typical PK profile, reaching concentrations exceeding 10×IC90 over the study period after repeated administration at a dose of 200 mg / kg. Figure 15B shows CD226 expression levels on circulating CD4 T cells normalized to pre-dose levels, as measured by flow cytometry using a specific antibody. NB1088 increased CD226 surface expression levels by up to 1.5-fold in the 50 mg / kg and 200 mg / kg repeated dose groups, and continued to increase in the 200 mg / kg repeated dose group. These data suggest that NB1088 is involved in and can regulate CD226 expression on CD4 T cells in cynomolgus monkeys.

[0196] Example 11 – Expression of human PVR among different tumor types We evaluated the expression levels of PVR in human cancers of different origins. PVR expression was detected by standard immunohistochemistry using the commercially available rabbit monoclonal antibody clone D3G7H and cancer tissue microarrays. Staining was digitized, intensity quantified, and H-scores were calculated within and across indications. Figure 16 shows elevated PVR expression levels in most of the analyzed indications at various frequencies. Elevated PVR expression was observed in liver cancer, colon cancer, adrenal cancer, uterine cancer, testicular cancer, squamous cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer, bladder cancer, prostate cancer, bile duct cancer, skin cancer, HNSCC cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, and melanoma. These data suggest that PVR contributes to tumor progression in multiple indications of human cancer.

[0197] Example 12 – Design of humanized antibodies A humanized IgG antibody was designed based on one of the variants having a heavy chain VH4 and a light chain VK2, respectively. An exemplary VK2 sequence is set forth in SEQ ID NO: 49. An exemplary VH4 sequence of hIgG4(S241P) is set forth in SEQ ID NO: 50, and an exemplary sequence of hIgG1 is set forth in SEQ ID NO: 51. Additionally, exemplary nucleotide sequences optimized for expressing the amino acid sequences in CHO cells were designed as follows: for VK2, the nucleotide sequence is set forth in SEQ ID NO: 52 or SEQ ID NO: 53. for IgG4 VH4, the nucleotide sequence is set forth in SEQ ID NO: 54 or SEQ ID NO: 55.

[0198] Example 12 –CAR-T cells expressing scFv derived from humanized anti-PVR antibody variants are specifically activated in the presence of tumor cells CAR-T constructs were designed based on the H4K2-NTX-1088C and H3K4-NTX-1034C mutants. The amino acid sequences of the ScFv molecules are set forth in SEQ ID NOs: 56 and 57, respectively. Parental Jurkat cells or Jurkat cells overexpressing anti-hPVR CAR-T (40K / well) were incubated with A549 or MDA-231 breast cancer cells (PVR-positive) at a 1:1 E:T ratio for 24 hours. As shown in Figure 18, both CAR-T drivers elicited the secretion of hundreds of pg of IL2 in the presence of the indicated targets, whereas parental Jurkat cells showed no detectable IL2 secretion. IL2 secretion was quantified using Biolegend hIL2 (cat431804). These results demonstrate that the αPVR-based CAR-T driver is highly functional in inducing T cell activation in the presence of PVR-expressing target cells.

[0199] To examine CAR-T tumor cell killing, 200K A549 or MDA-231 cells were plated in 12-well plates with CAR-T-PVR variants (NTX-1088C or NTX1034C) at an E:T ratio (based on GFP positivity) of 0.4 and 0.8–1, respectively, in NK medium for 72 hours. Tumor cell killing was assessed using a standard CTG protocol (Promega G9241). As shown in Figures 19A–19C, both PVR variants exhibited a more than two-fold increase in killing of MDA-231 cells and an eight-fold increase in killing of A549 cells compared to activated PBMCs. These findings strongly suggest that the αPVR CAR-T construct significantly increases killing of PVR-expressing targets.

[0200] Example 13 – Efficient killing of hematologic target cells by αPVR CAR-T CAR-T constructs were designed based on the mutants H4K2-NTX-1088C and H3K4-NTX-1034C, and the scFv sequences are set forth in SEQ ID NOs: 56 and 57, respectively.

[0201] To investigate CAR-T hematological tumor cell killing, 20K K562 cells / well were plated in 96-well plates at an E:T ratio of 3.4 to 0.22:1 in RPMI supplemented with 100 IU / IL-2 / ml for 18 hours. Tumor cell killing was assessed by flow cytometry. Both NTX-1034C and NTX-1088C were highly effective in eliminating targets at higher E:T ratios. The clear advantage of NTX-1088C over NTX-1034C at lower E:T ratios is likely due to the moderate levels of PVR expressed on K562 cells. These results suggest that αPVR CAR-T may be effective against hematological tumors expressing PVR.

Claims

1. A humanized antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 10-12; ii. a set of four heavy chain framework (FR) sequences, namely: (A) FR-H1 selected from the group consisting of SEQ ID NOs: 18, 22, and 26; (B) FR-H2 selected from the group consisting of SEQ ID NOs: 19, 23, and 28; (C) FR-H3 selected from the group consisting of SEQ ID NOs: 20, 24, 27, and 29; and (D) FR-H4 selected from the group consisting of SEQ ID NOs: 21 and 25. wherein the light chain variable region comprises: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 13-15; ii. a set of four light chain (LC) framework (FR) sequences, namely: (A) FR-L1 selected from the group consisting of SEQ ID NOs: 30 and 34; (B) FR-L2 selected from the group consisting of SEQ ID NOs: 31 and 37; (C) FR-L3 selected from the group consisting of SEQ ID NOs: 32, 35, and 36; and (D) FR-L4, which is SEQ ID NO:

33. wherein the humanized antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (PVR, CD155).

2. A humanized antibody or antigen-binding fragment thereof described in claim 1, wherein the heavy chain variable region comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:

9.

3. A humanized antibody or antigen-binding fragment thereof described in any one of claims 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain variable region comprises an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:

9.

4. A humanized antibody or antigen-binding fragment thereof described in any one of claims 1 to 3, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

2.

5. A humanized antibody or its antigen-binding fragment described in any one of claims 1 to 4, wherein the humanized antibody inhibits binding of PVR to at least one of TIGIT, CD96, and CD226.

6. A nucleic acid encoding a humanized antibody or its antigen-binding fragment described in any one of claims 1 to 5.

7. A chimeric antigen receptor (CAR) comprising a combination of a heavy chain variable region sequence and a light chain variable region sequence of a humanized antibody described in any one of claims 1 to 5.

8. A CAR cell which is a T cell or NK cell containing the CAR described in claim 7.

9. A pharmaceutical composition comprising a humanized antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, or a CAR cell described in claim 8, and a pharmaceutically acceptable excipient, carrier, or diluent.

10. A humanized antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, a CAR cell described in claim 8, or a pharmaceutical composition described in claim 9, for use in increasing surface expression and / or signal transduction of CD226 on CD8+ T cells or NK cells.

11. A humanized antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, a CAR cell described in claim 8, or a pharmaceutical composition described in claim 9, for use in treating cancer in an individual.

12. A humanized antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, a CAR cell described in claim 9, or a pharmaceutical composition described in claim 9, used in combination with an inhibitor of PD-1, PD-L1, CTLA-4, or CD112R signaling.

13. The humanized antibody or antigen-binding fragment thereof, CAR cell, or pharmaceutical composition described in claim 12, wherein the inhibitor of PD-1 signaling is an antibody or fragment thereof that binds to PD-1, PD-L1, or PD-L2.

14. The humanized antibody or antigen-binding fragment thereof, CAR cell, or pharmaceutical composition of claim 12, wherein the antibody or fragment thereof that binds to PD-1 is selected from pembrolizumab, nivolumab, AMP-514, tislelizumab, spartalizumab, and PD-1-binding fragments thereof.

15. The humanized antibody or antigen-binding fragment thereof, CAR cell, or pharmaceutical composition of claim 12, wherein the antibody or fragment thereof that specifically binds to PD-L1 or PD-L2 is selected from durvalumab, atezolizumab, avelumab, BMS-936559, or FAZ053, or PD-L1 and PD-L2-binding fragments thereof.

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