Antibodies against poliovirus receptor (PVR) and uses thereof

Abstract

The present application relates to antibodies against poliovirus receptor (PVR) and uses thereof. The present invention provides humanized antibodies and antigen binding fragments thereof that bind to human poliovirus receptor (PVR). The antibodies can be used to treat tumors or cancers.

Description

[0001] This application is a divisional application of an application filed on October 7, 2020, with application number 202080071097.9, and invention name “Antibodies against poliovirus receptor (PVR) and their uses”. Field of the Invention

[0002] The present invention belongs to the field of immunotherapy and relates to humanized antibodies that specifically bind to human poliovirus receptor (PVR), comprising a specific set of CDR and framework sequences, as well as pharmaceutical compositions comprising these humanized antibodies and uses thereof. Background of the Invention

[0003] The poliovirus receptor (PVR), also known as CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. It has previously been described as a tumor antigen and a potential target for therapeutic intervention because its expression is upregulated in neuroectodermal cancers, including glioblastoma multiforme, medulloblastoma, and colorectal cancer (Solecki et al., J. Biol. Chem. 2002, 277: 25697-700) and pancreatic cancer (Nishiwada et al., Anticancer Res. 2015, 35(4): 2287-97). It is also known that PVR enhances serum-induced Ras-Raf-MEK-ERK signaling activation, upregulates cyclins D2 and E, and downregulates p27Kip1, ultimately shortening the G0 / G1 phase of the cell cycle (Kakunaga 2004, J. Biological Chemistry, 279, 36419-36425). Therefore, blocking PVR on tumor cells is expected to reduce their viability. PVR also plays a key role in angiogenesis and is thought to regulate VEGF-induced angiogenesis by controlling the interaction of vascular endothelial growth factor receptor 2 (VEGFR2) with integrin α(v)β(3) and the VEGFR2-mediated Rap1-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012, 110(5), 716-26). In addition, PVR complexes with IGF1R and participates in tyrosine-protein kinase Met (cMet) signaling and blocks complex formation, reducing cell viability and angiogenesis (Lee et al., Scientific Reports 2014, 20, 4, 7139).

[0004] In recent years, it has become apparent that PVR is a key immune checkpoint ligand (Brilc PK et al. 2019 Cell Mol Immunology). PVR is upregulated in both malignant cells and tumor-infiltrating myeloid cells in humans and mice. PVR- / - mice exhibit reduced tumor growth and metastasis through upregulation of DNAM-1 (CD226) and enhanced effector function of CD8+ T and NK cells, respectively. Blocking programmed cell death protein 1 (PD-1) or blocking both PD-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA4) is more effective in the setting of PVR restriction, indicating the clinical potential of combination therapy using PD-1 / PD-L1 and PVR blockade (LI XY et al. JCI 2018). In addition, in the clinical setting, the expression of PD-L1 and PVR is independently regulated, which allows patients treated with anti-PD-1 antibodies to be stratified into four groups according to the expression levels of PD-L1 and PVR. High PVR expression in patients with low PD-L1 expression enriches for non-responders. This was further verified using a genetically engineered cancer model. These findings support the importance of PVR as a key immune checkpoint in tumor immunotherapy (Lee BR et al. JCI. Insight 2020). By injecting cancer cells into mouse tails and measuring lung metastasis, PVR was shown to participate in metastasis. It has been shown that PVR upregulated in cancer cells interacts with its trans receptor in platelets in trans, and this trans interaction enhances the metastasis of cancer cells to the lungs (Morimoto et al., Oncogene (2008) 27, 264-273).

[0005] WO2017149538, filed by one of the inventors of the present invention, discloses mouse antibodies and fragments thereof that bind to PVR, as well as polynucleotide sequences encoding these antibodies and hybridoma cells that produce these antibodies.

[0006] US Patent Application No. 20070041985 discloses molecules that specifically bind to at least one intracellular or extracellular domain of PVR, wherein the molecules have the ability to modulate receptor-mediated adhesion, trafficking and / or invasion behavior of cells expressing PVR or any derivatives thereof.

[0007] U.S. Patent Application No. 20090215175 provides molecules (e.g., small compounds, oligonucleotides, polypeptides, antibodies, and antibody fragments) that modulate PVR functions necessary for cell adhesion, transport, invasion, and / or metastatic potential. The molecules can be used to treat cells with metastatic potential, metastasis, and cancer.

[0008] There is an unmet need to provide humanized antibodies that recognize human PVR that are safer, more effective, and useful diagnostically and therapeutically for diseases involving PVR expression. SUMMARY OF THE INVENTION

[0009] According to some embodiments, humanized antibodies are described herein that specifically bind to the human poliovirus receptor (PVR; CD155) and prevent PVR from binding to at least one of the following ligands: T cell immune receptor (TIGIT) of Ig and ITIM domains, CD96, and CD226 (DNAM-1). The humanized antibodies of the present invention are selected from a larger collection of antibody clones and 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, biophysical properties, and improved expression. Since PVR binds to CD226, the surface expression of CD226 on T and NK cells is downregulated, and the activity of CD226 in stimulating T and NK cells and tumor cell killing is reduced, the antibodies of the present invention can restore the expression and / or activity of CD226 on these cells. The correct expression and function of CD226 allow immune cells, especially CD8+ T cells and NK cells, to enhance the killing of tumors.

[0010] By combining a specific set of CDR sequences with human framework sequences and introducing specific mutations into these sequences, improved antibodies with modified variable regions are generated, thereby creating a large collection of humanized antibodies. The newly designed humanized variable regions retain residues that are critical for maintaining antibody conformation and binding affinity, while having a minimal incidence of potential T-cell epitopes, thereby minimizing the risk of adverse immune responses to the antibody. The antibodies disclosed herein are designed based on factors including homology, T-cell epitopes, key residues, and predicted structure.

[0011] Unexpectedly, variants with a specific human framework in combination with a point mutation from glutamic acid to asparagine in the last residue of light chain variable region CDR2 (position 56 according to Kabat numbering) showed strong affinity for human PVR and improved immunoreactivity.

[0012] Several humanized antibody variants according to the present invention were found to be particularly suitable for chimeric antigen receptor (CAR) applications due to their lower affinity, which may facilitate targeting of highly expressing PVR tumor cells without targeting normal tissues.

[0013] Advantageously, several humanized antibody variants according to the present invention have improved producibility and can be produced in exceptionally high yields compared to other variants.

[0014] It has been disclosed that the humanized antibodies described herein demonstrate high potency in cytotoxic T and NK cell stimulation and in treating cancer in humanized mouse models, including in vivo models of pancreatic and lung cancer.

[0015] Thus, in some embodiments, the present invention provides highly specific, non-immunogenic, humanized antibodies against human PVR with improved affinity, activity, and / or reproducibility.

[0016] According to one aspect, the present invention provides a humanized antibody or a fragment thereof that specifically binds to the human poliovirus receptor (PVR, CD155), wherein the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region, the variable region having an amino acid sequence that is 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 wherein the light chain comprises a variable region, the variable region having an amino acid sequence that is 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.

[0017] According to some embodiments, the antibody comprises a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence, wherein the heavy chain variable region amino acid sequence comprises the CDR sequences listed in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and the light chain variable region amino acid sequence comprises the CDR sequences listed in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

[0018] According to some embodiments, the humanized antibody or fragment thereof is an IgG monoclonal antibody. According to 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.

[0019] According to some embodiments, the humanized antibody or fragment thereof comprises a human IgG4 constant region having an S228P (also referred to as S241P) substitution in the hinge region.

[0020] According to some embodiments, the humanized antibody or fragment thereof is a monoclonal antibody, Fab, F(ab)2, a single domain antibody, or a single chain variable fragment (scFv).

[0021] 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 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 the light chain variable region comprises 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 LQ(S / P)EDFA(D / T)YFCQQYSRYPLTFGQGT KLEIK (SEQ ID NO:48).

[0022] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain variable region comprising:

[0023] i. a set of three CDR sequences comprising the sequences listed in SEQ ID No.10-12; and

[0024] ii. A set of four heavy chain framework (FR) sequences: (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.

[0025] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a light chain variable region comprising:

[0026] i. a set of three CDR sequences comprising the sequences listed in SEQ ID No.13-15; and

[0027] 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 is SEQ ID NO: 33.

[0028] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising:

[0029] i. a set of three CDR sequences comprising the sequences listed in SEQ ID No.10-12; and

[0030] ii. a set of four heavy chain (HC) framework (FR) sequences: (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;

[0031] And the light chain variable region comprises:

[0032] i. a set of three CDR sequences comprising the sequences listed in SEQ ID No.13-15; and

[0033] ii. A set of four light chain (LC) framework (FR) 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 is SEQ ID NO: 33.

[0034] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 certain embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 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.

[0035] According to some embodiments, the humanized antibody comprises a combination of a heavy chain variable region and a light chain variable region, wherein the combination is selected from the group consisting of:

[0036] i. the heavy chain variable region sequence listed in SEQ ID NO: 1 and the light chain variable region sequence listed in SEQ ID NO: 2;

[0037] ii. the heavy chain variable region sequence set forth in SEQ ID NO: 4 and the light chain variable region sequence set forth in SEQ ID NO: 8;

[0038] iii. the heavy chain variable region sequence set forth in SEQ ID NO: 5 and the light chain variable region sequence set forth in SEQ ID NO: 2;

[0039] iv. the heavy chain variable region sequence set forth in SEQ ID NO: 5 and the light chain variable region sequence set forth in SEQ ID NO: 8;

[0040] v. the heavy chain variable region sequence set forth in SEQ ID NO: 4 and the light chain variable region sequence set forth in SEQ ID NO: 2;

[0041] vi. the heavy chain variable region sequence listed in SEQ ID NO: 1 and the light chain variable region sequence listed in SEQ ID NO: 8;

[0042] vii. 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: 2; and

[0043] viii. The heavy chain variable region sequence listed in SEQ ID NO: 6 and the light chain variable region sequence listed in SEQ ID NO: 8.

[0044] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody 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 variable region comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO:2.

[0045] According to 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.

[0046] According to some embodiments, the humanized antibody inhibits binding of PVR to at least one of TIGIT, CD96, and CD226.

[0047] According to some embodiments, the antibody inhibits the binding of PVR to TIGIT, CD96, and CD226.

[0048] 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 having 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 having at least 90% identity.

[0049] According to some embodiments, the humanized antibody comprises the light chain sequence set forth in SEQ ID NO:49.

[0050] According to some embodiments, the humanized antibodies exhibit improved antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) compared to other known antibodies.

[0051] According to another aspect of the present invention, a polynucleotide sequence encoding a humanized antibody or an antigen-binding fragment thereof is provided.

[0052] According to some embodiments, a polynucleotide sequence encoding the amino acid sequence of the heavy chain variable region, the light chain variable region, or both as described above is provided.

[0053] According to some embodiments, 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 is provided.

[0054] According to some embodiments, 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 is provided.

[0055] According to some embodiments, the polynucleotide encodes a humanized antibody or antibody fragment thereof, the humanized antibody 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 a heavy chain variable region and a light chain variable region represents a separate embodiment of the present invention.

[0056] 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. Each possibility represents a separate embodiment of the invention.

[0057] 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. Each possibility represents a separate embodiment of the invention.

[0058] In another 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.

[0059] Also described are cell lines comprising nucleic acids encoding antibodies of the present invention. Cell lines are used to express humanized antibodies or fragments thereof as described herein. In certain embodiments, the cell line is a mammalian cell line, such as a Chinese hamster ovary (CHO) cell line.

[0060] According to some embodiments, the cell line is bacterial, plant, murine (eg, NSO and SP2 / 0), rat (eg, YB2 / 0), hamster (eg, BHK and CHO), or human (eg, PER.C6).

[0061] According to one aspect, the present invention provides a chimeric antigen receptor (CAR), the chimeric antigen receptor (CAR) includes an extracellular portion (binding domain), including any humanized antibody or fragment thereof as described herein. According to some embodiments, there is provided a CAR comprising a combination of heavy chain variable region sequence and light chain variable region sequence as described above, the CAR having a unique combination of CDR and framework sequence and improved binding and other properties.

[0062] According to some embodiments, the CAR comprises a combination of a heavy chain variable region comprising an amino acid sequence having 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 comprising an amino acid sequence having 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.

[0063] According to some embodiments, the CAR comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region 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.

[0064] According to some embodiments, the CAR comprises a combination of a humanized antibody heavy chain variable region and a light chain variable region, wherein the combination is selected from the group consisting of:

[0065] i. the heavy chain variable region sequence listed in SEQ ID NO: 1 and the light chain variable region sequence listed in SEQ ID NO: 9;

[0066] ii. the heavy chain variable region sequence set forth in SEQ ID NO: 3 and the light chain variable region sequence set forth in SEQ ID NO: 9;

[0067] iii. the heavy chain variable region sequence set forth in SEQ ID NO: 4 and the light chain variable region sequence set forth in SEQ ID NO: 9;

[0068] iv. the heavy chain variable region sequence set forth in SEQ ID NO: 5 and the light chain variable region sequence set forth in SEQ ID NO: 9; and

[0069] 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.

[0070] According to some embodiments, the CAR comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 5, and 6 and a light chain variable region sequence set forth in SEQ ID NO: 9, a transmembrane domain, and an intracellular T cell signaling domain.

[0071] Also provided according to the present invention is a single chain variable fragment (scFv) comprising the heavy chain variable region and the light chain variable region of an antibody as described herein. According to certain embodiments, there is a hinge region between the variable regions.

[0072] According to some embodiments, the amino acid sequence of the scFv is set forth below: a sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, and analogs thereof having at least 90% sequence similarity to any of said sequences.

[0073] According to some embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NO:56 and SEQ ID NO:57.

[0074] According to some embodiments, CAR comprises an scFv sequence and at least one protein domain selected from the group consisting of a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3ζ (CD3Z, Zetta) domain. According to some embodiments, CAR comprises a CD8 Stalk domain. According to some embodiments, CAR comprises a CD28 TM domain. According to some embodiments, CAR comprises a CD3Z domain. According to some embodiments, CAR comprises a 41BB domain. According to a specific embodiment, CAR comprises a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

[0075] According to some embodiments, CAR comprises a scFv sequence containing a PVR binding site of any antibody disclosed above, and at least one domain selected from the group consisting of: a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain. According to a specific embodiment, CAR comprises a scFv sequence containing a PVR binding site of any antibody disclosed above, and a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

[0076] According to some embodiments, a lymphocyte engineered to express a CAR described herein is provided. According to some embodiments, a T cell engineered to express a CAR described herein is provided. According to another embodiment, a NK cell engineered to express a CAR described herein is provided.

[0077] According to a specific embodiment, an engineered T cell is provided, which expresses a 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 any of said sequences; a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

[0078] According to one aspect, the present invention provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one lymphocyte comprising a CAR described herein.

[0079] According to another aspect, the present invention provides a pharmaceutical composition comprising the humanized antibody or antigen-binding fragment described herein and a pharmaceutically acceptable excipient, carrier or diluent.

[0080] The compositions of the present invention can be administered to a subject in need thereof using any mode of administration, including parenteral and enteral modes of administration.

[0081] According to some embodiments, the pharmaceutical composition is formulated for injection or infusion. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. According to some embodiments, the pharmaceutical composition is formulated for intratumoral administration.

[0082] According to some embodiments, the humanized antibody or antigen-binding fragment thereof or pharmaceutical composition is for use in increasing the surface expression and / or signaling of CD226 on CD8+ and CD4+ T cells.

[0083] According to an embodiment, the humanized antibody or its antigen-binding fragment or pharmaceutical composition is used to treat a cancer in an individual. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of lung cancer, colon cancer, glioblastoma, adrenal cancer, uterine cancer, head and neck cancer, pancreatic cancer, and breast cancer. Each possibility represents a separate embodiment of the present invention.

[0084] According to some embodiments, the cancer is a hematological cancer.

[0085] 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.

[0086] According to some embodiments, the individual is a human.

[0087] According to some embodiments of the present invention, the use further comprises using an agent that downregulates the activity or expression of an immune co-inhibitory receptor.

[0088] 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 (CD 160), LAIR1, SIGLEC10, CD112R, CD112, ILT-4 and 2B4. Each possibility represents a separate embodiment of the present invention.

[0089] According to some embodiments of the present invention, the use further comprises use in combination with an anti-endothelial growth factor receptor (EGFR) antibody.

[0090] According to another aspect, the present invention provides a method for increasing surface expression and / or signaling of CD226 in CD8+ and CD4+ T cells of 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 as described herein. In certain embodiments, the CD8+ T cells are tumor-infiltrating CD8+ T cells.

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

[0092] According to 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 its antigen-binding fragment or a pharmaceutical composition, and a PD-1 signaling inhibitor, a PD-L1 signaling inhibitor, a CTLA-4 signaling inhibitor, or a CD112R signaling inhibitor. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of: glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, or lung cancer. In certain embodiments, the PD-1 signaling inhibitor is an antibody or fragment thereof that binds to PD-1. In certain embodiments, the antibody or fragment thereof that binds to PD-1 is Pembrolizumab, Nivolumab, AMP-514, Tislelizumab, Spartalizumab, or their PD-1 binding fragments. In certain embodiments, the PD-1 signaling inhibitor is an antibody that specifically binds to PD-L-1 or PD-L-2. In certain embodiments, antibodies that specifically bind to PD-L1 or PD-L2 include durvalumab, atezolizumab, avelumab, BMS-936559 or FAZ053, or their PD-L1 or PD-L2 binding fragments. In certain embodiments, the PD-1 signaling inhibitor includes an Fc-fusion protein that binds to PD-1, PD-L1 or PD-L2. In certain embodiments, the Fc-fusion protein includes AMP-224 or its PD-1 binding fragment. In certain embodiments, the PD-1 signaling inhibitor includes a small molecule inhibitor of PD-1, PD-L1 or PD-L2.In certain embodiments, small molecule inhibitors of PD-1, PD-L1, or PD-L2 signaling include one or more of the following: 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-[[(2-methyl-1,1'-biphenyl]-3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS202); (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-phenylindole; L-α-glutamyl Amine, 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-piperidinylcarboxylic acid; glycinamide, N-(2-mercaptoacetyl)-L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteine-, cyclic (1→14)-thioether; or derivatives or analogs thereof.

[0093] Also described herein is a method for preparing a composition for treating a cancer in an individual suffering from cancer, the method comprising mixing a humanized antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, and lung cancer. Also described herein is a method for producing a humanized antibody or an 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 its antigen-binding fragment.

[0094] According to one aspect, the present invention also 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 as described herein.

[0095] According to another aspect, the present invention also provides a method for determining or quantifying PVR expression, the method comprising contacting a biological sample with an antibody or antibody fragment as described herein, and measuring the level of complex formation.

[0096] According to some embodiments, the method for detecting or quantifying PVR expression comprises the following steps:

[0097] i. incubating the sample with an antibody specific for PVR or an antibody fragment thereof comprising at least an antigen-binding portion thereof;

[0098] ii. Detecting bound PVR using a detectable probe.

[0099] According to some embodiments, the method further comprises the following steps:

[0100] iii. comparing the amount of (ii) with a standard curve obtained from reference samples containing known amounts of PVR; and

[0101] iv. Calculate the amount of PVR in the sample from the standard curve.

[0102] According to some embodiments, the method includes indicating that the subject has a PVR-positive cancer if the PVR amount is higher than a control or a given reference.

[0103] According to some specific embodiments, the sample is a body fluid or a solid tissue.In some embodiments, the method is performed in vitro or ex vivo.

[0104] Also provided is a kit for measuring PVR expression in a biological sample, the kit comprising at least one antibody or antibody fragment as described herein and means for measuring PVR expression. In some embodiments, the kit further comprises instructional materials for use of the kit.

[0105] Additional embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description and the accompanying drawings, which set forth illustrative examples of the principles utilizing the features described herein, wherein:

[0107] Figure 1A-1B Affinity and competition assays of the .N56 substitution. Figure 1A The N56E and N56D variants improved the affinity of PVR binding. Biacore assays show the relative and absolute affinity of the N56 substitution (hIgG4 variant) for PVR-plus HIS tag (Sino Cat. no. 10109-H08H). Affinity changes of >25% were considered significant. (*) Relative K for each variant D By replacing K D Divided by the K of the parental N56 variant (VH0VK0) D to confirm. Figure 1B The potency of chimeric antibodies (5B9 wild-type-WT, with IgG4 (S241P) HC) whose variable regions carry LC CDR2 sequences with a single amino acid substitution (to remove a deamidation site) was compared. 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.

[0108] Figure 2A-2B The N56E and N56D variants have improved cross-reactivity for monkey PVR binding. To assess maximal binding, cells expressing PVR were stained with a saturating dose (10 μg / ml) of the N56 variant monoclonal antibody (shown on the X-axis) and the mean fluorescence intensity (MFI) values were determined by FACS analysis. The MFI of each variant was divided by the MFI of the parent antibody (K0) to calculate the fold change. Figure 2A )NCI-H1975 (human lung) cells and ( Figure 2B ) Vero (African green monkey kidney) cells. Affinity changes of >25% were considered significant.

[0109] Figure 3 .N56E and N56T variants improve NK activation. To characterize the N56 substitution variants, CD107a induction assays were performed using human NK cells from healthy donors and MDA-MB-231 as target cells at a ratio of 2:1. Ab was added at 600pM. K0 was the parent clone (N56). All variants resulted in significant CD107a induction (>240% higher than isotype IgG1). In addition, N56 substitutions of N56E & N56T significantly improved CD107a induction compared to K0. (*p<0.04, **p<0.01).

[0110] Figure 4 .N56E and N56T variants improve CD8 T cell proliferation. In order to characterize the N56 variant, T cell proliferation assays were performed. In the presence of 2.5ul / ml PHA-L and using fresh human PBMCs labeled with CFSE, A549 cancer cells were used with a 4:1 effector-target ratio. All N56 variant monoclonal antibodies (X-axis) were added at 4ug / ml, and the co-culture was incubated for 96 hours. The results of FACS analysis of CD8+ T cells gated are presented. The MFI of the IgG-treated group was divided by the MFI of each variant to calculate the relative MFI of CFSE labeling. Due to the increase in proliferation, the CFSE signal is reduced, and the Y-axis depicts the reciprocal value of the ratio. N56E and N56T variants significantly increase CD8 T cell proliferation compared to the parental clone (K0), indicated by #. (*p<0.05, #<0.04, **p<0.01).

[0111] Figure 5A-5B Graph showing the affinity of humanized variants for human PVR as measured by surface plasmon resonance (SPR) ( Figure 5A ) or their binding to the surface of HEK 293 cells expressing PVR as measured by flow cytometry ( Figure 5B ). Absolute and relative results are shown using the chimeric N56E mutant parent antibody (N56E VH0 / Vk0) as a baseline.

[0112] Figure 6A-6B The expression levels of humanized variants are shown ( Figure 6A ) and similarity to human variable domain germline sequences ( Figure 6B ). The titers of each variant after transient expression in HEK 293EBNA cells are shown ( Figure 6A ). The variable domain sequence identities of the humanized heavy and light chain variants to human germline sequences are shown ( Figure 6B ).

[0113] Figure 7A-7B The biophysical properties of the humanized lead variant NB1088 (right panel) were compared with the humanized variant NB941 carrying the LC CDR25B9 WT sequence (left panel). Figure 7A and Figure 7B It shows that NB1088 has a high activity at low pH ( Figure 7A ) or at 40℃ in high concentration ( Figure 7B ) After the stress test, the production of acidic species decreased over time.

[0114] Figures 8A-8D Schematic diagram of the EC of NB1088 binding to PVR 50( Figure 8A ); NB1088 inhibits PVR-TIGIT binding IC 50 ( Figure 8B ) ; NB1088 inhibits PVR-CD96 binding IC 50 ( Figure 8C ); and IC of NB1088 inhibiting PVR-CD226 binding 50 ( Figure 8D ).

[0115] Figure 9A-9B The figure shows that a single NB1088 ( Figure 9A and Figure 9B ) and in combination with pembrolizumab to inhibit PD1 ( Figure 9B ) in an antigen-specific T cell assay (human papillomavirus; HPV) ( Figure 9A ) or nonspecific allogeneic T cell assay ( Figure 9B ) increased interferon-γ release in the tumor / T cell co-culture system. **p<0.01, one-way Anova.

[0116] Figures 10A-10B The figure shows that NB1088, when combined with an endothelial growth factor receptor (EGFR)-binding antibody, increases antibody-dependent cellular cytotoxicity (ADCC) in the EGFR-expressing breast cancer cell line A549. Figure 10A ), and this is associated with increased interferon-γ release ( Figure 10B ) (*p<0.05, **p<0.01, one-way Anova).

[0117] Figures 11A-11B The figure shows that tumor cell lines expressing PVR (A549 or CaSki) can induce CD8 T cells ( Figure 11A ) and NK cells ( Figure 11B ), which could be restored by NB1088 but not by anti-TIGIT ( Figure 11A and Figure 11B ). Figure 11A Shown are results obtained using either an antigen-specific T cell assay (human papillomavirus; HPV) or a nonspecific allogeneic T cell assay in a co-culture system.

[0118] Figures 12A-12B Figure 1 shows the NB1088-dependent increase in IFN-γ release by allogeneic or antigen-responsive CD8+ T cells ( Figure 12A ) or NB1088 in the presence of EGFR blockade to induce antibody-dependent cellular cytotoxicity (ADCC)-responsive NK cells ( Figure 12B) were CD226-dependent, and in both cases, NB1088 showed superior activity for TIGIT inhibition (both Abs at 10 ug / ml).

[0119] Figures 13A-13E The figure shows the effect of NB1088 in a humanized mouse model of pancreatic cancer ( Figure 13A )(comparable to pembrolizumab (anti-PD-1, given at standard doses established for these models) Figure 13B )) and humanized mouse models of lung adenocarcinoma ( Figure 13C , humanized mice) as monotherapy, and in this lung adenocarcinoma model, efficacy depended on the presence of human immune cells ( Figure 13D , non-humanized mice) and is associated with increased expression of CD226 on tumor-infiltrating CD8+ T cells ( Figure 13E ). (*p<0.05, ***p<0.001, by two-way Anova).

[0120] Figures 14A-14D and Figure 13C and Figure 13E The model is related and illustrated that NB1088 increases interferon gamma positive ( Figure 14A ) and interferonγ / CD107a double positive ( Figure 14B )CD8 TIL cells, and NB1088 increased the frequency of interferonγ+ / CD226+ double-positive CD8 T cells ( Figure 14D ), but did not increase the frequency of interferonγ+ / CD226- single-positive CD8 T cells ( Figure 14C ). *p=0.0210***<0.0001, unpaired T test.

[0121] Figures 15A-15B Graph depicting the pharmacokinetics of NB1088 in cynomolgus monkeys treated with different doses of the antibody once (20 mg / kg dose and 50 mg / kg dose) or four times at one-week intervals (200 mg / kg dose) ( Figure 15A ) and corresponding pharmacodynamic changes in CD226 surface expression on circulating CD4 T cells ( Figure 15B ).

[0122] Figure 16 Shown is the expression of human PVR in biopsies across different cancer types as measured by immunohistochemistry and assessed by the H-score.

[0123] Figure 17 is a general schematic drawing of a CAR-T construct. The scFv comprises the heavy and light chains (VH and VL, respectively) of a humanized antibody according to the present invention.

[0124] Figure 18 The figure shows that Jurkat cells overexpressing the αPVR CAR-T construct secreted interleukin 2 (IL2) more robustly than parental Jurkat cells. Parental Jurkat cells or Jurkat cells overexpressing the αPVR CAR-T construct 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. IL2 secretion was quantified using Biolegend hIL-2 (cat. 431804). In the presence of the indicated targets, both CAR-T drivers increased IL2 secretion by more than 100-fold compared to parental Jurkat cells.

[0125] Figures 19A-19C The figure shows that anti-PVR (αPVR) CAR-T increased target cell killing. Figure 19A ) or MDA-231( Figure 19B ) cells (200K cells each) were plated in 12-well plates containing CAR-T-PVR variants NTX-1088C and NTX-1034C in NK culture medium at an E:T ratio of 0.4 and 0.8 to 1 (based on GFP positivity) for 72 hours. Tumor cell killing was assessed using a standard CTG protocol (Promega G9241).

[0126] Figure 20 Figure 1 shows the effective killing of blood target cells by αPVR CAR-T. K562 (an AML model) cells were incubated with the αPVR CAR-T variants NTX-1088C and NTX-1034C in RPMI + IL-2 for 18 hours at the E:T ratio indicated on the X-axis. Tumor cell killing was assessed using flow cytometry. Significant target cell killing was observed for both CAR-T agents. Detailed Description of the Invention

[0127] The present invention provides humanized monoclonal antibodies that recognize 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 thus being safe for use in humans. The antibodies of the present invention are characterized in that they have unique CDR sequences and novel humanized framework sequences and designs. More specifically, the monoclonal antibodies provided by the present invention have specific combinations of CDRs and non-completely humanized framework sequences and have unique properties and have improved safety and efficacy over known anti-PVR antibodies.

[0128] Some of the variants described herein have increased manufacturability and are expressed at higher levels compared to other humanized PVR antibodies comprising the same CDR regions. Also disclosed herein are methods of using these antibodies to treat cancer in an individual.

[0129] In the following description, certain specific details are listed in order to provide a thorough understanding of the various embodiments. However, those skilled in the art will understand that the embodiments provided can be practiced without these details. Unless the context requires otherwise, in the specification and the appended claims, the word "comprise" and its variations such as "comprises" and "comprising" should be interpreted in an open, inclusive sense, that is, "including but not limited to". As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural prompts. It should also be noted that the term "or" is generally used in its sense including "and / or" unless the context clearly dictates otherwise. In addition, the titles provided herein are for convenience only and do not explain the scope or meaning of the claimed embodiments.

[0130] The term "PVR" as used herein refers to the poliovirus receptor, also referred to as CD155 (cluster of differentiation 155) according to some embodiments, 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 comprising three Ig-like domains, specifically a V-like domain on the outermost side and two C2-like domains thereafter. The humanized antibodies described herein have affinity for human PVR (hPVR). In some embodiments, the antibodies have a certain affinity for PVR proteins from other animals, particularly primates. Advantageously, affinity for other primates, such as African green monkeys, enables further testing of the safety and efficacy of humanized antibodies in non-clinical trials. No affinity was observed for PVR from evolutionarily distant animals, such as rodents.

[0131] As used herein, the term "about" refers to an amount that approaches the stated amount by 10% or less.

[0132] 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 compositions and methods can be used to treat. According to some embodiments, the individual is a mammal. According to some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. According to some embodiments, the individual is a human.

[0133] As used herein, the term "combination" or "combination therapy" can refer to the concurrent administration of the items to be combined or the sequential administration of the items to be combined. As described herein, when the combination refers to the sequential administration of items, the items can be administered in any time order.

[0134] The terms "cancer" and "tumor" refer to the physiological condition in mammals characterized by uncontrolled cell growth. Cancer is a disease in which a group of cells exhibits uncontrolled or unwanted growth. Cancer cells can also spread to other locations, which can lead to the formation of metastases. For example, cancer cells can spread within the body through the lymph or blood. Uncontrolled growth, invasion, and metastasis formation are also known as the malignant characteristics of cancer. These malignant characteristics distinguish cancer from benign tumors, which typically do not invade or metastasize.

[0135] As used herein, the term "effective amount" refers to the amount of a therapeutic agent that causes a biological effect when administered to a mammal. Biological effects include, but are not limited to, 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 animals carrying tumors. A "therapeutic amount" is a concentration of a drug calculated to exert a therapeutic effect. A therapeutic amount covers a dosage range that can cause a therapeutic response in an individual population. The mammal can be a human individual. The human individual can have or be suspected of having or suffering from a tumor.

[0136] As used herein, "checkpoint inhibitors" refer to drugs that inhibit biological molecules ("checkpoint molecules") produced by an organism that negatively regulate the anti-tumor / 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.

[0137] The antibodies provided include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies and multireactive antibodies) and antibody fragments.Antibodies include antibody conjugates and molecules comprising antibodies, such as chimeric molecules.Therefore, antibodies include but are not limited to full length, and fragments and parts thereof that retain their binding specificity, such as any specific binding portion thereof, including fragments and parts with any number of immunoglobulin classes and / or isotypes (for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen binding) fragments or their specific binding portions, including but not limited to Fab, F(ab')2, Fv and scFv (single chain or related entities).Monoclonal antibodies are typically one of the compositions of substantially homogeneous antibodies; therefore, except for possible naturally occurring mutations that may exist in small amounts, any single antibody included in the monoclonal antibody composition is identical.Polyclonal antibodies are products comprising different antibodies of different sequences, typically for two or more different determinants (epitopes).Monoclonal antibodies can include human IgG1 constant regions. The monoclonal antibody may comprise a human IgG4 constant region.

[0138] The term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including complete antibodies and their functional (antigen binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (sFv or scFv) and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term covers genetically engineered and / or other modified forms of immunoglobulins, such as intrabodies, peptibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific antibodies, such as bispecific antibodies, double antibodies, three antibodies and four antibodies, tandem double scFv, tandem three scFv. Unless otherwise indicated, the term "antibody" should be understood to cover its functional antibody fragments. The term also covers complete 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.

[0139] 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, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("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. ("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 Plückthun 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 modelling 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.

[0140] The boundaries of a given CDR or FR can vary according to the scheme used to identify them. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, wherein insertions are accommodated by inserting letters (e.g., "30a"), and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects.

[0141] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, each domain comprising four conserved framework regions (FRs) and three CDRs (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single V H or V L A V domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by using a V domain from an antibody that binds to the antigen. H or V L domains to screen for complementary V L or V H Libraries of domains (see, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0142] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule, other than an intact antibody, that comprises a portion of an intact antibody and 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 specific embodiments, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as an scFv.

[0143] As used herein, the term "antigen" refers to a molecule or portion of a molecule that is capable of eliciting antibody formation and is specifically bound by an antibody. An antigen may have one or more epitopes. Specific binding as mentioned above means that an antigen reacts with its corresponding antibody in a highly selective manner and does not react with a large number of other antibodies that may be elicited by other antigens. The antigen according to some embodiments of the present invention is human PVR.

[0144] Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, antibodies are fragments produced by recombination, such as fragments comprising arrangements that are not naturally occurring, such as fragments having two or more antibody regions or chains connected by synthetic linkers (e.g., polypeptide linkers), and / or fragments that are not produced by enzymatic digestion of naturally occurring intact antibodies. According to some embodiments, the antibody fragment is a scFv.

[0145] "Humanized" antibodies are antibodies in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. Humanized antibodies may optionally include 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 humanized non-human antibody, typically to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. According to some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0146] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or using human antibody repertoires or other non-human sources of human antibody encoding sequences, including human antibody libraries. The term does not include humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as antibodies in which all or substantially all CDRs are non-human.

[0147] 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 antibodies and antibody chains provided and other peptides, such as linkers and binding peptides, can include amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. According to some embodiments, the polypeptide may contain modifications with respect to the natural or native sequence, as long as the protein retains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through mutations in the host producing the protein or errors due to PCR amplification.

[0148] The percentage of sequence identity (%) about the reference polypeptide sequence is after aligning the sequences and introducing room to realize maximum sequence identity percentage when necessary, and without considering any conservative substitution as a part of sequence identity, the percentage of the amino acid residue in the candidate sequence that is identical with the amino acid residue in the reference polypeptide sequence. The comparison for determining amino acid sequence identity percentage can be realized in various known ways, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. It is possible to determine the appropriate parameters for comparing sequences, including the algorithm required for realizing maximum comparison on the full length of the compared sequences. However, for the purposes of this paper, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been registered in the U.S. Copyright Office (USCopyrightOffice, Washington DC, 20559) together with user documentation, and it is registered with U.S. Copyright Registration Number TXU510087 in the U.S. Copyright Office. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., and can also be compiled from source code. The ALIGN-2 program should 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 are not modified.

[0149] In the case of amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a specific % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0150] When used herein to describe an amino acid sequence or a nucleic acid sequence, the terms "homologous," "homology," or "percent homology" relative to a reference sequence can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, as modified by Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The percent homology of a sequence can be determined using the most recent version of BLAST as of the filing date of this application.

[0151] In some embodiments, it is envisioned that the amino acid sequence variants of the antibodies provided herein are different from the polypeptides specifically disclosed herein in terms of one or more substitutions, deletions, additions and / or insertions. Such variants can be naturally occurring, or can be produced synthetically, for example, by modifying one or more of the above polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptide as described herein and / or using any of many known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The 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 and / or insertions and / or substitutions of residues from the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, provided that the final construct has desired characteristics such as antigen binding.

[0152] In some embodiments, antibody variants with one or more amino acid substitutions are provided. The sites of interest for mutagenesis by substitution include CDRs and FRs. Amino acid substitutions can be introduced into the antibody of interest, and the desired activity of the product can be screened, for example, to retain / improve antigen binding, reduce immunogenicity, or improve antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0153] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, wherein the substitutions, insertions, or deletions do not substantially reduce the binding of the antibody to the antigen. For example, conservative substitutions may be made in the CDRs that do not substantially reduce binding affinity. Such changes may be outside of CDR "hot spots." In variant V H and V L In some embodiments of the sequence, each CDR is invariant.

[0154] Changes (e.g., substitutions) can be made in CDRs, for example to improve antibody affinity. During somatic maturation, such changes may be made in CDRs encoding high mutation rate codons (see, for example, Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and the binding affinity of the resulting variants can be tested. Affinity maturation (e.g., using error-prone PCR, chain shuffling, CDR randomization or oligonucleotide-directed mutagenesis) can be used to improve antibody affinity (see, for example, Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (2001)). The CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, for example, Cunningham and Wells Science, 244: 1081-1085 (1989)). In particular, CDR-H3 and CDR-L3 are often targeted. Alternatively or additionally, a crystal structure of an antigen-antibody complex is used 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. Variants can be screened to determine whether they contain the desired properties.

[0155] Amino acid sequence insertions and deletions include amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing 100 or more residues, and insertions and deletions within the sequence of a single or more than one amino acid residue. Examples of terminal insertions include antibodies with an N-terminal methionine residue. Other insertion variants of antibody molecules include fusions of the N- or C-terminus of an antibody with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody. Examples of insertion variants within the sequence of an antibody molecule include insertions of 3 amino acids in the light chain. Examples of terminal deletions include antibodies with 7 or fewer amino acids missing at the light chain terminal.

[0156] In some embodiments, the antibodies provided herein have a dissociation constant (K) of 0.05 for the antibody target human poliovirus receptor (CD155). D ) 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 smaller, for example, 10 -8 M to 10 -13 M, for example, 10 - 9 M to 10 -13 M) K D Can be measured by any suitable assay. In certain embodiments, KD can be measured using a surface plasmon resonance (SPR) assay (e.g., using or ) to measure.

[0157] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby producing Fc region variants. The Fc region herein is the C-terminal region of the immunoglobulin heavy chain containing at least a portion of the constant region. The Fc region includes native sequence Fc regions and variant Fc regions. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc regions) comprising modifications (e.g., replacements) at one or more amino acid positions.

[0158] In some embodiments, the antibodies of the present disclosure are variants with some, but not all, effector functions, making them desirable candidates for applications in which the half-life of the antibody in vivo 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 reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Non-limiting examples of in vitro assays for evaluating ADCC activity of molecules of interest are described in U.S. Patents 5,500,362 and 5,821,337. Alternatively, non-radioactive assays (e.g., ACTI TM and CytoTox Non-radioactive cytotoxicity assays.) Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0159] The antibodies may have increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, US2005 / 0014934). Such antibodies may comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn, and include those having substitutions at one or more of the following 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 examples of Fc region variants are also contemplated (see, eg, Duncan & Winter, Nature 322:738-40 (1988); US Patent Nos. 5,648,260 and 5,624,821; and WO 94 / 29351).

[0160] In some embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. According to some embodiments, the substituted residues occur at accessible sites of the antibody. Reactive thiol groups can be positioned at sites for conjugation to other moieties, such as drug moieties or linker drug moieties, to produce immunoconjugates. In some embodiments, any one or more of the following residues may be substituted with 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.

[0161] In some embodiments, provided herein is an antibody that can be further modified to include known and available other non-protein moieties. Suitable parts for antibody derivatization include but are not limited to water-soluble polymers. The limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly (n vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polyoxypropylene / ethylene oxide copolymer, polyoxyethylene polyol (for example, glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have advantages in manufacturing due to its stability in water. Polymer can have any molecular weight and can be branched or non-branched. The number of polymers attached to the antibody can be different, and if two or more polymers are attached, polymer can be identical or different molecules.

[0162] The antibodies described herein can be encoded by nucleic acids. Nucleic acids are a type of polynucleotide comprising two or more nucleotide bases. In certain embodiments, nucleic acids are components of vectors that can be used to transfer a polynucleotide encoding a polypeptide 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 attached. One type of vector is a genomic integrating vector, or "integrating vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, such as 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 such as promoters, enhancers, and polyadenylation signals used to control transcription can be derived from genes of mammals, microorganisms, viruses, or insects. The ability to replicate in a host is typically conferred by an origin of replication, and selection genes that facilitate recognition of transformants may also be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses, can be used. Plasmid vectors can be linearized for integration into a chromosomal location. The vector may comprise a sequence that directs site-specific integration into a defined location or set of restriction sites in the genome (eg, AttP-AttB recombination). Additionally, the vector may comprise a sequence derived from a transposable element.

[0163] The nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform or otherwise make suitable cells transgenic for the nucleic acids, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell cultures are known in the art. In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells are cell lines that can be used to produce antibodies, and are Chinese hamster ovary (CHO) cells, NSO mouse myeloma cells or PER. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic site of a cell that can be used to produce the antibody. In certain embodiments, described herein is a method for preparing an antibody comprising culturing a cell containing a nucleic acid encoding the antibody under in vitro conditions sufficient to allow production and secretion of the antibody.

[0164] In certain embodiments, a master cell bank is described herein 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 certain embodiments, the cryoprotectant comprises glycerol. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antibody integrated at a genomic location, the 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 certain embodiments, the cryoprotectant comprises glycerol. In certain embodiments, the master cell bank is contained in a suitable vial or container capable of withstanding liquid nitrogen freezing.

[0165] Also described herein are methods for preparing antibodies described herein. Such methods include incubating cells or cell lines comprising nucleic acids encoding antibodies in a cell culture medium under conditions sufficient to allow expression and secretion of the antibody, and further gathering the antibody in the crops from the cell culture medium. The crops may also include one or more purification steps to remove living cells, cell debris, non-antibody proteins or polypeptides, undesirable salts, buffers, and culture 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.

[0166] Antibodies Described in This Article

[0167] In certain aspects, anti-human PVR (anti-hPVR) antibodies or antigen-binding fragments thereof are described herein. In certain embodiments, the antibodies or antigen-binding fragments thereof bind to human PVR at the PVR-TIGIT interface. In certain embodiments, the anti-hPVR antibodies or antigen-binding fragments thereof can compete with any one or more of TIGIT, CD96, and CD226.

[0168] In certain embodiments, the humanized antibody or antigen-binding fragment thereof binds to the EC of human PVR. 50 In certain embodiments, the EC of hPVR antibody 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 Between about 5 nM and 1 nM, between about 5 nM and about 2 nM, between about 4 nM and about 2 nM, between about 4 nM and about 3 nM, or between about 3 nM and about 2 nM.

[0169] Half-maximal effective concentration (EC 50 ) refers to the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time.

[0170] According to some embodiments, the antibody is a recombinant antibody. According to specific embodiments, the antibody is a recombinant humanized monoclonal antibody.

[0171] 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.

[0172] 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.

[0173] According to some embodiments, the humanized antibody or antigen-binding fragment thereof is NB1088 (SEQ ID NO: 1 and SEQ ID NO: 2).

[0174] In one aspect, the present invention describes a humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein 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, wherein the antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0175] In another aspect, described herein is a humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein 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, wherein the antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0176] In certain embodiments, described herein is a humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein 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, wherein the antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0177] In certain embodiments, described herein is a humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein 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, wherein the antibody or antigen-binding fragment binds to human poliovirus receptor (CD155). Each possibility represents a separate embodiment of the invention.

[0178] In certain embodiments, described herein is a humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 1, and wherein the light chain comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence set forth in SEQ ID NO: 2, wherein the antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0179] In certain embodiments, described herein is a humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1, and wherein the light chain comprises an amino acid sequence that is at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, wherein the antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0180] In certain embodiments, described herein is a humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises 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 wherein the light chain comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 2, wherein the antibody or antigen-binding fragment thereof binds to the human poliovirus receptor (CD155).

[0181] 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 the heavy chain sequence set forth in SEQ ID NO: 6 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: 6 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: 6 and the light chain sequence set forth in SEQ ID NO: 9.

[0182] 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 the light chain comprises 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)GSGSGTDFTLTISSLQ(S / P)EDFA(D / T)YFCQQYSRYPLTFGQGTKLEIK (SEQ ID NO:48).

[0183] According to some embodiments, the heavy chain comprises the amino acid sequence listed in SEQ ID NO:47, wherein position 11 is L, or position 20 is I, or position 37 is I, or position 38 is K, or position 48 is I, or position 68 is A, or position 77 is D, or position 79 is T, or position 82 is Q, or position 85 is S, or position 87 is T, or position 91 is S, or position 114 is T, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0184] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, or position 20 is I, or position 37 is I, or position 38 is K, or position 48 is I, or position 68 is A, or position 77 is D, or position 79 is T, or position 82 is E, or position 85 is R, or position 87 is R, or position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0185] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, or position 20 is V, or position 37 is V, or position 38 is R, or position 48 is M, or position 68 is V, or position 77 is S, or position 79 is A, or position 82 is E, or position 85 is R, or position 87 is R, or position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0186] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, or position 20 is V, or position 37 is I, or position 38 is K, or position 48 is I, or position 68 is V, or position 77 is S, or position 79 is T, or position 82 is E, or position 85 is R, or position 87 is R, or position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0187] According to some embodiments, the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, or position 20 is V, or position 37 is V, or position 38 is R, or position 48 is I, or position 68 is V, or position 77 is S, or position 79 is T, or position 82 is E, or position 85 is R, or position 87 is R, or position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0188] According to some embodiments, the light chain comprises the amino acid sequence listed in SEQ ID NO:48, wherein position 3 is M, or position 10 is F, or position 24 is K, or position 34 is V, or position 46 is L, or position 60 is D, or position 63 is T, or position 80 is S, or position 85 is D, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0189] According to some embodiments, the light chain comprises the amino acid sequence listed in SEQ ID NO:48, wherein position 3 is Q, or position 10 is S, or position 24 is K, or position 34 is V, or position 46 is L, or position 60 is D, or position 63 is S, or position 80 is P, or position 85 is D, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0190] According to some embodiments, the light chain comprises the amino acid sequence listed in SEQ ID NO:48, wherein position 3 is Q, or position 10 is S, or position 24 is R, or position 34 is V, or position 46 is L, or position 60 is S, or position 63 is S, or position 80 is P, or position 85 is T, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0191] According to some embodiments, the light chain comprises the amino acid sequence listed in SEQ ID NO:48, wherein position 3 is Q, or position 10 is S, or position 24 is R, or position 34 is A, or position 46 is L, or position 60 is S, or position 63 is S, or position 80 is P, or position 85 is T, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0192] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain comprising the amino acid sequence set forth in SEQ ID NO:47, wherein position 11 is V, or position 20 is V, or position 37 is V, or position 38 is R, or position 48 is I, or position 68 is V, or position 77 is S, or position 79 is T, or position 82 is E, or position 85 is R, or position 87 is R, or 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, or position 10 is S, or position 24 is K, or position 34 is V, or position 46 is L, or position 60 is D, or position 63 is S, or position 80 is P, or position 85 is D, or any combination thereof.

[0193] According to further embodiments, the heavy chain CDR1 sequence is GYTFSNYWIE (SEQ ID NO: 58).

[0194] 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.

[0195] According to some embodiments, the human constant region of the antibody is selected from the group consisting of: human IgG1 and human IgG4.

[0196] 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 having 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 having at least 90% identity.

[0197] According to some embodiments, the humanized antibody comprises the light chain sequence set forth in SEQ ID NO:49.

[0198] Treatment

[0199] In certain embodiments, disclosed herein are anti-hPVR antibodies that can be used to treat cancer or tumors. Treatment refers to a method that seeks to improve or alleviate the condition being treated. For cancer, treatment includes, but is not limited to, reducing tumor volume, reducing the growth of tumor volume, increasing progression-free survival, or overall life expectancy. In certain embodiments, treatment affects remission of the cancer being treated. In certain embodiments, treatment includes the use of a prophylactic or maintenance dose intended to prevent the recurrence or progression of a previously treated cancer or tumor. It is understood by those skilled in the art that not all individuals will respond equally or completely to an administered treatment, yet such individuals are considered to be treated.

[0200] In certain embodiments, the anti-hPVR antibodies and antigen-binding fragments described herein are used in the manufacture of a medicament for treating a PVR-positive cancer or a medicament for use in a method for treating a PVR-positive cancer.

[0201] In certain embodiments, the anti-hPVR antibodies or antigen-binding fragments described herein are used to treat cancers or tumors that are refractory to treatment with checkpoint inhibitors as a monotherapy. Refractory cancer refers to a cancer / tumor that develops progressive disease despite treatment with a single checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a PD-L2 inhibitor. In certain embodiments, the PD-1 inhibitor, PD-L1 inhibitor, 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 certain embodiments, the PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor is a PD-L2 Fc fusion protein (e.g., AMP-224). In certain embodiments, the PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor comprises an antibody or PD-L1 binding fragment that specifically binds to PD-L1 (CD274). In certain embodiments, the antibody or antigen-binding fragment that specifically binds to PD-L1 (CD274) comprises durvalumab (MEDI 4376), atezolizumab, avelumab, BMS-936559, or FAZ053, or a PD-L1 (CD274) binding fragment thereof. In certain embodiments, the PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor comprises an antibody or PD-L2 binding fragment thereof that specifically binds to PD-L2 (CD273).In certain embodiments, the PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor comprises one or more small molecule inhibitors such as 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-glutamic acid N-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-piperidinylcarboxylic acid; glycinamide, N-(2-mercaptoacetyl)-L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteine-, cyclic (1→14)-thioether; or derivatives or analogs thereof.

[0202] In certain embodiments, an anti-hPVR antibody or antigen-binding fragment thereof is used in combination with a PD-1 inhibitor, a PD-L1 inhibitor, or a PD-L2 inhibitor. In certain embodiments, the PD-1 inhibitor, the PD-L1 inhibitor, or the 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 their PD-1 (CD279) binding fragments. In certain embodiments, the PD-1 inhibitor, the PD-L1 inhibitor, or the PD-L2 inhibitor is a PD-L2 Fc fusion protein (e.g., AMP-224). In certain embodiments, the PD-1 inhibitor, the PD-L1 inhibitor, or the PD-L2 inhibitor includes an antibody or PD-L-1 binding fragment that specifically binds to PD-L-1 (CD274). In certain embodiments, antibodies or antigen-binding fragments that specifically bind to PD-L-1 (CD274) include durvalumab (MEDI 4376), atezolizumab, avelumab, BMS-936559, or FAZ053, or PD-L-1 (CD274) binding fragments thereof. In certain embodiments, PD-1 inhibitors, PD-L1 inhibitors, or PD-L2 inhibitors include antibodies or PD-L2 binding fragments thereof that specifically bind to PD-L2 (CD273).In certain embodiments, the PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor comprises one or more small molecule inhibitors such as 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-glutamic acid N-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-piperidinylcarboxylic acid; glycinamide, N-(2-mercaptoacetyl)-L-phenylalanyl-N-methyl-L-alanyl-L-aspartyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophanyl-L-seryl-L-tryptophanyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteine-, cyclic (1→14)-thioether; or derivatives or analogs thereof.

[0203] In certain embodiments, an anti-hPVR antibody or antigen-binding fragment thereof is used in combination with an EGFR inhibitor or an EGFR-binding antibody.

[0204] In certain embodiments, anti-hPVR antibodies or antigen-binding fragments thereof are used to treat cancer or tumors. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor is a blood cancer or tumor. In certain embodiments, the cancer or tumor includes tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles, and / or liver. In certain embodiments, tumors treatable with the antibodies of the invention include adenomas, adenocarcinomas, angiosarcomas, astrocytomas, epithelial carcinomas, germ cell tumors, glioblastomas, gliomas, hemangioendotheliomas, angiosarcomas, hematomas, hepatoblastomas, leukemias, lymphomas, medulloblastomas, melanomas, neuroblastomas, osteosarcomas, retinoblastomas, rhabdomyosarcomas, sarcomas, and / or teratomas. 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, astrocytic tumor, Bartholin gland carcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, bile duct carcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, focal nodular hyperplasia, gastrinoma, germ cell tumor, glioblastoma, glucagonoma, angioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatocellular carcinoma, insulinite, intraepithelial neoplasia, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung cancer, lymphoblastic leukemia, Blood cancer, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, neurilemoma, medulloblastoma, medullary epithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid 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 cancer, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar cancer, vasodilatory peptide tumor, and Wilms' tumor.In certain embodiments, tumors / cancers to be treated with one or more antibodies of the present invention include brain cancer, 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 mammary ductal carcinoma and / or mammary epithelial carcinoma. In certain embodiments, cancers treated with 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 certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes colon cancer. In certain embodiments, the cancer treated includes glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer is refractory to other treatments. In specific embodiments, the cancer treated is recurrent. In specific embodiments, the cancer is recurrent / refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.

[0205] It will be apparent to one of ordinary skill in the art that a therapeutically effective amount of a molecule according to the invention will depend, inter alia, on the schedule of administration, the unit dose of the molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the administered molecule, its persistence in the blood circulation, and the judgment of the treating physician.

[0206] In certain embodiments, the antibody can be administered to a subject in need thereof by any route suitable for administering a pharmaceutical composition containing the antibody, such as, for example, subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered according to an appropriate dosage schedule, for example, once a week, twice a week, once a month, twice a month, once every two weeks, once every three weeks, or once a month. In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. A therapeutically effective amount includes an amount sufficient to alleviate one or more symptoms associated with the disease or disorder to be treated.

[0207] The antibodies of the present invention can be used in CAR-based adoptive immunotherapy (adoptive immunotherapies), which utilizes engineered lymphocytes containing CAR to treat cancer. The CAR-T system is described herein as a non-limiting example.

[0208] T cell therapy utilizes chimeric antigen receptors (CARs) to treat cancer or tumors (i.e., CAR-T cell therapy). CAR-T cell therapy is a cellular immunotherapy that includes administering genetically engineered T cells that act on tumor cells and cause apoptosis of tumor cells to cancer patients. Genetically engineered T cells are prepared by expressing CARs on T cells using gene transfer technology, wherein the CARs have antibody variable regions (VL and VH) combined with intracellular domains (fragments such as CD3ζ chain sequences). CAR is a general term for a chimeric protein in which the light and heavy chains of the variable regions of monoclonal antibodies specific for tumor antigens are interconnected and then connected to the T cell receptor (TCR) chain on the C-terminal side.

[0209] According to some embodiments, the CAR comprises at least one protein domain selected from the group consisting of a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3 zeta domain. According to some embodiments, the CAR comprises a CD8 Stalk domain. According to some embodiments, the CAR comprises a CD28 TM domain. According to some embodiments, the CAR comprises a CD3 zeta signaling domain. According to some embodiments, the CAR comprises a 41BB domain. According to specific embodiments, the CAR comprises a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3 zeta domain.

[0210] According to some embodiments, there is provided a chimeric antigen receptor (CAR) comprising a heavy chain variable region (VH) and a light chain variable region (VL) according to the present invention. According to certain embodiments, there is provided a genetically modified lymphocyte expressing CAR on its surface. According to some specific embodiments, there is provided a genetically modified T cell (CAR-T cell) expressing CAR on its surface.

[0211] According to some embodiments, the CAR comprises a combination of a heavy chain variable region comprising an amino acid sequence having 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 comprising an amino acid sequence having 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.

[0212] According to some embodiments, the CAR comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region 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.

[0213] According to some embodiments, the CAR comprises a combination of a humanized antibody heavy chain variable region and a light chain variable region, wherein the combination is selected from the group consisting of:

[0214] i. the heavy chain variable region sequence listed in SEQ ID NO: 1 and the light chain variable region sequence listed in SEQ ID NO: 9;

[0215] ii. the heavy chain variable region sequence set forth in SEQ ID NO: 3 and the light chain variable region sequence set forth in SEQ ID NO: 9;

[0216] iii. the heavy chain variable region sequence set forth in SEQ ID NO: 4 and the light chain variable region sequence set forth in SEQ ID NO: 9;

[0217] iv. the heavy chain variable region sequence set forth in SEQ ID NO: 5 and the light chain variable region sequence set forth in SEQ ID NO: 9; and

[0218] 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.

[0219] According to some embodiments, the CAR comprises a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 5, and 6 and a light chain variable region sequence set forth in SEQ ID NO: 9, a transmembrane domain, and an intracellular T cell signaling domain.

[0220] According to some embodiments, CAR comprises an scFv sequence listed 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 any of the sequences provided. According to specific aspects, the present invention provides cells comprising CAR described herein. According to some embodiments, the cell expresses or is capable of expressing the CAR of the present invention. According to some embodiments, the cell is a lymphocyte. According to some embodiments, the cell is selected from T cells and natural killer (NK) cells.

[0221] According to some embodiments, a lymphocyte engineered to express a CAR described herein is provided. According to some embodiments, a T cell engineered to express a CAR described herein is provided.

[0222] According to additional embodiments, NK cells engineered to express the CAR described herein are provided.

[0223] The present invention also discloses methods for diagnosing and prognosing cancer.

[0224] According to one aspect, the present invention provides a method for diagnosing and / or prognosing cancer or infectious disease in a subject, the method comprising the step of determining the expression level of PVR in a biological sample of the subject using at least one antibody described herein.

[0225] Pharmaceutically acceptable excipients, carriers and diluents

[0226] In certain embodiments, the anti-PVR antibodies of the present disclosure are contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. The carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in an amount suitable to achieve the desired exposure.

[0227] In certain embodiments, the antibody of the present disclosure is suspended in a sterile solution and used. In certain embodiments, the solution comprises approximately 0.9% NaCl. In certain embodiments, the solution comprises approximately 5.0% dextrose. In certain embodiments, the solution also comprises one or more of the following: 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; polyols / disaccharides / polysaccharides, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, such as glycine or arginine; antioxidants, such as ascorbic acid, methionine; or chelating agents, such as EDTA or EGTA.

[0228] Typically, the antibody of the present invention and its antigen-binding portion thereof comprising antibody or comprising another polypeptide fragment and conjugate including peptide mimetics are suspended in sterile saline solution for therapeutic use. Pharmaceutical composition can be optionally formulated to control the release of active ingredient (molecule comprising the antigen-binding portion of antibody) or prolong its presence in the patient's system. A large number of suitable drug delivery systems are known, and include, for example, implantable drug release systems, hydrogels, hydroxymethyl cellulose, microcapsules, liposomes, microemulsions, microspheres, etc. Controlled release products can be prepared by using polymer composites or adsorption molecules according to the present invention. For example, biocompatible polymers include a matrix of poly(ethylene-co-vinyl acetate), and a matrix of a polyanhydride copolymer of stearic acid dimer and sebacic acid. The rate at which the molecule according to the present invention, i.e., the antibody or antibody fragment, is released from such a matrix depends on the molecular weight of the molecule, the amount of the molecule in the matrix, and the size of the dispersed particles.

[0229] In certain embodiments, the antibodies of the present disclosure are freeze-dried for transport / storage and reconstructed before administration. In certain embodiments, freeze-dried antibody formulations include swelling agents such as mannitol, sorbitol, sucrose, trehalose, dextran 40 or a combination thereof. Freeze-dried formulations may be contained in a vial made of glass or other suitable non-reactive materials. Antibodies, when formulated, whether reconstructed, may be buffered at a certain pH (generally less than 7.0). In certain embodiments, the pH may be between 4.5 and 6.5, between 4.5 and 6.0, between 4.5 and 5.5, between 4.5 and 5.0 or between 5.0 and 6.0.

[0230] In certain embodiments, lymphocytes with CAR described herein are transported / stored before use. When not used immediately, cells are typically cryopreserved. Cryopreservation methods and storage media suitable for cells with CAR are known in the art, see, for example, Wang, et al., 2019 May; 21(5): 566-578.

[0231] Also described herein are kits comprising one or more antibodies described herein and one or more additional components selected from the group consisting of: instructions for use; a diluent, an excipient, a carrier, and an administration device in a suitable container. In some embodiments, the kit comprises a means for measuring PVR expression in a human.

[0232] In certain embodiments, described herein is a method of preparing a cancer treatment comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the disclosure. In certain embodiments, described herein is a method of preparing a cancer treatment for storage or transport comprising lyophilizing one or more antibodies of the disclosure. Example

[0233] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0234] Example 1 - N56E and N56D variants improve PVR binding affinity

[0235] The variable region of the chimeric anti-PVR antibody 5B9 disclosed in WO2017149538 carries a deamidated sequence (asparagine-glycine) in the light chain CDR2 (WASSRHNG, SEQ ID NO: 17). Seven chimeric variants were generated by introducing a point mutation at residue asparagine N56. To evaluate the binding affinity of the N56 substitution variants, wild-type (WT) and substitution variant IgG4 (S241P) monoclonal antibodies were immobilized on a protein A capture chip. Binding of the analyte PVR conjugated to a histidine tag (PVR-HIS, Sino Cat. no. 10109-H08H) was tested. Dilution range: 5-point two-fold dilutions from 50 nM to 3.125 nM. Conditions used: 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. Regeneration: 10 mM glycine pH 1.5. Analysis: 1:1 binding. Relative K for each substitution D By replacing K D Divided by the K of the parental N56 variant (VH0VK0) D It was noted that the affinity of the N56E (Asp) and N56D (Glu) variants was significantly improved (>25%) ( Figure 1A The binding of chimeric variants to human PVR expressed on HEK 293EBNA cells was determined by flow cytometry using the parental 5B9 antibody (WT, Figure 1B ) were evaluated in a competition assay.

[0236] Example 2 - N56E and N56D variants improve cross-reactivity for monkey PVR binding.

[0237] The binding of the N56 substitution variant antibodies to cell-bound human PVR (protein id: Q92692) and chlorocebus PVR (African green monkey, protein id: UniProtKB-P32506) was examined. Figure 2A Depicted are the relative binding of all variants added at saturating concentrations (10 ug / ml) to NCI-H1975 cells expressing human PVR. Figure 2BThe relative binding of all variants added at saturating concentrations (10 ug / ml) to Vero cells expressing green monkey PVR is depicted. For detection, goat anti-human-647 antibody (Jackson ImunomeResearch 109-606-088) was used at a dilution of 1:250. Cell binding of the Abs was analyzed by FACS. The MFI of each variant was divided by the MFI of the parent antibody (KO) to calculate the fold change. A significant (>25%) increase in cross-reactivity was observed for the N56E and N56D variants.

[0238] Example 3 - N56E and N56T variants improve NK activation

[0239] NK cells from healthy donors were incubated at 37°C with an E:T ratio of 2:1 for 2 hours in the presence of selected N56 substitution variants and target breast cancer cell lines (MDA-MB-231). NK cell activation was measured by inducing CD107a surface expression, and the fold change (Y axis) of each variant over control IgG was calculated. All monoclonal antibodies were used at 600pM (0.09ug / ml). (*p<0.04, **p<0.01 by two-tailed student t test). Figure 3 As shown, the N56E and N56T variants displayed improved NK activation compared to KO, as demonstrated by increased expression of CD107a.

[0240] Example 4 - N56E and N56T variants improve CD8 T cell proliferation

[0241] Human PBMCs were fluorescently labeled with CFSE (C34554 ThermoFischer) and incubated with A549 target breast cancer cells and 4ug / ml of the indicated antibody variants in the presence of 2.5ul / ml PHA-L (Roche). Immune cells were collected after 96 hours of incubation, stained with anti-human CD8, and analyzed by FACS. Cell proliferation of CD8+T cells was assessed by CFSE signal intensity. The CFSE level of IgG-treated cells was set to 1. The results are presented as the fold increase in proliferation relative to this control. Since the increase in proliferation leads to a decrease in CFSE signal, the Y-axis depicts the reciprocal value of this ratio. The experiments were performed in quadruplicate; the results for a single PBMC donor are shown. The data show that in the presence of tumor cells, the effect of variants N56E and N56T on CD8+T cell proliferation is significantly stronger than that of the parental antibody ( Figure 4 ; by two-tailed student t test, *p<0.05, **p<0.01).

[0242] Example 5 - Identification of humanized 5B9 variants with improved manufacturability

[0243] The N56E antibody variant performed best in competition assays and was selected as the lead variant for humanization. Based on structural analysis, a large set of preliminary sequence segments were identified for the creation of 5B9 humanized variants. iTope was used for in silico analysis of peptides binding to human MHC class II alleles. TM technique (Perry et al. 2008) and using TCED of T cell epitopes associated with known antibody sequences TM (Bryson et al. 2010), these segments were selected and analyzed. TM The sequence segments of significant hits (hit) are discarded. This obtains a group of segments reduced, and as described above, further analyzes these combinations to ensure that the connection between the segments does not contain potential T cell epitopes. The sequence segments selected are assembled into complete V region sequences without significant T cell epitopes. Then 5 heavy chains (VH1 to VH5) and 4 light chains (containing N56E substitutions) (Vκ1 to Vκ4) sequences are selected.

[0244] Table 1. Variable region and CDR sequences

[0245]

[0246] Table 2. Framework (non-CDR) sequences of humanized heavy chain variable regions.

[0247]

[0248] Table 3. Framework (non-CDR) sequences of humanized light chain variable regions.

[0249]

[0250] All variants were tested for binding by SPR ( Figure 5A ) and tested by flow cytometry for cell surface PVR binding of variants with 2x the affinity of the parent antibody ( Figure 5B ). All variants, except those containing Vk4, showed very similar binding to the parental murine / human chimeric molecule carrying the N56E substitution (IgG4(S241P)N56E_VH0 / Vκ0). Note that humanization removes the N-linked glycosylation at position N20 of the FR1 light chain.

[0251] To select lead candidates, expression levels after transient expression in HEK 293EBNA cells, as well as similarity to human germline sequences were considered (Figure 6). Figure 6AThe titers of all variants after transient transfection are summarized. Variant VH4 / Vk2 showed the highest expression titer and had a high percentage of sequence identity with the human germline gene ( Figure 6B Finally, the VH4 / Vk2 variant (NB1088) was evaluated for manufacturability and compared to a variant designated NB0941, which is identical to NB1088 but has the original LC CDR2 of 5B9 with deamidation capability (WASSRHNG). The biophysical properties of NB0941 and NB1088 were determined. Figure 7A and 7B As shown, high pH stress and incubation at 40°C revealed changes in capillary isoelectric focusing (cIEF), specifically an increase in the percentage of acidic species, likely due to deamidation. These changes were more pronounced in NB0941 compared to NB1088. Therefore, NB1088, with its optimized immunogenicity, expression and binding profiles, and desirable biophysical properties, was selected as the lead humanized variant for functional analysis.

[0252] Given the fact that normal tissues express PVR at minimal levels, the observed decrease in affinity in some variants is particularly advantageous when designing CAR drivers. Figure 16 ) showed that PVR is overexpressed in various tumors, allowing PVR-driven CAR-T to effectively target these tumors. Potential safety issues can be easily addressed by anti-PVR variants with "down-regulated" affinity, as described by Liu et al. (Cancer Research, 2015; Vol. 75, No. 17).

[0253] Example 6 - NB1088 inhibits the binding of PVR to TIGIT, CD96 and CD226

[0254] The ability of NB1088 to block the binding of TIGIT, CD96, and CD226 to PVR was tested. Isolated CHO (Chinese Hamster Ovary) cells stably expressing human PVR were incubated with the indicated concentrations of NB1088 on ice for 20 minutes, followed by the addition of biotinylated recombinant TIGIT, CD96, or CD226-Fc at 10 μg / ml and incubated on ice for another 120 minutes. 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 NB1088 showed an EC of approximately 3.3 nmol / L. 50 Binding to PVR. NB1088 competes with TIGIT, CD96, or CD226 for the IC of PVR binding. 50are 1.1nM, 1.1nM and 1.9nM respectively, such as Figures 8B to 8D shown.

[0255] Example 7 - NB1088 stimulates cytotoxic T cells and NK cells

[0256] The ability of NB1088 to stimulate T and NK cell activity in vitro was determined. Using an antigen-specific human papillomavirus (HPV) assay, 30,000 HPV+ human cervical epidermoid carcinoma cell lines (CaSki cells) and 30,000 HPV-specific CD8 T cells were incubated overnight with 10 μg / ml control IgG or NB1088. Interferon gamma release was measured in the supernatant using a human interferon gamma-specific MSD system. Figure 9A As shown, NB1088 increases interferon gamma release by HPV-specific human CD8+ (cytotoxic) T cells when incubated with HPV+ CaSki cells. To test CD8 T cell activity in an allogeneic system, PBMCs were pre-activated for three days with phytohemagglutinin (PHA) and interleukin 2 (IL2), rested overnight in the absence of PHA / IL2, and then CD8 T cells were isolated using a magnetic negative separation procedure. 10,000 A549 tumor cells and 100,000 healthy donor CD8+ T cells were co-cultured overnight in the presence of 100U / ml IL2 and 1ug / ml anti-CD28 antibody. As Figure 9B As shown, NB1088 increased interferon-γ release from CD8+ T cells to a greater extent than anti-PD-1 (pembrolizumab), and the combination of NB1088 with anti-PD-1 antibody further increased interferon-γ release.

[0257] The effect of NB1088 on antibody-dependent cellular cytotoxicity (ADCC) was also determined. NK cells from normal donors were isolated from PBMCs that had been rested overnight using a magnetic negative separation procedure. 10,000 PVR+ and EGFR+ A549 tumor cells and 50,000 NK cells were incubated with: control IgG; control IgG and the anti-EGFR antibody cetuximab (5 μg / ml); or cetuximab and NB1088. The activity of NK cells in mediating antibody-dependent cellular cytotoxicity or interferon gamma release was determined by analyzing the viability of adherent A549 cells after co-culture and removal of NK cells using cell titer luminescence, or by analyzing the supernatant by MSD as above. Figure 10A and Figure 10B As shown, NB1088, when incubated with cetuximab, enhanced NK cell-mediated killing of A549 cells and interferon-γ release.

[0258] Example 8 - NB1088 restores CD226 expression and activity on CD8 T and NK cells

[0259] The ability of NB1088 to affect CD226 function was determined. CD226 (DNAM-1) is a cell surface glycoprotein receptor expressed by NK and T cells that serves as a ligand for PVR and aids in tumor killing by CD8+ T and NK cells. Functionally, CD226 is antagonized by the inhibitory molecules TIGIT and CD96 expressed on T and NK cells. Therefore, the increase in CD226 function caused by NB1088 suggests that NB1088 enhances the activity of T cells and NK cells and has broad anti-tumor activity. The effect of NB1088 on CD226 expression and function was tested in antigen-specific and allogeneic co-culture systems as described above. Figure 11A and Figure 11B As shown, co-culture of CD8 T cells and NK cells with PVR+ target cells resulted in a significant reduction in the surface expression of CD226 on CD8 T cells and NK cells. Regardless of the co-culture system, NB1088 restored the cell surface expression of CD226 on CD8 T cells or NK cells ( Figure 11A and Figure 11B ), whereas anti-TIGIT did not restore. Using the antigen-specific and allogeneic co-culture systems described above with minor modifications, the functional consequences of increased CD226 expression on T and NK cells after NB1088 treatment were evaluated ( Figure 12A and Figure 12B Increased CD226 expression after NB1088 treatment was associated with significantly higher levels of interferon-γ release compared with control IgG or anti-TIGIT treatment ( Figure 12A and Figure 12B The superior T cell and NK cell activity of NB1088 treatment is at least partially mediated by CD226 activity. Anti-CD226 (DX11, 20ug / ml) inhibits both allogeneic and antigen-stimulated CD8 T cells ( Figure 12A ) and NK cells ( Figure 12B ) NB1088-dependent interferon-γ release after A549 co-culture was reduced to the levels observed with anti-TIGIT. These data demonstrate that NB1088 improves T and NK cell activity by increasing CD226 expression and / or function beyond TIGIT blockade.

[0260] Example 9 - NB1088 Monotherapy Efficacy in a Humanized Mouse Tumor Xenograft Model

[0261] The ability of NB1088 to kill tumors was determined in the humanized mouse models A549 (lung adenocarcinoma) or HPAF (pancreatic cancer). 6Tumor cells (A549 or HPAF) were mixed with activated human peripheral blood mononuclear cells at a 1:1 ratio in matrigel and implanted subcutaneously into the flank of immunodeficient NOD / SCID mice (12 animals per condition). Figure 13A and 13B As shown in Figure 2, NB1088 can reduce tumor volume at least as much as the anti-PD-1 antibody pembrolizumab. NB1088 can also reduce tumor volume in the A549 / PBMC model ( Figure 13C ), but could not reduce tumor volume in A549 cells alone ( Figure 13D In the A549 / PBMC model, reductions in tumor volume were associated with increased CD226 expression on CD8 T cells isolated from NB1088-treated tumors. Figure 13E The effects of NB1088 on CD8 T cell effector function were also assessed ex vivo ( Figure 14A and Figure 14B ). Digested tumor single cell suspensions were stimulated with anti-CD28 / anti-CD3 in the presence of brefeldin A and anti-CD107a at 37°C for 5 hours. After stimulation, cells were stained by standard surface / intracellular staining methods to detect interferon gamma production by CD8+ T cells using flow cytometry. Figure 14A and Figure 14B As shown, NB1088 increased the total interferon gamma positive ( Figure 14A ) and multifunctional interferon γ / CD107a double positive ( Figure 14B ) tumor-derived CD8 T cells. In addition, the increased frequency of interferon-γ-positive CD8+ T cells in NB1088-treated tumors was entirely derived from CD226-positive CD8+ T cells ( Figure 14C and Figure 14D ), indicating an important in vivo contribution of CD226 function to the antitumor activity of NB1088.

[0262] Example 10 - NB1088 Pharmacokinetics in Cynomolgus Monkeys and Pharmacodynamic Changes in CD226 Expression on CD4 T Cells

[0263] The pharmacokinetic properties of NB1088 were measured in cynomolgus monkeys (2 female monkeys / dose group) following single or 4×1 weekly IV bolus injections at 2 mg / kg, 50 mg / kg, or 200 mg / kg dose levels. Changes in CD226 expression on circulating peripheral CD4 T cells were also evaluated. Figure 15AThe plasma concentration (μg / ml) of NB1088 is shown as a function of time (hours) and dose. IC90 and 10x IC90 were calculated based on an in vitro efficacy assay using cynomolgus monkey PBMCs. Following repeated dosing at the 200 mg / kg dose level, NB1088 exhibited a typical PK profile and reached concentrations greater than 10x IC90 for the duration of the study. Figure 15B CD226 expression levels on circulating CD4 T cells, normalized to pre-dose levels, are shown, as measured by flow cytometry using a specific antibody. NB1088 increased CD226 surface expression levels by 1.5-fold in the 50 mg / kg and 200 mg / kg repeat-dose groups, and this elevation remained in the 200 mg / kg repeat-dose group. These data suggest that NB1088 can engage and regulate CD226 expression on CD4 T cells in cynomolgus monkeys.

[0264] Example 11 - Expression of human PVR across different tumor types

[0265] The expression levels of PVR were evaluated in human cancers of various origins. PVR expression was detected by standard immunohistochemistry using the commercially available rabbit monoclonal antibody clone D3G7H and cancer tissue microarrays. The staining was digitized and the intensity was quantified to calculate H-scores within and across indications. Figure 16 PVR expression levels were shown to be elevated at varying frequencies in most of the indications analyzed. PVR was shown to be elevated in the following: 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.

[0266] Example 12 - Design of humanized antibodies

[0267] Humanized IgG antibodies were designed based on a variant having a heavy chain VH4 and a light chain VK2. An exemplary VK2 sequence is listed in SEQ ID NO:49. An exemplary VH4 sequence of hIgG4(S241P) is listed in SEQ ID NO:50, and an exemplary VH4 sequence of hIgG1 is listed in SEQ ID NO:51. In addition, exemplary nucleotide sequences optimized for amino acid sequence expression in CHO cells are designed as follows: for VK2, the nucleotide sequence is listed in SEQ ID NO:52 or SEQ ID NO:53. For VH4 of IgG4, the nucleotide sequence is listed in SEQ ID NO:54 or SEQ ID NO:55.

[0268] Example 12 - CAR-T cells expressing scFv derived from humanized anti-PVR antibody variants are specifically activated in the presence of tumor cells

[0269] CAR-T constructs were designed based on variants H4K2-NTX-1088C and H3K4-NTX-1034C. The amino acid sequences of the scFv molecules are listed in SEQ ID No: 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 an E:T ratio of 1:1 for 24 h. Figure 18 As shown, in the presence of the indicated targets, both CAR-T drivers resulted in the secretion of hundreds of pg of IL2, while no IL2 secretion was detected by the parental Jurkat cells. IL2 secretion was quantified using Biolegend hIL2 (cat 431804). These results indicate that αPVR-based CAR-T drivers are highly functional in inducing T cell activation in the presence of PVR-expressing target cells.

[0270] To examine the tumor cell killing of CAR-T, 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 of 0.4 and 0.8 to 1 (based on GFP positivity) for 72 hours in NK culture medium. Tumor cell killing was assessed using a standard CTG protocol (Promega G9241). Figures 19A-19C As shown, both PVR variants exhibited a greater than 2-fold increase in killing of MDA-231 cells and a greater than 8-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.

[0271] Example 13-αPVR CAR-T effectively kills hematological target cells.

[0272] CAR-T constructs were designed based on variants H4K2-NTX-1088C and H3K4-NTX-1034C. The scFv sequences are listed in SEQ ID Nos: 56 and 57, respectively.

[0273] To examine CAR-T's killing of hematologic tumor cells, 20K / well K562 cells were plated into 96-well plates alone or with CAR-T-PVR variants (NTX-1088C or NTX1034C) at E:Ts ranging from 3.4 to 0.22 to 1 for 18 hours in RPMI supplemented with 100 IU / IL-2 / ml. Tumor cell killing was assessed by flow cytometry. Both NTX-1034C and NTX-1088C were extremely effective at eliminating targets at higher E:Ts. The apparent superiority of NTX-1088C over NTX-1034C at low E:Ts may be due to the moderate expression of PVR on K562. These results suggest that αPVR CAR-T can effectively target hematologic tumors expressing PVR.

[0274] The present invention also provides the following items:

[0275] 1. A humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises an amino acid sequence that is 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 wherein the light chain variable region comprises an amino acid sequence that is 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, wherein the antibody or antigen-binding fragment thereof binds to a human poliovirus receptor (CD155).

[0276] 2. The humanized antibody or antigen-binding fragment thereof according to item 1, wherein the heavy chain variable region comprises the amino acid sequence listed in SEQ ID NO: 47.

[0277] 3. The humanized antibody or antigen-binding fragment thereof according to item 1, wherein the light chain variable region comprises the amino acid sequence listed in SEQ ID NO: 48.

[0278] 4. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the heavy chain variable region comprises the amino acid sequence listed in SEQ ID NO: 47; and the light chain variable region comprises the amino acid sequence listed in SEQ ID NO: 48.

[0279] 5. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the heavy chain variable region comprises:

[0280] i. a set of three CDR sequences comprising the sequences listed in SEQ ID No.10-12; and

[0281] ii. A set of four heavy chain framework (FR) sequences: (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.

[0282] 6. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the light chain variable region comprises:

[0283] i. a set of three CDR sequences comprising the sequences listed in SEQ ID No.13-15; and

[0284] 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 is SEQ ID NO: 33.

[0285] 7. The humanized antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is 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 wherein the light chain variable region comprises an amino acid sequence that is 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.

[0286] 8. The humanized antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is 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 wherein the light chain variable region comprises an amino acid sequence that is 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.

[0287] 9. The humanized antibody or antigen-binding fragment thereof according to item 1, 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 wherein 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.

[0288] 10. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 and 9, wherein the heavy chain variable region comprises the amino acid sequence listed in SEQ ID NO: 1 and the light chain variable region comprises the amino acid sequence listed in SEQ ID NO: 2.

[0289] 11. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 10, wherein the humanized antibody or antigen-binding fragment thereof is an IgG antibody.

[0290] 12. The humanized antibody or antigen-binding fragment thereof according to item 11, wherein the humanized antibody or antigen-binding fragment thereof comprises an IgG4 heavy chain constant region or an IgG1 heavy chain constant region.

[0291] 13. The humanized antibody or antigen-binding fragment thereof according to item 12, wherein the humanized antibody or antigen-binding fragment thereof comprises an IgG4 heavy chain constant region having an alteration in which a serine residue replaces a proline at position 228 of the IgG4 heavy chain constant region.

[0292] 14. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 10, wherein the humanized antibody or antigen-binding fragment thereof is Fab, F(ab)2, a single domain antibody or a single chain variable fragment (scFv).

[0293] 15. A single-chain variable fragment (scFv), which is a single-chain variable fragment (scFv) of a humanized antibody according to any one of items 1 to 14, comprising an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, and an analog thereof having at least 90% sequence similarity to any of said sequences.

[0294] 16. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 15, wherein the humanized antibody inhibits PVR binding to at least one of TIGIT, CD96 and CD226.

[0295] 17. A nucleic acid encoding the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16.

[0296] 18. A cell line comprising the nucleic acid according to item 17.

[0297] 19. The cell line according to item 18, wherein the cell line is a Chinese hamster ovary cell line.

[0298] 20. A chimeric antigen receptor (CAR), comprising a combination of a heavy chain variable region sequence and a light chain variable region sequence of the humanized antibody according to any one of items 1 to 16.

[0299] 21. The CAR of claim 20, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity with 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 having at least 90% sequence identity with 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.

[0300] 22. The CAR according to item 21, wherein the CAR comprises an scFv comprising an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57, and analogs thereof having at least 90% sequence similarity to any one of the sequences.

[0301] 23. The CAR according to any one of items 20 to 22, comprising at least one domain selected from the group consisting of: a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

[0302] 24. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16 or the CAR according to any one of items 20 and 23, and a pharmaceutically acceptable excipient, carrier or diluent.

[0303] 25. The pharmaceutical composition according to item 24, formulated for intravenous administration.

[0304] 26. The pharmaceutical composition according to item 24, formulated for intratumoral administration.

[0305] 27. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16, the CAR according to item 20 or 23, or the pharmaceutical composition according to any one of items 24 to 26, for use in increasing the surface expression and / or signaling of CD226 on CD8+ T cells.

[0306] 28. The humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16, the CAR according to item 20 or 23, or the pharmaceutical composition according to any one of items 24 to 26, for use in treating cancer in an individual.

[0307] 29. The humanized antibody or antigen-binding fragment thereof or CAR or pharmaceutical composition for use according to item 28, wherein the cancer comprises a solid tumor.

[0308] 30. The humanized antibody or antigen-binding fragment thereof or CAR or pharmaceutical composition for use according to claim 28, wherein the cancer is selected from the group consisting of lung cancer, colon cancer, adrenal cancer, uterine cancer, head and neck cancer, pancreatic cancer and breast cancer.

[0309] 31. A method for increasing the surface expression and / or signaling of CD226 in CD8+ T cells of an individual, the method comprising administering to the individual a therapeutically effective amount of a humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16, a CAR according to item 20 or 23, or a pharmaceutical composition according to any one of items 24 to 26.

[0310] 32. The method according to claim 31, wherein the CD8+ T cells are tumor-infiltrating CD8+ T cells.

[0311] 33. A method of treating cancer in an individual having cancer, the method comprising administering to the individual a therapeutically effective amount of the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16, the CAR according to item 20 or 23, or the pharmaceutical composition according to any one of items 24 to 26.

[0312] 34. A method for treating cancer in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16, a CAR according to item 20 or 23, or a pharmaceutical composition according to any one of items 24 to 26, and a PD-1 signaling inhibitor, a PD-L1 signaling inhibitor, a CTLA-4 signaling inhibitor, or a CD112R signaling inhibitor.

[0313] 35. The method of any one of items 33 and 34, wherein the cancer comprises a solid tumor.

[0314] 36. The method of item 33, wherein 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.

[0315] 37. The method according to any one of items 33 and 34, wherein the cancer is a hematological cancer.

[0316] 38. The method according to any one of items 34 to 37, wherein the PD-1 signaling inhibitor is an antibody or a fragment thereof that binds to PD-1.

[0317] 39. The method according to item 38, 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.

[0318] 40. The method according to any one of items 34 to 37, wherein the PD-1 signaling inhibitor is an antibody that specifically binds to PD-L1 or PD-L2.

[0319] 41. The method according to item 40, wherein the antibody 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.

[0320] 42. The method of any one of items 34 to 37, wherein the PD-1 signaling inhibitor comprises a small molecule inhibitor of PD-1, PD-L1 or PD-L2.

[0321] 43. A method for preparing a composition for treating cancer in an individual in need thereof, the method comprising mixing the humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16 and a pharmaceutically acceptable excipient, carrier or diluent.

[0322] 44. The method of claim 43, wherein the cancer comprises a solid tumor.

[0323] 45. The method of item 44, wherein 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.

[0324] 46. The method of claim 43, wherein the cancer is a hematological cancer.

[0325] 47. A method for producing a humanized antibody or antigen-binding fragment thereof according to any one of items 1 to 16, the method comprising incubating a cell line containing a nucleic acid encoding the humanized antibody or antigen-binding fragment thereof in a cell culture medium under conditions sufficient to allow expression and secretion of the humanized antibody or antigen-binding fragment thereof.

[0326] 48. A method of 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 or fragment thereof according to any one of items 1-16.

[0327] 49. The method according to item 48, comprising the step of diagnosing or prognosing cancer in the subject if the expression of PVR is higher than a control or reference value.

[0328] 50. A kit comprising at least one antibody or antibody fragment according to any one of items 1 to 16, and means for measuring PVR expression.

Claims

1. A humanized antibody or antigen-binding fragment thereof against PVR, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 47; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

48.

2. The humanized antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region and the light chain variable region comprise: i. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; or ii. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; or iii. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; or iv. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; or v. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8; or vi. a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 2; or vii. A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 6, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

8. 3 . The humanized antibody or antigen-binding fragment thereof according to claim 1 , wherein the humanized antibody or antigen-binding fragment thereof comprises an IgG4 heavy chain constant region or an IgG1 heavy chain constant region.

4. The humanized antibody or antigen-binding fragment thereof according to any one of claims 1 or 2, wherein the humanized antibody or antigen-binding fragment thereof is Fab, F(ab)2, a single domain antibody or a single chain variable fragment (scFv). 5 . A nucleic acid encoding at least one chain or at least one region of the humanized antibody or antigen-binding fragment thereof according to claim 1 , or a cell line comprising the nucleic acid.

6. A chimeric antigen receptor (CAR), comprising a combination of a heavy chain variable region sequence and a light chain variable region sequence of the humanized antibody according to any one of claims 1 to 4.

7. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the CAR according to claim 6, and a pharmaceutically acceptable excipient, carrier or diluent.

8. The humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the CAR according to claim 6, or the pharmaceutical composition according to claim 7, for use in treating cancer in an individual, or for use in increasing the surface expression and / or signaling of CD226 on CD8+ T cells.

9. A method for producing a humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the method comprising incubating a cell line containing a nucleic acid encoding the humanized antibody or antigen-binding fragment thereof in a cell culture medium under conditions sufficient to allow expression and secretion of the humanized antibody or antigen-binding fragment thereof.

10. A method of 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 or fragment thereof according to any one of claims 1 to 4.

11. A humanized antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises an amino acid sequence that is 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 wherein the light chain variable region comprises an amino acid sequence that is 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, wherein the antibody or antigen-binding fragment thereof binds to human poliovirus receptor (CD155).

12. 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 or fragment thereof according to claim 11. 13 . A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment thereof according to claim 11 .

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

15. A chimeric antigen receptor (CAR) comprising the heavy and light chains of a humanized antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises an amino acid sequence that is 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 wherein the light chain variable region comprises an amino acid sequence that is 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, wherein the antibody or antigen-binding fragment thereof binds to a human poliovirus receptor (CD155).

16. The CAR according to claim 15, comprising a CD8 Stalk domain, a CD28 TM domain, a 41BB domain and a CD3Z domain.

17. The CAR according to claim 15, for use in treating cancer.

Images