EA045980B1 - ANTIBODIES AGAINST THE POLIO VIRUS RECEPTOR (PVR) AND THEIR APPLICATION - Google Patents

Description

Field of technology to which the present invention relates

The present invention relates to the field of immunotherapy and relates to humanized antibodies that specifically bind to the human poliovirus receptor (PVR) containing specific sets of CDRs and framework sequences. The invention also relates to pharmaceutical compositions containing said humanized antibodies and their uses.

BACKGROUND OF THE INVENTION

The poliovirus receptor (PVR), also called CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. It has previously been described as a tumor antigen and as a potential therapeutic target because its expression is increased in neuroectodermal cancers, including glioblastoma multiforme, medulloblastoma, and colorectal carcinoma (Solecki et al., J. Biol. Chem. 2002, 277: 25697-700), as well as in pancreatic cancer (Nishiwada et al., Anticancer Res. 2015, 35(4): 2287-97). PVR is also known to enhance serum-induced activation of the Ras-Raf-MEK-ERK signaling pathway by activating cyclins D2 and E and suppressing p27Kipl, which ultimately shortens the duration of the G0/G1 phase of the cell cycle. (Kakunaga 2004, J. Biological Chemistry, 279, 36419-36425. For this reason, blocking PVR on tumor cells is expected to reduce their viability. PVR also plays a critical role in angiogenesis and is thought to regulate VEGF-induced angiogenesis by controlling receptor interaction vascular endothelial growth factor 2 (VEGFR2) with a(v)P(3) integrin and the VEGFR2-mediated Rap1-Akt signaling pathway (Kinugasa et al., 2012, Circ Res. 2012, 110(5),716-26). In addition, PVR forms a complex with IGF1R and is involved in protein tyrosine kinase Met (cMet) signaling, and blocking complex formation reduced cell viability and angiogenesis (Lee et al., Scientific Reports 2014, 20, 4, 7139).

In recent years, it has become apparent that PVR is an important immune checkpoint ligand (Brilc PK et al. 2019 Cell Mol Immunology). PVR expression is increased in both malignant cells and tumor-infiltrating myeloid cells in humans and mice. PVR−/mice exhibit reduced tumor growth and metastasis through activation of DNAM-1 (CD226) and increased effector function of CD8+ T and NK cells, respectively. Blockade of programmed cell death protein-1 (PD-1) or both PD-1 and cytotoxic T-lymphocyte antigen 4 (CTLA4) was more effective in settings where the number of PVRs was limited, suggesting the clinical potential of combination therapy using PD-1. 1/PD-L1 and PVR blockade (LI XY et.al JCI2018). Moreover, in clinical settings, the expression of PD-L1 and PVR is independently regulated, which allowed patients treated with anti-PD-1 antibody therapy to be divided into 4 groups depending on the expression levels of PD-L1 and PVR. High PVR expression in patients with low PD-L1 expression improved treatment non-responders. This was further confirmed using a genetically engineered cancer model. These results support the importance of PVR as an important immune checkpoint in tumor immunotherapy (Lee BR et al. JCI. Insight, 2020). The involvement of PVR in metastasis was demonstrated by injecting cancer cells into the tail of mice and measuring lung metastases. It has been shown that activated PVR in cancer cells cross-talks with its counter-receptor in platelets, and that such cross-talk enhances cancer cell metastasis to the lung (Morimoto et al., Oncogene (2008) 27, 264-273).

Publication WO 2017149538 of one of the authors of the present invention describes murine antibodies and fragments thereof that bind to PVR, as well as the polynucleotide sequences encoding them and the hybridoma cells that produce these antibodies.

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

US Patent Application Publication No. 20090215175 proposes molecules (eg, small molecule chemicals, oligonucleotides, polypeptides, antibodies, and antibody fragments) that modulate PVR functions required for cell adhesion, migration, invasion, and/or metastatic potential. These molecules can be used for treatment by targeting cells with metastatic potential, metastasis and cancer.

There is an unmet need to develop humanized antibodies that recognize human PVR, which are safer and more effective and can be used for diagnostic and therapeutic purposes in diseases associated with PVR expression.

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Summary of the present invention

Disclosed herein, in accordance with some embodiments of the invention, are humanized antibodies that specifically bind to the human poliovirus receptor (PVR; CD155) and prevent PVR from binding to at least one of the ligands, T-cell immunoreceptor with Ig and ITIM domains (TIGIT) , CD96 and CD226 (DNAM-1). The humanized antibodies of the present invention, selected from a large collection of antibody clones, have improved properties compared to other known anti-PVR antibodies. These improved properties include, but are not limited to: reduced immunogenicity potential, improved binding affinity, activity and biophysical properties, and improved expression. Since binding of PVR to CD226 results in suppression of CD226 expression on the surface of T and NK cells and a decrease in the activity of CD226 in stimulating T and NK cells and killing tumor cells, the antibodies of the present invention can restore the expression and/or activity of CD226 on these cells. Proper expression and function of CD226 ensures more effective tumor killing by immune cells, in particular CD8+ T cells and NK cells.

By combining certain sets of human CDR region sequences and human framework sequences and introducing certain mutations into these sequences to produce improved antibodies with modified variable regions, a large collection of humanized antibodies has been obtained. The newly designed humanized variable regions retain residues critical to maintaining antibody conformation and binding affinity, while at the same time having the lowest frequency of potential T cell epitopes, thereby minimizing the risk of an unwanted immune response to antibodies. The antibodies described here were designed taking into account factors such as homology, T cell epitopes, key residues and predicted structures.

Variants with a combination of specific human framework regions and a point mutation of glutamic acid to asparagine in the last residue of the light chain variable region CDR2 (position 56 according to Kabat numbering) unexpectedly demonstrate strong affinity for human PVR and improved immune activity.

Several variants of the humanized antibodies of the invention have been found to be particularly suitable for use in chimeric antigen receptors (CARs) due to their lower affinity, which may be useful for targeting tumor cells with high PVR expression without targeting normal tissues.

Several variants of the humanized antibodies of the present invention provide the advantage of improved processability and can be produced in exceptionally high yield compared to other variants.

The humanized antibody described herein has now been disclosed to be highly effective in stimulating cytotoxic T and NK cells and in treating cancer in humanized mouse models, including in vivo models of pancreatic cancer and lung cancer.

Thus, the present invention provides, in some embodiments, highly specific non-immunogenic humanized anti-human PVR antibodies having improved affinity, activity and/or reproducibility.

According to one aspect of the present invention, a humanized antibody is provided that specifically binds to the human poliovirus receptor (PVR, CD155) or a fragment thereof comprising at least an antigen binding site, wherein the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable a region with 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 a variable region with 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 .

In some embodiments, the antibody comprises a heavy chain variable region amino acid sequence comprising the CDR sequences corresponding to SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, and a light chain variable region amino acid sequence comprising the CDR sequences corresponding to SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

In some embodiments, the humanized antibody or fragment thereof is an IgG monoclonal antibody. In accordance with some embodiments of the invention, the humanized monoclonal antibody comprises a heavy chain constant region selected from IgG4 and IgG1. In some embodiments, the humanized antibody or fragment thereof is of the IgG4 subclass. In some embodiments, the humanized antibody or antigen binding fragment thereof is of the IgG1 subclass.

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In accordance with some embodiments of the invention, 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.

In accordance with some embodiments of the invention, the humanized antibody or fragment thereof is a monoclonal antibody, Fab, F(ab) ₂ , single domain antibody, or single chain variable fragment (scFv).

In accordance with some embodiments of the invention, the humanized antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence

QVQLVQSGAE(L/V)KKPGASVK(I/V)SCKATGYTFSNYWIEW(I/V)(K/R)QAPGQGLEW(I/M)GEIFPGSG RINFNEKFKGR(A/V)TFTADTSI(D/S)T(T/A)YM( Q/E)LS(S/R)L(T/R)SDD(S/T)AVYYCARTKIYGNSFDY WGQGT(T/L)VTVSS (SEQ ID NO: 47); and a light chain variable region containing the amino acid sequence

DI(M/Q)MTQSPS(F/S)LSASVGDRVTITC(K/R)ASQDVGTAV(V/A)WYQQKPGKAPK(L/S)LIYWASSRHE GVP(D/S)RF(T/S)GSGSGTDFTLTISSLQ(S/P)EDFA (D/T)YFCQQYSRYPLT FGQGT KLEIK (SEQ ID NO: 48).

In some embodiments, the humanized antibody or fragment thereof comprises a heavy chain variable region comprising: i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 10-12; and ii) a set of four heavy chain framework region (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.

In some embodiments, the humanized antibody or antigen binding fragment thereof comprises a light chain variable region comprising:

i) a set of three CDR sequences containing the sequences presented in SEQ ID NO: 13-15; and 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 being SEQ ID NO: 33.

In some embodiments, the humanized antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises:

i) a set of three CDR sequences containing the sequences presented in SEQ ID NO: 10-12; and 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;

(D) FR-H4 selected from the group consisting of SEQ ID NOs: 21 and 25;

and the light chain variable region contains:

i) a set of three CDR sequences containing the sequences presented in SEQ ID NO: 13-15; and ii) a set of four light chain (LC) framework regions (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 being SEQ ID NO: 33.

In accordance with some embodiments of the invention, the heavy chain variable region of the humanized monoclonal antibody contains 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 contains 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. B in accordance with some embodiments of the invention, the heavy chain variable region of the humanized monoclonal antibody contains 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 of at least

- 3 045980 at least approximately 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 some embodiments, the heavy chain variable region humanized monoclonal antibody contains 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 contains 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 some 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.

In 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:

i) the heavy chain variable region sequence shown in SEQ ID NO: 1 and the light chain variable region sequence shown in SEQ ID NO: 2;

ii) the heavy chain variable region sequence shown in SEQ ID NO: 4 and the light chain variable region sequence shown in SEQ ID NO: 8;

iii) the heavy chain variable region sequence shown in SEQ ID NO: 5 and the light chain variable region sequence shown in SEQ ID NO: 2;

iv) the heavy chain variable region sequence shown in SEQ ID NO: 5 and the light chain variable region sequence shown in SEQ ID NO: 8;

v) the heavy chain variable region sequence shown in SEQ ID NO: 4 and the light chain variable region sequence shown in SEQ ID NO: 2;

vi) the heavy chain variable region sequence shown in SEQ ID NO: 1 and the light chain variable region sequence shown in SEQ ID NO: 8;

vii) the heavy chain variable region sequence shown in SEQ ID NO: 6 and the light chain sequence shown in SEQ ID NO: 2; and viii) the heavy chain variable region sequence set forth in SEQ ID NO: 6 and the light chain variable region sequence set forth in SEQ ID NO: 8.

In accordance with some embodiments of the invention, the heavy chain variable region of the humanized monoclonal antibody contains an amino acid sequence that is at least about 90% identical to the sequence of SEQ ID NO: 1, and the light chain variable region contains an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1. identical to the sequence of SEQ ID NO: 2.

In accordance with some embodiments of the invention, the heavy chain variable region of the humanized monoclonal antibody contains an amino acid sequence identical to SEQ ID NO: 1, and the light chain variable region contains an amino acid sequence identical to SEQ ID NO: 2.

In some embodiments, the humanized antibody inhibits the binding of PVR to at least one of TIGIT, CD96, and CD226.

In some embodiments, the antibody inhibits the binding of PVR to TIGIT, CD96, and CD226.

In accordance with some embodiments of the invention, the humanized antibody is an IgG4 antibody containing the heavy chain sequences set forth in SEQ ID NO: 49, or a sequence that is at least 90% identical thereto. In accordance with some embodiments of the invention, the humanized antibody is an IgG1 antibody comprising the heavy chain sequence set forth in SEQ ID NO: 50, or a sequence at least 90% identical thereto.

In some embodiments, the humanized antibody comprises the light chain sequence set forth in SEQ ID NO: 49.

In accordance with some embodiments of the invention, the humanized antibody exhibits improved antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) compared to other known antibodies.

Polynucleotide sequences encoding a humanized antibody or antigen binding fragment thereof are provided in accordance with another aspect of the invention.

In accordance with some embodiments of the invention, a polynucleotide sequence is provided encoding the amino acid sequences of the heavy chain variable region, the light chain variable region, or both, described above.

In accordance with some embodiments of the invention, there is provided a polynucleotide

- 4 045980 encoding a heavy chain variable region containing 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.

In accordance with some embodiments of the invention, there is provided 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 .

In accordance with some embodiments of the invention, the polynucleotide encodes a humanized antibody or fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 5 and SEQ ID NO: 6; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. Each combination of heavy and light chain variable regions is a separate embodiment of the invention.

In accordance with some embodiments of the invention, the polynucleotide sequence encoding the heavy chain variable region of a humanized antibody 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.

In accordance with some embodiments of the invention, the polynucleotide sequence encoding the light chain variable region of a humanized antibody 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.

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

A cell line containing nucleic acids encoding the antibodies of the present invention is also described. The cell line is designed to express a humanized antibody or fragment thereof, as described herein. In some embodiments, the cell line is a mammalian cell line, such as a Chinese hamster ovary (CHO) cell line.

In accordance with some embodiments of the invention, the cell line is a line of bacterial, plant, murine (eg, NSO and Sp2/0), rat (eg, YB2/0), hamster (eg, BHK and CHO) or human cells (eg, , PER.C6).

In accordance with yet another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising an extracellular portion (binding domain) containing any of the humanized antibodies or fragments thereof described herein. In accordance with some embodiments of the invention, a CAR is provided that contains a combination of the heavy and light chain variable region sequences described above, having a unique combination of CDR and framework sequences, improved binding, and other properties.

In accordance with some embodiments of the invention, the CAR contains a combination of heavy and light chain variable regions, wherein the heavy chain variable region contains an amino acid sequence that is at least 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 contains an amino acid sequence that is at least 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.

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

In some embodiments, the CAR comprises a combination of heavy and light chain variable regions of a humanized antibody, wherein the combination is selected from the group consisting of:

i) the heavy chain variable region sequence shown in SEQ ID NO: 1 and the light chain variable region sequence shown in SEQ ID NO: 9;

ii) the heavy chain variable region sequence shown in SEQ ID NO: 3 and the light chain variable region sequence shown in SEQ ID NO: 9;

iii) the heavy chain variable region sequence set forth in SEQ ID NO: 4, and

- 5 045980 sequence of the variable region of the light chain presented in SEQ ID NO: 9;

iv) the heavy chain variable region sequence shown in SEQ ID NO: 5 and the light chain variable region sequence shown in SEQ ID NO: 9; And

v) the heavy chain variable region sequence shown in SEQ ID NO: 6 and the light chain variable region sequence shown in SEQ ID NO: 9.

In accordance with some embodiments of the invention, 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 presented in SEQ ID NO: 9 , transmembrane domain and intracellular T cell signaling domain.

The present invention also provides a single chain variable region (scFv) comprising the heavy chain and light chain variable regions of the antibodies described herein. In accordance with some embodiments of the invention, a hinge region is located between the variable regions. In accordance with some embodiments of the invention, the amino acid sequence of the scFv is selected from SEQ ID NO: 56, SEQ ID NO: 57, and an analogue thereof that is at least 90% identical to any of these sequences.

In some embodiments, the CAR comprises amino acid sequences represented by any of SEQ ID NO: 56 and SEQ ID NO: 57.

In accordance with some embodiments of the invention, the CAR comprises a 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 CD3Z domain (CD3Z, Zetta). In accordance with some embodiments of the invention, the CAR contains a CD8 Stalk domain. In some embodiments, the CAR contains a CD28 TM domain. In some embodiments, the CAR contains a CD3Z domain. In some embodiments, the CAR contains a 41BB domain. In accordance with specific embodiments of the invention, the CAR contains a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

In accordance with some embodiments of the invention, the CAR contains a scFv sequence containing the PVR binding site of any antibody described 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. In accordance with specific embodiments of the invention, the CAR contains a scFv sequence containing the PVR binding site of any antibody described above, and a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3Z domain.

In accordance with some embodiments of the invention, a lymphocyte engineered to express a CAR described herein is provided. In accordance with some embodiments of the invention, a T cell engineered to express a CAR described herein is provided. In accordance with additional embodiments of the invention, there is provided an NK cell engineered to express a CAR described herein.

In accordance with specific embodiments of the invention, there is provided an engineered T cell expressing a scFv sequence selected from the group consisting of: SEQ ID NO: 56, SEQ ID NO: 57, or an analogue thereof that is at least 90% identical to any of the above sequences; CD8 Stelk domain, CD28 TM domain, 41BB domain and CD3Z domain.

In accordance with yet another aspect, the present invention provides a method of treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of at least one CAR-containing lymphocyte described herein. In accordance with yet another aspect, the present invention provides a pharmaceutical composition comprising a humanized antibody or antigen binding fragment described herein and a pharmaceutically acceptable excipient, carrier or diluent.

Any route of administration, including parenteral and enteral routes of administration, can be used to deliver the compositions of the present invention to a subject in need thereof.

In accordance with some embodiments of the invention, the pharmaceutical composition is formulated for injection or infusion. In accordance with some embodiments of the invention, the pharmaceutical composition is for intravenous administration. In some embodiments of the invention, the pharmaceutical composition is for intratumoral administration.

In accordance with some embodiments of the invention, a humanized antibody or antigen binding fragment or pharmaceutical composition thereof is provided for use in enhancing the surface expression and/or signaling of CD226 on CD8+ and CD4+ T Cells.

In accordance with embodiments of the invention, the humanized antibody or antigen binding fragment thereof, or pharmaceutical composition is provided for use in the treatment of cancer in an individual. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group with

- 6 045980 worth 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 invention.

In some embodiments, the cancer is a hematologic cancer.

In accordance with some embodiments of the invention, the hematologic cancer is selected from leukemia, 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.

In some embodiments of the invention, the individual is a human.

In accordance with some embodiments of the invention, the use further includes the use of an agent that inhibits the activity or expression of an immune coinhibitory receptor.

In some embodiments, the immune coinhibitory receptor is selected from the group consisting of PD-1, PD-L1, TIGIT, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, CD96, BY55 (CD160), LAIR1, SIGLEC10, CD112R, CD112, ILT-4 and 2B4. Each possibility represents a separate embodiment of the invention.

In accordance with some embodiments of the invention, use also includes use in combination with an anti-endothelial growth factor receptor (EGFR) antibody.

In accordance with another aspect of the present invention, there is provided a method of increasing the surface expression and/or signaling of CD226 in CD8+ and CD4+ T cells of an individual, comprising administering to the individual a therapeutically effective amount of a humanized antibody or antigen binding fragment thereof, or a pharmaceutical composition described herein. In some embodiments, the CD8+ T cells are tumor-infiltrating CD8+ T cells.

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

In accordance with another aspect, the present invention provides a method of treating cancer in an individual suffering from cancer, comprising administering to the individual a therapeutically effective amount of a humanized antibody or antigen binding fragment thereof or a pharmaceutical composition and an inhibitor of PD-1, PD-L1, CTLA-4 or CD112R signaling . In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is selected from the group consisting of glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, or lung cancer. In some embodiments, the PD-1 signaling inhibitor is an antibody or fragment thereof that binds to PD-1. In some embodiments, the PD-1 binding antibody or fragment thereof is pembrolizumab, nivolumab, AMP-514, tislelizumab, spartalizumab, or a PD-1 binding fragment thereof. In some embodiments, the PD-1 signaling inhibitor is an antibody that specifically binds to PD-L1 or PD-L2. In some embodiments, an antibody that specifically binds PD-L1 or PD-L2 includes durvalumab, atezolizumab, avelumab, BMS936559 or FAZ053 or a PD-L1 or PD-L2 binding fragment thereof. In some embodiments, the PD-1 signaling inhibitor comprises an Fc fusion protein that binds PD1, PD-L1, or PD-L2. In some embodiments, the Fc fusion protein comprises AMP-224 or a PD-1 binding fragment thereof. In some embodiments, the PD-1 signaling inhibitor comprises a small molecule inhibitor of PD-1, PD-L1, or PD-L2. In some embodiments, the small molecule inhibitor of PD-1, PD-L1, or PD-L2 signaling contains one or more of the following compounds: #{2-[({2-methoxy-6-[(2methyl[1,1' -biphenyl]-3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}-acetamide (BMS 202); (2-((3cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5-methylbenzyl)-Oserine hydrochloride; (2R,4R)-1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin6-yl)-2 -methylbenzyl)-hydroxy)benzyl)-4-hydroxypyrrolidine-2-carboxylic acid; 3-(4,6-dichloro-1,3,5- 7 045980 triazin-2-yl)-1-phenylindole; 3-(4,6-dih.tor-1,3,5-triazin-2-yl)-1-(|)enyl-111-indole; L-a-glutamine; N6,N6bis(k-seryl-k-asparaginyl-k-threonyl-k-seryl-k-a-glutamyl-k-seryl-k-phenylalanyl)-k-lysyl-cphenylalanyl-k-arginyl-k-valyl-k -threonyl-c-glutaminyl-c-leucyl-c-alanyl-c-prolyl-c-lysyl-calanyl-c-glutaminyl-c-isoleucyl-c-lysyl; (2 S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1'-biphenyl]-3 yl)methoxy]-phenyl]methyl]-2-piperidinecarboxylic acid; glycinamide; H-(2-mercaptoacetyl)-cphenylalanyl-N-methyl-c-alanyl-c-asparaginyl-c-prolyl-c-histidyl-c-leucyl-N-methylglycyl-ctryptophyl-c-seryl-c-tryptophil-N- methyl-c-norleucyl-N-methyl-c-norleucyl-c-arginyl-c-cysteinyl-. cyclic (1^14)-thioether; or their derivatives or analogues.

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

In yet another aspect, the present invention provides a method for diagnosing or prognosticating cancer in a subject, comprising determining the level of PVR expression in a biological sample of the subject using at least one humanized antibody, fragment or scFv described herein.

In accordance with yet another aspect, the present invention also provides a method for detecting or quantifying PVR expression, comprising contacting a biological sample with an antibody or antibody fragment described herein and measuring the level of complex formation.

According to some embodiments of the invention, a method for detecting or quantifying PVR expression includes the following steps:

i) incubating the sample with an antibody specific for PVR, or a fragment thereof containing at least an antigen-binding portion;

ii) detecting the associated PVR using a detectable probe.

In accordance with some embodiments of the invention, the method further includes the following steps:

iii) comparing the amount of (ii) with a standard curve obtained from a standard sample containing a known amount of PVR; and iv) calculating the amount of PVR in the sample from the standard curve.

In accordance with some embodiments of the invention, the method includes identifying a subject as having a PVR-positive cancer if the amount of PVR is higher than a control or predetermined value.

In accordance with some specific embodiments of the invention, the sample is a body fluid or solid tissue. In some embodiments of the invention, the method is carried out in vitro or ex-vivo.

Also provided is a kit for measuring PVR expression in a biological sample, comprising at least one antibody or antibody fragment described herein and a means for measuring PVR expression. In some embodiments of the invention, the kit further includes instructions for use of the kit.

Additional embodiments and the full scope of the present invention will become apparent from the detailed description below. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from the detailed description.

Description of drawings

The new features described herein are set forth in detail in the accompanying claims. A better understanding of the characteristics and advantages of the features described herein will be obtained by reference to the following detailed description, which sets forth illustrative examples employing the principles of the features described herein, and which are accompanied by the following drawings.

Fig. 1A-1B. Affinity and competitive analysis of N56 substitutions.

Fig. 1A. Improved PVR binding affinity of N56E and N56D variants. Biacore analysis, by

- 8 045980 showing the relative and absolute affinity of N56 substitutions (hIgG4 variants) to PVR-HIS (Sino Cat. no. 10109-H08H). Affinity shifts >25% were considered significant. (*) The relative K _D s for each variant were determined by dividing the K _D of the replacement by the K _D of the original N56 variant (VH0VK0).

In fig. 1B shows a comparison of the effectiveness of chimeric antibodies (5B9 wild type (WT) with HC from IgG4 (S241P)) with variable regions containing the CDR2 LC sequence with single amino acid substitutions (to remove the deamidation site). Activity was measured in a competition assay using parent 5B9. The relative IC ₅₀ was determined for each variant by dividing its IC ₅₀ by the IC ₅₀ of the wild-type chimeric antibody tested in parallel.

Fig. 2A-2B. Improved binding cross-reactivity of simian PVR with N56E and N56D variants. To assess maximal binding, a saturating dose (10 μg/mL) of the N56 monoclonal antibody variant (shown on the x-axis) was used to stain PVR-expressing cells, and mean fluorescence intensity (MFI) values were determined by FACS analysis. The fold change was calculated by dividing the MFI of each variant by the MFI of the parent antibody (K0). Fig. 2A. NCI-H1975 cells (human lung) and Fig. 2B. Vero cells (African green monkey kidney). Affinity shifts >25% were considered significant.

Fig. 3. Improved NK cell activation by N56E and N56T variants. To characterize N56 substitution variants, a CD107a induction assay was performed using human NK cells from a healthy donor and MDA-MB-231 as target cells in a 2:1 ratio. Antibodies were added at a concentration of 600 pM. K0 is the original (parent) clone (N56). All variants resulted in significant induction of CD107a (>240% compared to the IgG1 isotype). In addition, N56 substitutions such as N56E and N56T significantly improved CD107a induction compared with K0. (*p<0.04, **p<0.01).

Fig. 4. Improved proliferation of CD8 T cells by N56E and N56T variants. T cell proliferation assays were performed to characterize N56 variants. A549 cancer cells were used at an effector/target (E:T) ratio of 4:1 in the presence of 2.5 μl/ml PHA-L and using fresh CFSE-labeled human PBMCs. All monoclonal antibodies of the N56 variant (X axis) were added at 4 μg/ml, and the co-culture was incubated for 96 hours. Results of FACS analysis of CD8+ T cells are presented. The relative MFI of the CFSE tag was calculated by dividing the MFI of the IgG-treated group by the MFI of each variant. Since increased proliferation leads to decreased CFSE signal, the y-axis displays the inverse of this relationship. The N56E and N56T variants significantly increased CD8 T cell proliferation compared to the parental clone (K0) labeled #. (*p<0.05, #<0.04, **p<0.01).

Fig. 5A-5B illustrate the affinity of the humanized variants for human PVR measured using surface plasmon resonance (SPR) (Fig. 5A) or their binding to the surface of PVR-expressing HEK293 cells as measured by flow cytometry (Fig. 5B). Absolute and relative results are shown using the chimeric mutant parent antibody N56E (N56E VH0/Vk0) as a reference point.

In fig. 6A-6B show the expression levels (FIG. 6A) and similarity of the humanized variants to human germline variable domain sequences (FIG. 6B). The titers of individual variants after transient expression in HEK293 EBNA cells are shown (Fig. 6A). The variable region sequence of the humanized heavy and light chain variants is shown to be identical to the human germline sequence (Fig. 6B).

In fig. 7A-7B compare the biophysical properties of the humanized variant NB1088 (right column) with the humanized variant NB941 containing the LC CDR2 5B9 WT sequence (left column). In fig. 7A and FIG. Figure 7B shows the decrease in acidic species formation in NB1088 over time following stress testing at low pH (Figure 7A) or at high concentration at 40°C (Figure 7B).

Fig. 8A-8D illustrate EC ₅₀ for binding of NB1088 to PVR (FIG. 8A); IC ₅₀ for inhibition of PVR-TIGIT binding by NB1088 (Fig. 8B); IC50 for inhibition of PVR-CD96 binding by NB1088 (Fig. 8C) and _IC50 for inhibition of PVR-CD226 binding by NB1088 (Fig. 8D).

In fig. 9A-9B show that NB1088 alone (FIGS. 9A and 9B) and in combination with PD1 inhibition by pembrolizumab (FIG. 9B) increases interferon gamma release in a tumor/T cell coculture system using an antigen-specific T assay -cells (human papillomavirus; HPV) (Fig. 9A) or a nonspecific allogeneic T cell assay (Fig. 9B). **p<0.01 one-tailed ANOVA.

In fig. 10A-10B show that, in combination with an endothelial growth factor receptor (EGFR) binding antibody, NB1088 increases antibody-dependent cellular cytotoxicity in the EGFR-expressing breast cancer cell line A549 (Fig. 10A), and that this coincides with increased interferon-gamma release (Fig. 10B), (*p<0.05, **p<0.01 one-way Anova).

Fig. 11A-11B illustrate that tumor cell lines expressing PVR (A549 or CaSki) can induce downregulation of CD226 expressed on CD8 T cells (FIG. 11A) and NK cells (FIG. 11B), which can be rescued by NB1088. but not anti-TIGIT (Fig. 11A

- 9 045980 and B). In fig. 11A shows results obtained in a co-culture system using an antigen-specific T-cell (human papillomavirus; HPV) assay or a non-specific allogeneic T-cell assay.

In fig. 12A-12B show NB1088-dependent increase in interferon-gamma release by alloy or antigen-sensitive CD8+ T cells (Fig. 12A), or NB1088-dependent induction of antibody-dependent cellular cytotoxicity (ADCC)-sensitive NK cells in the presence of EGFR blockade (Fig. 12B) is dependent on CD226 activity, and in both cases NB1088 exhibits higher activity compared to TIGIT inhibition (both antibodies at 10 μg/ml).

In fig. 13A-13E show that NB1088 is effective as monotherapy in a humanized mouse model of pancreatic cancer (Fig. 13A), competing with pembrolizumab (anti-PD-i administered at the standard dose established for these models) (Fig. 13B), and in a humanized mouse model of lung adenocarcinoma (Fig. 13C, humanized mice), and that in this model of lung adenocarcinoma, efficacy is dependent on the presence of human immune cells (Fig. 13D, non-humanized mice) and correlates with increased expression of CD226 on tumor infiltrating CD8+ T -cells (Fig. 13E). (*p<0.05, ***p<0.001, two-way Anova).

Fig. 14A-14D refer to the model in FIG. 13C and 13E, and show that NB1088 increases the frequency of interferon-gamma positive (Fig. 14A) and interferon-gammaα/CD107a double positive (Fig. 14B) CD8 T cells, and that NB1088 increases the frequency of interferon-gamma+/CD226+ double positive CD8 T cells (Fig. 14D), but not interferon-gamma+/CD226- single positive CD8 T cells (Fig. 14C). *p=0.0210 ***<0.0001 unpaired TTEST.

Fig. 15A-15B illustrate the pharmacokinetics of NB1088 (FIG. 15A) and the corresponding pharmacodynamic changes in surface expression of CD226 (FIG. 15B) on circulating cynomolgus CD4 T cells that were treated with various doses of the antibody once (for doses of 20 and 50 mg/kg) or 4 times with an interval of one week (for a dose of 200 mg/kg).

In fig. Figure 16 shows human PVR expression in biopsies of various cancer types measured by immunohistochemistry and scored by H score.

Fig. 17 is a general schematic representation of CAR-T designs. The scFv fragment includes the heavy and light chains (VH and VL, respectively) of the humanized antibodies of the invention.

Fig. 18 illustrates the potent secretion of interleukin 2 (IL2) by Jurkat cells overexpressing the aPVR CAR-T construct compared to parental Jurkat cells. Parental Jurkat cells or Jurkat cells overexpressing aPVR CAR-T constructs H4K2-NTX1088C or H3K4-NTX1034C (40 thousand cells/well) were incubated with A549 or MDA-231 cells at an E:T ratio of 1:1 for 24 hours. Secretion IL2 was quantified using Biolegend hIL-2 (cat. 431804). Both CAR-T drivers increased IL2 secretion more than 100-fold compared to parental Jurkat cells in the presence of these targets.

Fig. 19A-19C illustrate increased killing of target cells by anti-PVR (aPVR) CAR-T. A549 (Fig. 19A) or MDA-231 (Fig. 19B) cells (200 thousand cells each) were placed in a 12-well plate with CAR-T-PVRNTX-1088C and NTX-1034C variants in NK medium for 72 hours, at ratios E :T 0.4 and 0.8 to 1, respectively (based on GFP positivity). Tumor cell death was assessed using a standard CTG protocol (Promega G9241).

Fig. 20 illustrates the effective killing of hematological target cells by aPVR CAR-T. K562 cells (AML model) were incubated with aPVR CAR-T variants NTX-1088C and NTX1034C in RPMI+IL-2 for 18 h at E:T ratios indicated on the x-axis. Tumor cell death was assessed by flow cytometry. Significant target cell killing was observed for both CAR-T drivers.

Detailed Disclosure of the Present Invention

The present invention relates to humanized monoclonal antibodies that recognize the poliovirus receptor (PVR). Advantageously, the antibodies of the invention are almost completely humanized, avoiding the risk of an unwanted immune response to the antibodies, and are therefore safe for use in humans in vivo. The antibodies of the present invention are characterized by a unique CDR sequence as well as novel humanized framework sequences and design. More specifically, the monoclonal antibodies of the present invention have certain combinations of CDRs and non-humanized framework sequences and have unique properties and improved safety and efficacy compared to known anti-PVR antibodies.

Some of the variants described herein have improved reproducibility and higher expression levels compared to other humanized anti-PVR antibodies containing the same CDR regions. Also disclosed herein are methods of using these antibodies to treat cancer in an individual.

To provide a thorough understanding of the various embodiments of the invention, certain specific details are set forth in the following description. However, a person skilled in the art

- 10 045980 technology it will be clear that the above embodiments can be implemented in practice without these details. Unless the context otherwise requires, throughout the following specification and claims, the word contain and its variants, such as contains and containing, are to be construed in an open, inclusive sense, that is, including, but not limited to. As used in this specification and the accompanying claims, the singular forms a, an and the include references to the plural unless the context clearly requires otherwise. It should also be noted that the term or is generally used with its meaning including and/or, unless the context clearly indicates otherwise. In addition, the headings set forth herein are for convenience only and are not intended to construe the scope or spirit of the claimed embodiments.

As used herein, the term PVR refers to the poliovirus receptor, also known as CD155 (cluster of differentiation 155), Protein ID: Q92692. In some embodiments, PVR is a transmembrane glycoprotein with an N-terminal signal sequence, three extracellular immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic tail. It has a molecular mass of approximately 80 kDa and a structure consisting of three Ig-like domains, with two C2-like domains located behind the outermost V-like domain. The humanized antibodies described herein have affinity for human PVR (hPVR). In some embodiments, the antibodies have some affinity for PVR proteins of other animals, particularly primates. Preferably, the affinity for other primates, such as the African green monkey, makes it possible to further test the humanized antibodies for safety and effectiveness in non-clinical trials. No affinity for PVR has been observed in more evolutionarily distant animals such as rodents.

As used herein, the term approximately refers to an amount that differs from the specified amount by 10% or less.

As used herein, the term individual, patient or subject refers to individuals diagnosed with, suspected of having, or at risk of developing at least one disease for which the described compositions and method are useful. In some embodiments of the invention, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In some embodiments of the invention, the individual is a human.

As used herein, the term combination or combination treatment can refer to either simultaneous or sequential administration of drugs that are to be in combination. As described herein, when the combination refers to sequential administration of drugs, the drugs can be administered in any temporal order.

The terms cancer and tumor refer to a mammalian physiological condition characterized by dysregulation of cell growth. Cancer is a class of diseases in which a group of cells exhibit uncontrolled or unwanted growth. Cancer cells can also spread to other places, which can lead to the formation of metastases. The spread of cancer cells in the body can occur, for example, through lymph or blood. Uncontrolled growth, intrusion and formation of metastases are also called malignant properties of cancer. These malignant properties distinguish cancer from benign tumors, which usually do not invade or metastasize.

As used herein, the term effective amount refers to the amount of a therapeutic agent that produces a biological effect when administered to a mammal. Biological effects include, but are not limited to: inhibition or blockade of receptor-ligand interaction (eg, PVR-TIGIT, PD-1-PD-L1/PD-L-2), inhibition of signaling pathway, reduction of tumor growth, reduction of tumor metastasis, or increasing the life expectancy of an animal with a tumor. The therapeutic amount is the concentration of a drug calculated to produce a therapeutic effect. A therapeutic amount covers the range of doses capable of producing a therapeutic response in a population of individuals. A mammal can be a human. A human individual may suffer from, suspect or be affected by a tumor.

As used herein, the term checkpoint inhibitor refers to a drug that inhibits a biological molecule (checkpoint molecule) produced by the body that negatively regulates the antitumor/anticancer activity of T cells in the body. 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, SEACAM1, NOX2, CD112R and CD112.

Antibodies provided may be monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies and polyactive antibodies) and antibody fragments.

Antibodies include antibody conjugates and antibody-containing molecules such as chimeric

- 11 045980 molecules. Thus, an antibody includes, but is not limited to, a full-length antibody, as well as fragments and portions of an antibody that retain binding specificity, such as any specific binding portion thereof, including antibodies containing any number of such portions, immunoglobulin classes, and/or isotypes (e.g., IgG1 , IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to the following: Fab, F(ab') ₂ , Fv and scFv (single chain or similar fragment). A monoclonal antibody is typically an antibody in a composition of substantially homogeneous antibodies; thus, any individual antibodies contained in a monoclonal antibody composition are identical, except for possible naturally occurring mutations that may be present in minute quantities. A polyclonal antibody is a preparation that includes various antibodies with different sequences, which are usually directed against two or more different determinants (epitopes). The monoclonal antibody may contain a human IgG1 constant region. The monoclonal antibody may contain a human IgG4 constant region.

The term antibody is used here in its broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and their functional (antigen-binding) fragments, including antigen binding sites Fab fragments, F(ab') ₂ fragments, Fab' fragments, Fv fragments, recombinant IgG fragments (rIgG), single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibody fragments (eg, sdAb, sdFv, nanobody). The term includes genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptide antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific, e.g. bispecific antibodies, diabodies, triantbodies and tetrabodies, tandem di-scFv, tandem tri -scFv. Unless otherwise indicated, the term antibody should be understood to include functional antibody fragments. The term also covers intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD. The antibody may contain a human IgG1 constant region. The antibody may contain a human IgG4 constant region.

The terms complementarity determining region and CDR, which are synonymous with hypervariable region or HVR, are known in the art and refer to non-contiguous amino acid sequences in the variable regions of an antibody that are responsible for antigen specificity and/or binding affinity. Typically, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). It is known in the art that framework regions and FRs refer to those portions of the heavy and light chain variable regions that are not CDRs. Typically, there are four FRs in each full-length heavy chain variable region (FR-H1, FRH2, FR-H3 and FR-H4) and four FRs in each full-length light chain variable region (FR-H4). L1, FR-L2, FR-L3 and FR-L4). The precise amino acid sequence boundaries of a particular CDR or FR region can be readily determined using any of a number of well-known systems, including those described by Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering system), Al-Lazikani et al., (1997) JMB 273,927-948 (Chothia numbering system); 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 system); 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 system); Honegger A and Pluckthun 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 system); and Whitelegg NR and Rees AR, WAM: an improved algorithm for modeling antibodies on the WEB, Protein Eng. 2000 Dec; 13(12):819-24 (AbM numbering system). In some embodiments, the CDRs of antibodies described herein can be determined by a method selected from Kabat, Chothia, EVIGT, Aho, AbM, or combinations thereof.

The boundaries of a given CDR or FR may vary depending on the system used for identification. For example, Kabat numbering is based on structural alignments, whereas Chothia numbering is based on structure information. The numbering of both Kabat and Chothia systems is based on the most common sequence lengths of antibody regions, with insertions indicated by additional letters, such as 30a, and deletions appearing in some antibodies. The two systems place certain insertions and deletions (insertion-deletion mutations) in different locations, resulting in differences in numbering. The Contact system is based on the analysis of complex crystal structures and is similar in many aspects to the Chothia numbering system.

The term variable region or variable domain refers to the heavy or light chain domain of an antibody that is involved in the binding of the antibody to an antigen. Variable domains of severe

- 12 045980 chain and light chain (VH and VL, respectively) of a native antibody typically have similar structures, with each domain containing four conserved framework regions (FR) and three CDR regions (see, for example, Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to provide antigen-binding specificity. In addition, antibodies that bind a particular antigen can be isolated using the VH or VL domain of the antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, for example, Portolano et al., J. Immunol 150 :880-887 (1993), Clarkson et al., Nature 352:624-628 (1991)).

Antibodies provided include antibody fragments. An antibody fragment refers to a molecule, other than an intact antibody, that contains a portion of an intact antibody that binds an 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 (eg scFv or sFv); and multispecific antibodies formed from antibody fragments. In specific embodiments, the antibodies are single chain antibody fragments containing a heavy chain variable region and/or a light chain variable region, such as scFv.

As used herein, the term antigen refers to a molecule or part of a molecule capable of causing the formation of antibodies and specifically binding to an antibody. An antigen may have one or more epitopes. The specific binding mentioned above means that the antigen will react in a highly selective manner with its corresponding antibody, but not with the many other antibodies that can be induced by other antigens. The antigen in accordance with some embodiments of the present invention is a human PVR.

Antibody fragments can be produced by a variety of methods, including, but not limited to, proteolytic cleavage of the intact antibody, as well as production of antibodies by recombinant host cells. In some embodiments, the antibodies are recombinantly produced fragments, such as fragments comprising an arrangement that does not occur in nature; such as fragments with two or more antibody regions or chains connected by synthetic linkers, for example, polypeptide linkers; and/or those that are not obtained by enzymatic cleavage of a naturally occurring intact antibody. In accordance with some embodiments of the invention, the antibody fragments are scFv.

A humanized antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FR. The humanized antibody may optionally include at least a portion of the antibody constant region derived from a human antibody. A humanized form of a non-human antibody refers to a variant of a non-human antibody that has been humanized, typically to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. In accordance with some embodiments of the invention, certain FR residues in a humanized antibody are replaced with corresponding residues of a non-human antibody (eg, the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

A human antibody is an antibody with an amino acid sequence corresponding to that produced by a human or a human cell or non-human source that uses human antibody repertoires or other sequences encoding human antibodies, including human antibody libraries. The term does not include humanized forms of non-human antibodies containing non-human antigen-binding regions, such as antibodies in which all or substantially all of the CDRs are non-human.

The terms polypeptide and protein are used interchangeably to refer to a polymer composed of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains, and other peptides, such as linkers and binding peptides, may include natural and/or non-natural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. According to some embodiments of the invention, modification of the polypeptide with respect to its native or natural sequence can be carried out while the protein retains the desired activity. These modifications may be intentional, such as the result of site-directed mutagenesis, or they may be accidental, such as the result of mutations in the hosts that produce the proteins or errors due to amplification during PCR.

Percentage (%) sequence identity with respect to the reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the sequence of the reference polypeptide obtained

- 13 045980 nal after sequence alignment and, if necessary, insertion of gaps to achieve the maximum percentage of sequence identity, with any conservative substitutions not considered identical. Alignment to determine percent amino acid sequence identity can be achieved by various known methods, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Suitable parameters for sequence alignment can be determined, including algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this document, % amino acid sequence identity values are obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison program was created by Genentech, Inc., and the source code was filed along with user documentation with the United States Copyright Office, Washington, DC 20559, where the program was registered under U.S. Copyright Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. For use on the UNIX operating system, including digital UNIX. V4.0D, ALIGN-2 program must be compiled. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In situations where ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of A with respect to amino acid sequence B (which can otherwise be thought of as a given amino acid sequence A that has or contains a certain percentage of amino acid sequence identity with respect to, in comparison with or against a given amino acid sequence B) is calculated as follows: 100 times X/Y, where X is the number of amino acid residues counted as identical by the ALIGN-2 sequence alignment program, which performs the alignment of A and B, and where Y - the total number of amino acid residues in B. It should be understood that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the % identity of the amino acid sequence of A with respect to B will not equal the % identity of the amino acid sequence of B with respect to A. Unless specifically stated otherwise, all % amino acid sequence identity values used here were obtained as described in the previous paragraph using the ALIGN-2 computer program.

The terms homologous, homology, or percent homology, when used herein to describe an amino acid sequence or nucleic acid sequence in comparison to a reference sequence, can be defined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264 -2268, 1990, as modified as Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is included in the basic local alignment search programs (BLAST) of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent sequence homology can be determined using the most recent version of BLAST as of the filing date of this application.

Some embodiments of the invention are directed to amino acid sequence variants of the antibodies provided herein. A variant typically differs from the polypeptide directly described herein by one or more substitutions, deletions, additions and/or insertions. Such variants may occur naturally or may be produced synthetically, for example, by modifying one or more of the above polypeptide sequences of the invention and assessing one or more biological activities of the polypeptide as described herein and/or using any of the known techniques. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Variants of the amino acid sequence of an antibody can be obtained 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 in the amino acid sequences of the antibody. Any combination of deletions, insertions and substitutions can be used to produce the final construct, provided that the resulting construct has the desired characteristics, such as antigen binding.

In some embodiments, the invention provides antibody variants containing one or more amino acid substitutions. Regions of interest for substitution mutagenesis include CDR and FR. Amino acid substitutions can be introduced into the antibody of interest, and the products are screened for the desired activity, such as preserved/improved antigen binding, reduced immunogenicity, or improved antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

In some embodiments, substitutions, insertions or deletions may be made in one or more CDRs, wherein the substitutions, insertions or deletions do not significantly reduce the binding of the antibody to the antigen. For example, conservative substitutions can be made in the CDRs that do not significantly reduce binding affinity. Such changes may be outside the hot zones

- 14,045,980 CDR points. In some variants of the VH and VL sequences, none of the CDR regions are changed.

Changes (eg, substitutions) can be made to the CDR, for example, to improve antibody affinity. Such changes can be made to codons encoding CDRs with high mutation rates during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting variant can be tested for binding affinity . Affinity maturation (eg, using error-prone PCR, strand shuffling, CDR randomization, or oligonucleotide-directed mutagenesis) can be used to improve antibody affinity (see, eg, Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2001 )). CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, for example, Cunningham and Wells Science, 244:1081-1085 (1989)). The most common targets are CDR-H3 and CDR-L3. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify points of contact between antibody and antigen. Such contact residues and adjacent residues may be selected as candidates for replacement, or excluded. Variants can be screened to determine whether they have the desired properties.

Amino acid sequence insertions and deletions include amino and/or carboxyl terminus fusions to polypeptides ranging in length from one residue to one hundred or more residues, as well as insertions and deletions of one or more amino acid residues within a sequence. Examples of terminal inserts include an antibody with an N-terminal methionine. Other insertions into the antibody molecule include fusing the N- or C-terminus of the antibody with an enzyme (eg, for ADEPT) or polypeptide that increases the half-life of the antibody in serum. Examples of insertion variants within the sequence of antibody molecules include the insertion of 3 amino acids in the light chain. Examples of terminal deletions include an antibody with a deletion of 7 or fewer amino acids at the end of the light chain.

In some embodiments, the dissociation constant (KD) for the antibody provided herein and the antibody target, human poliovirus receptor (CD155), is approximately 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 (for example, 10 ^-8 M or less, for example, from 10 ^-8 M up to 10 ^-13 M, for example, from 10 ^-9 M to 10 ^-13 M). KD can be measured by any suitable method. In some embodiments, KD can be measured using surface plasmon resonance (SPR) (eg, using BIACORE®-2000 or BIACORE®-3000).

In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby forming a variant Fc region. The Fc region here is the C-terminal region of the immunoglobulin heavy chain, which contains at least part of the constant region. The Fc region includes native sequence Fc regions and Fc region variants. The Fc region variant may comprise a human Fc region sequence (eg, human IgG1, IgG2, IgG3, or IgG4 Fc region) including an amino acid modification (eg, substitution) at one or more amino acid positions.

In some embodiments, the antibodies herein are variants that possess some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the antibody is important, while some effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/loss of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody does not bind to FcyR (thus likely to have no ADCC activity) but retains the ability to bind to FcRn. Non-limiting examples of in vitro assays for assessing the ADCC activity of a molecule of interest are described in US Pat. No. 5,500,362 and US Pat. No. 5,821,337. Alternatively, non-radioactive assay methods (eg, the ACTI™ and CytoTox 96® non-radioactive cytotoxicity methods) can be used. Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

Antibodies may have an increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, for example, US 2005/0014934). Such antibodies may contain an Fc region with one or more substitutions that improve binding of the Fc region to FcRn, and include antibodies with substitutions at one or more 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, for example, US patent 7371826). Other examples of Fc region variants are also discussed (see, for example, Duncan & Winter, Nature 322:738-40 (1988); US Pat. Nos. 5,648,260 and US 5,624,821; and WO 94/29351).

In some embodiments, it may be desirable to create antibodies with

- 15 045980 engineered cysteine, for example thioMAB, in which one or more residues are replaced by cysteine residues. In accordance with some embodiments of the invention, the substituted residues are located in accessible regions of the antibody. To create an immunoconjugate, reactive thiol groups can be located at sites of conjugation with other moieties, such as drug moieties or drug-linker moieties. In some embodiments, any one or more of the following residues may be replaced by cysteine: V205 (Kabat numbering) light chain; A118 (EU numbering) heavy chain; and S400 (EU numbering) heavy chain Fc region.

In some embodiments, the antibody provided herein may be further modified to contain additional non-protein moieties that are known and available. Fragments suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to: polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene copolymer and maleic anhydride, polyamino acids (either homopolymers or random copolymers) and dextran or poly(vinylpyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody may vary, and if two or more polymers are attached, they may be the same or different molecules.

The antibodies described herein may be encoded by nucleic acid. A nucleic acid is a type of polynucleotide made up of two or more nucleotide bases. In some embodiments, the nucleic acid is a component of a vector that can be used to carry 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 associated. One type of vector is a genome-integrated vector or an integrated vector that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an episomal vector, for example a nucleic acid capable of extrachromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as expression vectors. Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. Regulatory elements contained in expression vectors, such as promoters, enhancers, polyadenylation signals for transcriptional regulation, can be derived from mammalian, microbial, viral or insect genes. Additionally, the ability to replicate in the host, typically provided by the origin of replication, and a selection gene may be included to facilitate recognition of transformants. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like can be used. Plasmid vectors can be linearized for integration into the chromosome. Vectors may contain sequences that direct site-specific integration to a specific position or limited set of genomic regions (eg, AttP-AttB recombination). In addition, the vectors may contain sequences derived from mobile genetic elements.

The nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform or otherwise transform a suitable cell transgenic for the nucleic acid, thereby producing antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell culture are known in the art. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a cell line suitable for antibody production, such as a Chinese hamster ovary (CHO) cell, an NS0 mouse myeloma cell, or a PER.C6® cell. In some embodiments, the nucleic acid encoding the antibody is integrated into the genomic locus of a cell suitable for producing antibodies. In some embodiments, the invention provides a method for producing an antibody, comprising culturing a cell containing a nucleic acid encoding the antibody under in vitro conditions sufficient to permit production and secretion of said antibody.

In some embodiments described herein, a cell master bank is provided comprising: (a) a mammalian cell line containing a genome-integrated nucleic acid encoding an antibody described herein; and (b) a cryoprotectant. In some embodiments, the cryoprotectant contains glycerol. In some embodiments, the cell master bank comprises: (a) a cell line

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CHO containing a genome-integrated nucleic acid encoding an antibody with (i) a heavy chain amino acid sequence represented by any of SEQ ID NO: 1, 3, 4, 5 or 6; and (ii) a light chain amino acid sequence represented by any of SEQ ID NO: 2, 7, 8 or 9; and (b) a cryoprotectant. In some embodiments, the cryoprotectant contains glycerol. In some embodiments, the cell master bank is contained in a suitable vial or container capable of withstanding freezing in liquid nitrogen.

Also described herein are methods for producing the antibody described herein. Such methods include incubating a cell or cell line containing a nucleic acid encoding an antibody in a cell culture medium under conditions sufficient for expression and secretion of the antibody, and then isolating the antibody from the cell culture medium. The recovery may further include one or more purification steps to remove live cells, cellular debris, non-antibody proteins or polypeptides, unwanted salts, buffers, and media components. In some embodiments, additional purification steps include centrifugation, ultracentrifugation, Protein A, Protein G, Protein A/G, or Protein L, and/or ion exchange chromatography.

Antibodies described herein

In a certain aspect, an anti-human PVR (anti-hPVR) antibody or an antigen-binding fragment thereof is described herein. In some embodiments, the antibody or antigen-binding fragment thereof binds to the human PVR at the PVR-TIGIT interface. In some embodiments, the anti-hPVR antibody or antigen binding fragment thereof is capable of competing with any one or more of TIGIT, CD96, and CD226.

In some embodiments, the EC ₅₀ of the humanized antibody or antigen binding fragment thereof for binding to human PVR is less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 , 0.05 or 0.01 nM. In some embodiments, the EC ₅₀ of the anti-hPVR antibody for binding to PVR is in the range of about 5 nM to about 1 nM, about 5 nM to about 2 nM, about 4 nM to about 2 nM, about 4 nM to about 3 nM nM, or from about 3 nM to about 2 nM.

The half-maximal effective concentration (EC ₅₀ ) refers to the concentration of an antibody that produces a response equal to half the value between baseline and maximum after a specified exposure time.

In some embodiments, the antibody is a recombinant antibody. According to specific embodiments of the invention, the antibody is a recombinant humanized monoclonal antibody.

In 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.

In 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.

In accordance with some embodiments of the invention, the humanized antibody or antigen binding fragment thereof is NB1088 (SEQ ID NO: 1 and SEQ ID NO: 2).

One aspect described herein relates to 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 a 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 contains 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 its antigen-binding fragment binds to the human poliovirus receptor (CD155).

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

In some embodiments, provided 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 the sequence selected

- 17 045980 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 its antigen-binding fragment binds to the human poliovirus receptor (CD155).

In some embodiments, provided 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 at least about 95, 96, 97, 98 or 99% amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and the light chain contains an amino acid sequence of at least approximately 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 thereof binds to the human poliovirus receptor (CD155). Each possibility represents a separate embodiment of the invention.

In some embodiments, provided herein is a humanized antibody or antigen binding fragment thereof, wherein said 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 98% identical to SEQ ID NO: 1. and the light chain contains an amino acid sequence at least about 98% identical to SEQ ID NO: 2, and the antibody or antigen binding fragment thereof binds to the human poliovirus receptor (CD155).

In some embodiments, provided herein is a humanized antibody or antigen binding fragment thereof, wherein said 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 99% identical to SEQ ID NO: 1. and the light chain contains an amino acid sequence at least about 99% identical to SEQ ID NO: 2, and the antibody or antigen binding fragment thereof binds to the human poliovirus receptor (CD155).

In some embodiments, the invention provides herein 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 that of SEQ ID NO: 1 and the light chain comprises an amino acid sequence a sequence identical to that of SEQ ID NO: 2, wherein the antibody or antigen binding fragment thereof binds to the human poliovirus receptor (CD155).

In accordance with some embodiments of the invention, the antibody or antigen binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 1 and a light chain sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen binding fragment thereof contains the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigen binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 1. and a light chain sequence set forth in SEQ ID NO: 9. In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 3 and a light chain sequence set forth in SEQ ID NO: 2 In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 3 and a light chain sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen binding fragment thereof comprises the antigen binding fragment comprises the heavy chain sequence set forth in SEQ ID NO: 3 and the light chain sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigen binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 8. 3, and the light chain sequence set forth in SEQ ID NO: 9. In accordance with some embodiments of the invention, 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. In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 4 and a light chain sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen binding fragment thereof comprises the antigen binding fragment comprises the heavy chain sequence set forth in SEQ ID NO: 4 and the light chain sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigen binding fragment thereof comprises the sequence

- 18 045980 the heavy chain set forth in SEQ ID NO: 4, and the light chain sequence set forth in SEQ ID NO: 9. In some embodiments, the antibody or antigen binding fragment thereof comprises the heavy chain sequence set forth in SEQ ID NO: 9. 5, and the light chain sequence set forth in SEQ ID NO: 2. In 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. In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 5 and a light chain sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigen binding fragment thereof contains the heavy chain sequence set forth in SEQ ID NO: 5 and the light chain sequence set forth in SEQ ID NO: 9. In 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. In 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 In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 6 and a light chain sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain sequence set forth in SEQ ID NO: 8. the chain shown in SEQ ID NO: 6, and the light chain sequence shown in SEQ ID NO: 9.

In some embodiments, the humanized antibody or heavy chain antigen binding fragment thereof comprises the amino acid sequence QVQLVQSGAE(L/V)KKPGASVK(I/V)SCKATGYTFSNYWIEW(I/V)(K/R)QAPGQGLEW(I/M)GEIFPGSG RINFNEKFKGR(A/V)TFTADTSI(D/S)T(T/A)YM(Q/E)LS(S/R)L(T/R)SDD(S/T)AVYYCARTKIYGNSFDY WGQGT(T/ L)VTVSS (SEQ ID NO: 47); and the light chain contains the amino acid sequence DI(M/Q)MTQSPS(F/S)LSASVGDRVTITC(K/R)ASQDVGTAV(V/A)WYQQKPGKAPK(L/S)LIYWASSRHE GVP(D/S)RF(T/S)GSGSGTDFTLTISSLQ (S/P)EDFA(D/T)YFCQQYSRYPLTFGQGTKLEIK (SEQ ID NO: 48).

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

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

In 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 in position 48 there is M, or in position 68 there is V, or in position 77 there is S, or in position 79 there is A, or in position 82 there is E, or in position 85 there is R, or in position 87 there is R, or in position 91 is T, or position 114 is L, or any combination thereof. Each possibility represents a separate embodiment of the invention.

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

- 19 045980

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

In accordance with some embodiments of the invention, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 48, where 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 invention.

In some embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 48, where 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 invention.

In some embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 48, where 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 invention.

In some embodiments, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 48, where 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 invention.

In some embodiments, the humanized antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 47 where position 11 is V, or position 20 there is V, or at position 37 there is V, or at position 38 there is R, or at position 48 there is I, or at position 68 there is V, or at position 77 there is S, or at position 79 there is T, or at position 82 there 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 contains the amino acid sequence set forth in SEQ ID NO: 48, where 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.

In accordance with additional embodiments of the invention, the heavy chain CDR1 sequence is GYTFSNYWIE (SEQ ID NO: 58).

In accordance with some embodiments of the invention, the human constant regions of the antibody are selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

In accordance with some embodiments of the invention, the human antibody constant regions are selected from the group consisting of human IgG1 and human IgG4.

In some embodiments, the humanized antibody is an IgG4 antibody comprising the heavy chain sequences set forth in SEQ ID NO: 49 or a sequence that is at least 90% identical thereto. In some embodiments, the humanized antibody is an IgG1 comprising the heavy chain sequence set forth in SEQ ID NO: 50 or a sequence at least 90% identical thereto.

In some embodiments, the humanized antibody comprises the light chain sequence set forth in SEQ ID NO: 49.

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Therapeutic methods

In some embodiments of the invention disclosed herein, anti-hPVR antibodies useful for treating cancer or tumor are provided. Treatment refers to a method aimed at improving or alleviating the condition being treated. For cancer, treatment includes, but is not limited to: reducing tumor volume, reducing tumor growth, increasing progression-free survival or overall life expectancy. In some embodiments, the treatment will affect remission of the cancer being treated. In some embodiments, treatment includes administration of a prophylactic or maintenance dose designed to prevent recurrence or progression of a previously treated cancer or tumor. Those skilled in the art will appreciate that not all individuals will respond or fail to respond equally to treatment, but those individuals will nonetheless be considered to have received treatment.

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

In some embodiments, an anti-hPVR antibody or antigen-binding fragments thereof described herein are intended to treat a cancer or tumor that is refractory to treatment with a checkpoint inhibitor as monotherapy. Refractory cancer is a cancer/tumor that develops progressive disease despite treatment with a single checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a PD-1, PD-L1, or PD-L2 inhibitor. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is an antibody or antigen binding fragment that specifically binds to PD-1 (CD279), including pembrolizumab, nivolumab, AMP-514, spartalizumab, tislelizumab (BGB- A317) or their PD-1(CD279)-binding fragment. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is a PD-L2-Fc fusion protein (eg, AMP-224). In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor comprises an antibody or PD-L1 binding fragment that specifically binds to PD-L1 (CD274). In some embodiments, an antibody or antigen-binding fragment that specifically binds to PD-L1 (CD274) includes durvalumab (MEDI 4376), atezolizumab, avelumab, BMS-936559, or FAZ053, or a PDL1(CD274)-binding fragment thereof. In some embodiments, a PD1, PD-L1, or PD-L2 inhibitor comprises an antibody or PD-L2 binding fragment thereof that specifically binds PD-L2 (CD273). In some embodiments, the PD-1, PDL1, or PD-L2 inhibitor comprises one or more small molecule inhibitors, such as H-{2-[({2-methoxy6-[(2-methyl[1,1'-biphenyl]- 3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS202); (2-((3cyanobenzyl)oxy)-4-((3 -(2,3-dihydro-benzo[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 -(2,3dihydrobenzo[b][1,4]dioxin-6-yl)-2 -methylbenzyl)oxy)benzyl)-4-hydroxy-pyrrolidine-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-1Nindol; L-a-glutamine; N^6-bis(L-seryl-E-asparaginyl-E-threonyl-E-seryl-E-a-glutamyl-E-seryl-Ephenylalanyl)-E-lysyl-E-phenylalanyl-E-arginyl-E-valyl -E-threonyl-E-glutaminyl-E-leucyl-E-alanyl-prolyl-E-lysyl-E-alanyl-E-glutaminyl-E-isoleucyl-L-lysyl; (2S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1'biphenyl]-3-yl)methoxy]phenyl]methyl]-2-piperidinecarboxylic acid; glycinamide; N-(2mercaptoacetyl)-E-phenylalanyl-Nmethyl-E-alanyl-E-asparaginyl-E-prolyl-E-histidyl-E-leucyl-N methylglycyl-E-tryptophil-E-seryl-E-tryptophil-Nmethyl -E-norleucyl-Nmethyl-E-norleucyl-Earginyl-L-cysteinyl-cyclic (1 ^ 14)-thioether; or their derivative or analogue.

In some embodiments, an anti-hPVR antibody or antigen binding fragments thereof are provided for use in combination with a PD-1, PD-L1, or PDL2 inhibitor. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is an antibody or antigen binding fragment that specifically binds to PD-1 (CD279), including pembrolizumab, nivolumab, AMP-514, spartalizumab, tislelizumab (BGB- A317), or their PD1(CD279)-binding fragment. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor is a PD-L2-Fc fusion protein (eg, AMP-224). In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor comprises an antibody or PD-L1 binding fragment that specifically binds to PD-L1 (CD274). In some embodiments, an antibody or antigen binding fragment that specifically binds to PD-L1 (CD274) includes durvalumab (MEDI4376), atezolizumab, avelumab, BMS-936559 or FAZ053, or a PD-L1 (CD274) binding fragment thereof. In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor comprises an antibody or PD-L2-binding fragment thereof that specifically binds PD-L2 (CD273). In some embodiments, the PD-1, PD-L1, or PD-L2 inhibitor comprises one or more small molecule inhibitors, such as

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F{2-[({2-methoxy-6-[(2-methyl[1,G-biphenyl]-3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS 202); (2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)5-methylbenzyl)- O-serine hydrochloride; (2R,4R)-1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3dihydrobenzo[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-1Nindol; L-a-glutamine; J.J>-bis(G-seryl-G-asparaginyl-G-threonyl-G-seryl-G-a-glutamyl-G-seryl-Gphenylalanil)-G-lysyl-G-phenylalanyl-G-arginyl-G- valyl-G-threonyl-G-glutaminyl-G-leucyl-G-alanylG-prolyl-G-lysyl-G-alanyl-G-glutaminyl-G-isoleucyl-G-lysyl; (2S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1'biphenyl]-3-yl)methoxy]phenyl]methyl]-2-piperidinecarboxylic acid; glycinamide, N-(2mercaptoacetyl)-G-phenylalanyl-Gmethyl-G-alanyl-G-asparaginyl-G-prolyl-G-histidyl-G-leucyl-G methylglycyl-G-tryptophil-G-seryl-G-tryptophil-Gmethyl -G-norleucyl-Gmethyl-G-norleucyl-Garginyl-G-cysteinyl-cyclic (1^14)-thioether; or their derivative or analogue.

In some embodiments, an anti-hPVR antibody or antigen binding fragments thereof are provided for use in combination with an EGFR inhibitor or an EGFR binding antibody.

In some embodiments, anti-hPVR antibodies or antigen binding fragments thereof are for the treatment of cancer or tumor. In some embodiments, the cancer or tumor is a solid cancer or tumor. In some embodiments, the cancer or tumor is a hematologic cancer or tumor. In some embodiments, the cancer or tumor includes cancer of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone brain, blood, thymus, uterus, testicles and/or liver. In some embodiments, tumors that can be treated with antibodies of the invention include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and/or teratoma. In some 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 tumors, Bartholin's gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoma , carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia, gastronoma, germline tumors, glioblastoma, glucagonoma, hemangioblastoma stoma, hemangioendothelioma, hemangioma , hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinitis, intraepithelial neoplasia, intraepithelial squamous neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung carcinoma, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelial tumor, membrane tumor nerve, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, blastic carcinoma of the prostate, renal cell carcinoma, re tinoblastoma, rhabdomyosarcoma , sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue cancer, somatostatin-secreting tumor, epidermoid carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal/vulvar cancer, VIPPoma and Wilms tumor. In some embodiments, the tumor/cancer treatable with one or more antibodies of the invention includes brain cancer, head and neck cancer, colorectal carcinoma, acute myeloid leukemia, pre-B cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell cancer and non-small cell cancer, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma and breast cancer, preferably ductal breast cancer and/or breast carcinoma. In some embodiments, the cancer that is treated with the antibodies of the present invention includes glioblastoma. In some embodiments, the cancer that is treated with one or more antibodies of the present invention includes pancreatic cancer. In some embodiments, the cancer that is treated with one or more antibodies of the present invention includes ovarian cancer. In some embodiments, the cancer that is treated with one or more antibodies of the present invention includes lung cancer. In some embodiments of the invention, cancer,

- 22 045980 which is treated with one or more antibodies of the present invention includes prostate cancer. In some embodiments, the cancer that is treated with one or more antibodies of the present invention includes colon cancer. In some embodiments, the cancer being treated includes glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In a certain embodiment of the invention, the cancer is not responsive to other treatment. In a certain embodiment of the invention, the cancer being treated is recurrent. In a certain embodiment, the cancer is relapsed/refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.

It will be apparent to those skilled in the art that the therapeutically effective amount of a molecule of the present invention will depend on, among other things, the schedule of administration, the unit dosage 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 stability in the blood circulation and the opinion of the attending physician.

In some embodiments, antibodies can be administered to a subject in need thereof by any route suitable for administering pharmaceutical compositions containing antibodies, such as subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral, etc. In some embodiments, the antibodies are administered intravenously. In some embodiments, the antibodies are administered subcutaneously. In some embodiments, the antibodies are administered intratumorally. In some embodiments, the antibodies are administered at a suitable dosage schedule, for example, weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once a month, and the like. In some embodiments, the antibodies are administered once every three weeks. Antibodies can be administered in any therapeutically effective amount. In some embodiments, the therapeutically acceptable amount is in the range of from about 0.1 mg/kg to about 50 mg/kg. In some embodiments, the therapeutically acceptable amount is in the range of from about 1 mg/kg to about 40 mg/kg. In some embodiments, the therapeutically acceptable amount is in the range of from about 5 mg/kg to about 30 mg/kg. Therapeutically effective amounts include amounts sufficient to relieve one or more symptoms associated with the disease or illness being treated.

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

T cell therapy uses a chimeric antigen receptor (CAR) to treat cancer or tumors (CAR-T cell therapy). CAR-T cell therapy is a cellular immunotherapy that involves injecting a cancer patient with genetically modified T cells that target tumor cells and cause apoptosis of the tumor cells. Genetically engineered T cells are produced by expressing in a T cell a CAR containing the antibody variable regions (VL and VH) in combination with an intracellular domain, such as a fragment of the CD3ζ chain sequence, using a gene transfer method. CAR is a general term for a chimeric protein in which the variable regions of the light and heavy chains of a tumor antigen-specific monoclonal antibody are linked to each other and then linked to the T cell receptor (TCR) chain at the end.

In 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 CD3ζ domain. In some embodiments, the CAR contains a CD8 Stalk domain. In some embodiments, the CAR contains a CD28 TM domain. In some embodiments, the CAR contains a CD3Z signaling domain. In some embodiments, the CAR contains a 41BB domain. In some embodiments, the CAR comprises a CD8 Stalk domain, a CD28 TM domain, a 41BB domain, and a CD3ζ domain.

According to some embodiments of the invention, a chimeric antigen receptor (CAR) is provided comprising a heavy chain variable region (VH) and a light chain variable region (VL) of the invention. According to some embodiments of the invention, a genetically modified lymphocyte is provided that expresses a CAR on its surface. According to some embodiments of the invention, a genetically modified T cell is provided that expresses a CAR on its surface (CAR-T cell).

In some embodiments, the CAR comprises a combination of heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90, 92, 94, 96, or 98% identical to the sequence.

- 23 045980 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 contains an amino acid sequence that is at least 90, 92, 94, 96, or 98% 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 some embodiments, the CAR comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID nO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID nO : 6, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

In some embodiments, the CAR comprises a combination of heavy and light chain variable regions of a humanized antibody, wherein the combination is selected from the group consisting of:

i) the heavy chain variable region sequence shown in SEQ ID NO: 1 and the light chain variable region sequence shown in SEQ ID NO: 9;

ii) the heavy chain variable region sequence shown in SEQ ID NO: 3 and the light chain variable region sequence shown in SEQ ID NO: 9;

iii) the heavy chain variable region sequence shown in SEQ ID NO: 4 and the light chain variable region sequence shown in SEQ ID NO: 9;

iv) the heavy chain variable region sequence shown in SEQ ID NO: 5 and the light chain variable region sequence shown in SEQ ID NO: 9; And

v) the heavy chain variable region sequence shown in SEQ ID NO: 6 and the light chain variable region sequence shown in SEQ ID NO: 9.

In 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 SEQ ID NO: 9, transmembrane domain and intracellular T cell signaling domain.

In some embodiments, the CAR comprises the scFv sequence set forth in SEQ ID NO: 56 or SEQ ID NO: 57, or an analogue thereof, that has at least 90%, 92%, 94%, 96%, or 98% sequence similarity to any of these sequences. In accordance with a certain aspect, the present invention relates to a cell containing a CAR described herein. In some embodiments, the cell expresses or is capable of expressing a CAR of the present invention. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is selected from a T cell and a natural killer (NK) cell.

According to some embodiments of the invention, a lymphocyte is provided that is engineered to express a CAR described herein. According to some embodiments of the invention, a T cell engineered to express a CAR described herein is provided.

In accordance with additional embodiments of the invention, an NK cell engineered to express a CAR described herein is provided.

The present invention also discloses methods for diagnosing and prognosticating cancer.

In accordance with one aspect, the present invention provides a method for diagnosing and/or prognosticating cancer or an infectious disease in a subject, comprising the step of determining the level of PVR expression in a biological sample of the subject using at least one antibody described herein.

Pharmaceutically acceptable excipients, carriers and diluents

In some embodiments, the anti-PVR antibodies of the present disclosure are included in a pharmaceutical composition containing one or more pharmaceutically acceptable excipients, carriers, and diluents. The carrier(s) must be pharmaceutically acceptable in the sense that it must be compatible with the other ingredients of the composition and not be unduly harmful to the recipient. The active agent is provided in an amount effective to achieve the desired pharmacological effect as described above and in an amount suitable to achieve the desired effect.

In some embodiments, antibodies as described herein are administered as a suspension in a sterile solution. In some embodiments, the solution contains about 0.9% NaCl. In some embodiments, the solution contains about 5.0% dextrose. In some embodiments, the solution further contains one or more buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20) and poloxamer 188; polyol/disaccharide/polysaccharides, for example glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose and dextran 40; amino acids, such as glycine or arginine; antioxidants, for example, ascorbic acid, methionine; or

- 24 045980 chelating agents, for example EDTA or EGTA.

Typically, the antibodies, fragments thereof, and conjugates of the present invention, comprising an antigen-binding moiety of the antibody or containing another polypeptide, including a peptidomimetic, are suspended in sterile saline for therapeutic use. Alternatively, pharmaceutical compositions can be formulated to provide controlled release of the active ingredient (the molecule containing the antigen-binding portion of the antibody) or prolong its presence in the patient's body. Numerous suitable drug delivery systems are known, which include, for example, implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release formulations can be formulated using polymers to complex or adsorb the molecule of the present invention. For example, biocompatible polymers include ethylene-vinyl acetate copolymer matrices and polyanhydride copolymer stearic acid-sebaric acid dimer matrices. The rate of release of the molecule of the present invention from such a matrix, i.e. antibody or antibody fragment depends on the molecular weight of the molecule, the amount of the molecule in the matrix and the size of the dispersed particles.

In some embodiments, antibodies as described herein are delivered/stored in lyophilized form and reconstituted prior to administration. In some embodiments, lyophilized antibody formulations contain an excipient such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or combinations thereof. The lyophilized formulation may be contained in a vial of glass or other suitable non-reactive material. As-formulated antibodies, whether reconstituted or unreconstituted, may be buffered at a specific pH, typically less than 7.0. In some embodiments, the pH may be from 4.5 to 6.5, from 4.5 to 6.0, from 4.5 to 5.5, from 4.5 to 5.0, or from 5.0 to 6 ,0.

In some embodiments, lymphocytes carrying a CAR described herein are transported/stored prior to use. Cells are usually cryopreserved if they are not used immediately. Cryopreservation methods and storage media suitable for CAR-bearing cells are known in the art, see, for example, Wang, et al. 2019 Mau;21(5):566-578. Also described herein are kits containing one or more antibodies described herein in a suitable container and one or more additional components selected from: instructions for use; diluent, excipient, carrier and administration device. In some embodiments, the kit contains means for measuring human PVR expression.

Some embodiments of the invention described herein relate to a method of preparing an agent for treating cancer, comprising mixing one or more pharmaceutically acceptable excipients, carriers or diluents and an antibody as described herein. Some embodiments of the invention described herein relate to a method of preparing a cancer treatment agent for storage or transportation, comprising lyophilizing one or more antibodies as described herein.

Examples

The following illustrative examples represent embodiments of the compositions and methods described herein and should not be construed as limiting in any way.

Example 1: Improved PVR Binding Affinity by N56E and N56D Variants.

The variable region of the chimeric anti-PVR antibody 5B9 disclosed in WO 2017149538 includes a deamidation sequence (asparagine-glycine) in the light chain CDR2 (WASSRHNG, SEQ ID NO: 17). Seven chimeric variants were obtained by introducing a point mutation at the position of the asparagine residue N56. To assess the binding affinity of N56 substitution variants, wild-type (WT) and IgG4 substitution variant (S241P) monoclonal antibodies were immobilized on a Protein A capture chip. Binding was tested for the PVR analyte conjugated to a histidine tag (PVR-HIS, Sino Cat. No. 10109-H08H). Dilution range: five points with 2-fold dilution from 50 nM to 3.125 nM. Conditions used: Instrument: Biacore T200 (serial number 1909913) with Biacore T200 Evaluation Software V2.0.1. Working buffer: HBS-P+, 300 mM NaCl, 1 mg/ml BSA. Flow rate: 30 µl/min. Association: 350 s, dissociation: 800 s. Recovery: 10 mM glycine, pH 1.5. Analysis: 1:1 binding. The relative KD for each variant with substitution was established by dividing the KD of the variant with substitution by the KD of the parent variant N56 (VH0VK0). Significant (>25%) improvements in affinity were noted for the N56E (Asp) and N56D (Glu) variants (Fig. 1A). Binding of the chimeric variants to human PVR expressed on HEK293 EBNA cells was assessed by flow cytometry in a competition assay using the parental antibody 5B9 (WT, Fig. 1B).

Example 2: Improved cross-reactivity for simian PVR binding to N56E and N56D variants.

We studied the binding of antibody variants with the N56 substitution to cells that bind human PVR (protein id: Q92692Q92692) and chlorocebus (African green monkey, protein id: UniProtKB

- 25 045980

P32506). In fig. 2A shows the relative binding of all variants added at saturating concentration (10 μg/ml) to NCI-H1975 cells expressing human PVR. In fig. 2B shows the relative binding of all variants added at saturating concentration (10 μg/ml) to Vero cells expressing Chlorocebus PVR. For detection, goat anti-human antibody 647 (Jackson immunoassay 109-606-088) was used at a dilution of 1:250. Antibody binding of cells was analyzed by FACS. The fold change was calculated by dividing the MFI of each variant by the MFI of the parent antibody (K0). A significant (>25%) increase in cross-reactivity was observed for the N56E and N56D variants.

Example 3: Improvement of NK cell activation by N56E and N56T variants.

NK cells from healthy donors were incubated in the presence of selected N56 substitution variants and the target breast cancer cell line (MDA-MB-231) at an E:T (effector:target) ratio of 2:1 for 2 h at 37°C. NK cell activation was measured by induction of CD107a surface expression, and the fold change relative to control IgG was calculated for each variant (y-axis). All monoclonal antibodies were used at a concentration of 600 pM (0.09 μg/ml). (*p<0.04, **p<0.01 by two-sided Student's t -test). As shown in FIG. 3, N56E and N56T variants showed improved NK cell activation compared to K0, as evidenced by increased CD107a expression.

Example 4: Enhanced CD8 T Cell Proliferation by N56E and N56T Variants.

Human PBMCs were fluorescently labeled with CFSE (C34554 ThermoFischer) and incubated with targeted A549 breast cancer cells in the presence of 2.5 μl/ml PHA-L (Roche) and the indicated antibody variants at a concentration of 4 μg/ml. After incubation for 96 hours, immune cells were collected, stained with anti-human CD8, and analyzed by FACS. Cellular proliferation of CD8+ T cells was assessed by CFSE signal intensity. CFSE levels for IgG-treated cells were set to 1. Results were reported as fold increase in proliferation compared to that control. Since increased proliferation leads to decreased CFSE signal, the y-axis displays the inverse of this relationship. Experiments were carried out in four repetitions. Results for one PBMC donor are shown. The findings suggest that the N56E and N56T variants have a significantly stronger effect on CD8+ T cell proliferation in the presence of tumor cells compared to the parent antibody (Fig. 4; *p<0.05, **p<0.01 by two-tailed Student's t-test).

Example 5: Identification of a humanized 5B9 variant with improved reproducibility.

The N56E antibody variant performed best in the competition assay and was selected as the lead variant for humanization. Based on structural analysis, a large preliminary set of sequence segments was identified and used to generate humanized 5B9 variants. These segments were selected and analyzed using iTope™ technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al. 2008) and using the TCED™ (T cell epitope database) of known T cell epitopes related to antibody sequences (Bryson et al 2010). Sequence segments that were identified as non-human germline fragments binding to human MHC class II, or those that showed significant overlap with the TCED™ base, were discarded. This resulted in a reduced set of segments, and their combinations were further analyzed as described above to ensure that segment junctions did not form potential T cell epitopes. Selected segment sequences were assembled into complete V region sequences that were devoid of significant T cell epitopes. Five heavy chain sequences (VH1 to VH5) and 4 light chain sequences (containing the N56E substitution) (Vk1 to Vk4) were then selected.

Table 1

Variable regions and CDR sequences

SEQ ID NO: Subsequence Chain 1 QVQLVQSGAEVKKPGASVKVSCKATGYTFSNYWIEWVR QAPGQGLEWIGEIFPGSGRINFNEKFKGRVTFTADTSISTT YMELSRLRSDDTAVYYCARTKIYGNSFDYWGQGTLVTVS S VH4

- 26 045980

2 DIQMTQSPSSSLSASVGDRVTITCKASQDVGTAVVWYQQK PGKAPKLLIYWASSRHEGVPDRF SSGSGSGTDFTLTIS SLQP EDFADYFCQQYSRYPLTFGQGTKLEIK VK2 3 QVQLVQSGAELKKPGASVKISCKATGYTFSNYWIEWIKQ APGQGLEWIGEIFPGSGRINFNEKFKGR ATFTADTSIDTTYMQLSSLTSDDSAVYYCARTKIYGNSFD YWGQGTTVTVSS VH1 4 QVQLVQSGAEVKKPGASVKISCKATGYTFSNYWIEWIKQ APGQGLEWIGEIFPGSGRINFNEKFKGRATFTADTSIDTTY MELSRLRSDDTAVYYCARTKIYGNSFDYWGQGTLVTVSS VH2 5 QVQLVQSGAEVKKPGASVKVSCKATGYTFSNYWIEWIKQ APGQGLEWIGEIFPGSGRINFNEKFKGR VTFTADTSISTTY MELSRLRSDDTAVYYCARTKIYGNSFDYWGQGTLVTVSS VH3 6 QVQLVQSGAEVKKPGASVKVSCKATGYTFSNYWIEWVR QAPGQGLEWMGEIFPGSGRINFNEKFKGRVTFTADTSIST AYMELSRLRSDDTAVYYCARTKIYGNSFDYWGQGTLVT vss VH5 7 DIMMTQSPSFLSASVGDRVTITCKASQDVGTAVVWYQQK PGKAPKLLIYWASSRHEGVPDRFTGSGSGTDFTLTISSLQS EDFADYFCQQYSRYPLTFGQGTKLEIK VK1 8 DIQMTQSPSSLSSASVGDRVTITCRASQDVGTAVVWYQQK PGKAPKLLIYWASSRHEGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQYSRYPLTFGQGTKLEIK VK3 9 DIQMTQSPSSLSASVGDRVTITCRASQDVGTAVAWYQQK PGKAPKSLIYW AS SRHEGVPSRF SGSSGSGTDFTLTIS SLQP EDFATYFCQQYSRYPLTFGQGTKLEIK VK4 10 NYWIE HCCDR1 AND EIFPGSGRINFNEKFKG HCCDR2 12 TKIYGNSFDY HCCDR3 13 X1ASQDVGTAVX2 Xi=K or R, X ₂ =V/A LCCDR1 14 WASSRHE LCCDR2 15 QQYSRYPLT LCCDR3 16 QVQLQQSGAELMKPGASVKISCKATGYTFSNYWIEWIKQ RPGHGLEWIGEIFPGSGRINFNEKFKGKATFTADTSSDTTY MQLSSLTSADSAVYYCARTKIYGNSFDYWGQGTTLTVSP VHO 5B9 17 DIMMTQSHKFMSTSVGDRVNITCKASQDVGTAVVWYQQ KPGQSPKLLIYWASSRHNGVPDRFTGSGSGTDFTLTISNV QSEDLSDYFCQQYSRYPLTFGAGTKLELK VKO 5B9

- 27 045980

Table 2 Sequences of framework regions (non-CDRs) of humanized heavy chain variable regions

Chain FR-H1 FR-H2 FR-H3 FR-H4 VH4 QVQLVQSGAEV KKPGASVKVSC KATGYTFS (SEQIDNO: 18) WVRQAPGQGLE WIG (SEQ ID NO: 19) RVTFTADTSISTT YMELSRLRSDDT AVYYCAR (SEQ ID NO: 20) WGQGTLVTVSS (SEQ ID NO: 21) VH 1 QVQLVQSGAEL KKPGASVKISCK ATGYTFS (SEQIDNO: 22) WIKQAPGQGLE WIG (SEQ ID NO: 23) RATFTADTSIDT TYMQLSSLTSDD SAVYYCAR (SEQ ID NO: 24) WGQGTTVTVSS (SEQ ID NO: 25) VH2 QVQLVQSGAEV KKPGASVKISCK ATGYTFS (SEQ ID NO: 26) WIKQAPGQGLE WIG (SEQ ID NO: 23) RATFTADTSIDT TYMELSRLRSDD TAVYYCAR (SEQ ID NO: 27) WGQGTLVTVSS (SEQ ID NO: 21) VH3 QVQLVQSGAEV KKPGASVKVSC KATGYTFS (SEQ ID NO: 18) WIKQAPGQGLE WIG (SEQ ID NO: 23) RVTFTADTSISTT YMELSRLRSDDT AVYYCAR (SEQ ID NO: 20) WGQGTLVTVSS (SEQ ID NO: 21) VH5 QVQLVQSGAEV KKPGASVKVSC KATGYTFS (SEQ ID NO: 18) WVRQAPGQGLE WMG (SEQ ID NO: 28) RVTFTADTSIST AYMELSRLRSD DTAVYYCAR (SEQ ID NO: 29) WGQGTLVTVSS (SEQ ID NO: 21)

Table 3

Framework sequences (not CDRs) of humanized ______________ light chain variable regions ____________

Chain FR-L1 FR-L2 FR-L3 FR-L4 VK2 DIQMTQSPSSLS ASVGDRVTITC (SEQ ID NO: 30) WYQQKPGKAPK LLIY (SEQIDNO: 31) GVPDRFSGSGSG TDFTLTISSLQPE DFADYFC (SEQ ID NO: 32) FGQGTKLEIK (SEQ ID NO: 33) VK 1 DIMMTQSPSFLS ASVGDRVTITC (SEQ ID NO: 34) WYQQKPGKAPK LLIY (SEQIDNO: 31) GVPDRFTGSGSG TDFTLTISSLQSE DFADYFC (SEQ ID NO: 35) FGQGTKLEIK (SEQ ID NO: 33) VK3 DIQMTQSPSSLS ASVGDRVTITC (SEQ ID NO: 30) WYQQKPGKAPK LLIY (SEQIDNO: 31) GVPSRFSGSGSG TDFTLTISSLQPE DFATYFC (SEQ ID NO: 36) FGQGTKLEIK (SEQ ID NO: 33) VK4 DIQMTQSPSSLS ASVGDRVTITC (SEQ ID NO: 30) WYQQKPGKAPK SLIY (SEQ ID NO: 37) GVPSRFSGSGSG TDFTLTISSLQPE DFATYFC (SEQ ID NO: 36) FGQGTKLEIK (SEQ ID NO: 33)

All variants were tested for binding by SPR (Fig. 5A), and variants with 2-fold different affinities from the parent antibody were tested for binding to cell surface PVR using flow cytometry (Fig. 5B). With the exception of the Vk4-containing variants, all variants showed binding levels similar to the parental chimeric (mouse/human) molecule containing the N56E substitution (IgG4 (S241P) N56E_VHO/VKO). It was noted that humanization resulted in the removal of an N-glycosylation site at position N20 of the FR1 region of the light chain.

Expression levels after transient expression in HEK293 EBNA cells and similarity to the human germline sequence were taken into account to select the lead candidate (Fig. 6). In fig. Figure 6A summarizes the titers of all variants after transient transfection. Option VH4/Vk2 showed sa

- 28 045980 high expression titers and has a high percentage of sequence identity with human germline genes (Fig. 6B). Finally, the reproducibility of the VH4/Vk2 variant (NB1088) was assessed in comparison with a variant identical to NB1088 but with the parent deamidating competent LC CDR2 from 5B9 (WASSRHNG), designated NB0941. The biophysical properties of NB0941 and NB1088 were determined. As shown in FIG. 7A and 7B, high pH stress and incubation at 40°C revealed changes in capillary isoelectric focusing (cIEF), particularly an increase in the percentage of acidic species, possibly due to deamidation. These changes were more pronounced in NB0941 compared to NB1088. Therefore, NB1088, with its optimized immunogenicity, expression and binding profile, as well as desirable biophysical properties, was selected as the lead humanized variant for functional analysis.

The reduction in affinity observed in some embodiments is particularly useful in CAR driver development given the fact that normal tissues express minimal levels of PVR. The results (Figure 16) show that PVR is overexpressed in a variety of tumors, allowing PVR-driven CAR-T to effectively target these tumors. The potential safety issue can be easily addressed by using affinity-reduced anti-PVR variants as described by Liu et al. (Cancer research, 2015; Volume 75, Issue 17).

Example 6 NB1088 inhibits PVR binding to TIGIT, CD96 and CD226.

NB1088 was tested for its ability to block the binding of TIGIT, CD96, and CD226 to PVR. Dissociated CHO (Chinese hamster ovary) cells stably expressing human PVR were incubated with NB1088 at the indicated concentrations for 20 min on ice, followed by the addition of 10 μg/ml biotinylated recombinant TIGIT, CD96, or CD226-Fc, respectively, and kept on ice for an additional 120 min. After washing, surface-bound NB1088 was detected using a secondary antibody conjugated to anti-human Alexa-488, and biotinylated proteins were detected using Alexa647-conjugated streptavidin and analyzed using flow cytometry. In fig. 8A shows that NB1088 binds to PVR with an EC ₅₀ of about 3.3 nM. The IC ₅₀ for NB1088 to compete with TIGIT, CD96, or CD226 for binding to PVR is 1.1, 1.1, and 1.9 nM, respectively, as shown in FIG. 8B-8D.

Example 7: NB1088 stimulates cytotoxic T and NK cells.

The ability of NB1088 to stimulate T and NK cell activity in vitro was determined. Using an antigen-specific human papillomavirus (HPV)-based technique, 30,000 HPV+ human cervical epidermoid carcinoma cell line cells (CaSki cells) and 30,000 HPV-specific CD8 T cells were co-incubated with control IgG or NB1088 at a concentration of 10 μg/ml for nights. The release of interferon gamma into the supernatant was determined using the human interferon gamma-specific MSD system. As shown in FIG. 9A, NB1088 increased interferon-gamma release from human HPV-specific CD8+ (cytotoxic) T cells when incubated with HPV+ CaSki cells. To test CD8 T cell activity in an allogeneic system, PBMCs were preactivated for three days with phytohemagglutinin (PHA) and interleukin 2 (TL2), maintained overnight in the absence of PHA/IL2 before isolating CD8 T cells using a negative isolation technique. using magnetic separation. 10,000 A549 tumor cells and 100,000 healthy donor CD8+ T cells were cocultured overnight in the presence of 100 U/ml IL2 and 1 μg/ml anti-CD28 antibody. As shown in FIG. 9B, NB1088 increased the release of interferon gamma from CD8+ T cells to a greater extent than the anti-PD-1 antibody (pembrolizumab), and it was further increased by the combination of NB1088 and the anti-PD-1 antibody.

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

Example 8: NB1088 restores CD226 expression and activity in CD8 T and NK cells.

The ability of NB1088 to influence 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 promotes tumor killing by CD8+ T and NK cells. In terms of its function, it is opposed by TIGIT and CD96, which are inhibitory molecules expressed on T and NK cells. Thus, the gain of CD226 function due to NB1088 indicates that

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NB1088 will enhance T and NK cell activity and have broad spectrum antitumor activity. The effect of NB1088 on CD226 expression and function was tested in antigen-specific and allogeneic coculture systems as described above. As shown in FIG. 11A and 11B, coculture of CD8 T cells and NK cells with PVR+ target cells resulted in a strong reduction in the surface expression of CD226 on CD8 T cells and NK cells. NB1088 restored CD226 expression on the cell surface of CD8 T cells or NK cells, regardless of the coculture system (Fig. 11A and B), in contrast to anti-TIGIT. The functional consequences of increased CD226 expression on T and NK cells following NB1088 treatment were assessed using the antigen-specific and allogeneic coculture systems described above with minor modifications (Fig. 12A and B). Increased expression of CD226 correlated with significantly higher levels of interferon-gamma release following treatment with NB1088 compared to treatment with control IgG or anti-TIGIT (Fig. 12A and B). The superior T and NK cell activity upon NB1088 treatment was at least partially mediated by CD226 activity. Anti-CD226 (DX11, 20 μg/ml) reduced NB1088-dependent interferon gamma release by both allo- and antigen-stimulated CD8 T cells (Fig. 12A) and NK cells after coculture with A549 (Fig. 12B) to levels observed with anti-TIGIT. These data demonstrate that NB1088 improves T and NK cell activity compared with TIGIT blockade by increasing CD226 expression and/or function.

Example 9 Efficacy of NB1088 monotherapy in humanized tumor xenograft mouse models.

The ability of NB1088 to kill A549 (lung adenocarcinoma) or HPAF (pancreatic) tumors was determined in a humanized mouse model. Briefly, 5 x 10 ⁶ tumor cells (A549 or HPAF) were mixed in Matrigel with human activated peripheral blood mononuclear cells in a 1:1 ratio and implanted subcutaneously in the flank of immunodeficient NOD/SCID mice (12 animals per condition). As shown in FIG. 13A and 13B, NB1088 was able to reduce tumor volume at least as well as the anti-PD-1 antibody pembrolizumab. NB1088 was also able to reduce tumor volume in the A549/PBMC model (Fig. 13C), but not when A549 cells alone were used (Fig. 13D). In the A549/PBMC model, decreased tumor volume correlated with increased expression of CD226 on CD8 T cells isolated from tumors treated with NB1088 (Fig. 13E). The effect of NB1088 on CD8 T cell effector function ex vivo was also assessed (FIG. 14A and B). Suspensions of lysed single cells from tumors were stimulated with anti-CD28/anti-CD3 in the presence of brefeldin A and αanti-CD107a for 5 h at 37°C. After stimulation, cells were stained using standard surface/intracellular staining techniques to assess interferon gamma production by CD8+ T cells using flow cytometry. As shown in FIG. 14A and 14B, NB1088 increased the frequency of total interferon gamma positive (Fig. 14A) and multifunctional interferon gamma/CD107a double positive (Fig. 14B) tumor-derived CD8 T cells. In addition, the increased frequency of interferon gamma positive CD8+ T cells in tumors treated with NB1088 was derived exclusively from CD226 positive CD8+ T cells (Figures 14C and 14D), suggesting an important contribution of CD226 function in vivo to the antitumor activity of NB1088.

Example 10: Pharmacokinetics of NB1088 and pharmacodynamic changes in CD226 expression on cynomolgus CD4 T cells.

The pharmacokinetic properties of NB1088 were measured after a single or 4x1 weekly intravenous bolus injection at doses of 2, 50, or 200 mg/kg in cynomolgus monkeys (2 female monkeys per dose). In addition, changes in CD226 expression on circulating peripheral CD4 T cells were assessed. In fig. 15A shows plasma concentrations of ⁴ ® 1088 (µg/ml) as a function of time (hours) and dose. IC90 and 10x _IC90 were calculated from in vitro activity assays using the cynomolgus monkey PBMC assay. NB1088 exhibited a typical pharmacokinetic profile and achieved concentrations above 10x IC90 throughout the study after a repeat dose of 200 mg/kg. In fig. 15B shows CD226 expression levels on circulating CD4 T cells, normalized to predose, measured by flow cytometry with specific antibodies. NB1088 increased CD226 surface expression levels by up to 1.5-fold in the 50 mg/kg dose group and the 200 mg/kg repeat dose group, and remained elevated in the 200 mg/kg repeat dose group. These data indicate that NB1088 can trigger and modulate CD226 expression on cynomolgus CD4 T cells.

Example 11: Expression of human PVR in different tumor types.

PVR expression levels were assessed for human cancers of various origins. PVR expression was determined by standard immunohistochemistry methods using a commercially available rabbit monoclonal antibody clone D3G7H and cancer tissue microarrays. Staining was digitized and intensity quantified to calculate H-scores within and between trials. In fig. Figure 16 shows increased levels of PVR expression in most cancers, pro

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Claims (15)

analyzed in different replicates. Increased expression of PVR has been shown in liver cancer, colon cancer, adrenal cancer, uterine cancer, testicular cancer, squamous cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer, bladder cancer, prostate cancer, cholangiocarcinoma, skin cancer, HNSCC cancer , breast cancer, pancreatic cancer, non-small cell lung cancer and melanoma. These data suggest the contribution of PVR to tumor progression in various types of human cancer. Example 12 Construction of a Humanized Antibody. Humanized IgG antibodies were constructed based on one of the variants containing the heavy and light chains of VH4 and VK2, respectively. An example VK2 sequence is presented in SEQ ID NO: 49. An example VH4 sequence for hIgG4 (S241P) is presented in SEQ ID NO: 50, and for hIgG1 in SEQ ID NO: 51. Next, the following nucleotide sequences were obtained, optimized for the expression of amino acids sequences in CHO cells: for VK2 the nucleotide sequence is given in SEQ ID NO: 52 or SEQ ID NO: 53. For VH4 IgG4 the nucleotide sequence is given in SEQ ID NO: 54 or SEQ ID NO: 55. Example 12a. CAR-T cells expressing scFv derived from humanized variants of anti-PVR antibodies are specifically activated in the presence of tumor cells. The CAR-T design was developed from the H4K2-NTX-1088C and H3K4-NTX-1034C variants. The amino acid sequences of scFv are shown in SEQ ID NO: 56 and 57, respectively. Parental Jurkat cells or Jurkat cells overexpressing anti-hPVR CAR-T (40 k/well) were incubated with A549 or MDA-231 breast cancer cells (PVR positive) at an E:T ratio of 1:1 for 24 h. As shown in FIG. 18, both CAR-T drivers resulted in the secretion of hundreds of picograms of IL2, whereas IL2 secretion in parental Jurkat cells was undetectable in the presence of these targets. IL2 secretion was quantified using Biolegend hIL2 (cat. 431804). These results suggest that the aPVR-based CAR-T driver is highly functional in inducing T cell activation in the presence of PVR-expressing target cells. To study the destruction of tumor cells by CAR-T, 200 thousand A549 or MDA-231 cells were seeded into a 12-well plate with CAR-T-PVR variants (NTX-1088C or NTX1034C) at E:T 0.4 and 0.8 to 1 (based on positive GFP), respectively, in NK medium for 72 hours. Tumor cell death was assessed using a standard CTG protocol (Promega G9241). As shown in FIG. 19A-19C, both PVR variants demonstrated a greater than 2-fold increase in MDA-231 cell killing and an 8-fold increase in A549 cell killing compared to activated PBMCs. These results strongly suggest that aPVR CAR-T constructs significantly enhance the killing efficiency of PVR-expressing targets. Example 13: Effective killing of hematological target cells using aPVR CAR-T. The CAR-T design was developed from the H4K2-NTX-1088C and H3K4-NTX-1034C variants. The scFv sequences are shown in SEQ ID NO: 56 and 57, respectively. To test hematologic tumor cell killing by CAR-T, 20k/well K562 cells were seeded in 96-well plates either alone or with CAR-TPVR variants (NTX-1088C or NTX1034C) at E:T ranging from 3. 4 to 0.22 to 1, in RPMI supplemented with 100 IU IL-2/ml, and incubated for 18 hours. Tumor cell death was assessed using flow cytometry. Both the NTX-1034C and NTX-1O88C were extremely effective at destroying targets at high E:T values. The clear advantage of NTX-1088C over NTX-1034C at lower E:T is likely due to the moderate levels of PVR expressed on K562. These results suggest that aPVR CAR-T may be effective against PVR-expressing hematologic tumors. CLAIM 1. A humanized antibody that specifically binds to the human poliovirus receptor (PVR), or an antigen binding fragment thereof, comprising a heavy chain and a light chain, wherein the heavy chain variable region contains the amino acid sequence set forth in SEQ ID NO: 47, and the light chain variable region contains amino acid sequence shown in SEQ ID NO: 48. 2. A humanized antibody or an antigen-binding fragment thereof according to claim 1, where the heavy chain variable region contains: i) a set of three CDR sequences containing the sequences presented in SEQ ID NO: 10-12; and ii) a set of four heavy chain framework region (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 - 31 045980 (D) FR-H4, selected from the group consisting of SEQ ID NOs: 21 and 25. 3. A humanized antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1 or 2, wherein the light chain variable region comprises: i) a set of three CDR sequences containing the sequences presented in SEQ ID NO: 13-15; and ii) a set of four light chain framework sequences: (A) FR-L1 selected from the group consisting of SEQ ID NOs: 30 and 34; (B) FR-L2 selected from the group consisting of SEQ ID NOs: 31 and 37; (C) FR-L3 selected from the group consisting of SEQ ID NOs: 32, 35 and 36; and (D) FR-L4, which is SEQ ID NO: 33. 4. The humanized antibody or antigen binding fragment thereof according to claim 1, wherein the heavy chain variable region contains 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 contains 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. 5. The humanized antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region contains 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 contains 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. 6. A humanized antibody or antigen binding fragment thereof according to any one of claims 1 to 5, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1 and the light chain variable region contains the amino acid sequence set forth in SEQ ID NO: 2 . 7. A humanized antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the humanized antibody is an IgG antibody; or wherein the humanized antibody comprises an IgG4 or IgG1 heavy chain constant region. 8. The humanized antibody or antigen-binding fragment thereof according to claim 7, wherein the humanized antibody comprises an IgG4 heavy chain constant region comprising a substitution of a serine residue for a proline at position 228 of said IgG4 heavy chain constant region. 9. The humanized antibody or antigen binding fragment thereof according to any one of claims 1 to 5, wherein the humanized antibody or antigen binding fragment thereof is a single chain variable fragment (scFv) containing an amino acid sequence selected from SEQ ID NO: 56, SEQ ID NO: 57 , or an analogue thereof, at least 90% identical to any of the specified sequences. 10. The humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the humanized antibody inhibits the binding of poliovirus receptor (PVR) to at least one of T-cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 and CD226. 11. Nucleic acid encoding a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10. 12. A chimeric antigen receptor (CAR) comprising a combination of heavy and light chain variable region sequences of a humanized antibody according to any one of claims 1 to 10. 13. A pharmaceutical composition containing a humanized antibody or an antigen-binding fragment thereof according to any one of claims 1 to 10 or a CAR according to claim 12, as well as a pharmaceutically acceptable excipient, carrier or diluent. 14. Use of a humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a CAR according to claim 12, or a pharmaceutical composition according to claim 13 to increase the surface expression and/or signaling of CD226 in CD8+ T or NK cells. 15. Use of a humanized antibody or antigen binding fragment thereof according to any one of claims 1 to 10, a CAR according to claim 12 or a pharmaceutical composition according to claim 13 for the treatment of cancer in an individual. -