Anti-PVRIG antibodies, pharmaceutical compositions thereof and uses thereof

Abstract

The present invention provides an anti-PVRIG antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3. In the antibody heavy chain variable region, the amino acid sequence of HCDR1 is as shown in SEQ ID NO:9, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:10, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:11, and in the antibody light chain variable region, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:12, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:13, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:14.

Description

[Technical field]

[0001] The present invention belongs to the field of biomedicine and relates to anti-PVRIG antibodies, pharmaceutical compositions thereof and uses thereof. [Background technology]

[0002] Poliovirus receptor-related immunoglobulin domain-containing protein (PVRIG; also known as CD112R), a member of the PVR family with an IgV structural domain in its extracellular region and an immunoreceptor tyrosine-based inhibitor motif (ITIM) in its intracellular region, is a novel immunoinhibitory receptor expressed on natural killer (NK) cells and T cells.

[0003] Poliovirus receptor-related 2 (PVRL2, CD112, nectin-2) is an adhesion molecule involved in cell-cell junctions and is overexpressed in some cancers. PVRL2 has been identified as the only functional ligand for PVRIG, which binds with high affinity to PVRIG and inhibits immune activation of T cells and NK cells. PVRL2 is also a ligand for the coactivation receptor DNAX accessory molecule-1 (DNAM-1, CD226) and weakly binds to another inhibitory receptor with Ig and ITIM domains - T cell immunoreceptor (TIGIT). Thus, similar to the immunosuppressive mechanism of TIGIT, PVRIG, a member of the same family, may also act as an immunosuppressant by blocking the activation signal transmitted by CD226 through competitive binding to its ligand PVRL2.

[0004] PVRIG or its ligand PVRL2 is highly expressed in lung, kidney, endometrial, breast and skin cancers and their microenvironments, and PVRIG was also highly expressed on NK cells from prostate cancer patients (Whelan S, et al. Cancer Immunol Res. 2019;7(2):257-268.). Additionally, PVRIG is co-expressed with exhaustion markers TIGIT and PD-1 on CD8+ T cells, suggesting that PVRIG expression has some tumor specificity and may be associated with the activation / exhaustion state of TILs (Whelan S, et al. Cancer Immunol Res. 2019;7(2):257-268.). International Publication WO2021180205A1 reported that anti-PVRIG antibodies can be used to treat lung, breast, ovarian, kidney, gastric, endometrial and head and neck cancers.

[0005] Studies in preclinical mouse tumor models have shown that PVRIG depletion or blockade restores T / NK cell activity and promotes antitumor immune responses. CD8+ T cells isolated from Pmel-1 TCR-CD112R-deficient mice exhibit enhanced degranulation and cytokine secretion. MC38 tumor growth is slower in CD112R-deficient mice compared to wild-type mice, and CD8 +TILs show improved immune effects and upregulated transcriptome profiles of inflammation / cytotoxicity-related genes. Literature also reported that PVRIG antibody or the combination of PVRIG gene knockout mice and PD-L1 antibody provides better therapeutic efficacy than PD-L1 antibody alone against MC38 and CT26 mouse tumor models (Murter B, et al. Cancer Immunology Research, 2019.). Feng Xu et al. demonstrated that blocking the interaction of PVRIG with CD112 using an antibody improves the cytotoxicity of NK cells against tumor cells, and further combination with TIGIT blocking monoclonal antibody further improves the trastuzumab-mediated killing of breast cancer cells by NK cells (Xu F, et al. Cancer Immunology Immunotherapy Cii, 2017). In knockout mouse models, PVRIG antibodies reduced tumor growth in TIGIT- / - mice, and tumor growth control was further improved in PVRIG and TIGIT double knockout mice compared to single gene knockout mice (RJ Sullivan, et al, ASCO-SITC Clinical Immuno-Oncology Symposium, 2020).

[0006] Preclinical data suggests that PVRIG antibody can effectively stimulate anti-tumor immune responses in certain cancers, and it has been found that it can exert some synergistic effects with PD-1 / L1 and TIGIT antibodies, enhance the function of T / NK cells, improve anti-tumor responses, and slow tumor growth. PVRIG as a therapeutic target is expected to improve the therapeutic efficacy of existing antibody drugs (e.g., anti-TIGIT and anti-PD-1 / L1 antibodies), providing advantages over other drug targets and combination therapies in the clinic. COM701, developed by Compugen, is the world's first clinically approved PVRIG antibody, and is currently undergoing phase I trials in combination with TIGIT monoclonal antibody (COM902), and has signed an agreement with BMS to develop a phase I trial of the triple combination of COM701+BMS-986207+nivolumab. In addition, GSK4381562, manufactured by Surface Oncology and licensed by GSK, and JS009, manufactured by China's Junshi Biosciences, are due for clinical application by the end of 2021, and Amgen is also developing a PVRIG monoclonal antibody, which is currently in preclinical development.

[0007] Thus, there is a need in the art to develop new high affinity, selective, biologically active anti-PVRIG antibodies for the treatment of disease, particularly cancer. Summary of the Invention

[0008] After thorough research, the inventors of the present application obtained a novel anti-PVRIG antibody. The inventors surprisingly found that the anti-PVRIG antibody of the present invention exhibits excellent affinity and biological activity, and has potential for anti-tumor effect. This led to the following invention.

[0009] One aspect of the invention is an anti-PVRIG antibody or antigen-binding fragment thereof, comprising: the antibody comprises a heavy chain variable region comprising HCDR1 to HCDR3 and a light chain variable region comprising LCDR1 to LCDR3; The heavy chain variable region of the antibody is HCDR1 having the amino acid sequence shown in SEQ ID NO:9, HCDR2 having the amino acid sequence shown in SEQ ID NO:10, and HCDR3 having the amino acid sequence shown in SEQ ID NO:11; HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 17; HCDR1 having the amino acid sequence shown in SEQ ID NO: 21, HCDR2 having the amino acid sequence shown in SEQ ID NO: 22, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 23, or HCDR1 having the amino acid sequence shown in SEQ ID NO:27, HCDR2 having the amino acid sequence shown in SEQ ID NO:28, and HCDR3 having the amino acid sequence shown in SEQ ID NO:29. Including, The light chain variable region of the antibody is LCDR1 having the amino acid sequence shown in SEQ ID NO: 12, LCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 14; LCDR1 having the amino acid sequence shown in SEQ ID NO: 18, LCDR2 having the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 20; LCDR1 having the amino acid sequence shown in SEQ ID NO: 24, LCDR2 having the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 26, or LCDR1 having the amino acid sequence shown in SEQ ID NO: 30, LCDR2 having the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 32. The present invention relates to an anti-PVRIG antibody or an antigen-binding fragment thereof, comprising:

[0010] In some embodiments of the present invention, The heavy chain variable region of the antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO:9, an HCDR2 having the amino acid sequence shown in SEQ ID NO:10, and an HCDR3 having the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region of the antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO:12, an LCDR2 having the amino acid sequence shown in SEQ ID NO:13, and an LCDR3 having the amino acid sequence shown in SEQ ID NO:14. The heavy chain variable region of the antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region of the antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO: 18, an LCDR2 having the amino acid sequence shown in SEQ ID NO: 19, and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 20. The heavy chain variable region of the antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO:21, an HCDR2 having the amino acid sequence shown in SEQ ID NO:22, and an HCDR3 having the amino acid sequence shown in SEQ ID NO:23, and the light chain variable region of the antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO:24, an LCDR2 having the amino acid sequence shown in SEQ ID NO:25, and an LCDR3 having the amino acid sequence shown in SEQ ID NO:26. or The heavy chain variable region of the antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO:27, an HCDR2 having the amino acid sequence shown in SEQ ID NO:28, and an HCDR3 having the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region of the antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO:30, an LCDR2 having the amino acid sequence shown in SEQ ID NO:31, and an LCDR3 having the amino acid sequence shown in SEQ ID NO:32. Anti-PVRIG antibodies or antigen-binding fragments thereof are provided.

[0011] The variable regions of the light and heavy chains determine the binding to the antigen, and the variable region of each chain contains three hypervariable regions called complementarity determining regions (CDRs), of which the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3. In some embodiments of the present invention, HCDR1-HCDR3 and LCDR1-LCDR3 are defined or numbered according to US Patent Publication No. 20210380669A1 or the literature Lu et al. (Deamidation and isomerization liability analysis of 131 clinical-stage antibodies, MABS, 2019, VOL. 11, NO. 1, 45-57, DOI: 10.1080 / 19420862.2018.1548233).

[0012] In some embodiments of the invention, the heavy chain variable region has an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, An anti-PVRIG antibody, or antigen-binding fragment thereof, is provided, wherein the light chain variable region has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8.

[0013] In some embodiments of the present invention, The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:1, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:2. The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:3, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:4. The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:5, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:6. or The heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:7, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:8. Anti-PVRIG antibodies or antigen-binding fragments thereof are provided.

[0014] In some embodiments of the present invention, the heavy chain constant region of the antibody is an Ig gamma-1 chain C region or an Ig gamma-4 chain C region, and the light chain constant region is an Ig kappa chain C region; Preferably, the heavy chain constant region of the antibody has the amino acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, and the light chain constant region of the antibody has the amino acid sequence shown in SEQ ID NO: 36. Anti-PVRIG antibodies or antigen-binding fragments thereof are provided.

[0015] In some embodiments of the invention, an anti-PVRIG antibody or antigen-binding fragment thereof is provided, wherein the heavy chain constant region of the antibody comprises a mutation represented by the EU numbering system as a L234A mutation and a L235A mutation (abbreviated as LALA). In the present invention, unless otherwise specified, the letter before the site represents the amino acid before the mutation, and the letter after the site represents the amino acid after the mutation.

[0016] In some embodiments of the invention, an anti-PVRIG antibody or antigen-binding fragment thereof is provided, which is selected from the group consisting of a Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody and diabody.

[0017] In some embodiments of the invention, an anti-PVRIG antibody or antigen-binding fragment thereof is provided, wherein the antibody comprises non-CDR regions, wherein the non-CDR regions are derived from an antibody of a species other than mouse, for example human.

[0018] In some embodiments of the invention, the antibody has an EC 50 is less than 3 nM, less than 2.9 nM or less than 2.8 nM, preferably 50 is the value measured by flow cytometry assay when anti-PVRIG antibody or antigen-binding fragment thereof was provided.

[0019] Another aspect of the invention pertains to an isolated nucleic acid molecule encoding an anti-PVRIG antibody, or antigen-binding fragment thereof, according to any embodiment of the invention.

[0020] A further aspect of the present invention pertains to a vector comprising the isolated nucleic acid molecule of the present invention.

[0021] A further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a vector of the invention.

[0022] A further aspect of the invention relates to a conjugate comprising an antibody and a conjugate moiety, wherein the antibody is an anti-PVRIG antibody or antigen-binding fragment thereof according to any one of the embodiments of the invention, and the conjugate moiety is a detectable label, preferably the conjugate moiety is a radioisotope, a fluorescent substance, a colored substance or an enzyme.

[0023] Another aspect of the invention is a kit comprising an anti-PVRIG antibody according to any embodiment of the invention, or a conjugate of the invention, Preferably, the kit further comprises a second antibody that specifically recognizes the antibody, and optionally the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a colored substance or an enzyme.

[0024] Another aspect of the invention relates to a pharmaceutical composition comprising an anti-PVRIG antibody or antigen-binding fragment according to any embodiment of the invention, and one or more pharma- ceutically acceptable excipients.

[0025] In some embodiments of the invention, the pharmaceutical composition further comprises one or more anti-TIGIT antibodies, Preferably, the pharmaceutical composition further comprises one or more anti-PD-1 antibodies or one or more anti-PD-L1 antibodies.

[0026] In some embodiments of the present invention, the molar ratio of the anti-PVRIG antibody or antigen-binding fragment thereof to the anti-TIGIT antibody is (1:5) to (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1. and / or Pharmaceutical compositions are provided in which the molar ratio of the anti-PVRIG antibody or antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody is (1:5) to (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1.

[0027] In one or more embodiments of the present invention, the anti-TIGIT antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3; the heavy chain variable region of the anti-TIGIT antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 42, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 43, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 44; and the light chain variable region of the anti-TIGIT antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO: 45, an LCDR2 having the amino acid sequence shown in SEQ ID NO: 46, and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; Preferably, a pharmaceutical composition is provided in which the heavy chain variable region of the anti-TIGIT antibody has the amino acid sequence shown in SEQ ID NO:40 and the light chain variable region has the amino acid sequence shown in SEQ ID NO:41.

[0028] In one or more embodiments of the invention, the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3; the heavy chain variable region of the anti-PD-1 antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:50, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:51, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:52, and the light chain variable region of the anti-PD-1 antibody comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:53, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:54, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:55; Preferably, a pharmaceutical composition is provided in which the heavy chain variable region of the anti-PD-1 antibody has the amino acid sequence set forth in SEQ ID NO:48, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:49.

[0029] In one or more embodiments of the present invention, a pharmaceutical composition is provided, wherein the unit dose of the pharmaceutical composition, calculated from the mass of the bispecific antibody contained therein, is 100 mg to 1000 mg, 200 mg to 800 mg, 200 mg to 500 mg, 300 mg to 600 mg, 400 mg to 500 mg, or 450 mg.

[0030] In some embodiments of the invention, a pharmaceutical composition is provided, wherein the active ingredient of the pharmaceutical composition consists of an anti-PVRIG antibody or an antigen-binding fragment thereof.

[0031] In some embodiments of the present invention, a pharmaceutical composition is provided, wherein the active ingredients of the pharmaceutical composition consist of an anti-PVRIG antibody or an antigen-binding fragment thereof and an anti-TIGIT antibody.

[0032] In some embodiments of the present invention, a pharmaceutical composition is provided, wherein the active ingredients of the pharmaceutical composition consist of an anti-PVRIG antibody or an antigen-binding fragment thereof, an anti-TIGIT antibody, and an anti-PD-1 antibody.

[0033] Another aspect of the invention is a combination product comprising a first product and a second product packaged separately, The first article of manufacture comprises an anti-PVRIG antibody or antigen-binding fragment thereof according to any embodiment of the invention, or a conjugate of the invention; The second article comprises at least one anti-TIGIT antibody; Preferably, the method further comprises a third article of manufacture, the third article of manufacture comprising at least one anti-PD-1 antibody or at least one anti-PD-L1 antibody; Preferably, the first product, the second product and the third product each independently also contain one or more pharma- ceutically acceptable excipients; Preferably, it relates to a combination product further comprising a package insert.

[0034] In some embodiments of the present invention, The molar ratio of the anti-PVRIG antibody or antigen-binding fragment thereof to the anti-TIGIT antibody is (1:5) to (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1. and / or Combination products are provided in which the molar ratio of the anti-PVRIG antibody or antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody is (1:5) to (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1.

[0035] In some embodiments of the present invention, the anti-TIGIT antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3; the heavy chain variable region of the anti-TIGIT antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 42, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 43, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 44, and the light chain variable region of the anti-TIGIT antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO: 45, an LCDR2 having the amino acid sequence shown in SEQ ID NO: 46, and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; Preferably, a combination product is provided in which the heavy chain variable region of the anti-TIGIT antibody has the amino acid sequence shown in SEQ ID NO:40 and the light chain variable region has the amino acid sequence shown in SEQ ID NO:41.

[0036] In some embodiments of the invention, the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 to HCDR3 and the light chain variable region comprises LCDR1 to LCDR3; the heavy chain variable region of the anti-PD-1 antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:50, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:51, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:52, and the light chain variable region of the anti-PD-1 antibody comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:53, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:54, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:55; Preferably, a combination product is provided in which the heavy chain variable region of the anti-PD-1 antibody has the amino acid sequence set forth in SEQ ID NO:48 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:49.

[0037] Another aspect of the invention is the use of an antibody or an antigen-binding fragment thereof according to any embodiment of the invention, or a conjugate of the invention, in the manufacture of a medicament for the treatment or prevention of a tumor, comprising: Preferably, the tumor is one or more selected from the group consisting of colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0038] An antibody or antigen-binding fragment thereof according to any embodiment of the invention or a conjugate of the invention for use in the treatment or prevention of a tumor, comprising: Preferably, the tumor is one or more selected from the group consisting of colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer, the antibody or antigen-binding fragment thereof according to any embodiment of the invention, or the conjugate of the invention.

[0039] Another aspect of the invention is a method of treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof according to any embodiment of the invention, or a conjugate of the invention, Preferably, the tumor is one or more selected from the group consisting of colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0040] In one or more embodiments of the present invention, The single dose of the anti-PVRIG antibody is 0.1 to 100 mg, preferably 1 to 10 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg) per kilogram of body weight, or the single dose of the anti-PVRIG antibody of the present invention is 10 to 1000 mg (e.g., about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg) per kilogram of body weight for a subject, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg, Preferably, administration is performed every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, a method for treating and / or preventing malignant tumors is provided, in which administration is carried out by intravenous infusion or intravenous injection.

[0041] In some regimens, administration of the anti-PVRIG antibody is in 2-week (14 day) or 3-week (21 day) cycles, preferably with the anti-PVRIG antibody administered intravenously on day 1 (D1) of each cycle. For example, the anti-PVRIG antibody is administered once every 2 weeks (q2w) or once every 3 weeks (q3w). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0043] As used herein, when referring to the amino acid sequence of PVRIG (NCBI GenBank ID: NP_076975.2), it includes the full-length PVRIG protein or its extracellular domain, and also includes fusion proteins of PVRIG, such as fragments fused to mouse or human IgG Fc protein fragments (mFc or hFc). However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) in the amino acid sequence of PVRIG protein may occur naturally or may be artificially introduced without affecting its biological function. Thus, in the present invention, the term "PVRIG protein" or "PVRIG" is intended to include all such sequences, including the sequences shown and natural or artificial variants thereof. Furthermore, when describing a sequence fragment of PVRIG protein, it includes not only the sequence fragment but also the corresponding sequence fragment in its natural or artificial variant.

[0044] As used herein, when referring to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2), it includes the full-length TIGIT protein or a functional fragment thereof, and also includes fusion proteins of TIGIT, such as fragments fused to mouse or human IgG Fc protein fragments (mFc or hFc). However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions and / or additions) in the amino acid sequence of TIGIT protein may occur naturally or may be artificially introduced without affecting its biological function. Thus, in the present invention, the term "TIGIT protein" or "TIGIT" is intended to include all such sequences, including the sequences shown and natural or artificial variants thereof. Furthermore, when describing a sequence fragment of TIGIT protein, it includes not only the sequence fragment but also the corresponding sequence fragment in its natural or artificial variant.

[0045] As used herein, "EC50 The term "50% maximal effect concentration" refers to the concentration that produces 50% of the maximal effect.

[0046] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, defining the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also containing a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant regions of antibodies may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subclassified into highly variable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of each heavy / light chain pair (VH and VL), respectively, form the antibody binding site.The assignment of amino acids to regions or domains is as defined in Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917, Chothia et al, Nature, 1989; 342:878-883, or according to the IMGT numbering system, see the definitions D301 to D307 in Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF [J]. Nucleic acids research, 2009; 38(suppl_1). The term "antibody" is not limited to any particular method of producing antibodies, including, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0047] As used herein, the term "antigen-binding fragment", also known as "antigen-binding portion", refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen. See generally Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibody fragments (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability.

[0048] As used herein, the term "Fd fragment" refers to a V H and C H The term "Fv fragment" refers to an antibody fragment consisting of one domain, the V domain of a single arm of an antibody. L and V H The term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)), and the term "Fab fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)). L , V H , C L and CH1 domain, and the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by disulfide bridges in the hinge region.

[0049] In some cases, the antigen-binding fragment of the antibody is L and V HSingle chain antibodies (e.g., scFv) are monovalent molecules in which the domains pair together via a linker that allows them to produce a single polypeptide chain (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules have the general structure: NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L The linker may have -COOH. A suitable linker of the prior art consists of the GGGGS amino acid sequence repeat or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but also variants thereof (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

[0050] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a V H and V LIt is a bivalent antibody in which the domains are expressed on a single polypeptide chain. However, the linker used is too short to allow pairing of the two domains on one chain, so the domains are forced to pair with complementary domains on another chain, creating two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448(1993), and Poljak RJ et al., Structure 2:1121-1123(1994)).

[0051] In other cases, the antigen-binding fragment of an antibody is a "bispecific antibody" which refers to a conjugate formed from a first antibody (fragment) and a second antibody (fragment) or antibody analog via a linker, and the conjugation methods include, but are not limited to, chemical reaction, gene fusion, and enzyme catalysis. The antigen-binding fragment of an antibody can be a "multispecific antibody", which includes, for example, trispecific antibodies and tetraspecific antibodies, the former being antibodies with three different types of antigen-binding specificities and the latter being antibodies with four different types of antigen-binding specificities. For example, a designed ankyrin repeat protein (DARPin) is conjugated to an IgG antibody, scFv-Fc antibody fragment, or a combination thereof, e.g., CN104341529A. An anti-IL-17a fynomer is conjugated to an anti-IL-6R antibody, e.g., WO2015141862A1.

[0052] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibody ADI-56127, ADI-56204, ADI-56249 or ADI-56145 provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as the intact antibody.

[0053] The terms "mcAb" and "monoclonal antibody" refer to an antibody or antibody fragment from a highly homogeneous population of antibody molecules, i.e., identical except for spontaneous mutations that may occur spontaneously. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies, as opposed to a monoclonal antibody, usually contain at least two or more different antibodies, which usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using hybridoma technology, first reported by Kohler et al. (Koehler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. nature, 1975; 256(5517):495), but can also be obtained using recombinant DNA technology (see, for example, U.S. Pat. No. 4,816,567).

[0054] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained when all or part of the CDRs of a human immunoglobulin (receptor antibody) are replaced by the CDRs of a non-human antibody (donor antibody), which may be a non-human (e.g., mouse, rat, or rabbit) antibody with the predicted specificity, affinity, or reactivity. In addition, some amino acid residues in the framework region (FR) of the receptor antibody can also be replaced by the corresponding amino acid residues of a non-human antibody or by amino acid residues of other antibodies to further improve or optimize the performance of the antibody. For further details on humanized antibodies, see, e.g., Jones et al., Nature, 1986; 321:522-525; Reichmann et al., Nature, 1988; 332:323-329; Presta, Curr. Op. Struct. Biol. 1992; 2:593-596; and Clark M. Antibody humanization: a case of the 'Emperor's new clothes'? [J]. Immunol. Today, 2000; 21:397-402.

[0055] As used herein, the term "isolated" or "isolated" refers to being obtained by artificial means from a natural state. When an "isolated" substance or component occurs in nature, it may result from a change in its natural environment, or the substance is separated from its natural environment, or both. For example, a non-isolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with a high degree of purity isolated from this natural state is called "isolated". The term "isolated" or "isolated" does not exclude mixtures of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0056] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell via transformation, transduction or transfection so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages, such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40). Vectors may contain various expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain an origin of replication site.

[0057] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell, such as Escherichia coli or Bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus, an insect cell, such as an S2 Drosophila cell or Sf9, or an animal cell, such as a fibroblast, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a GS cell, a BHK cell, a HEK293 cell or a human cell.

[0058] As used herein, the terms "monoclonal antibody" and "McAb" have the same meaning and are used interchangeably, and the terms "polyclonal antibody" and "PcAb" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0059] As used herein, the term "pharmaceutical acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient. This is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, auxiliary agents, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80, and ionic strength enhancers include, but are not limited to, sodium chloride.

[0060] As used herein, the term "effective amount" refers to an amount sufficient to at least partially obtain a desired effect. For example, a prophylactically effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease (e.g., a tumor), and a therapeutically effective amount is an amount sufficient to cure or at least partially prevent a disease and its complications in a patient already suffering from the disease.

[0061] As used herein, the terms "hybridoma" and "hybridoma cell line" are interchangeable and references to "hybridoma" and "hybridoma cell line" also include subclones and progeny of the hybridoma.

[0062] In the present invention, unless otherwise specified, the terms "first" (e.g., first product) and "second" (e.g., second product), as well as the term "third" (e.g., third product) are used for distinction or clarity of expression, and do not imply any typical procedural order.

[0063] Advantageous Effects of the Invention The present invention achieves one or more of the following technical effects described in items (1) to (4). (1) The antibodies of the present invention can bind to PVRIG with high affinity. (2) The antibody of the present invention is likely to have a synergistic effect with an anti-TIGIT antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody. (3) The antibody of the present invention is effective in treating and / or preventing tumors, such as colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain cancer, urothelial cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B lymphoma, plasma cell cancer, prostate cancer, and testicular cancer, among others. (4) The antibodies of the present invention have low levels of toxicity and side effects. [Brief description of the drawings]

[0064] [Figure 1] Figures 1A-B show binding curves of anti-PVRIG antibodies to human PVRIG overexpressed on CHO cells. [Diagram 2] Figures 2A-B show binding curves of anti-PVRIG antibodies to cynomolgus PVRIG overexpressed on CHO cells. [Diagram 3] Figures 3A-B show binding curves of anti-PVRIG antibodies to mouse PVRIG overexpressed on CHO cells. [Figure 4] Figures 4A-B show curves of anti-PVRIG antibodies blocking binding of human PVRIG to human CD112 overexpressed on CHO cells. [Diagram 5]Figures 5A-B show curves for anti-PVRIG antibodies blocking binding of mouse CD112 to mouse PVRIG protein. [Figure 6] FIG. 6 shows binding curves of anti-PVRIG antibodies to PVRIG on activated human primary T cells. [Figure 7] FIG. 7 shows the curves of combination of anti-PVRIG antibody with anti-TIGIT antibody, blocking the PVRIG / CD112 and TIGIT / CD155 signaling pathways. [Figure 8] Figure 8 shows the curves of combination of anti-PVRIG antibody with anti-TIGIT and anti-PD-L1 antibodies blocking the PVRIG / CD112, TIGIT / CD155 and PD-1 / PD-L1 signaling pathways. [Figure 9-1] Figures 9A to 9C show the results of ADCC assays against CHO cells expressing human PVRIG, cynomolgus monkey PVRIG and mouse PVRIG, sequentially mediated by anti-PVRIG antibodies. [Figure 9-2] Same as above [Figure 10] Figure 10 shows the efficacy of anti-PVRIG antibodies in a CT-26 tumor-bearing wild-type mouse model. [Figure 11] FIG. 11 shows the efficacy of anti-PVRIG antibodies in the NDG mouse model inoculated with a mixture of A375 and human PBMCs. [Figure 12] FIG. 12 shows the efficacy of anti-PVRIG antibody in combination with anti-TIGIT antibody in the NDG mouse model inoculated with a mixture of A375 and human PBMC. [Figure 13] Figure 13 shows the efficacy of anti-PVRIG antibody in combination with anti-TIGIT and anti-PD-1 antibodies in an NDG mouse model inoculated with a mixture of A375 and human PBMCs. [Figure 14] Figure 14 shows the in vivo half-life curves of anti-PVRIG antibodies in mice. EXAMPLES

[0065] Specific Model for Implementing the Invention The embodiments of the present invention will be described in detail with reference to the following examples, but it will be appreciated by those skilled in the art that the examples are presented by way of illustration and are not intended to limit the scope of the invention as claimed. If specific conditions are not given in the examples, conventional conditions or conditions recommended by the manufacturer were followed. If no manufacturer is given, the reagents or equipment used are conventional commercial products.

[0066] Antibody Atezolizumab (ATE): anti-PD-L1 monoantibody, trade name: Tecentriq, Roche.

[0067] The following control antibodies used in the examples were expressed and purified from HEK293 cells. COM701 is an anti-human PVRIG antibody produced by Compugen and expressed by HEK293 cells, having light chain variable region sequences and heavy chain variable region sequences corresponding to SEQ ID NO: 1372 and SEQ ID NO: 1380 in U.S. Pat. No. 10,227,408 B2. Mab46 is an anti-human PVRIG antibody manufactured by Surface Oncology and expressed by HEK293 cells, having light chain variable region and heavy chain variable region sequences corresponding to sequence numbers 912 and 918 in U.S. Patent Application Publication No. 20200040081A1. Tiragolumab is an anti-human TIGIT antibody from Genentech expressed by HEK293 cells with light and heavy chain sequences derived from WHO Drug Information, Vol. 31, No. 2, 2017, Proposed INN. List 117, CAS number 1918185-84-8.

[0068] Example 1: Antibody sequence design, expression and purification 1.1 Antibody sequence design Based on the yeast display antibody library (Adimab), amplification and optimization were performed according to existing methods (see patent applications WO2009036379, WO2010105256 and WO2012009568). The yeast cells obtained by screening were induced to secrete and produce anti-PVRIG antibodies (full-length IgG) by shaking at 30°C for 48 hours. After completing the induced expression, the yeast cells were removed by centrifugation at 1300 rpm for 10 minutes and the supernatant was collected. Protein A was used to purify the anti-PVRIG antibodies in the supernatant, and elution was performed with an acetic acid solution at pH 2.0, and anti-PVRIG antibodies ADI-56127, ADI-56204, ADI-56249 and ADI-56145 were collected. Meanwhile, antibodies ADI-56127, ADI-56204, ADI-56249, and ADI-56145 were digested with papain, and the corresponding antibody Fab fragments were purified using Kappa Select (GE Healthcare).

[0069] The variable region sequences of the four antibodies ADI-56127, ADI-56204, ADI-56249, and ADI-56145 are shown below. ADI-56127 heavy chain variable region sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTESVDAISWVRQAPGQGLEWMGDIIPFFDTDYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGGTSWTHFFDLWGRGTLVTVSS (SEQ ID NO: 1) ADI-56127 light chain variable region sequence: EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGGGTKVEIK (SEQ ID NO: 2) ADI-56204 heavy chain variable region sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYGIYWVRQAPGQGLEWMGHIYAYSGDTDYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGSPTYEHFNWFDPWGQGTLVTVSS (SEQ ID NO: 3) ADI-56204 light chain variable region sequence: DIQMTQSPSSLSASVGDRVTITCQASDDISDYLNWYQQKPGKAPKLLIYDASSLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYGNLPITFGGGTKVEIK (SEQ ID NO: 4) ADI-56249 heavy chain variable region sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTFAISWVRQAPGQGLEWMGDIIPIFGSNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPLEGKVSPFDIWGQGTMVTVSS (SEQ ID NO: 5) ADI-56249 light chain variable region sequence: DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSTGYNYLDWYLQKPGQSPQLLIYVGSTRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQERQFPITFGGGTKVEIK (SEQ ID NO: 6) ADI-56145 heavy chain variable region sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWVGWISSYDASTKYSQELQGRVTMTTDTSTSTAYMELRSLRSEDTAVYYCARGSPIYEHFNWFDPWGQGTLVTVSS (SEQ ID NO: 7) ADI-56145 light chain variable region sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDSSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQLSHHPLTFGGGTKVEIK (SEQ ID NO: 8)

[0070] The CDRs for the above antibodies are listed below: (1)ADI-56127 HCDR1: GTESVDAIS (SEQ ID NO: 9) HCDR2: DIPFFDTDYAQKFQG (SEQ ID NO: 10) HCDR3: AREGGTSWTHFFDL (SEQ ID NO: 11) LCDR1: RASQSVSSSYLA (SEQ ID NO: 12) LCDR2: GASSRAT (SEQ ID NO: 13) LCDR3: QQYGSSPIT (SEQ ID NO: 14)

[0071] (2)ADI-56204 HCDR1: YTFTHYGIY (SEQ ID NO: 15) HCDR2: HIIAYSGDTDYAQKLQG (SEQ ID NO: 16) HCDR3: ARGSPTYEHFNWFDP (SEQ ID NO: 17) LCDR1: QASDDISDYLN (SEQ ID NO: 18) LCDR2: DASSLHT (SEQ ID NO: 19) LCDR3: QQYGNLPIT (SEQ ID NO: 20)

[0072] (3)ADI-56249 HCDR1: GTFSTFAIS (SEQ ID NO: 21) HCDR2: DIIPIFGSNYAQKFQG (SEQ ID NO: 22) HCDR3: ARGPLEGKVSPFDI (SEQ ID NO: 23) LCDR1: RSSQSLLHSTGYNYLD (SEQ ID NO: 24) LCDR2: VGSTRAS (SEQ ID NO: 25) LCDR3: MQERQFPIT (SEQ ID NO: 26)

[0073] (4)ADI-56145 HCDR1: YTFTSYGIS (SEQ ID NO: 27) HCDR2: WISSYDASTKYSQELQG (SEQ ID NO: 28) HCDR3: ARGSPIYEHFNWFDP (SEQ ID NO: 29) LCDR1: RASQSVSSYLA (SEQ ID NO: 30) LCDR2: DSSNRAT (SEQ ID NO: 31) LCDR3: QQLSHHPLT (SEQ ID NO: 32)

[0074] 1.2 Expression and purification of antibodies and their variants For the antibodies ADI-56127, ADI-56204 and the control antibody Mab46, all of their heavy chain variable regions (VH) were constructed in the wild-type human IgG1 heavy chain constant region (amino acid sequence shown in SEQ ID NO: 33). In addition, for the antibodies ADI-56127, ADI-56204, ADI-56249, ADI-56145 and the control antibody Mab46, all of their heavy chain variable regions (VH) were constructed in the heavy chain constant region modified by L234A, L235A (amino acid sequence shown in SEQ ID NO: 34), respectively, to constitute a complete "VH-CH1-CH2-CH3" antibody heavy chain. For the control antibody COM701, its heavy chain variable region was constructed in the human IgG4 heavy chain constant region (amino acid sequence shown in SEQ ID NO: 35). In addition, the light chain variable regions (VL) of all the above antibodies were constructed in the human immunoglobulin kappa light chain constant region (amino acid sequence shown in SEQ ID NO: 36). The antibodies were transiently expressed and purified by HEK293 expression system, the specific procedure was as follows: pcDNA3.1 vector containing the antibody heavy and light chains was transferred into HEK293 cells by chemical transfection, which was then cultured at 37°C, 8% CO2 for 7 days. The cell liquid was collected and centrifuged at 13,000 rpm for 20 minutes. The supernatant was collected and purified with protein A, and the antibody purity was detected by SEC while controlling the endotoxin content. The produced antibodies were named ADI-56127-G1, ADI-56127-G1LALA, ADI-56204-G1, ADI-56204-G1LALA, ADI-56249-G1LALA, ADI-56145-G1LALA, Mab46-G1, Mab46-G1LALA and COM701-G4.

[0075] Wild type human IgG1 heavy chain constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 33) Heavy chain constant region modified by L234A, L235A: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 34) Human IgG4 heavy chain constant region: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 35) Human immunoglobulin kappa light chain constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 36)

[0076] Example 2: Affinity detection The binding and dissociation rate constants (K ) of the antibody and its variants or Fab fragments obtained in Example 1 to human, cynomolgus monkey and mouse PVRIG were measured using biofilm layer optical interference technology (ForteBio). D ) values ​​were determined. ForteBio affinity measurements were performed according to existing methods (Este, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013.5(2):p270-8). The amino acid sequences of the extracellular domains of human, cynomolgus monkey, and mouse PVRIG are shown in SEQ ID NOs:37 to 39, respectively.

[0077] Amino acid sequence of the extracellular domain of human PVRIG: TPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDPGLSAPPTPAPILRAD (SEQ ID NO: 37) Amino acid sequence of the extracellular domain of cynomolgus PVRIG: TPEVWVQVQMEATELSSFTVHCGFLGPGSISLVTVSWGGPDGAGGTKLAVLHPELGTRQWAPARQARWETQSSISLALEDSGASSPFANTTFCCKFASFPEGSWESCGSLPPSSDPGLSAPPTPVPILRAD (SEQ ID NO: 38) Amino acid sequence of the extracellular domain of mouse PVRIG: SPEVWVQVQMEATNLSSFSVHCGVLGYSLISLVTVSCEGFVDAGRTKLAVLHPEFGTQQWAPARQAHWETPNSVSVTLTMGQSKARSSLANTTFCCEFVTFPHGSRVACRDLHRSDPGLSAPTPALNLQAD (SEQ ID NO: 39)

[0078] Measurement of monovalent affinity of intact antibody (full length IgG obtained from Adimab) against human PVRIG-his protein: The sensor was equilibrated offline for 20 min in analysis buffer, then detected online for 120 s to establish a baseline, and intact PVRIG antibody was loaded to a thickness of 1 nm onto the AHQ sensor for affinity detection. The antibody-loaded sensor was incubated in 100 nM PVRIG-his antigen until the plateau phase, then the sensor was transferred to analysis buffer and incubated for at least 2 min for measurement of dissociation rates. Kinetic analysis was performed using a 1:1 binding model.

[0079] Measurement of monovalent affinity of candidate antibody Fab fragments (Fab fragments obtained from full-length IgG treated with papain) for human, cynomolgus and mouse PVRIG-Fc proteins: The sensors were equilibrated offline for 20 min in analysis buffer, then detected online for 120 s to establish a baseline, and PVRIG-Fc antibodies were loaded to a thickness of 1 nm onto the AHQ sensor for affinity detection. The antigen-loaded sensors were incubated in 100 nM Fab solution until the plateau phase, then the sensors were transferred to analysis buffer and incubated for at least 2 min for measurement of dissociation rates. Kinetic analysis was performed using a 1:1 binding model.

[0080] Measurement of bivalent affinity of intact antibodies to cynomolgus and mouse PVRIG-Fc proteins: The sensors were equilibrated offline for 20 minutes in analysis buffer, then detected online for 120 seconds to establish a baseline, and intact PVRIG antibody was loaded to a thickness of 1 nm onto the AHQ sensor for affinity detection. The antibody-loaded sensor was continued to be loaded for 10 minutes in a high concentration of irrelevant intact antibody to saturate the Fc binding sites on the AHQ sensor. The saturated sensor was incubated in 100 nM PVRIG-Fc antigen until the plateau phase, then the sensor was transferred to analysis buffer and incubated for at least 2 minutes for measurement of dissociation rates. Kinetic analysis was performed using a 1:1 binding model.

[0081] In the above determination method, the K D The values ​​are shown in Table 1.

[0082] [Table 1]

[0083] As can be seen from the results in Table 1, ADI-56127 and ADI-56249 have cross-binding activity with mouse PVRIG.

[0084] Example 3: Binding and blocking activity of anti-PVRIG antibodies to CHO cells overexpressing human / cynomolgus / mouse PVRIG 3.1 Flow cytometry-based detection of binding activity of anti-PVRIG antibodies and their variants to human / cynomolgus / mouse PVRIG overexpressed on CHO cells Specifically, human PVRIG-overexpressing CHO cells (CHO-huPVRIG cells), cynomolgus PVRIG-overexpressing CHO cells (CHO-cynoPVRIG cells), and mouse PVRIG-overexpressing CHO cells (CHO-muPVRIG cells) were generated by transfection of pCHO1.0 vector (purchased from Invitrogen) with cDNAs encoding human PVRIG, cynomolgus PVRIG, and mouse PVRIG, respectively, cloned into the multiple cloning site (MCS), followed by pressure screening. The overexpressing cells were expanded and adjusted to an appropriate cell density, and then added to a 96-well flow cytometry plate. After centrifugation, serially diluted test samples were added to the cells, and the cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS, and the corresponding fluorescent secondary antibodies diluted to the appropriate concentrations were added to the cells, and then the cells were incubated at 4°C for 30 minutes and washed twice with PBS. The cells were resuspended in PBS and detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated. GraphPad software was used for graphical analysis to determine the EC 50 The results are shown in Table 2, A to B of Fig. 1, A to B of Fig. 2, and A to B of Fig. 3.

[0085] 3.2 Flow cytometry-based detection of blocking activity of anti-PVRIG antibodies and their variants on binding of human PVRIG to human CD112 overexpressed on CHO cells Specifically, human CD112-overexpressing CHO cells (CHO-huCD112 cells) were generated by transfection with pCHO1.0 vector (purchased from Invitrogen) carrying a cDNA encoding human CD112 cloned into the multiple cloning site (MCS) and subsequent pressure screening. CHO-huCD112 cells were expanded to 2 × 10 6 The cells were adjusted to a cell density of 100 cells / mL and added to a 96-well flow plate at 100 μL / well and centrifuged for later use. Purified monoclonal antibodies were serially diluted in PBS and the diluted samples were added to a 96-well flow plate at 60 μL / well. Then, 1 μg / mL human PVRIG protein with mouse IgG2a Fc tag was added at 60 μL / well, mixed and incubated at 4°C for 30 minutes. The above incubated samples were added to a 96-well flow plate containing CHO-huCD112 cells at 100 μL / well, incubated at 4°C for 30 minutes and washed twice with PBS. APC-conjugated goat anti-mouse IgG antibody diluted 100-fold in PBS was added at 100 μL / well, incubated at 4°C for 30 minutes and washed twice with PBS. The cells were resuspended in PBS at 100 μL / well and detected on a Cytoflex flow cytometer and the corresponding MFI was calculated. The results are shown in Table 2 and in FIGS.

[0086] 3.3 Detection of blocking activity of anti-PVRIG antibodies and their variants against binding of mouse PVRIG protein to mouse CD112 protein by ELISA Specifically, mouse CD112-his protein diluted to a final concentration of 1 μg / mL in 1× coating buffer was added to the ELISA plate at 100 μL / well, covered with film, and coated overnight at 4° C. The coating solution in the ELISA plate was discarded, the ELISA plate was washed three times with 1× PBST, and blocked in 200 μL / well of 5% BSA / PBS at room temperature for 2 hours. During the blocking period, the test samples were serially diluted with 1% BSA / PBS to a final volume of 60 μL / well. Biotin-labeled mouse PVRIG huFc protein diluted to 2 μg / mL in 1% BSA / PBS was added to the sample wells at 60 μL / well, mixed, and incubated at room temperature for 1 hour. The blocking solution in the ELISA plate was discarded, and the sample mixture was added at 100 μL / well and incubated at room temperature for 2 hours. The above co-incubated samples were discarded, the ELISA plate was washed three times with 1x PBST, and SA-HRP diluted in 1% BSA / PBS was added at 100 μL / well and incubated at room temperature for 1 h. The SA-HRP diluent was discarded, the ELISA plate was washed three times with 1x PBST, ELISA color development solution was added at 100 μL / well, incubated at room temperature for 1-3 min, ELISA stop solution was added at 50 μL / well, and absorbance values ​​were read at 450 nm. Concentration-absorbance binding curves were obtained using GraphPad software, and the results are shown in Table 2 and Figure 5A-B.

[0087] [Table 2]

[0088] The following conclusions can be drawn from Table 2, Figures 1A to 1B, Figures 2A to 2B, and Figures 3A to 3B. (1) the binding activity of ADI-56249-G1LALA, ADI-56145-G1LALA, ADI-56204-G1, ADI-56204-G1LALA, ADI-56127-G1 and ADI-56127-G1LALA to human PVRIG protein overexpressed on the surface of CHO cells is better than that of the control molecules Mab46-G1, Mab46-G1LALA and COM701-G4; (2) the binding activity of ADI-56249-G1LALA, ADI-56204-G1, ADI-56204-G1LALA, ADI-56127-G1 and ADI-56127-G1LALA to cynomolgus monkey PVRIG protein overexpressed on the surface of CHO cells is better than that of the control molecules Mab46-G1, Mab46-G1LALA and COM701-G4; (3) ADI-56127-G1 and ADI-56127-G1LALA show significant binding to mouse PVRIG protein overexpressed on the surface of CHO cells, with binding activity comparable to that of the control molecules Mab46-G1 and Mab46-G1LALA.

[0089] The following conclusions can be drawn from Table 2, FIGS. 4A to 4B, and FIGS. 5A to 5B. (1) the activity of ADI-56145-G1LALA, ADI-56204-G1, ADI-56204-G1LALA, ADI-56127-G1 and ADI-56127-G1LALA in blocking the binding of human PVRIG to human CD112 protein overexpressed on the surface of CHO cells is comparable to that of the control molecules Mab46-G1, Mab46-G1LALA and COM701-G4; (2) Some of the anti-PVRIG candidates (ADI-56127-G1 and ADI-56127-G1LALA) that bind to mouse PVRIG protein overexpressed on the surface of CHO cells can significantly block the binding of mouse PVRIG to mouse CD112 protein. The blocking activity was superior to that of the control molecules Mab46-G1 and Mab46-G1LALA.

[0090] Example 4: Binding of anti-PVRIG antibodies to PVRIG on the surface of primary T cells The binding activity of the anti-PVRIG antibody of the present invention to PVRIG on the surface of activated T cells was detected based on a flow cytometry detection method.

[0091] Specifically, human PBMCs were sorted according to the experimental protocol provided by STEMCELL (Stemcell, Catalog No.: #17951C) to obtain human total T cells. T cells were sorted at 1.0 × 10 in X-VIVO15 medium (purchased from Lonza, Catalog No.: 04-418Q) supplemented with 1 μL of IL-2 stock solution (1,000,000 IU) and simultaneously added with CD3 / CD28 Dynabeads (purchased from Gibco, Catalog No.: 11132D) at a ratio of 1:1 (beads to cells). 6 The cells were adjusted to a concentration of 1000 cells / mL and cultured for 48 hours in a 37°C, 5% CO2 incubator. The activated T cells were adjusted to an appropriate cell density and added to a 96-well flow cytometry plate. After centrifugation, serially diluted test samples were added and incubated at 4°C for 30 minutes. Washing was performed twice with PBS, fluorescent secondary antibodies diluted to appropriate concentrations were added, incubated at 4°C for 30 minutes, and washed twice with PBS. The cells were resuspended in PBS, detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated.

[0092] The results are shown in Figure 6. The results showed that the anti-PVRIG antibodies ADI-56127-G1 and ADI-56127-G1LALA of the present invention can bind to PVRIG molecules on the surface of activated T cells, and that their binding activity is better than that of the control molecules Mab46-G1, Mab46-G1LALA, and COM701-G4.

[0093] Example 5: Detection of the blocking effect of the combination of anti-PVRIG antibody with anti-TIGIT antibody in a luciferase reporter system In order to detect the blocking activity of the combination of anti-PVRIG antibody and anti-TIGIT antibody at the cellular level, the following luciferase reporter system was constructed in this example. Briefly, the CHO-K1 cell line (CHO-K1-CD155-CD112) overexpressing human CD155, human CD112 and OKT-3scFv and the Jurkat cell line (Jurkat-TIGIT-PVRIG-luc) overexpressing human TIGIT, human PVRIG and the accompanying NF-AT luciferase reporter gene were constructed by transfecting cells with lentivirus. Then, related experiments were carried out using this reporter system.

[0094] Specifically, CHO-K1-CD155-CD112 functional cells were obtained by digestion, adjusted to the desired cell density, added to a 96-well white-bottom plate at 100 μL / well, and cultured overnight for attachment. The next day, Jurkat-TIGIT-PVRIG-luc effector cell suspension was prepared, and the test samples were serially diluted with reaction medium. The white-bottom plate was removed, the culture supernatant was discarded by pipetting, and the above diluted samples were added to the white-bottom plate at 40 μL / well, and Jurkat-TIGIT-PVRIG-luc effector cell suspension was added at 40 μL / well simultaneously, and incubation was performed in a 37° C., 5% CO2 incubator for 6 hours. After incubation, Bio-Glo™ reagent was added to the cells at 80 μL / well, and the luminescence signal value was read using a multifunctional microplate reader.

[0095] The results are shown in Figure 7. The results show that the combination of the anti-PVRIG antibodies ADI-56127-G1LALA, ADI-56145-G1LALA, ADI-56204-G1LALA and ADI-56249-G1LALA of the present invention with the anti-TIGIT antibody tiragolumab at a molar ratio of 1:1 can release a PVRIG inhibitory signal mediated by CD112 and a TIGIT inhibitory signal mediated by CD155, improving the level of luciferase signal to a higher value, and the activity is comparable to that of the control molecule COM701-G4.

[0096] Example 6: Detection of the blocking effect of combination of anti-TIGIT and anti-PD-L1 antibodies in a luciferase reporter system In order to detect the blocking activity of anti-PVRIG antibody combined with anti-TIGIT and anti-PD-L1 antibody at the cellular level, the following luciferase reporter system was constructed in this example. Briefly, based on Example 5, lentivirus was used to infect CHO-K1-CD155-CD112 to overexpress PD-L1 to obtain CHO-K1-CD155-CD112-PD-L1 functional cells, and lentivirus was used to infect Jurkat-TIGIT-PVRIG-luc to overexpress PD-1 to obtain Jurkat-TIGIT-PVRIG-PD-1-luc effector cell suspension, and this reporter system was used in subsequent experiments.

[0097] Specifically, CHO-K1-CD155-CD112-PD-L1 functional cells were obtained by digestion, adjusted to the desired cell density, and added to a 96-well white-bottom plate at 100μL / well and cultured overnight for attachment. The next day, Jurkat-TIGIT-PVRIG-PD-1-luc effector cell suspension was prepared, and the test samples were serially diluted with reaction medium. The white-bottom plate was removed, the culture supernatant was discarded by pipetting, and the above diluted samples were added to the white-bottom plate at 40μL / well, and Jurkat-TIGIT-PVRIG-PD-1-luc effector cell suspension was added at 40μL / well at the same time, and incubation was performed for 6 hours in a 37℃, 5% CO2 incubator. After incubation, Bio-Glo™ reagent was added to the cells at 80μL / well, and the luminescence signal value was read using a multi-function microplate reader.

[0098] The results are shown in Figure 8. The results show that the combination of the anti-PVRIG antibodies ADI-56127-G1LALA, ADI-56145-G1LALA, ADI-56204-G1LALA and ADI-56249-G1LALA of the present invention with the anti-TIGIT antibody tiragolumab and the anti-PD-L1 antibody atezolizumab (ATE) in a 1:1:1 molar ratio can further improve the luciferase signal by blocking the inhibitory signals mediated by PVRIG / CD112, TIGIT / CD155, and PD-1 / PD-L1, respectively, with the activity being comparable to that of the control molecule COM701-G4.

[0099] Example 7: Detection of in vitro ADCC activity of anti-PVRIG antibodies The in vitro ADCC activity of the anti-PVRIG antibody of the present invention was detected based on the luciferase reporter gene system.

[0100] Specifically, Jurkat-NFAT-luciferase-CD16 ADCC effector cells (purchased from Promega) were expanded and cultured at 4 × 10 cells / ml in RPMI 1640 medium containing 10% low IgG FBS. 6 CHO-huPVRIG cells, CHO-cynoPVRIG cells and CHO-muPVRIG cells were resuspended in RPMI1640 medium containing 10% low IgG FBS to a concentration of 1.6 × 10 6 The cells were diluted to 100 μL / mL. The above three cell suspensions were mixed with Jurkat-NFAT-luciferase-CD16 cells at a 1:1 ratio, respectively, and added at 50 μL / well into a sterile 96-well white bottom plate. The antibody samples to be tested were added serially diluted in RPMI1640 medium containing 10% low IgG FBS. The co-incubation was carried out at 37°C and 5% CO2 for 6 hours. After incubation, the cells were removed and equilibrated at room temperature for 5 minutes, and Bio-Glo™ reagent was added thereto at 100 μL / well, and the luminescence signal value was read using a multi-function microplate reader.

[0101] The results are shown in Figure 9A-C. The results showed that the anti-PVRIG antibodies ADI-56204-G1 and ADI-56127-G1 of the present invention can mediate in vitro ADCC activity against CHO-huPVRIG cells, CHO-cynoPVRIG cells and CHO-muPVRIG cells in a luciferase reporter system. ADI-56204-G1 mediated stronger ADCC activity against CHO-huPVRIG cells and CHO-cynoPVRIG cells than the control molecule Mab46-G1, while the ADCC activity mediated by ADI-56127-G1 against CHO-huPVRIG cells, CHO-cynoPVRIG cells and CHO-muPVRIG cells was comparable to that of the control molecule Mab46-G1.

[0102] Example 8: In vivo pharmacodynamics study of anti-PVRIG antibodies in wild-type Balb / c mice In this experiment, wild-type Balb / c mice were inoculated with CT-26 colon cancer cells (purchased from Gempharmtech Co., Ltd.) to determine the anti-tumor effect of the anti-PVRIG antibody of the present invention.

[0103] Specifically, a suspension of CT-26 cells was prepared, approximately 5 × 10 cells in 0.1 mL. 5 A CT-26 tumor-bearing mouse model was established by subcutaneous injection of cells into the right inguinal region of mice. The average tumor volume was approximately 100 mm. 3 When the tumor reached the target tumor volume, the mice were divided into groups and intraperitoneally injected with PBS or different doses of therapeutic antibodies at the same administration volume. Each group consisted of 6 mice. The changes in tumor volume and body weight of the mice in each group were monitored, and the monitoring frequency was every 2-3 days for 2-3 weeks. The dosage and administration method are shown in Table 3.

[0104] [Table 3]

[0105] The results are shown in Figure 10. The results showed that the anti-PVRIG antibody ADI-56127-G1 of the present invention could significantly inhibit the growth of CT26 tumors in mice.

[0106] Example 9: In vivo pharmacodynamics study of anti-PVRIG antibodies in NDG mice inoculated with a mixture of A375 and human PBMCs In this experiment, an A375 huPBMC model was established in B-NDG mice inoculated with a mixture of A375 (purchased from Addexbio, Catalog No. C0020004, malignant human melanoma cells) and human PBMC cells (Milestone Biotechnologies, A10S033014 / PB100C) to determine the antitumor effect of the anti-PVRIG antibody of the present invention. In this regard, a humanized tumor mouse model with a partial recombinant human immune system was generated by inoculating human immune cells (PBMC) into immune-deficient mice.

[0107] Specifically, A375 cells were first mixed 1:1 with human PBMCs to form a 0.1 mL cell suspension, which was then injected subcutaneously into the right groin of mice to establish the A375 huPBMC model. The mean tumor volume was approximately 30–50 mm. 3 When the tumor reached the target tumor volume, the mice were divided into groups and intraperitoneally injected with PBS or different doses of therapeutic antibodies at the same administration volume. Each group consisted of 6 mice. The changes in tumor volume and body weight of the mice in each group were monitored, and the monitoring frequency was every 2-3 days for 2-3 weeks. The dosage and administration method are shown in Table 4.

[0108] [Table 4]

[0109] The results are shown in Figure 11. The results showed that the anti-PVRIG antibody ADI-56127-G1 of the present invention could significantly inhibit the growth of A375 tumors in mice, and the anti-tumor activity was superior to that of the control molecules Mab46-G1 and COM701-G4.

[0110] Example 10: In vivo pharmacodynamics testing of anti-PVRIG antibodies in combination with anti-TIGIT antibodies in NDG mice inoculated with a mixture of A375 and human PBMCs In this experiment, a human PBMC-A375 tumor-bearing mouse model was first established by subcutaneous mixed inoculation (the modeling procedure was the same as in Example 9). The average tumor volume was approximately 60 mm 3 When the tumor reached the target tumor volume, the mice were divided into groups and intraperitoneally injected with PBS or different doses of therapeutic antibodies at the same administration volume. Each group consisted of 6 mice. The changes in tumor volume and body weight of the mice in each group were monitored, and the monitoring frequency was every 2-3 days for 2-3 weeks. The dosage and administration method are shown in Table 5.

[0111] [Table 5]

[0112] The results are shown in Figure 12. The results showed that the combined administration of the anti-PVRIG antibody ADI-56127-G1 of the present invention and an anti-TIGIT monoclonal antibody had a more significant tumor-suppressing effect than monotherapy with ADI-56127-G1 or anti-TIGIT-mAb.

[0113] The sequence of the anti-TIGIT monoclonal antibody used in this example is shown below. Amino acid sequence of VH of anti-TIGIT antibody: QLQLQESGPGLVKPAETLSLTCTVSGGSISSYDHYWTWIRQPPGKGLEWIGTVYYSGSTFHNPSLKSRVTIPVDTSKNQFSLKLSSVTAADTAVYYCARVGPDVSHPPFDYWGQGTLVTVSS (SEQ ID NO: 40) Amino acid sequence of the VL of the anti-TIGIT antibody: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPITFGGGTKVEIK (SEQ ID NO: 41) The CDRs for the above anti-TIGIT antibodies are listed below: HCDR1: GSISSYDHYWT (SEQ ID NO: 42) HCDR2: TVYYSGSTFHNPSLKS (SEQ ID NO: 43) HCDR3: ARVGPDVSHPPFDY (SEQ ID NO: 44) LCDR1: RASQSISSYLN (SEQ ID NO: 45) LCDR2: AASSLQS (SEQ ID NO: 46) LCDR3: QQSYSTPIT (SEQ ID NO: 47)

[0114] The heavy chain constant region of the anti-TIGIT monoclonal antibody used in this example was the wild-type human IgG1 heavy chain constant region (SEQ ID NO: 33).

[0115] The light chain constant region of the anti-TIGIT monoclonal antibody used in this example was the human kappa light chain constant region (SEQ ID NO: 36).

[0116] Example 11: In vivo pharmacodynamics study of anti-PVRIG antibodies in combination with anti-PD-1 and anti-TIGIT antibodies in NDG mice inoculated with a mixture of A375 and human PBMCs In this experiment, a human PBMC-A375 tumor-bearing mouse model was first established by subcutaneous mixed inoculation (the modeling procedure was the same as in Example 9). The average tumor volume was approximately 200 mm 3 When the tumor reached the target tumor volume, the mice were divided into groups and intraperitoneally injected with PBS or different doses of therapeutic antibodies at the same administration volume. Each group consisted of 6 mice. The changes in tumor volume and body weight of the mice in each group were monitored, and the monitoring frequency was every 2-3 days for 2-3 weeks. The dosage and administration method are shown in Table 6.

[0117] [Table 6]

[0118] As shown in Figure 13, the results showed that the combined administration of the anti-PVRIG antibody ADI-56127-G1 of the present invention, the anti-TIGIT monoclonal antibody and the anti-PD-1 antibody could effectively inhibit tumor growth, and the tumor-suppressing activity against A375 was significantly greater than that of other treatment groups.

[0119] The sequence of the anti-TIGIT monoclonal antibody used in this example corresponds to that in Example 10.

[0120] The sequences of the anti-PD-1 monoclonal antibodies used in this example are shown below. Amino acid sequence of VH of anti-PD-1 antibody QVQLVQSGAEVKKPGASVKVSCKASGYTFTEYYIYWVRQAPGQGLEWIGGINPSNGGTNFNEKFKPRVTMTVDTSTSTAYMELSSLRSEDTAVYYCTVRDFRFDKGFKYWGQGTLVTVSS (SEQ ID NO: 48) Amino acid sequence of VL of anti-PD-1 antibody EIVLTQSPATLSLSPGERATLSCRASKSVSTSGLNYVHWYQRKPGQAPRLLIYLGSYLDSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSWELPLTFGGGTKVEIK (SEQ ID NO: 49)

[0121] The six CDRs for the above anti-PD-1 antibodies are listed below. HCDR1: FTEYYIY (SEQ ID NO: 50) HCDR2: NPSNGGTNFNEKFKP (SEQ ID NO:51) HCDR3: RDFRFDKGFKY (SEQ ID NO:52) LCDR1: SKSVSTSGLNYVH (SEQ ID NO:53) LCDR2: SYLDS (SEQ ID NO:54) LCDR3: SWELPLT (SEQ ID NO:55)

[0122] The heavy chain constant region of the anti-PD-1 monoclonal antibody used in this example was an IgG1 heavy chain constant region modified by L234A and L235A (sequence number 34).

[0123] The light chain constant region of the anti-PD-1 monoclonal antibody used in this example was the human kappa light chain constant region (SEQ ID NO: 36).

[0124] Example 12: In vivo half-life study of anti-PVRIG antibodies in mice The in vivo half-life of ADI-56127-G1, an anti-PVRIG antibody of the present invention, was detected in mice by a single tail vein injection method.

[0125] Specifically, half male and half female Balb / c mice were used in this experiment and were kept in a 12 / 12-hour light / dark controlled environment at a temperature of 24°C ± 2°C and humidity of 40%-70% with free access to water and food. On the day of the experiment, Balb / c mice received a single tail vein injection of monoclonal antibody molecules at a dose of 10 mg / kg. Blood collection time points: Blood was collected from the mouse's orbit at 5 min, 0.5 h, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, 336 h, and 504 h after administration. The whole blood samples were left to stand at 2°C-8°C for 30 min and centrifuged at 12,000 rpm for 5 min to collect serum. The collected serum was centrifuged at 2°C-8°C for 5 min at 12,000 rpm and stored at -80°C. The amount of monoclonal antibody molecules in serum was detected by ELISA.

[0126] The results are shown in Figure 14. The results showed that the in vivo half-life of the anti-PVRIG antibody of the invention ADI-56127-G1 after a single injection in mice was 160 hours.

[0127] Although the specific embodiments of the present invention have been described in detail, it is understood that those skilled in the art can make various modifications and changes in the details based on all the teachings disclosed, and these modifications are within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-PVRIG antibody or antigen-binding fragment thereof, the antibody comprising a heavy chain variable region comprising HCDR1-HCDR3 and a light chain variable region comprising LCDR1-LCDR3; The heavy chain variable region of the antibody comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:9, HCDR2 having the amino acid sequence shown in SEQ ID NO:10, and HCDR3 having the amino acid sequence shown in SEQ ID NO:11; HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 17; HCDR1 having the amino acid sequence set forth in SEQ ID NO:21, HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:23, or HCDR1 having the amino acid sequence shown in SEQ ID NO:27, HCDR2 having the amino acid sequence shown in SEQ ID NO:28, and HCDR3 having the amino acid sequence shown in SEQ ID NO:

29. Including, The light chain variable region of the antibody comprises: LCDR1 having the amino acid sequence shown in SEQ ID NO: 12, LCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 14; LCDR1 having the amino acid sequence shown in SEQ ID NO: 18, LCDR2 having the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 20; an LCDR1 having the amino acid sequence set forth in SEQ ID NO:24, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:25, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:26; or LCDR1 having the amino acid sequence shown in SEQ ID NO: 30, LCDR2 having the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 32; An anti-PVRIG antibody or an antigen-binding fragment thereof.

2. The heavy chain variable region of the antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:9, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:10, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:11, and the light chain variable region of the antibody comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:12, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:13, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:

14. The heavy chain variable region of the antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 15, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 16, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 17, and the light chain variable region of the antibody comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 18, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:

20. The heavy chain variable region of the antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:21, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:22, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:23, and the light chain variable region of the antibody comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:24, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:25, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:

26. or The heavy chain variable region of the antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:27, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:28, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:29, and the light chain variable region of the antibody comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:30, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:31, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:

32. The anti-PVRIG antibody or antigen-binding fragment thereof according to claim 1.

3. the heavy chain variable region has an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7; The light chain variable region has an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:

8. The anti-PVRIG antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

2. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

4. The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

6. or The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

8. The anti-PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. the heavy chain constant region of the antibody is an Ig gamma-1 chain C region or an Ig gamma-4 chain C region, and the light chain constant region is an Ig kappa chain C region; Preferably, the heavy chain constant region of the antibody has the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, and the light chain constant region of the antibody has the amino acid sequence set forth in SEQ ID NO:

36. The anti-PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. the heavy chain constant region of the antibody comprises a mutation represented by the EU numbering system as a L234A mutation and a L235A mutation; The anti-PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. Fab, Fab', F(ab') ２ , Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody and diabody; The anti-PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. the antibody comprises non-CDR regions, the non-CDR regions being derived from an antibody of a species other than mouse, e.g., human; The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The antibody exhibited EC values ​​of binding to human PVRIG protein overexpressed on the surface of CHO cells. ５０ is less than 3 nM, less than 2.9 nM, or less than 2.8 nM, preferably ５０ is a value measured by a flow cytometry assay method.

10. An isolated nucleic acid molecule encoding the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A vector comprising the isolated nucleic acid molecule of claim 10.

12. 12. A host cell comprising the isolated nucleic acid molecule of claim 10 or the vector of claim 11.

13. A conjugate comprising an antibody and a conjugate moiety, wherein the antibody is an anti-PVRIG antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, and the conjugate moiety is a detectable label, preferably a radioisotope, a fluorescent substance, a colored substance or an enzyme.

14. A kit comprising an anti-PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or a conjugate according to claim 13, Preferably, the kit further comprises a second antibody that specifically recognizes the antibody, and optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a colored substance or an enzyme.

15. A pharmaceutical composition comprising an anti-PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or a conjugate according to claim 13, optionally further comprising one or more pharma- ceutical acceptable excipients.

16. further comprising one or more anti-TIGIT antibodies, Preferably, the antibody further comprises one or more anti-PD-1 antibodies or one or more anti-PD-L1 antibodies.

16. The pharmaceutical composition of claim 15.

17. the molar ratio of the anti-PVRIG antibody or antigen-binding fragment thereof to the anti-TIGIT antibody is (1:5) to (5:1); and / or the molar ratio of the anti-PVRIG antibody or antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody is (1:5) to (5:1); 17. A pharmaceutical composition according to claim 15 or 16.

18. the anti-TIGIT antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3; the heavy chain variable region of the anti-TIGIT antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 42, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 43, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 44, and the light chain variable region of the anti-TIGIT antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO: 45, an LCDR2 having the amino acid sequence shown in SEQ ID NO: 46, and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; Preferably, the heavy chain variable region of the anti-TIGIT antibody has the amino acid sequence shown in SEQ ID NO: 40, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

41.

18. A pharmaceutical composition according to claim 16 or 17.

19. the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3; the heavy chain variable region of the anti-PD-1 antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:50, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:51, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:52, and the light chain variable region of the anti-PD-1 antibody comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:53, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:54, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:55; Preferably, the heavy chain variable region of the anti-PD-1 antibody has the amino acid sequence set forth in SEQ ID NO: 48, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:

49. The pharmaceutical composition according to any one of claims 16 to 18.

20. A combination product comprising a first product and a second product packaged separately, The first product comprises an anti-PVRIG antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, or a conjugate according to claim 13; said second article comprising at least one anti-TIGIT antibody; Preferably, the combination product further comprises a third product, the third product comprising at least one anti-PD-1 antibody or at least one anti-PD-L1 antibody; Preferably, said first product, said second product and said third product each independently also contain one or more pharma- ceutically acceptable excipients; Preferably, said combination product further comprises a package insert.

21. the molar ratio of the anti-PVRIG antibody or antigen-binding fragment thereof to the anti-TIGIT antibody is (1:5) to (5:1); and / or the molar ratio of the anti-PVRIG antibody or antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody is (1:5) to (5:1); 21. The combination product of claim 20.

22. the anti-TIGIT antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3; the heavy chain variable region of the anti-TIGIT antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 42, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 43, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 44, and the light chain variable region of the anti-TIGIT antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO: 45, an LCDR2 having the amino acid sequence shown in SEQ ID NO: 46, and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 47; Preferably, the heavy chain variable region of the anti-TIGIT antibody has the amino acid sequence shown in SEQ ID NO: 40, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

41.

22. A combination product according to claim 20 or 21.

23. the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3; the heavy chain variable region of the anti-PD-1 antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:50, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:51, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:52, and the light chain variable region of the anti-PD-1 antibody comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:53, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:54, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:55; Preferably, the heavy chain variable region of the anti-PD-1 antibody has the amino acid sequence set forth in SEQ ID NO: 48, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:

49. A combination product according to any one of claims 20 to 22.

24. Use of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9 or a conjugate according to claim 13 in the manufacture of a medicament for the treatment or prevention of a tumor, comprising: Preferably, the tumor is one or more selected from the group consisting of colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

25. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, or a conjugate according to claim 13, for use in the treatment or prevention of a tumor, Preferably, the tumor is one or more selected from the group consisting of colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer. An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9, or a conjugate according to claim 13.

26. A method for treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the conjugate according to claim 13, Preferably, the tumor is one or more selected from the group consisting of colon cancer, melanoma, lung cancer, kidney cancer, endometrial cancer, breast cancer, skin cancer, ovarian cancer, gastric cancer, head and neck cancer, liver cancer, brain tumor, urothelial cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer; Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer.

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