Anti-TIGIT-anti-PVRIG bispecific antibodies, pharmaceutical compositions thereof and uses thereof

Abstract

The present invention belongs to the field of biomedicine and relates to an anti-TIGIT-anti-PVRIG bispecific antibody, and its pharmaceutical composition and use. Specifically, the present invention relates to a bispecific antibody comprising a first protein functional region targeting PVRIG and a second protein functional region targeting a target different from PVRIG (e.g., TIGIT), wherein the first protein functional region is an anti-PVRIG immunoglobulin or an antigen-binding fragment thereof, and the heavy chain variable region of the anti-PVRIG immunoglobulin comprises HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PVRIG immunoglobulin comprises LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 24. The bispecific antibody of the present invention has good antitumor effect.

Description

[Technical field]

[0001] The present invention belongs to the field of biomedicine and relates to an anti-TIGIT-anti-PVRIG bispecific antibody, a pharmaceutical composition thereof and uses thereof. [Background technology]

[0002] T cell immunoreceptor with Ig and ITIM domains (also known as TIGIT, WUCAM, Vstm3, VSIG9) is a novel immunoinhibitory receptor expressed by activated CD8+ and CD4+ T cells, natural killer (NK) cells, regulatory T cells (Tregs) and follicular helper T cells.

[0003] 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 NK cells and T cells.

[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] TIGIT / PVRIG participates in a complex regulatory network. CD226 (DNAM-1) is an activating receptor on T / NK cells that mediates activation signals by binding to CD155 and CD112. TIGIT and PVRIG are inhibitory receptors on T / NK cells that are upregulated upon cell activation and independently transduce inhibitory signals by binding to the ligands CD155 and CD112, respectively, while also competing with CD226 for ligand binding, thereby blocking its stimulatory signals and achieving negative regulation of immune cell function.

[0006] The researchers found that separate blockade of TIGIT / CD155 and PVRIG / CD112 interactions with antibodies could improve NK cell cytotoxicity against tumor cells, and that a combination of PVRIG and a TIGIT-blocking monoclonal antibody could further improve the killing effect of NK cells against breast cancer cells (Xu F, et al. Blockade of CD112R and TIGIT signaling sensitizes human natural killer cell functions. Cancer Immunol Immunother. 2017 Oct;66(10):1367-1375.). In vitro T cell function studies showed that TIGIT and PVRIG antibodies could increase T cell proliferation and IFN-γ secretion individually, while the combination of the two could produce additive or synergistic effects and further improve T cell function (Whelan S, et al. PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+ T-cell Function. Cancer Immunol Res. 2019 Feb;7(2):257-268.). 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 (Kathryn Logronio, et al. COM902, a Novel Therapeutic Antibody Targeting TIGIT Augments T Cell Function and the Activity of PVRIG Pathway Blockade In Vitro and In Vivo. SITC 2019.). These experimental data demonstrate that TIGIT / CD155 and PVRIG / CD112 are two independent and non-redundant T cell inhibitory pathways. Co-targeting both TIGIT and PVRIG holds great promise for better unleashing T cell activation signals generated by CD226 in cancer therapy.

[0007] SHR-2002, developed by Hengrui Pharmaceuticals, is the first and only TIGIT / PVRIG dual-targeted antibody of its kind to enter clinical development. It was approved for clinical development in early December 2021 and is currently in Phase I clinical trials for the treatment of cancer.

[0008] The simultaneous targeting of TIGIT and PVRIG has shown good results in both in vivo and in vitro experiments. Bispecific antibodies can specifically bind to two antigens or antigen epitopes at the same time, which are characterized by specificity and bifunctionality, and have become a research hotspot in the field of antibody engineering. However, the challenges faced in the development of bispecific antibodies, such as complex preclinical evaluation models, low expression, poor stability, cumbersome processes, and high variability in quality control, have affected the progress of bispecific antibody development.

[0009] Therefore, there remains an urgent need to develop bispecific antibodies that target TIGIT and PVRIG with good specificity, efficacy, and are easy to prepare. Summary of the Invention

[0010] After extensive research and creative work, the present inventors have obtained a novel bispecific antibody. The present inventors have surprisingly found that the bispecific antibody of the present invention exhibits excellent affinity and biological activity, and has the potential for anti-tumor effect. This has led to the following invention: One aspect of the invention is a bispecific antibody comprising: a first protein functional region targeting PVRIG; and PVRIG and a second protein functional region that targets a different target (e.g., TIGIT), the first protein functional domain is an anti-PVRIG immunoglobulin or an antigen-binding fragment thereof; The heavy chain variable region of the anti-PVRIG immunoglobulin comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:25, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:26, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:27; The light chain variable region of the anti-PVRIG immunoglobulin relates to a bispecific antibody comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO:22, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:23, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:24.

[0011] In some embodiments of the invention, the bispecific antibody is an anti-TIGIT-anti-PVRIG bispecific antibody, also referred to as anti-PVRIG-anti-TIGIT bispecific antibody, abbreviated as bispecific antibody of the invention.

[0012] In some embodiments of the invention, the heavy chain variable region of the anti-TIGIT immunoglobulin comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 13, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 15, and the light chain variable region of the anti-TIGIT immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 16, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 18. Or, A bispecific antibody is provided, wherein the heavy chain variable region of the anti-TIGIT immunoglobulin comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 19, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 20, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 21, and the light chain variable region of the anti-TIGIT immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 22, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0013] 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 to HCDR3 and LCDR1 to LCDR3 are defined or numbered according to US Patent Publication No. 20210380669A1 or 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).

[0014] In some embodiments of the invention, bispecific antibodies are provided whose antigen-binding fragments are each independently a single chain antibody or an IgG half molecule (IgG-HM).

[0015] In some embodiments of the present invention, the first protein functional domain is an anti-PVRIG immunoglobulin and the second protein functional domain is an anti-TIGIT single chain antibody; the heavy chain variable region of the anti-PVRIG immunoglobulin comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:25, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:26, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:27, and the light chain variable region of the anti-PVRIG immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:22, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:23, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:24; the heavy chain variable region of the anti-TIGIT single chain antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 13, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 15, and the light chain variable region of the anti-TIGIT immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 16, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 18; or A bispecific antibody is provided, in which the heavy chain variable region of the anti-TIGIT single chain antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 19, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 20, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 21, and the light chain variable region of the anti-TIGIT immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 22, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0016] In some embodiments of the present invention, the first protein functional domain is an anti-PVRIG single chain antibody and the second protein functional domain is an anti-TIGIT immunoglobulin; the heavy chain variable region of the anti-PVRIG single chain antibody comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:25, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:26, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:27, and the light chain variable region of the anti-PVRIG immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:22, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:23, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:24; the heavy chain variable region of the anti-TIGIT immunoglobulin comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 13, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 15, and the light chain variable region of the anti-TIGIT immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 16, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 18; or A bispecific antibody is provided, wherein the heavy chain variable region of the anti-TIGIT immunoglobulin comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 19, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 20, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 21, and the light chain variable region of the anti-TIGIT immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 22, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0017] In some embodiments of the present invention, The heavy chain variable region of the anti-PVRIG immunoglobulin or anti-PVRIG single chain antibody has the amino acid sequence shown in SEQ ID NO:5, The light chain variable region of the anti-PVRIG immunoglobulin or anti-PVRIG single chain antibody has the amino acid sequence shown in SEQ ID NO:4, Preferably, a bispecific antibody is provided in which the glycine at position 44 of the heavy chain variable region of the anti-PVRIG immunoglobulin or anti-PVRIG single chain antibody is substituted with a cysteine ​​and the glycine at position 100 of the light chain variable region of the anti-PVRIG immunoglobulin or anti-PVRIG single chain antibody is substituted with a cysteine.

[0018] In some embodiments of the present invention, the heavy chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single chain antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single chain antibody has the amino acid sequence shown in SEQ ID NO: 2; or the heavy chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single chain antibody has the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single chain antibody has the amino acid sequence shown in SEQ ID NO: 4; Preferably, a bispecific antibody is provided in which the glycine at position 44 of the heavy chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single chain antibody is replaced with cysteine, and the glycine at position 100 of the light chain variable region of the anti-TIGIT immunoglobulin or anti-TIGIT single chain antibody is replaced with cysteine.

[0019] In some embodiments of the invention, the first protein functional domain and the second protein functional domain are directly linked or linked via a linker; Preferably, the linker is (GGGGS)m, where m is a positive integer, e.g., 1, 2, 3, 4, 5, or 6; Preferably, a bispecific antibody is provided wherein the amino acid sequence of the linker is that shown in SEQ ID NO:6.

[0020] In some embodiments of the invention, bispecific antibodies are provided, wherein the number of first protein functional regions and second protein functional regions is each independently one, two, or more than two.

[0021] In some embodiments of the present invention, anti-TIGIT single chain antibodies (bimolecules) are linked to the C-terminus of each of the two heavy chains of anti-PVRIG immunoglobulin, or An anti-PVRIG single-chain antibody (bimolecule) is provided, which is a bispecific antibody bound to the C-terminus of each of the two heavy chains of an anti-TIGIT immunoglobulin.

[0022] In some embodiments of the present invention, the constant region of the anti-PVRIG or anti-TIGIT immunoglobulin is derived from a human antibody; Preferably, a bispecific antibody is provided, wherein the constant regions are independently selected from the group consisting of human IgG1, IgG2, IgG3, or IgG4 constant regions.

[0023] In some embodiments of the present invention, the heavy chain constant region of the anti-PVRIG or anti-TIGIT immunoglobulin is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region is a human Ig kappa chain C region; Preferably, a bispecific antibody is provided, wherein the heavy chain constant regions of the anti-PVRIG immunoglobulin and the anti-TIGIT immunoglobulin further comprise the mutations designated according to the EU numbering system as the L234A and L235A mutations (abbreviated as LALA).

[0024] 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.

[0025] In some embodiments of the invention, a bispecific antibody is provided which is a dimer or trimer formed of a peptide chain having the amino acid sequence set forth in SEQ ID NO:7 and a peptide chain having the amino acid sequence set forth in SEQ ID NO:8, or a dimer or trimer formed of a peptide chain having the amino acid sequence set forth in SEQ ID NO:9 and a peptide chain having the amino acid sequence set forth in SEQ ID NO:10.

[0026] Another aspect of the invention pertains to an isolated nucleic acid molecule encoding a bispecific antibody according to any embodiment of the invention.

[0027] A further aspect of the present invention pertains to a vector comprising the isolated nucleic acid molecule of the present invention.

[0028] 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.

[0029] A further aspect of the invention relates to a conjugate comprising a bispecific antibody and a conjugate moiety, wherein the bispecific antibody is a bispecific antibody according to any embodiment of the invention and wherein the conjugate moiety is a detectable label, preferably wherein the conjugate moiety is a radioisotope, a fluorescent substance, a colored substance or an enzyme.

[0030] Another aspect of the invention is a kit comprising a bispecific antibody according to any embodiment of the invention, or a conjugate of the invention, Preferably, the kit further comprises a second antibody capable of specifically binding to the bispecific antibody, optionally the second antibody further comprising a detectable label, such as a radioisotope, a fluorescent substance, a colored substance or an enzyme.

[0031] Another aspect of the invention is a pharmaceutical composition comprising a bispecific antibody according to any embodiment of the invention and one or more pharma- ceutically acceptable excipients, Preferably, the antibody further comprises at least one anti-PD-1 antibody, Preferably, the molar ratio of the bispecific antibody to the anti-PD-1 antibody is between (1:5) and (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1, and more preferably, it is 1:1.

[0032] 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: 36, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 37, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 38, 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: 39, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 40, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; 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:34, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:35.

[0033] In one or more embodiments of the 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.

[0034] Another aspect of the invention is a combination product comprising a first product and a second product packaged separately, the combination product comprising: the first article of manufacture comprises a bispecific antibody according to any embodiment of the invention; the second article comprises at least one anti-PD-1 antibody; Preferably, the first product and the second product each independently further comprise one or more pharma- ceutically acceptable excipients; Preferably, it relates to a combination product further comprising a package insert.

[0035] In some embodiments of the invention, combination products are provided in which the molar ratio of the bispecific antibody to the anti-PD-1 antibody is between (1:5) and (5:1), for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1, and more preferably 1:1.

[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: 36, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 37, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 38, 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: 39, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 40, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; 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:34, and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:35.

[0037] Another aspect of the invention is the use of a bispecific antibody 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, urinary tract cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell 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] A bispecific antibody according to any embodiment of the invention, or a conjugate according to the invention, or a pharmaceutical composition according to any embodiment 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, urinary tract cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell 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 bispecific antibody according to any embodiment of the invention, or the conjugate according to the invention, or the pharmaceutical composition according to any embodiment 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 a bispecific antibody according to any embodiment of the invention or a conjugate of the invention, or a pharmaceutical composition according to any embodiment 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, urinary tract cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell 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, A single dose of the bispecific 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 a single dose of the bispecific 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), preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg, per kilogram of body weight for a subject; 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 bispecific antibody is in 2-week (14 day) or 3-week (21 day) cycles, with the anti-PVRIG antibody preferably administered intravenously on day 1 (D1) of each cycle. For example, the bispecific 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, the term "EC50" refers to the 50% maximal effective concentration, meaning the concentration that causes 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 further 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 in the following order from amino terminus to carboxyl terminus: 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)), which is 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 an antibody fragment consisting of the VH and CH1 domains, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546(1989)), the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains, and the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by disulfide bridges on the hinge regions.

[0049] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv) in which the VL and VH domains pair to form a monovalent molecule 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 can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in the prior art consist of GGGGS amino acid sequence repeats or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, although variants thereof can also be used (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 bivalent antibody in which the VH and VL 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 that the domains are forced to pair with complementary domains on another chain to generate two antigen-binding sites (see, e.g., 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 antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., monoclonal antibodies ADI-56127, ADI-55796 or ADI-55812 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 antibodies are screened for specificity in the same manner as intact antibodies.

[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, 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 phage or M13 phage, 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 term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen that it targets. In one embodiment, an antibody that specifically binds to an antigen (or an antibody specific to an antigen) refers to an antibody that binds to an antigen with an affinity (KD) of less than about 10-5M, such as less than about 10-6M, less than about 10-7M, less than about 10-8M, less than about 10-9M, or less than about 10-10M or less.

[0059] As used herein, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the greater the affinity between the antibody and the antigen. Typically, an antibody binds to an antigen (e.g., a TIGIT protein) with a dissociation equilibrium constant (KD) of less than about 10-5M, e.g., less than about 10-6M, less than about 10-7M, less than about 10-8M, less than about 10-9M, or less than about 10-10M or less. KD can be determined using methods known to those skilled in the art, for example, using a Fortebio molecular interaction device.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In the present invention, unless otherwise specified, the terms "first" (e.g., first product) and "second" (e.g., second product) are used for distinction or clarity of expression, and do not imply any typical procedural order.

[0065] 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 bispecific antibody of the present invention can bind to TIGIT and PVRIG with high affinity. (2) The bispecific antibodies of the present invention are likely to have a synergistic effect with anti-PD-1 antibodies or anti-PD-L1 antibodies. (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 bispecific antibodies of the present invention have low levels of toxicity and side effects. [Brief description of the drawings]

[0066] [Figure 1] 1A to 1C show schematic structures of bispecific antibodies of the present invention. [Diagram 2] FIG. 2 shows the binding curves of the bispecific antibodies of the invention to human TIGIT overexpressed on CHO cells. [Diagram 3] FIG. 3 shows the binding curves of bispecific antibodies of the invention to cynomolgus TIGIT overexpressed on CHO cells. [Figure 4] FIG. 4 shows the binding curves of the bispecific antibodies of the invention to mouse TIGIT overexpressed on CHO cells. [Diagram 5]FIG. 5 shows the curves of bispecific antibodies of the invention blocking binding of human CD155 to human TIGIT overexpressed on CHO cells. [Figure 6] FIG. 6 shows the curves of bispecific antibodies of the invention blocking binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells. [Figure 7] FIG. 7 shows binding curves of bispecific antibodies of the invention to human PVRIG overexpressed on CHO cells. [Figure 8] FIG. 8 shows binding curves of bispecific antibodies of the invention to cynomolgus PVRIG overexpressed on CHO cells. [Figure 9] FIG. 9 shows the binding curves of bispecific antibodies of the invention to murine PVRIG overexpressed on CHO cells. [Figure 10] FIG. 10 shows a curve of a bispecific antibody of the invention blocking binding of human PVRIG to human CD112 overexpressed on CHO cells. [Figure 11] FIG. 11 shows the curves of bispecific antibodies of the invention blocking binding of mouse CD112 to mouse PVRIG protein. [Figure 12] FIG. 12 shows curves of a bispecific antibody of the invention simultaneously binding to human PVRIG and human TIGIT proteins. [Figure 13] FIG. 13 shows curves of bispecific antibodies of the invention blocking the PVRIG / CD112 and TIGIT / CD155 signaling pathways. [Figure 14A] Figures 14A-B show the curves of bispecific antibodies of the invention blocking the PVRIG / CD112, TIGIT / CD155 and PD-1 / PD-L1 signaling pathways in combination with anti-PD-1 / L1 antibodies. [Figure 14B] Same as above [Figure 15] FIG. 15 shows the efficacy of bispecific antibodies of the invention in the B-NDG mouse model inoculated with a mixture of A375 and human PBMCs. [Figure 16]Figures 16A-B show the efficacy of a bispecific antibody of the invention in combination with an anti-PD-1 antibody in the B-NDG mouse model inoculated with a mixture of A375 and human PBMCs. [Figure 17] FIG. 17 shows the in vivo half-life curves of bispecific antibodies of the invention in mice. [Figure 18] FIG. 18 shows the binding curves of the anti-TIGIT antibodies of the present invention to human TIGIT overexpressed on CHO cells. [Figure 19] FIG. 19 shows the binding curves of the anti-TIGIT antibodies of the present invention to cynomolgus TIGIT overexpressed on CHO cells. [Figure 20] FIG. 20 shows the binding curve of the anti-TIGIT antibody of the present invention to mouse TIGIT overexpressed on CHO cells. [Figure 21] FIG. 21 shows the curves of the anti-TIGIT antibodies of the present invention blocking the binding of human CD155 to human TIGIT overexpressed on CHO cells. [Figure 22] FIG. 22 shows the curve of the anti-TIGIT antibody of the present invention blocking the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells. [Figure 23] FIG. 23 shows the binding curves of the anti-TIGIT antibodies of the present invention to TIGIT on activated human primary T cells. [Figure 24] Figure 24 shows the binding curves of anti-PVRIG antibodies of the invention to human PVRIG overexpressed on CHO cells. [Diagram 25] FIG. 25 shows binding curves of anti-PVRIG antibodies of the invention to cynomolgus PVRIG overexpressed on CHO cells. [Figure 26] FIG. 26 shows the binding curves of anti-PVRIG antibodies of the invention to murine PVRIG overexpressed on CHO cells. [Figure 27] FIG. 27 shows a curve of an anti-PVRIG antibody of the invention blocking binding of human PVRIG to human CD112 overexpressed on CHO cells. [Figure 28]FIG. 28 shows a curve of an anti-PVRIG antibody of the invention blocking binding of mouse CD112 to mouse PVRIG protein. [Figure 29] Figure 29 shows the binding curves of anti-PVRIG antibodies of the invention to PVRIG on activated human primary T cells. [Diagram 30] Figure 30 shows drug efficacy of anti-PVRIG antibodies of the invention in an NDG mouse model inoculated with a mixture of A375 and human PBMCs. EXAMPLES

[0067] Specific Model for Implementing the Invention The present invention will be further described below with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, experimental methods that do not specify specific conditions usually follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0068] Antibody Atezolizumab: Anti-PD-L1 monoclonal antibody, trade name Tecentriq, Roche. Antibody pembrolizumab: Anti-PD-1 monoclonal antibody, trade name Keytruda, Merck.

[0069] 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 and heavy chain variable region sequences corresponding to sequence numbers 1372 and 1380 in U.S. Pat. No. 10,227,408. Mab46 is an anti-human PVRIG antibody from Surface Oncology 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 Publication No. 20200040081. 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 No. 1918185-84-8.

[0070] Example 1: Design and preparation of anti-TIGIT-anti-PVRIG bispecific antibodies In this example, the scFv of anti-PVRIG monoclonal antibody ADI-56127 was linked in tandem to the C-terminus of the heavy chain of anti-TIGIT monoclonal antibody ADI-55796 via a GGGGSGGGGSG linker (SEQ ID NO: 44) (other linkers could also be used, e.g., GGGGSGGGGS, SEQ ID NO: 6) by total gene synthesis (A in FIG. 1). The scFv of anti-TIGIT monoclonal antibody ADI-55796 was linked in tandem to the C-terminus of the heavy chain of anti-PVRIG monoclonal antibody ADI-56127 via a GGGGSGGGGSG linker (SEQ ID NO: 44) (other linkers could also be used, e.g., GGGGSGGGGS, SEQ ID NO: 6) by total gene synthesis (B in FIG. 1). The heavy chain of anti-TIGIT monoclonal antibody ADI-55812 (mutated in the CH3 region to form a "knob" structure) and the heavy chain of anti-PVRIG monoclonal antibody ADI-56127 (mutated in the CH3 region to form a "hole" structure) were assembled to form a "1+1" molecular structure by the "knob-into-hole" method (Figure 1C). Three different structures of anti-TIGIT / PVRIG bispecific antibodies, designated TP-007-008, TP-003-009, and TP-001-002-003, were formed, and their schematic structures are shown in Figure 1A-C. The scFvs of anti-TIGIT and anti-PVRIG antibodies were constructed with a "VH-linker-VL" bond, with the flexible linker peptide between VH and VL being GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 45) (other flexible linker peptides could also be used, e.g., GGGGSGGGGSGGGGSGGGGS, SEQ ID NO: 46). The glycine at position 44 in the heavy chain variable region and position 100 in the light chain variable region of anti-TIGIT monoclonal antibody ADI-55796 and anti-PVRIG monoclonal antibody ADI-56127 were mutated to cysteine ​​to improve the stability of the scFvs, respectively. All three bispecific antibodies were of human IgG1 subtype. The sequences of anti-TIGIT monoclonal antibodies ADI-55796 and ADI-55812, anti-PVRIG monoclonal antibody ADI-56127, and the bispecific antibodies formed are shown in Table 1 below.

[0071]

Table 1

[0072] Nucleic acids encoding the peptides of the antibodies were constructed separately in the expression frame of pcDNA3.1 by molecular cloning. The TP-007-008 bispecific antibody was produced by transfecting HEK293 cells with a pcDNA3.1 vector containing peptide chain #1 and peptide chain #2 of the anti-TIGIT / PVRIG bispecific antibody TP-007-008, the TP-003-009 bispecific antibody was produced by transfecting HEK293 cells with a pcDNA3.1 vector containing peptide chain #1 and peptide chain #2 of the anti-TIGIT / PVRIG bispecific antibody TP-003-009, and the TP-001-002-003 bispecific antibody was prepared by transfecting HEK293 cells with a pcDNA3.1 vector containing peptide chain #1, peptide chain #2 and peptide chain #3 of the anti-TIGIT / PVRIG bispecific antibody TP-001-002-003. The supernatant was collected after 5 days of cell culture, and the target protein was isolated and purified using Protein A magnetic beads (purchased from Genscript). The magnetic beads were resuspended in an appropriate volume (1-4 times the volume of the magnetic beads) of binding buffer (PBS + 0.1% Tween 20, pH 7.4), then added to the sample to be purified and incubated at room temperature for 1 hour with gentle shaking. The sample was placed on a magnetic rack (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. Elution buffer (0.1 M sodium citrate, pH 3.2) was added in a volume 3-5 times the volume of the magnetic beads, shaken at room temperature for 5-10 minutes, returned to the magnetic rack, collected, transferred to a collection tube containing neutralization buffer (1 M Tris, pH 8.54), and mixed. The three bispecific antibodies TP-007-008, TP-003-009 and TP-001-002-003 were purified, ultrafiltered with buffer replaced by PBS, molecular weights were confirmed by LC-MS and then used for subsequent in vitro and in vivo detection.

[0073] Example 2: Antibody affinity detection The binding and dissociation rate constants (KD) of the three bispecific antibodies obtained in Example 1 and their corresponding single-terminal anti-TIGIT monoclonal antibodies ADI-55796 and ADI-55812 and anti-PVRIG monoclonal antibody ADI-56127 against human and cynomolgus TIGIT and PVRIG were determined using biofilm layer optical interference technology (ForteBio). Fortebio's affinity measurements were performed according to a known method (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 and cynomolgus TIGIT and PVRIG are shown in Table 1.

[0074] Details are as follows: Measurement of monovalent affinity of antibodies to human, cynomolgus TIGIT-his and human PVRIG-his proteins: Sensors were equilibrated offline for 20 min in analysis buffer, then detected online for 120 s to establish a baseline, and intact antibodies were loaded onto AHQ sensors at a thickness of 1 nm for affinity detection. Antibody-loaded sensors were incubated in 100 nM target antigen until the plateau phase, then the sensors were transferred to analysis buffer and incubated for at least 2 min for dissociation rate measurements. Kinetic analysis was performed using a 1:1 binding model.

[0075] Measurement of bivalent affinity of antibodies to cynomolgus PVRIG-Fc protein: The sensors were equilibrated offline in analysis buffer for 20 min, then detected online for 120 s to establish a baseline, and intact antibody was loaded onto the AHQ sensor to reach a thickness of 1 nm for affinity detection. The antibody-loaded sensors were continued to be loaded for 10 min in a high concentration of unrelated intact antibody to saturate the Fc binding sites on the AHQ sensor. The saturated sensors were incubated in 100 nM of target antigen 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.

[0076] The KD values ​​of the bispecific antibodies and their corresponding single-terminal antibodies that bound to human and cynomolgus monkey TIGIT and PVRIG are shown in Table 2 below.

[0077] [Table 2]

[0078] The results show that the anti-TIGIT-anti-PVRIG bispecific antibodies TP-007-008 and TP-003-009 bind to human TIGIT and cynomolgus monkey TIGIT antigens with monovalent affinity similar to that of their monoterminal antibody ADI-55796; the anti-TIGIT-anti-PVRIG bispecific antibodies TP-001-002-003 bind to human TIGIT antigens with monovalent affinity similar to that of their monoterminal antibody ADI-55812; The bispecific antibodies TP-007-008, TP-003-009 and TP-001-002-003 were shown to bind to human PVRIG antigen with monovalent affinity similar to that of their monoterminal antibody ADI-56127, and the anti-TIGIT-anti-PVRIG bispecific antibodies TP-007-008, TP-003-009 and TP-001-002-003 were shown to bind to cynomolgus monkey PVRIG antigen with bivalent affinity similar to that of their monoterminal antibody ADI-56127.

[0079] Example 3: Binding and blocking activity of anti-TIGIT-anti-PVRIG bispecific antibodies on CHO cells overexpressing human / cynomolgus / mouse TIGIT 3.1 Flow cytometry-based detection of binding activity of anti-TIGIT-anti-PVRIG bispecific antibodies to human / cynomolgus / mouse TIGIT overexpressed on CHO cells Specifically, human TIGIT-overexpressing CHO cells (CHO-huTIGIT cells), cyno TIGIT-overexpressing CHO cells (CHO-cynoTIGIT cells), and mouse TIGIT-overexpressing CHO cells (CHO-muTIGIT cells) were generated by transfection of pCHO1.0 vector (purchased from Invitrogen) with cDNAs encoding human TIGIT, cyno TIGIT, and mouse TIGIT, respectively, cloned into the multiple cloning site (MCS), followed by pressure screening. The overexpressing cells were expanded, adjusted to an appropriate cell density, and added to a 96-well flow cytometry plate. After centrifugation, gradiently 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 graph analysis to obtain EC50 values. The results were shown in Table 3 and Figures 2 to 4.

[0080] [Table 3]

[0081] The results showed that the anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 of the present invention and its TIGIT-terminal monoclonal antibody ADI-55796 have similar binding activity to CHO cells overexpressing human / cynomolgus / mouse TIGIT.

[0082] 3.2 Flow cytometry-based detection of blocking activity of anti-TIGIT-anti-PVRIG bispecific antibodies against binding of human CD155 to human TIGIT overexpressed on CHO cells and against binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells Specifically, CHO-huTIGIT cells were expanded and adjusted to a cell density of 2 x 106 cells / mL, added to a 96-well flow plate at 100 μL / well, and centrifuged for further use. Purified antibodies were diluted 3-fold in PBS for a total of 12 concentration gradients starting from 400 nM. The diluted samples were added to the cell-loaded 96-well flow plate at 60 μL / well and incubated at 4 °C for 30 min. Human CD155 protein with mouse IgG2a Fc tag was then added at 60 μL / well to reach a final concentration of 2 μg / mL, incubated at 4 °C for 30 min, 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 min, and washed twice with PBS. Cells were resuspended in PBS at 100 μL / well, detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated.

[0083] CHO-muTIGIT cells were expanded and adjusted to a cell density of 2x106 cells / mL, added to a 96-well flow plate at 100μL / well, and centrifuged for further use. Purified antibodies were diluted 3-fold in PBS for a total of 12 concentration gradients starting from 400nM. The diluted samples were added to the cell-loaded 96-well flow plate at 60μL / well and incubated at 4℃ for 30min. Mouse CD155 protein with mouse IgG2a Fc tag was then added at 60μL / well to reach a final concentration of 2μg / mL, incubated at 4℃ for 30min, and washed twice with PBS. APC-conjugated goat anti-mouse IgG antibody diluted 100-fold in PBS was added at 100μL / well, incubated at 4℃ for 30min, and washed twice with PBS. Cells were resuspended in PBS at 100μL / well, detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated. GraphPad software was used for graphing analysis to obtain IC50 values.

[0084] The results are shown in Table 3 and Figures 5 to 6. The results showed that the activity of the anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 of the present invention in blocking both the binding of human CD155 to human TIGIT overexpressed on the surface of CHO cells and the binding of mouse CD155 to mouse TIGIT overexpressed on the surface of CHO cells was comparable to that of its TIGIT-terminal monoclonal antibody, ADI-55796.

[0085] Example 4: Binding and blocking activity of anti-TIGIT-anti-PVRIG bispecific antibodies on CHO cells overexpressing human / cynomolgus / mouse PVRIG 4.1 Flow cytometry-based detection of binding activity of anti-TIGIT-anti-PVRIG bispecific antibodies 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 graph analysis to obtain EC50 values. The results were shown in Table 4 and Figures 7-9.

[0086] [Table 4]

[0087] The results showed that the binding activity of the anti-TIGIT-anti-PVRIG bispecific antibody TP-003-009 of the present invention to human / cynomolgus / mouse PVRIG overexpressed on CHO cells was equivalent to that of its PVRIG-terminal monoantibody ADI-56127, and that the binding activity of the anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 of the present invention to human PVRIG overexpressed on CHO cells was equivalent to that of its PVRIG-terminal monoantibody ADI-56127.

[0088] 4.2 Flow cytometry-based detection of blocking activity of anti-TIGIT-anti-PVRIG bispecific antibodies against binding of human PVRIG to human CD112 overexpressed on CHO cells Specifically, CHO cells overexpressing human CD112 (CHO-huCD112 cells) were generated by transfection of pCHO1.0 vector (purchased from Invitrogen) with cDNA encoding human CD112 cloned into the multiple cloning site (MCS) followed by pressure screening. Purified antibodies were diluted in PBS and the diluted samples were added to a 96-well flow plate at 60 μL / well. 1 μg / mL human PVRIG protein with mouse IgG2a Fc tag was then added at 60 μL / well, mixed and incubated at 4°C for 30 minutes. CHO-huCD112 cells were expanded and adjusted to a cell density of 2×106 cells / mL, added to a 96-well flow plate at 100 μL / well, and the supernatant was discarded after centrifugation. The above co-incubated antibody-antigen mixture was added at 100 μL / well to a 96-well flow plate containing CHO-huCD112 cells, incubated at 4°C for 30 min, 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 min, and washed twice with PBS. Cells were resuspended at 100 μL / well in PBS and detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated. GraphPad software was used for graphical analysis to obtain IC50.

[0089] The results, as shown in Table 4 and Figure 10, show that the anti-TIGIT-anti-PVRIG bispecific antibodies of the present invention, TP-007-008 and TP-003-009, exhibit similar blocking activity to their PVRIG-terminal monoantibody, ADI-56127, in binding of human PVRIG to human CD112 overexpressed on CHO cells.

[0090] 4.3 Detection of blocking activity of anti-TIGIT-anti-PVRIG bispecific antibody against binding of mouse PVRIG protein to mouse CD112 protein based on ELISA method Specifically, mouse CD112-his protein diluted to a final concentration of 1 μg / mL in 1× ELISA coating buffer was added to a 96-well 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 with 200 μL / well of 5% BSA / PBS at room temperature for 2 hours. During the blocking period, the purified antibody to be tested was serially diluted with 1% BSA / PBS to a final volume of 60 μL / well. Biotin-labeled mouse PVRIG protein with mouse IgG2a Fc tag was added at 60 μL / well, mixed, and incubated at room temperature for 1 hour. The blocking solution in the ELISA plate was discarded, and the above antibody-antigen mixture was added at 100 μL / well and incubated at room temperature for 2 hours. The antibody-antigen mixture was discarded, the ELISA plate was washed 3 times with 1x PBST, and SA-HRP diluted in 1% BSA / PBS was added at 100μL / well and incubated at room temperature for 1 hour. The SA-HRP dilution was discarded, the ELISA plate was washed 3 times with 1x PBST, ELISA color solution was added at 100μL / well, incubated at room temperature for 1-3 minutes, ELISA stop solution was added at 50μL / well, and absorbance values ​​were read at 450nm. Concentration-absorbance binding curves and IC50 values ​​were obtained using GraphPad software.

[0091] The results are shown in Table 4 and Figure 11. The anti-TIGIT-anti-PVRIG bispecific antibody TP-003-009 of the present invention exhibits blocking activity against the binding of mouse PVRIG to mouse CD112 protein similar to that of the PVRIG-terminal monoclonal antibody ADI-56127.

[0092] Example 5: Simultaneous binding activity of anti-TIGIT-anti-PVRIG bispecific antibodies to human TIGIT and human PVRIG The simultaneous binding activity of the anti-TIGIT-anti-PVRIG bispecific antibody of the present invention to human TIGIT and human PVRIG proteins was detected based on an enzyme-linked immunosorbent assay (ELISA).

[0093] Specifically, human PVRIG protein was dissolved according to the instructions, diluted to 1 μg / mL with 1× ELISA coating solution, coated in 100 μL / well in a 96-well ELISA plate, covered with film, and left to stand overnight at 4°C. The coating solution was discarded, washed three times with 1× PBST, and 5% BSA / PBS was added at 200 μL / well and blocked at room temperature for 2 hours. The blocking solution was discarded, and the test antibody serially diluted with 1% BSA / PBS was added at 100 μL / well and incubated at room temperature for 2 hours. The antibody diluent was discarded, washed three times with 1× PBST, and biotin-labeled TIGIT protein diluted with 1% BSA / PBS was added at 100 μL / well so that the final concentration was 1 μg / mL, and incubated at room temperature for 1 hour. The antigen dilution solution was discarded, the plate was washed three times with 1x PBST, and SA-HRP diluted with 1% BSA / PBS was added at 100 μL / well and incubated at room temperature for 1 hour. The SA-HRP dilution solution was discarded, the plate was washed three times with 1x PBST, and ELISA color development solution was added at 100 μL / well and incubated at room temperature for 1-3 minutes. Then ELISA stop solution was added at 50 μL / well and the absorbance value was read at 450 nm. The concentration-absorbance value binding curve was plotted using GraphPad software.

[0094] The results are shown in Figure 12. All of the anti-TIGIT-anti-PVRIG bispecific antibodies of the present invention, TP-007-008, TP-003-009 and TP-001-002-003, were able to bind simultaneously to human TIGIT protein and human PVRIG protein.

[0095] Example 6: Blocking activity of anti-TIGIT-anti-PVRIG bispecific antibodies against TIGIT / CD155 and PVRIG / CD112 signaling pathways in a luciferase reporter gene system To further detect the simultaneous blocking activity of anti-TIGIT-anti-PVRIG bispecific antibody on TIGIT / CD155 and PVRIG / CD112 signaling pathways at the cellular level, the following luciferase reporter system was constructed in this example. Briefly, CHO-K1 cell line (CHO-K1-CD155-CD112-aAPC) overexpressing human CD155, human CD112 and anti-CD3scFv and Jurkat cell line (Jurkat-TIGIT-PVRIG-NF-AT-luc) overexpressing human TIGIT, human PVRIG and the accompanying NF-AT luciferase reporter gene were constructed by transfecting cells with lentivirus. Related experiments were then carried out using this reporter system.

[0096] Specifically, CHO-K1-CD155-CD112-aAPC 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-NF-AT-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, the above diluted samples were added to the white-bottom plate at 40 μL / well, and Jurkat-TIGIT-PVRIG-NF-AT-luc effector cell suspension was added at the same time at 40 μL / well, and incubation was performed for 6 hours in a 37° C., 5% CO2 incubator. Bio-Glo™ reagent was allowed to return to room temperature during this period. After incubation, the cells were equilibrated at room temperature for 5 min, Bio-Glo™ reagent was added to the cells at 80 μL / well, and the luminescence signal values ​​were read using a multifunctional microplate reader.

[0097] The results are shown in Figure 13. The results show that the anti-TIGIT-anti-PVRIG bispecific antibodies of the present invention, TP-007-008, TP-003-009 and TP-001-002-003, can all release TIGIT inhibitory signals mediated by CD155 and PVRIG inhibitory signals mediated by CD112, and improve the expression of luciferase reporter gene, and the blocking activity of TP-007-008 is equivalent to that of the combination of the two monoclonal antibodies.

[0098] Example 7: Synergistic blocking effect of anti-TIGIT-anti-PVRIG bispecific antibody and anti-PD-1 / L1 antibody in a luciferase reporter system In order to detect the synergistic blocking activity of anti-TIGIT-anti-PVRIG bispecific antibody and anti-PD-1 / L1 antibody at the cellular level, the following luciferase reporter gene system was constructed in this example. Briefly, based on Example 6, lentivirus was used to infect CHO-K1-CD155-CD112-aAPC to overexpress PD-L1 and obtain CHO-K1-CD155-CD112-PD-L1-aAPC functional cells, and lentivirus was used to infect Jurkat-TIGIT-PVRIG-NF-AT-luc to overexpress PD-1 and obtain Jurkat-TIGIT-PVRIG-PD-1-NF-AT-luc functional cells, and this reporter gene system was used in subsequent experiments.

[0099] Specifically, CHO-K1-CD155-CD112-PD-L1-aAPC functional cells were obtained by digestion, adjusted to the desired cell density, and added to a 96-well white-bottom plate at 100 μL / well and cultured overnight for attachment. The next day, Jurkat-TIGIT-PVRIG-PD-1-NF-AT-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-PD-1-NF-AT-luc effector cell suspension was added at the same time at 40 μL / well, and the incubation was carried out 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 was read using a multifunctional microplate reader.

[0100] The results are shown in Figures 14 to 14B. The results show that the combination of the anti-TIGIT-anti-PVRIG bispecific antibodies of the present invention, TP-007-008, TP-003-009 and TP-001-002-003, with the anti-PD-1 antibody pembrolizumab (Pembro) or the anti-PD-L1 antibody atezolizumab (ATE) at a 1:1 molar ratio can synergistically block downstream inhibitory signals mediated by PVRIG / CD112, TIGIT / CD155 and PD-1 / PD-L1, and further enhance luciferase signals.

[0101] Example 8: In vivo pharmacodynamics testing of anti-TIGIT-anti-PVRIG bispecific antibodies in B-NDG mice inoculated with a mixture of A375 and human PBMCs In this experiment, the A375 huPBMC model was established by inoculating B-NDG mice 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 anti-tumor effect of the anti-TIGIT-anti-PVRIG bispecific antibody of the present invention. In this regard, human immune cells (PBMC) were inoculated into immunodeficient mice to generate a humanized tumor mouse model with a partial recombinant human immune system.

[0102] Specifically, A375 huPBMC models were established by first mixing A375 cells with human PBMCs 1:1 to form a 0.1 mL cell suspension, which was then subcutaneously injected into the right groin. When the average tumor volume reached approximately 75 mm3, the mice were divided into groups and intraperitoneally injected with PBS or different doses but the same administration volume of therapeutic antibodies. Each group consisted of six mice. The changes in tumor volume and body weight of mice in each group were monitored, and the monitoring frequency was every 2-3 days for 2-3 weeks. The doses and administration methods are shown in Table 5.

[0103] [Table 5]

[0104] The results are shown in Figure 15. The results showed that the anti-TIGIT-anti-PVRIG bispecific antibodies of the present invention, TP-007-008 and TP-003-009, had significant antitumor effects, and the tumor-suppressing effects were equivalent to those of the combination of the two single-terminal antibodies.

[0105] Example 9: In vivo pharmacodynamics study of anti-TIGIT-anti-PVRIG bispecific antibody and anti-PD-1 monoclonal antibody combination in B-NDG mice inoculated with a mixture of A375 and human PBMCs In this experiment, an A375 huPBMC mouse model was established by subcutaneous mixed inoculation (the modeling procedure was the same as in Example 8). When the average tumor volume reached about 200 mm3, the mice were divided into groups and intraperitoneally injected with PBS or different doses but the same administration volume of therapeutic antibodies. 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.

[0106] [Table 6]

[0107] To further explore the dose-dependent efficacy of the combination of anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 and anti-PD-1 monoclonal antibody, the following experimental protocol was designed. An A375 huPBMC model was established by subcutaneous mixed inoculation (the modeling procedure was the same as in Example 8), and the mice were divided into groups when the average tumor volume reached about 300 mm3. Different doses of TP-007-008 in combination with a fixed dose of anti-PD-1 antibody were administered to 6 mice in each group by intraperitoneal injection. 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 7.

[0108] [Table 7]

[0109] As shown in Figure 16A, when the volume of the initially administered tumor reached 200mm3 or more, no significant tumor-inhibiting efficacy was observed in the group of the anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 of the present invention, anti-PD-1 monoclonal antibody, and the combination of anti-TIGIT monoclonal antibody and anti-PVRIG monoclonal antibody. Meanwhile, the combination of TP-007-008 and anti-PD-1 monoclonal antibody could significantly inhibit tumor growth, and thus had similar efficacy to the combination of anti-PD-1 monoclonal antibody, anti-TIGIT monoclonal antibody, and anti-PVRIG monoclonal antibody. As shown in Figure 16B, the combination of anti-TIGIT-anti-PVRIG bispecific antibody TP-007-008 and a fixed dose of PD-1 monoclonal antibody (3mg / kg) had a dose-dependent and significant tumor-inhibiting activity.

[0110] 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: 34) Amino acid sequence of VL of anti-PD-1 antibody EIVLTQSPATLSLSPGERATLSCRASKSVSTSGLNYVHWYQRKPGQAPRLLIYLGSYLDSGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSWELPLTFGGGTKVEIK (SEQ ID NO: 35)

[0111] The CDRs of the above anti-PD-1 antibodies are listed below. HCDR1: YTFTEYYIY (SEQ ID NO: 36) HCDR2: GINPSNGGTNFNEKFKP (SEQ ID NO: 37) HCDR3: TVRDFRFDKGFKY (SEQ ID NO:38) LCDR1: RASKSVSTSGLNYVH (SEQ ID NO: 39) LCDR2: LGSYLDS (SEQ ID NO: 40) LCDR3: QQSWELPLT (SEQ ID NO: 41)

[0112] 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. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 42)

[0113] The light chain constant region of the anti-PD-1 monoclonal antibody used in this example was a human kappa light chain constant region. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 43)

[0114] Example 10: In vivo half-life study of anti-TIGIT-anti-PVRIG bispecific antibodies in mice The in vivo half-life of TP-007-008, an anti-TIGIT-anti-PVRIG bispecific antibody of the present invention, was detected in mice by a single tail vein injection method.

[0115] Specifically, half male and half female Balb / c mice were used in this example and were kept in a 12 / 12 hour light / dark controlled environment at a temperature of 24°C ± 2°C and 40%-70% humidity with free access to water and food. On the day of the experiment, Balb / c mice received a single tail vein injection of the antibody at a dose of 10 mg / kg. Blood collection time points: blood was collected from the orbit at 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 336 hours, and 504 hours after administration. The whole blood samples were placed at 2°C-8°C for 30 minutes and centrifuged at 12,000 rpm for 5 minutes to collect serum. The collected serum was centrifuged at 2°C-8°C for 5 minutes at 12,000 rpm and stored at -80°C. The amount of bispecific antibody molecules in the serum was detected by ELISA (ELISA assay is the same as in Example 5).

[0116] The results are shown in Figure 17. The results showed that the in vivo half-life of the anti-TIGIT-anti-PVRIG bispecific antibody of the present invention, TP-007-008, after a single injection into mice was 103 hours.

[0117] Preparation Example 1: Preparation of anti-TIGIT monoclonal antibody Nucleic acids encoding the heavy chain variable region of an anti-TIGIT monoclonal antibody (amino acid sequences shown in SEQ ID NOs: 1-2) were constructed in the heavy chain constant region of wild-type human IgG1 and in the heavy chain constant region of human IgG1 modified by L234A and L235A, respectively. In addition, nucleic acids encoding the light chain variable region (amino acid sequences shown in SEQ ID NOs: 3-4) were constructed in the human immunoglobulin kappa light chain constant region. The anti-TIGIT monoclonal antibodies were named 55796-G1, 55796-G1LALA, 55812-G1 and 55812-G1LALA, respectively, and were transiently expressed and purified using a HEK293 expression system. The specific procedures were as follows. The pcDNA3.1 vector containing the heavy and light chains of the antibody was transferred into HEK293 cells by chemical transfection and cultured at 37°C and 8% CO2 for 7 days. The cell solution 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. Finally, anti-TIGIT monoclonal antibodies 55796-G1, 55796-G1LALA, 55812-G1 and 55812-G1LALA were obtained.

[0118] Test Example 1: Detection of affinity of anti-TIGIT monoclonal antibody Using biofilm layer optical interference technology (ForteBio), the binding and dissociation rate constants (KD) of the anti-TIGIT monoclonal antibody obtained in Example 1 against human, cynomolgus monkey, and mouse TIGIT were determined. ForteBio affinity measurement was performed according to an existing method (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 TIGIT are shown in Table 1 above.

[0119] Measurement of monovalent affinity of intact antibodies (full length IgG from Adimab) against human, cynomolgus and mouse TIGIT-his proteins: Sensors were equilibrated offline for 20 min in analysis buffer, then detected online for 120 s to establish a baseline, and intact TIGIT antibodies were loaded to a thickness of 1 nm onto the AHQ sensors for affinity detection. The antibody-loaded sensors were incubated in 100 nM TIGIT-his antigen 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.

[0120] The KD values ​​of the antibodies determined by the above method are shown in Table 8.

[0121] [Table 8]

[0122] The results in Table 8 indicate that (1) the monovalent affinity of the anti-TIGIT monoclonal antibody for human TIGIT-his protein is higher than that of the control molecule tiragolumab, (2) the monovalent affinity of the anti-TIGIT monoclonal antibody for cynomolgus monkey TIGIT-his protein is equivalent to that of the control molecule tiragolumab, and (3) the anti-TIGIT monoclonal antibodies 55796-G1 and 55796-G1LALA show cross-binding activity with mouse TIGIT.

[0123] Test Example 2: Binding and blocking activity of anti-TIGIT antibodies against CHO cells overexpressing human / cynomolgus monkey / mouse TIGIT 2.1 Flow cytometry-based detection of binding activity of anti-TIGIT antibodies to human / cynomolgus / mouse TIGIT overexpressed on CHO cells Specifically, human TIGIT-overexpressing CHO cells (CHO-huTIGIT cells), cynomolgus TIGIT-overexpressing CHO cells (CHO-cynoTIGIT cells) and mouse TIGIT-overexpressing CHO cells (CHO-muTIGIT cells) were generated by transfection of pCHO1.0 vector (purchased from Invitrogen) with cDNAs encoding human TIGIT, cynomolgus TIGIT and mouse TIGIT, 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 graph analysis to obtain EC50 values. The results were shown in Table 9 and Figures 18 to 20.

[0124] 2.2 Flow cytometry-based detection of blocking activity of anti-TIGIT antibodies against the binding of human CD155 to human TIGIT overexpressed on CHO cells and against the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells Specifically, CHO-huTIGIT cells were expanded and adjusted to a cell density of 2 x 106 cells / mL, added to a 96-well flow plate at 100 μL / well, and centrifuged for further use. Purified monoclonal antibodies were diluted 3-fold in PBS to a concentration gradient of 12 total concentrations starting from 400 nM. The diluted samples were added to the cell-loaded 96-well flow plate at 60 μL / well and incubated at 4 °C for 30 min. Then, human CD155 protein with mouse IgG2a Fc tag was added at 60 μL / well to a final concentration of 2 μg / mL, incubated at 4 °C for 30 min, 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 min, and washed twice with PBS. Cells were resuspended in PBS at 100 μL / well and detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated.

[0125] CHO-muTIGIT cells were expanded and adjusted to a cell density of 2×106 cells / mL, added to a 96-well flow plate at 100 μL / well, and centrifuged for further use. Purified antibodies were diluted 3-fold in PBS to a concentration gradient of 12 total concentrations starting from 400 nM. The diluted samples were added to the cell-loaded 96-well flow plate at 60 μL / well and incubated at 4° C. for 30 min. Mouse CD155 protein with mouse IgG2a Fc tag was then added at 60 μL / well to a final concentration of 2 μg / mL, incubated at 4° C. for 30 min, 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 min, and washed twice with PBS. Cells were resuspended in PBS at 100 μL / well, detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated. GraphPad software was used for graphing analysis to obtain IC50 values, and the results are shown in Table 9 and Figures 21-22.

[0126] [Table 9]

[0127] It can be seen from the results in Table 9 and Figures 18 to 20 that (1) the binding activity of the anti-TIGIT antibody of the present invention to human TIGIT overexpressed on the surface of CHO cells is better than that of the control molecule tiragolumab, (2) the binding activity of the anti-TIGIT antibody of the present invention to cynomolgus monkey TIGIT overexpressed on the surface of CHO cells is better than that of the control molecule tiragolumab, and (3) the anti-TIGIT antibodies 55796-G1 and 55796-G1LALA of the present invention show significant binding to mouse TIGIT protein overexpressed on the surface of CHO cells.

[0128] It can be seen from the results in Table 9 and Figures 21 to 22 that (1) the activity of the anti-TIGIT antibody of the present invention in blocking the binding of human CD155 to human TIGIT protein overexpressed on the surface of CHO cells is better than that of the control molecule, tiragolumab, and (2) the anti-TIGIT antibodies 55796-G1 and 55796-G1LALA, which showed binding to mouse TIGIT protein overexpressed on the surface of CHO cells, can also significantly block the binding of mouse CD155 to mouse TIGIT protein overexpressed on the surface of CHO cells.

[0129] Test Example 3: Binding of anti-TIGIT antibodies to TIGIT on the surface of primary T cells The binding activity of the anti-TIGIT antibody of the present invention to TIGIT on the surface of activated T cells was detected based on a flow cytometry detection method.

[0130] 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 adjusted to a concentration of 1.0×106 cells / mL using X-vivo 15 medium (purchased from Lonza, Catalog No.: 04-418Q) supplemented with 1 μL of IL-2 stock solution (1,000,000IU) and simultaneously added with CD3 / CD28 Dynabeads (purchased from Gibco, Catalog No.: 11132D) at a 1:1 ratio (beads to cells), and cultured for 48 hours in an incubator at 37°C and 5% CO2. 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. After washing twice with PBS, fluorescent secondary antibodies diluted to appropriate concentrations were added and incubated for 30 min at 4°C, and washed twice with PBS. Cells were resuspended in PBS and detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated.

[0131] The results are shown in Figure 23. The results showed that the anti-TIGIT antibodies of the present invention, 55796-G1, 55796-G1LALA, 55812-G1 and 55812-G1LALA, can bind to TIGIT molecules on the surface of activated T cells, and their binding activity is better than that of the control molecule, tiragolumab.

[0132] Preparation Example 2: Preparation of anti-PVRIG monoclonal antibody Nucleic acids encoding the heavy chain variable region of anti-PVRIG monoclonal antibody (amino acid sequence shown in SEQ ID NO:5) were constructed in the heavy chain constant region of wild-type human IgG1 and in the heavy chain constant region of human IgG1 modified by L234A and L235A, respectively. In addition, nucleic acids encoding the light chain variable region (amino acid sequence shown in SEQ ID NO:4) were constructed in the human immunoglobulin kappa light chain constant region. The anti-PVRIG monoclonal antibodies were named ADI-56127-G1 and ADI-56127-G1LALA, respectively, and were transiently expressed and purified using the HEK293 expression system. The specific procedures were as follows: The pcDNA3.1 vector containing the antibody heavy and light chains was transferred into HEK293 cells by chemical transfection and cultured at 37°C and 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 for endotoxin content.

[0133] Finally, anti-PVRIG monoclonal antibodies ADI-56127-G1 and ADI-56127-G1LALA were obtained.

[0134] Test Example 4: Binding and blocking activity of anti-PVRIG antibodies against CHO cells overexpressing human / cynomolgus monkey / mouse PVRIG 4.1 Flow cytometry-based detection of binding activity of anti-PVRIG antibodies 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 graph analysis to obtain EC50 values. The results were shown in Table 10 and Figures 24 to 26.

[0135] 4.2 Flow cytometry-based detection of blocking activity of anti-PVRIG antibodies against binding of human PVRIG to human CD112 overexpressed on CHO cells Specifically, CHO cells overexpressing human CD112 (CHO-huCD112 cells) were generated by transfection of pCHO1.0 vector (purchased from Invitrogen) with cDNAs encoding human CD112 cloned into the multiple cloning site (MCS) and subsequent pressure screening. CHO-huCD112 cells were expanded and adjusted to a cell density of 2×106 cells / mL, added to a 96-well flow plate at 100 μL / well, and centrifuged for further use. Purified monoclonal antibodies were serially diluted in PBS, and the diluted samples were added to a blank 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, and the mixture was mixed and incubated at 4°C for 30 minutes. The above incubated samples were added at 100 μL / well to a 96-well flow plate containing CHO-huCD112 cells, 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 at 100 μL / well in PBS and detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated. The results are shown in Table 10 and Figure 27.

[0136] 4.3 Detection of blocking activity of anti-PVRIG antibodies 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 with 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 so that the final volume was 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 above sample mixture was added at 100 μL / well and incubated at room temperature for 2 hours. The co-incubated samples were discarded, the ELISA plate was washed three times with 1xPBST, and SA-HRP diluted with 1% BSA / PBS was added at 100μL / well and incubated at room temperature for 1 hour. The SA-HRP diluent was discarded, the ELISA plate was washed three times with 1xPBST, ELISA color development solution was added at 100μL / well, incubated at room temperature for 1-3 minutes, ELISA stop solution was added at 50μL / well, and absorbance values ​​were read at 450nm. Concentration-absorbance binding curves were obtained using GraphPad software, and the results are shown in Table 10 and Figure 28.

[0137] [Table 10]

[0138] The following conclusions can be drawn from Table 10 and Figures 24 to 26. (1) the binding activity of 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-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.

[0139] From Table 10 and Figures 27 to 28, the following conclusions can be drawn. (1) the activity of 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) ADI-56127-G1 and ADI-56127-G1LALA could significantly block the binding of mouse PVRIG to mouse CD112 protein, and their blocking activity was superior to that of the control molecules Mab46-G1 and Mab46-G1LALA.

[0140] Test Example 5: 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.

[0141] 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 adjusted to a concentration of 1.0×106 cells / mL using 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 1:1 ratio (beads to cells), 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. After washing twice with PBS, fluorescent secondary antibodies diluted to appropriate concentrations were added and incubated for 30 min at 4°C, and washed twice with PBS. Cells were resuspended in PBS and detected on a Cytoflex flow cytometer, and the corresponding MFI was calculated.

[0142] The results are shown in Figure 29. The results showed that the anti-PVRIG antibodies of the present invention, ADI-56127-G1 and ADI-56127-G1LALA, can bind to PVRIG molecules on the surface of activated T cells, and have better binding activity than the control molecules Mab46-G1, Mab46-G1LALA, and COM701-G4.

[0143] Test Example 6: 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 partially recombinant human immune system was generated by inoculating human immune cells (PBMC) into immune-deficient mice.

[0144] Specifically, A375 cells were first mixed 1:1 with human PBMCs to form a 0.1 mL cell suspension, which was then subcutaneously injected into the right groin of mice to establish the A375 huPBMC model. When the average tumor volume reached approximately 30-50 mm3, the mice were divided into groups and intraperitoneally injected with PBS or different doses but the same administration volume of therapeutic antibodies. Each group consisted of six 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 11.

[0145] [Table 11]

[0146] The results are shown in Figure 30. 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.

[0147] 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. It is a bispecific antibody, The first protein functional domain that targets PVRIG, It includes a second protein functional region that targets a different target from PVRIG, The first protein functional region is anti-PVRIG immunoglobulin or its antigen-binding fragment, The heavy chain variable region of the anti-PVRIG immunoglobulin includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO:

27. The light chain variable region of the anti-PVRIG immunoglobulin is a bispecific antibody comprising LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

24.

2. The bispecific antibody according to claim 1, wherein the target different from PVRIG is TIGIT.

3. The second protein functional region is anti-TIGIT immunoglobulin or its antigen-binding fragment. The heavy chain variable region of the anti-TIGIT immunoglobulin includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 13, HCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 16, LCDR2 having the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

18. Or, The heavy chain variable region of the anti-TIGIT immunoglobulin includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

24. The bispecific antibody according to claim 1.

4. The bispecific antibody according to claim 1, wherein each antigen-binding fragment is independently a single-chain antibody or an IgG halve (IgG-HM).

5. The first protein functional region is an anti-PVRIG single-chain antibody, and the second protein functional region is an anti-TIGIT immunoglobulin. The heavy chain variable region of the anti-PVRIG single-chain antibody includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PVRIG immunoglobulin includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

24. The heavy chain variable region of the anti-TIGIT immunoglobulin includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 13, HCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 16, LCDR2 having the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 18, or The heavy chain variable region of the anti-TIGIT immunoglobulin includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

24. or The first protein functional region is anti-PVRIG immunoglobulin, and the second protein functional region is anti-TIGIT single-chain antibody. The heavy chain variable region of the anti-PVRIG immunoglobulin comprises HCDR1 having the amino acid sequence shown in SEQ ID NO: 25, HCDR2 having the amino acid sequence shown in SEQ ID NO: 26, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region of the anti-PVRIG immunoglobulin comprises LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

24. The heavy chain variable region of the anti-TIGIT single-chain antibody includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 13, HCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 16, LCDR2 having the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 18, or The heavy chain variable region of the anti-TIGIT single-chain antibody includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 19, HCDR2 having the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region of the anti-TIGIT immunoglobulin includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 22, LCDR2 having the amino acid sequence shown in SEQ ID NO: 23, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

24. The bispecific antibody according to claim 1.

6. The heavy chain variable region of the anti-PVRIG immunoglobulin or the anti-PVRIG single-chain antibody has the amino acid sequence shown in SEQ ID NO:

5. The light chain variable region of the anti-PVRIG immunoglobulin or the anti-PVRIG single-chain antibody has the amino acid sequence shown in SEQ ID NO:

4. The bispecific antibody according to claim 5.

7. The glycine at position 44 of the heavy chain variable region of the anti-PVRIG immunoglobulin or the anti-PVRIG single-chain antibody is substituted with cysteine, and the glycine at position 100 of the light chain variable region of the anti-PVRIG immunoglobulin or the anti-PVRIG single-chain antibody is substituted with cysteine. The bispecific antibody according to claim 6.

8. The heavy chain variable region of the anti-TIGIT immunoglobulin or the anti-TIGIT single-chain antibody has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region of the anti-TIGIT immunoglobulin or the anti-TIGIT single-chain antibody has the amino acid sequence shown in SEQ ID NO:

2. or The heavy chain variable region of the anti-TIGIT immunoglobulin or the anti-TIGIT single-chain antibody has the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region of the anti-TIGIT immunoglobulin or the anti-TIGIT single-chain antibody has the amino acid sequence shown in SEQ ID NO:

4. The bispecific antibody according to claim 5.

9. The bispecific antibody according to claim 8, wherein the glycine at position 44 of the heavy chain variable region of the anti-TIGIT immunoglobulin or the anti-TIGIT single-chain antibody is substituted with cysteine, and the glycine at position 100 of the light chain variable region of the anti-TIGIT immunoglobulin or the anti-TIGIT single-chain antibody is substituted with cysteine.

10. The bispecific antibody according to claim 1, having one or more of the following features (1) to (4). (1) The first protein functional region and the second protein functional region are directly bound or bound via a linker. (2) The first protein functional region and the second protein functional region are linked via a linker, the linker being (GGGGS)m, where m is 1, 2, 3, 4, 5, or 6. (3) The first protein functional region and the second protein functional region are linked via a linker, the amino acid sequence of the linker is as shown in SEQ ID NO:

6. (4) The number of the first protein functional region and the second protein functional region is independently 1, 2, or more than 2.

11. The bispecific antibody according to claim 5, having one or more of the following features (1) to (5). (1) The anti-TIGIT single-chain antibody is bound to the C-terminus of each of the two heavy chains of the anti-PVRIG immunoglobulin, or The anti-PVRIG single-chain antibody is bound to the C-terminus of each of the two heavy chains of the anti-TIGIT immunoglobulin. (2) The constant region of the anti-PVRIG immunoglobulin or the anti-TIGIT immunoglobulin is derived from a human antibody. (3) The steady-state region is independently selected from the group consisting of the steady-state regions of human IgG1, IgG2, IgG3, or IgG4. (4) The heavy chain constant region of the anti-PVRIG immunoglobulin or the anti-TIGIT immunoglobulin is the human Ig gamma-1 chain C region or the human Ig gamma-4 chain C region, and the light chain constant region is the human Ig kappa chain C region. (5) The heavy chain constant regions of the anti-PVRIG immunoglobulin and the anti-TIGIT immunoglobulin include mutations represented as L234A and L235A mutations according to the EU numbering system.

12. The bispecific antibody according to claim 1, which is a dimer or trimer formed from a peptide chain having the amino acid sequence shown in SEQ ID NO: 7 and a peptide chain having the amino acid sequence shown in SEQ ID NO: 8, or a dimer or trimer formed from a peptide chain having the amino acid sequence shown in SEQ ID NO: 9 and a peptide chain having the amino acid sequence shown in SEQ ID NO:

10.

13. An isolated nucleic acid molecule encoding the bispecific antibody described in Claim 1.

14. A vector comprising the isolated nucleic acid molecule described in Claim 13.

15. A host cell comprising an isolated nucleic acid molecule or vector according to claim 13, wherein the vector comprises the isolated nucleic acid molecule according to claim 13.

16. A conjugate comprising a bispecific antibody and a conjugate portion, wherein the bispecific antibody is the bispecific antibody according to any one of claims 1 to 12, the conjugate portion is a detectable label, and the conjugate portion is a radioisotope, a fluorescent substance, a coloring substance, or an enzyme.

17. A kit comprising a bispecific antibody according to any one of claims 1 to 12, A kit further comprising a second antibody capable of specifically binding to the bispecific antibody, wherein the second antibody further comprises a detectable label, the detectable label being a radioisotope, a fluorescent substance, a coloring agent, or an enzyme.

18. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 12 and one or more pharmaceutically acceptable excipients.

19. The pharmaceutical composition according to claim 18, further comprising at least one anti-PD-1 antibody.

20. The pharmaceutical composition according to claim 19, having one or more of the following features (1) to (3). (1) The molar ratio of the bispecific antibody to the anti-PD-1 antibody is (1:5) to (5:1) or 1:

1. (2) 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 includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 36, HCDR2 having the amino acid sequence shown in SEQ ID NO: 37, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 38, and the light chain variable region of the anti-PD-1 antibody includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 39, LCDR2 having the amino acid sequence shown in SEQ ID NO: 40, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

41. (3) The heavy chain variable region of the anti-PD-1 antibody has the amino acid sequence shown in SEQ ID NO: 34, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

35.

21. A combination product comprising a first product and a second product packaged separately, The first product comprises a bispecific antibody as described in any one of claims 1 to 12. The second product comprises at least one anti-PD-1 antibody. Combination product.

22. The combination product according to claim 21, having one or more of the following features (1) to (5). (1) The first product and the second product each independently further comprise one or more pharmaceutically acceptable excipients. (2) The combination product further includes an accompanying document, (3) The molar ratio of the bispecific antibody to the anti-PD-1 antibody is (1:5) to (5:1), or 1:

1. (4) 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 includes HCDR1 having the amino acid sequence shown in SEQ ID NO: 36, HCDR2 having the amino acid sequence shown in SEQ ID NO: 37, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 38, and the light chain variable region of the anti-PD-1 antibody includes LCDR1 having the amino acid sequence shown in SEQ ID NO: 39, LCDR2 having the amino acid sequence shown in SEQ ID NO: 40, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

41. (5) The heavy chain variable region of the anti-PD-1 antibody has the amino acid sequence shown in SEQ ID NO: 34, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

35.

23. Use of a bispecific antibody according to any one of claims 1 to 12 in the manufacture of a pharmaceutical product for the treatment or prevention of tumors.

24. 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, urinary tract cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer, or The tumor is lung cancer, and the lung cancer is either non-small cell lung cancer or small cell lung cancer. The use described in claim 23.

25. A bispecific antibody according to any one of claims 1 to 12, for use in the treatment or prevention of tumors.

26. 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, urinary tract cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer, or The tumor is lung cancer, and the lung cancer is either non-small cell lung cancer or small cell lung cancer. The bispecific antibody according to claim 25.

27. ​​A pharmaceutical composition for treating or preventing a tumor, comprising an effective amount of the bispecific antibody described in any one of claims 1 to 12.

28. 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, urinary tract cancer, bone tumor, bile duct cancer, rectal cancer, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, plasma cell carcinoma, prostate cancer, and testicular cancer, or The tumor is lung cancer, and the lung cancer is either non-small cell lung cancer or small cell lung cancer. The pharmaceutical composition according to claim 27.

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