Anti-PVRIG antibodies and uses thereof

Abstract

The present invention discloses anti-human PVRIG antibodies or antigen-binding fragments thereof and their use in the preparation of medicaments for treating diseases associated with the aberrant expression of PVRIG, such as cancer.

Description

[Technical Field]

[0001] The present invention relates to the biomedical field, and specifically to an anti-human PVRIG antibody or an antigen-binding fragment thereof and uses thereof. [Background technology]

[0002] Human PVRIG (poliovirus receptor-related immunoglobulin domain-containing protein), also known as CD112 receptor (CD112R), is a 34-kDa protein in the poliovirus receptor-like protein (PVR) family. This gene encodes a transmembrane protein consisting of an extracellular IgV domain, a transmembrane domain, and a long intracellular domain. PVRIG (CD112R) is primarily expressed on T cells and NK cells and plays a regulatory role in immune function. When PVRIG binds to its ligand PVRL2 (CD112, nectin-2) on antigen-presenting cells, it inhibits immune activation. Furthermore, PVRIG can exert its immunosuppressive effects by competing with CD226 for its ligand and blocking the stimulatory signal transduction. This mechanism is similar to that of its family member, TIGIT, and these two proteins may exert a synergistic effect in immunosuppression. Clinically, PVRL2 has been found to be overexpressed in multiple tumor types, including breast and ovarian cancer, suggesting that its interaction with PVRIG may be a major mechanism of immunosuppression in these diseases. Therefore, therapeutic strategies targeting PVRIG may be clinically applicable as monotherapy, dual antibody therapy, or combination therapy. Currently, the most advanced therapeutics are Compugen's COM-701 and Surface Oncology's SRF813, but these are still in clinical research and no commercially available products have yet been released.

[0003] To provide patients with more diverse treatment options, the present invention provides an antibody with higher affinity for recognizing PVRIG on the cell surface, and compared with SRF813 (Patent US20210253699A1), the antibody of the present invention can more potently inhibit the binding of PVRIG to CD112 at the cellular level. Furthermore, in vivo animal experiment data confirm that the antibody obtained by the present invention can effectively suppress the excessive proliferation of tumor cells. DISCLOSURE OF THE INVENTION

[0004] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and / or LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (2) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and / or LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 25, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26; or (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38, and / or LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 39, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 40, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 41. The present invention relates to an anti-human PVRIG antibody or antigen-binding fragment thereof, comprising:

[0005] In one particular embodiment of the present invention, (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, as well as LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (2) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 21, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 23, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 26; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38, as well as LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 39, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 40, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 41. The present invention provides an anti-human PVRIG antibody or antigen-binding fragment thereof, comprising:

[0006] In one embodiment of the present invention, comprising a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16; (2) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32, and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35; or (3) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47, and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50; Anti-human PVRIG antibodies or antigen-binding fragments thereof are provided.

[0007] In one embodiment of the present invention, comprising a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 47, and / or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 50; (2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32, and / or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35; or (3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and / or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14; Anti-human PVRIG antibodies or antigen-binding fragments thereof are provided.

[0008] In one embodiment of the present invention, comprising a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 50; (2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35; or (3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14; Anti-human PVRIG antibodies or antigen-binding fragments thereof are provided.

[0009] In one embodiment of the present invention, comprising a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16; (2) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35; or (3) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50; Anti-human PVRIG antibodies or antigen-binding fragments thereof are provided.

[0010] In one embodiment of the present invention, comprising a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:47, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:50; (2) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 32, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 35; or (3) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 10, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 14; Anti-human PVRIG antibodies or antigen-binding fragments thereof are provided.

[0011] In one embodiment of the present invention, comprising a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:47, and the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:50; (2) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 32, and the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 35; or (3) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 10, and the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 14; Anti-human PVRIG antibodies or antigen-binding fragments thereof are provided.

[0012] In one embodiment of the invention, the anti-human PVRIG antibody or antigen-binding fragment thereof is preferably a monoclonal antibody.

[0013] In one embodiment of the invention, the anti-human PVRIG antibody or antigen-binding fragment thereof is a humanized or human antibody or portion thereof.

[0014] In one embodiment of the invention, the anti-human PVRIG antibody or antigen-binding fragment thereof further comprises an Fc region.

[0015] In one embodiment of the invention, the anti-human PVRIG antibody or antigen-binding fragment thereof is an IgG1 or IgG4 antibody or portion thereof.

[0016] In one embodiment of the invention, the anti-human PVRIG antibody or antigen-binding fragment thereof further comprises an Fc region variant.

[0017] In one embodiment of the present invention, Fc variants comprise an amino acid sequence that differs from a "native" or "wild-type" Fc region sequence by virtue of at least one amino acid mutation compared to the native Fc region sequence. Preferably, Fc variants have at least one amino acid substitution compared to the native Fc region sequence or the Fc region sequence of a parent polypeptide, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native Fc region sequence or the Fc region sequence of a parent polypeptide. The Fc region variants herein preferably have at least about 80% homology with the native Fc region sequence and / or the Fc region sequence of the parent polypeptide, most preferably at least about 90% homology, and even more preferably at least about 95% homology.

[0018] In one embodiment of the invention, the Fc region variant is an IgG1 Fc variant sequence.

[0019] In one embodiment of the invention, the mutations in said IgG1 Fc variant occur at serine at position 239, alanine at position 330, or isoleucine at position 332.

[0020] In one embodiment of the invention, the mutations in said IgG1 Fc variant occur at serine at position 239, alanine at position 330, and isoleucine at position 332.

[0021] In one embodiment of the invention, the mutations in the IgG1 Fc variant comprise three point mutations: S239D, A330L, and I332E.

[0022] The Fc variants of the present invention are defined according to the modification of their constituent amino acids. Thus, for example, P329G is an Fc variant in which proline is substituted with glycine at position 329 relative to the parent Fc polypeptide, and numbering is according to the EU index. The identity of the wild-type amino acid may not be specified, in which case the variant is referred to as P329G. For all positions in the Fc region discussed in the present invention, numbering is according to the EU index. The EU index or the EU index or EU numbering scheme in Kabat refers to the EU numbering of antibodies (Edelman et al., Proc Natl Acad Sci USA 63 (1969) 78-85, the entirety of which is incorporated herein by reference).

[0023] In one embodiment of the invention, the antigen-binding fragment of the anti-human PVRIG antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, scFv, dAb, or CDR.

[0024] Unless otherwise specified, amino acid numbering of variable regions herein is determined according to the Kabat numbering convention.

[0025] Another aspect of the invention provides isolated nucleic acid molecules encoding the aforementioned anti-human PVRIG antibodies or antigen-binding fragments thereof.

[0026] A third aspect of the present invention provides an expression vector comprising the above-described nucleic acid molecule.

[0027] A fourth aspect of the present invention provides a host cell comprising an expression vector as described above.

[0028] A fifth aspect of the invention provides a method for preparing the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof, comprising culturing the aforementioned host cell, thereby expressing the nucleic acid molecule.

[0029] In one embodiment of the present invention, the aforementioned method of preparing an anti-human PVRIG antibody or antigen-binding fragment thereof further comprises recovering the anti-human PVRIG antibody or antigen-binding fragment thereof from the host cell culture.

[0030] A sixth aspect of the invention provides an antibody-drug conjugate comprising the aforementioned anti-human PVRIG antibody, or antigen-binding fragment thereof, conjugated to a therapeutic agent.

[0031] A seventh aspect of the invention provides a bispecific molecule comprising an anti-human PVRIG antibody, or antigen-binding fragment thereof, as described above, that targets an epitope of another antigen or is conjugated to a peptide of another antigen.

[0032] An eighth aspect of the invention provides a composition comprising the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof, or the aforementioned antibody-drug conjugate, or the aforementioned bispecific molecule. Optionally, the composition comprises a pharmaceutically acceptable carrier.

[0033] A ninth aspect of the invention relates to the use of an anti-human PVRIG antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, or a bispecific molecule, or a composition, as described above, in the preparation of a medicament for modulating an immune response in a subject.

[0034] A tenth aspect of the invention provides a method of modulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an anti-human PVRIG antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, or a bispecific molecule, or a composition.

[0035] An eleventh aspect of the invention provides an anti-human PVRIG antibody or antigen-binding fragment thereof as described above, or an antibody-drug conjugate as described above, or a bispecific molecule as described above, or a composition as described above, for use in modulating an immune response in a subject.

[0036] A twelfth aspect of the invention provides the use of an anti-human PVRIG antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, or a bispecific molecule, or a composition as described above, in the preparation of a medicament for inhibiting the growth of tumor cells in a subject.

[0037] A thirteenth aspect of the invention provides a method of inhibiting tumor cell growth in a subject, comprising administering to the subject a therapeutically effective amount of any of the above-described anti-human PVRIG antibodies or antigen-binding fragments thereof, or any of the above-described antibody-drug conjugates, or any of the above-described bispecific molecules, or any of the above-described compositions.

[0038] A fourteenth aspect of the invention provides an anti-human PVRIG antibody or antigen-binding fragment thereof as described above, or an antibody-drug conjugate as described above, or a bispecific molecule as described above, or a composition as described above, for use in inhibiting the growth of tumor cells in a subject.

[0039] In one specific embodiment of the invention, the tumor cells are tumor cells that overexpress PVRIG or PVRL2. In one specific embodiment of the invention, the tumor cells are selected from the group consisting of bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute bone marrow cancer, and the like. The cancer is selected from tumor cells of cancers including myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and any combination of the foregoing cancers.

[0040] A fifteenth aspect of the invention provides the use of an anti-human PVRIG antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, or a bispecific molecule, or a composition as described above, in the preparation of a medicament for treating a hyperproliferative disease.

[0041] A sixteenth aspect of the invention provides a method of treating a hyperproliferative disease, comprising administering to a subject a therapeutically effective amount of any of the above-described anti-human PVRIG antibodies or antigen-binding fragments thereof, or any of the above-described antibody-drug conjugates, or any of the above-described bispecific molecules, or any of the above-described compositions.

[0042] A seventeenth aspect of the invention provides a therapeutically effective amount of an anti-human PVRIG antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, or a bispecific molecule, or a composition, as described above, for use in the treatment of a hyperproliferative disease.

[0043] In one particular embodiment of the invention, said hyperproliferative disease is cancer, preferably a cancer that overexpresses PVRL2.

[0044] In one particular embodiment of the invention, the cancer is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute bone marrow cancer, or the like. The cancer is selected from myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and any combination of the foregoing cancers.

[0045] An eighteenth aspect of the present invention provides the use of an anti-human PVRIG antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, or a bispecific molecule, or a composition, as described above, in the preparation of a drug for inhibiting binding between a PVRIG protein and a CD112 protein.

[0046] A nineteenth aspect of the present invention provides a method for inhibiting binding of PVRIG protein to CD112 protein, comprising administering to a subject a therapeutically effective amount of the above-described anti-human PVRIG antibody or antigen-binding fragment thereof, or the above-described antibody-drug conjugate, or the above-described bispecific molecule, or the above-described composition.

[0047] A twentieth aspect of the present invention provides an anti-human PVRIG antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, or a bispecific molecule, or a composition as described above, for use in inhibiting the binding of a PVRIG protein to a CD112 protein.

[0048] definition As used herein, the term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions are further divided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The amino acid residues in the complementarity determining regions are generally, for example, residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable region and residues 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region (Kabat et al., Sequence of proteins of immunological interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)), or, for example, residues 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3) in the light chain variable region and residues 26-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3) in the heavy chain variable region (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987). The numbering convention for amino acids in this application is the Kabat numbering convention.

[0049] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human PVRIG). It has been demonstrated that the antigen-binding function of an antibody can be achieved by certain fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains; (ii) an F(ab')2 fragment, i.e., a bivalent fragment consisting of two Fab' fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of a single-arm VL and VH domains of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 241:544-546); and (vi) a CDR. Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by different genes but can be recombinantly linked with a synthetic linker to form a single linked chain, in which the VL and VH domains pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426, and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also encompassed by the term "antigen-binding fragment" of an antibody. Other forms of single-chain antibodies, such as bispecific antibodies, are also included (see, e.g., Holliger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448).

[0050] The term "humanized antibody" contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies are most often human immunoglobulins (recipient antibodies) in which CDR region residues from the recipient are replaced by CDR region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and performance. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not present in either the recipient or donor antibody. These modifications are intended to further improve antibody performance. Generally, humanized antibodies comprise substantially all of at least one (usually two) variable domain, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or substantially all of the FR sequences being human immunoglobulin sequences. When the VH and VL sequences of a humanized antibody are all derived from a non-human species, the humanized antibody is a chimeric antibody. The humanized antibody may optionally further comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For details, see Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0051] As used herein, the term "human antibody" is intended to include antibodies in which both the framework and CDR regions within the variable regions are derived from human germline immunoglobulin sequences. Additionally, if the antibody contains a constant region, the constant region also is derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0052] "Homology" is defined as the percentage of identical residues among amino acid sequence variants after aligning the sequences and introducing gaps (if necessary) to maximize the percentage of homology. Methods and computer programs for alignment are well known in the art.

[0053] An expression vector is a DNA molecule containing a gene to be expressed in a host cell. Usually, gene expression is placed under the control of specific regulatory elements, such as a constitutive promoter, an inducible promoter, a tissue-specific regulatory element, or an enhancer. Such a gene is said to be "operably linked" to the regulatory element.

[0054] A host cell is any prokaryotic or eukaryotic cell that contains an expression vector. The term also includes prokaryotic and eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell, or intracellularly of the host cell, as well as transgenic animals.

[0055] An antibody-drug conjugate refers to an anti-human PVRIG antibody or its antigen-binding fragment conjugated with a therapeutic agent such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. Cytotoxins include any substance that damages cells. Examples of cytotoxins and chemotherapeutic agents suitable for forming immunoconjugates are well known in the art. See, for example, WO05 / 103081.

[0056] A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. For example, a bispecific antibody can bind to two different epitopes of the human PVRIG protein. Other such antibodies may combine a human PVRIG binding site with a binding site of another antigen. Bispecific antibodies can be prepared as full-length antibodies or antigen-binding fragments (e.g., F(ab')2 bispecific antibodies).

[0057] The phrase "therapeutically effective amount" refers to an amount capable of producing a desired effect in a subject to which it is administered. The exact dosage will depend on the purpose of treatment, the age and body size of the subject to be treated, the route of administration, etc., and can be determined by one of ordinary skill in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). In the case of treating cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer.

[0058] As used herein, the term "test subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. [Brief explanation of the drawings]

[0059] [Figure 1] Figure 1 shows the results of a binding experiment between chimeric antibodies and soluble PVRIG. [Figure 2] Figure 1 shows the results of a binding experiment between humanized antibodies and soluble human PVRIG protein. [Figure 3] FIG. 1 shows the results of a binding experiment between humanized antibodies and soluble cynomolgus monkey PVRIG. [Figure 4] This figure shows the results of a binding experiment between PVRIG and humanized antibodies on the surface of cells overexpressing human PVRIG. [Figure 5] FIG. 1 shows the results of a binding experiment between PVRIG and a humanized antibody on the surface of cells overexpressing cynomolgus monkey PVRIG. [Figure 6] FIG. 1 shows the results of an ELISA assay of a humanized antibody that inhibits the binding of PVRIG protein to CD112 protein. [Figure 7] Fig. 10 shows the results of flow cytometry of a humanized antibody that inhibits the binding of PVRIG protein to CD112 cells. [Figure 8] FIG. 1 shows the results of a reporter gene assay of a humanized antibody that inhibits the binding of PVRIG protein to CD112 cells. [Figure 9A] FIG. 1 shows the results of chimeric antibodies inhibiting the volume change of transplanted tumors in A375 tumor-bearing mice. [Figure 9B] FIG. 1 shows the effect of chimeric antibodies on the body weight of A375 tumor-bearing mice. [Figure 9C] FIG. 1 shows the results of chimeric antibodies inhibiting changes in the volume of individual tumors implanted in A375 tumor-bearing mice. [Figure 10] This figure shows the effect of combined use of anti-PVRIG monoclonal antibody and anti-PD-1 monoclonal antibody on the volume of subcutaneously implanted tumors containing mixed PBMCs from human melanoma A375. [Figure 11] FIG. 1 shows the drug blood concentration-time curves following a single intravenous injection of anti-PVRIG monoclonal antibody in cynomolgus monkeys. DETAILED DESCRIPTION OF THE INVENTION

[0060] Example 1: Obtaining anti-human PVRIG monoclonal antibodies Healthy BALB / c mice were immunized with recombinant human PVRIG extracellular domain protein (amino acid sequence shown in SEQ ID NO: 51). After the first immunization, booster immunizations were administered every 14 days for a total of four immunizations. Mouse serum titers were detected by flow cytometry, and mice with high serum antibody titers were selected for cell fusion. Three days before fusion, PVRIG extracellular domain protein was injected into the tail vein for rush immunization. On the day of fusion, mice were euthanized, and their spleens were removed under aseptic conditions to prepare single-cell suspensions. SP2 / 0 cells and spleen cells were mixed at a 1:1 ratio and fused by electrofusion. The fused cells were seeded into 96-well cell culture plates, with each well containing one to three hybridoma cells. After adding selection medium (HAT medium), the cells were cultured in a 37% CO2 incubator and screened based on cell growth status for approximately 10 days.

[0061] Recombinant human PVRIG extracellular domain protein sequence: TPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDPGLSAPPTPAPILRAD (SEQ ID NO: 51) High-affinity antibodies were selected, and fragments of the variable regions of mouse immunoglobulin heavy and light chains were isolated by RNA extraction, reverse transcription, and PCR amplification, followed by sequencing. The final amino acid sequences of the heavy and light chain CDR regions and variable regions of the resulting clones 1B10E8, 21F10C10, and 43F7C9 were as follows: 1B10E8 Heavy chain CDR1: SYWMH (SEQ ID NO: 1) Heavy chain CDR2: EINPSNGLTNYNEQFRN (SEQ ID NO: 2) Heavy chain CDR3: HHGSTYYALDS (SEQ ID NO: 3) Heavy chain variable region sequence QVQLQQPGAELVKPGASVKLSCKASGISFPS YWMH WVRQRPGQDLEWIG EINPSNGLTNYNEQFRN KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR HHGSTYYALDS WGQGTSITVSS (SEQ ID NO: 7) 1B10E8 Light chain CDR1: KSSQSLLNSINQKNFLA (SEQ ID NO: 4) Light chain CDR2: FASTRES (SEQ ID NO: 5) Light chain CDR3: QQHYSTPPT (SEQ ID NO: 6) Light chain variable region sequence DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSINQKNFLA WYQQRPGQSPKLLVY FASTRES GVPDRFIGSGSGTDFLTISSVQAEDLADYFC QQHYSTPPT FGGGTKLEIK (SEQ ID NO: 8) 21F10C10 Heavy chain CDR1: NNFIE (SEQ ID NO: 17) Heavy chain CDR2: VINPGSGDANYNEKFKG (SEQ ID NO: 18) Heavy chain CDR3: HWLAY (SEQ ID NO: 21) Heavy chain variable region sequence QVQLQQSGAELVRPGTSVKVSCKASGYAFI NNFIE WVKQRPGQGLEWIG VINPGSGDANYNEKFKG KATLTADKSSTTAYIQLSSLTSDDSAVYFCAS HWLAY WGQGTLVTVSA (SEQ ID NO: 27) 21F10C10 Light chain CDR1: KSSQSLLSSGNQKNYLA (SEQ ID NO: 22) Light chain CDR2: GASTRES (SEQ ID NO: 24) Light chain CDR3: QNGHFYPYT (SEQ ID NO: 26) Light chain variable region sequence DIVMTQSPSSLSVSAGEKVTMSC KSSQSLLSSGNQKNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNGHFYPYT FGGGTKLEIK (SEQ ID NO: 28) 43H7C9 Heavy chain CDR1: SDYAWN (SEQ ID NO: 36) Heavy chain CDR2: YIRYNGNTNYNPSLKS (SEQ ID NO: 37) Heavy chain CDR3: IYYDYDGFVY (SEQ ID NO: 38) Heavy chain variable region sequence DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNRLEWMG YIRYNGNTNYNPSLKS RISITRDTSKNQFFLQLNSVTTEDTATYYCAI IYYDYDGFVY WGQGTLVTVSA (SEQ ID NO: 42) 43H7C9 Light chain CDR1: RASQNVYENLH (SEQ ID NO: 39) Light chain CDR2: FASDSIS (SEQ ID NO: 40) Light chain CDR3: LQLYSTPYT (SEQ ID NO: 41) Light chain variable region sequence DILLTQSPATLSVTPGETVSLSC RASQNVYENLH WYQQKSHRSPRLLIK FASDSIS GIPSRFFTGSGSGTDYTLSINSVKPEDEGIYYC LQLYSTPYT FGGGTELEIK (SEQ ID NO: 43) Note: In the above antibody sequences, the underlined sequences are the CDR sequences (determined and annotated according to the Kabat numbering system).

[0062] Example 2: Detection of affinity of chimeric antibodies by ELISA A human-mouse chimeric antibody expression vector was constructed based on the variable region sequences of the monoclonal antibody gene. VH and VL were separately constructed in eukaryotic expression vectors containing the hIgG1-kappa constant region. The resulting eukaryotic expression vectors were transiently transformed into HEK-293 cells and cultured for 6 days. The culture supernatants were collected and purified with protein A to obtain the target antibodies, designated 21F10C10-hIgG1 and 43F7C9-hIgG1, respectively. The affinity of these antibodies for recombinant PVRIG protein was determined by ELISA. Extracellular full-length PVRIG protein was diluted to a concentration of 500 ng / ml and applied to an ELISA plate. The plate was washed three times with PBST buffer and blocked with 200 μl of 2% BSA in PBS buffer at 37°C for 2 hours. After blocking, the plate was washed three times with PBST buffer. A 3-fold gradient dilution of the test antibody (0-33.3 nM) was added and incubated at 37°C for 2 hours. The plate was then washed three times with PBST buffer. Horseradish peroxidase-conjugated goat anti-human antibody was added and incubated at 37°C for 1 hour. The plate was then washed three times with PBST buffer. A TMB single-component color development solution was added for color development, and absorbance values ​​were read after the addition of stop solution. The resulting affinities are shown in Figure 1 and Table 1.

[0063] [Table 1]

[0064] EC2 binding of 21F10C10-hIgG1 and 43F7C9-hIgG1 to PVRIG protein 50 were 0.1222 nM and 0.2901 nM, respectively.

[0065] Example 3: Humanization of antibodies Homology modeling was performed using Discovery Studio and Schrodinger Antibody Modeling. Structural simulation and rational design yielded the human framework regions that were closest to the murine antibody framework regions.

[0066] The light and heavy chain CDRs were grafted onto the framework sequences of the corresponding light and heavy chain genes, resulting in the humanized antibodies Hab1B10E8, Hab21F10C10, and Hab43F7C9. Next, 3D models were constructed using Discovery Studio and Schrodinger Antibody Modeling to analyze whether there were any sites in the framework where mouse amino acids could be substituted with human amino acids that would affect binding and / or CDR conformation, and backmutations were performed. The sequences are shown in Tables 2 and 3 (in Tables 2 and 3, all CDR regions are numbered using the Kabat numbering convention). The resulting backmutations were combined and expressed, and the expressed antibody combinations are shown in Tables 4, 5, and 6.

[0067] [Table 2]

[0068] TIFF2025529238000003.tif222166

[0069] [Table 3]

[0070] TIFF2025529238000005.tif185166

[0071] [Table 4]

[0072] [Table 5]

[0073] [Table 6]

[0074] The antibody constant region sequences used in the examples are as follows: IgG1 constant region ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 51) IgG4 constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 52) The IgG1 constant region, Fc, contains the S239D-A330L-I332E mutation (abbreviated as DLE mutation). ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 53)

[0075] Example 4: Affinity detection of humanized anti-PVRIG antibodies 4.1 Detection of affinity between humanized antibodies and human PVRIG protein by Fortebio Using the HIS1K biosensor, human PVRIG-His protein was immobilized on the biosensor with a binding response threshold of 0.3 nM. After a 120-second baseline step, the sensor was immersed in anti-PVRIG antibody diluted in 0.02% PBST buffer. The starting antibody concentration was 25 nM, and seven gradients were set up with two-fold dilutions. The association and dissociation times of the immobilized material and analyte were 120 and 300 seconds, respectively. Antigen-antibody affinity data were calculated using regression analysis software based on the binding curves. Among all the antibodies tested, Hab1B10E8.6, Hab21F10C10.12, and Hab43F7C9.12 showed the highest affinity and few back mutations. The results are shown in Table 7.

[0076] [Table 7]

[0077] 4.2 Detection of affinity between humanized antibodies and human PVRIG protein by ELISA The affinity of the humanized antibodies for recombinant human PVRIG protein was measured by ELISA. PVRIG protein was diluted to 500 ng / ml and applied to an ELISA plate. After blocking, a 3-fold gradient dilution of the antibody (0-66.7 nM) was added and incubated with a goat anti-human secondary antibody. Colorimetric measurements were performed, and the affinities obtained by fitting the results using GraphPad are shown in Figure 2 and Table 8.

[0078] [Table 8]

[0079] EC of Hab1B10E8.6, Hab21F10C10.12, and Hab43F7C9.12 binding to humanized PVRIG protein 50 were 0.0869 nM, 0.1646 nM, and 0.1447 nM, respectively.

[0080] 4.3 Detection of affinity between humanized antibodies and cynomolgus monkey PVRIG protein by ELISA The affinity of the humanized antibodies for recombinant cynomolgus monkey (Cyno) PVRIG protein was measured by ELISA. Cyno-PVRIG protein was diluted to 500 ng / ml and applied to an ELISA plate. After blocking, a 3-fold gradient dilution of the antibody (0-33.3 nM) was added and incubated with a goat anti-human secondary antibody for 1 hour. The plate was then washed and subjected to colorimetric measurements. The affinities obtained by fitting the results using GraphPad are shown in Figure 3 and Table 9.

[0081] [Table 9] The EC50 values ​​of antibodies Hab1B10E8.6, Hab21F10C10.12, and Hab43F7C9.12 that bind to Cyno-PVRIG protein are the EC50 values ​​of the affinity of the antibodies to human PVRIG protein. 50 not much different from the value.

[0082] Example 5: Binding of humanized antibodies to cells overexpressing human PVRIG After expressing the Hab1B10E8.6, Hab21F10C10.12, Hab43F7C9.12, and SRF813 antibodies (the CDR region sequences are from clone 46 of US20210253699A1, the amino acid sequence of the HV is as set forth in SEQ ID NO: 912 of the same patent application, and the amino acid sequence of the LV is as set forth in SEQ ID NO: 918 of the same patent application), the ability of the humanized antibodies to bind to cell surface PVRIG was evaluated using a Jurkat-hPVRIG cell line overexpressing human PVRIG. 2 × 10 cells were cultured. 6 The cells were seeded into a 96-well plate at a density of 100 μl / ml per well. A 4-fold gradient dilution of the antibody (0-66.7 nM) was added and incubated at 4°C for 1 hour. A goat anti-human secondary antibody was added and the cells were labeled in the dark at 4°C for 45 minutes. After harvesting and washing, the cells were analyzed for fluorescence intensity by flow cytometry. The experimental results are shown in Figure 4 and Table 10. The results demonstrate that the humanized antibody can bind to the PVRIG protein expressed on the cell line in a dose-dependent manner, and that the affinity of Hab43F7C9.12 to recognize PVRIG on the cell surface is superior to that of the control molecule SRF813.

[0083] [Table 10]

[0084] Example 6: Binding of humanized anti-PVRIG antibodies to cells overexpressing cynomolgus monkey PVRIG To characterize the ability of the screened antibodies to recognize the cynomolgus monkey PVRIG molecule on the cell membrane, 293T cells capable of stably expressing full-length monkey PVRIG were constructed. 2 × 10 cells were cultured. 6The cells were seeded into a 96-well plate at a density of 100 μl / ml per well. The cells were washed three times with FACS buffer, and the supernatant was discarded. Gradient dilutions of antibody (0-2.5 nM) in PBS buffer were added and incubated at 4°C for 1 hour. The cells were washed three times with FACS buffer and then labeled with Alexa Fluor 488-conjugated goat anti-human IgG antibody in the dark for 45 minutes at 4°C. The cells were washed three times with FACS buffer, and the fluorescence intensity of the cells was measured by flow cytometry. The experimental results are shown in Figure 5 and Table 11. The experimental results demonstrate that the humanized antibody can bind to the monkey PVRIG protein expressed in the cell line in a dose-dependent manner.

[0085] [Table 11]

[0086] Example 7: Functional verification of anti-PVRIG antibodies The biological function of the PVRIG antibody was verified by ELISA blocking experiments, FACS blocking experiments, and reporter gene experiments.

[0087] 7.1 Antibodies inhibit binding of human PVRIG protein to human CD112 protein The ability of humanized PVRIG antibodies to inhibit binding between human CD112 and human PVRIG was measured using an ELISA method. CD112-His protein was diluted to a concentration of 1 μg / ml and coated on an ELISA plate. A 3-fold gradient dilution of antibody (0-133 nM) was mixed with PVRIG-hFc protein, added to the coated ELISA plate, and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human Fc antibody was added and incubated at 37°C for 1 hour. After washing the plate, a color development solution was added, followed by a stop solution 5 minutes later, and the results were read. The blocking results obtained by the antibodies were fitted using GraphPad and are shown in Figure 6 and Table 12. The results show that the humanized antibodies can effectively inhibit the binding of CD112 to PVRIG protein, and that Hab1B10E8.6, Hab21F10C10.12, and Hab43F7C9.12 have better ELISA inhibition ability than the control molecule SRF813.

[0088] [Table 12]

[0089] 7.2 Antibodies inhibit binding of PVRIG to 293T-PVRL2 cells The ability of the humanized antibodies to block PVRIG binding to CD112-overexpressing 293T cells was measured using flow cytometry. Logarithmically growing 293T-CD112 cells were digested and diluted to 1E6 / ml. 50 μl was added to each well of a flow cytometry plate, along with 25 μl of a 3-fold gradient diluted antibody sample (0–300 nM) and 25 μl of 1 μg / ml PVRIG-hFc protein. The plates were then incubated for 1 hour at 4°C. The cells were washed three times with FACS buffer and labeled with Alexa Fluor 488-conjugated goat anti-human IgG antibody in the dark for 50 minutes at 4°C. The cells were then washed three times with FACS buffer, and the fluorescence intensity of the cells was measured by flow cytometry. The blocking results are shown in Figure 7 and Table 13. The results demonstrate that the humanized antibodies dose-dependently block PVRIG binding to 293T-CD112 cells.

[0090] [Table 13]

[0091] 7.3 Detecting the inhibition of PVRIG binding to CD112 by humanized antibodies using reporter gene method Logarithmic growth phase CD112 / TCR Activator / CHO cells were digested, centrifuged, and adjusted to a cell density of 4E5 / ml. 100 μl / well of these cells were seeded into a white-walled, clear-bottom 96-well plate and cultured overnight at 37°C. The next day, the culture supernatant was removed, and 50 μl of antibody sample (0-66.7 nM) diluted 3-fold in RPMI 1640 medium was added. 50 μl of logarithmic growth phase effector cells stably expressing PVRIG were added, and the cells were incubated at a cell density of 4E5 / ml in a 37°C incubator for 6 hours. Detection was performed using the Bright-Glo™ luciferase assay system. The results for the reporter gene are shown in Figure 8 and Table 14. The results show that the humanized antibodies can effectively inhibit the binding of CD112 cells to PVRIG cells, and the ELISA inhibition abilities of Hab1B10E8.6, Hab21F10C10.12, and Hab43F7C9.12 are clearly superior to those of the control molecule SRF813-IgG1.

[0092] [Table 14]

[0093] Example 8: Biological activity of anti-PVRIG chimeric antibodies in vivo in animals The efficacy of 21F10C10-hIgG1 and 43H7C9-hIgG1 chimeric antibodies as monotherapy was evaluated in tumor xenograft models. 6 A375 melanoma cells and 5 x 10 5Human PBMCs were mixed and then inoculated into NCG immunodeficient mice at a 1:1 ratio with Matrigel. On the day of implantation, 21F10C10, 43H7C9 chimeric antibodies, and PBS control were intraperitoneally injected twice weekly at 20 mg / kg. In the control group, the mean tumor volume was 2387.34 ± 95.79 mm on day 28 of treatment. 3 Compared with the control group, the 43H7C9 group suppressed tumor growth, with a tumor inhibition rate of 43.93% and a tumor volume of 1338.48 ± 296.09 mm. 3 The 21F10C10 group suppressed tumor growth, with a tumor inhibition rate of 20.83% and a tumor volume of 1890.09 ± 568.21 mm. 3 (Figure 9A). Body weight changes between experimental groups were not significant (Figure 9B), indicating that treatment was well tolerated. Individual tumor volume results are shown in Figure 9C (multiple lines in Figure 9C represent multiple mice).

[0094] Example 9: Biological activity of anti-PVRIG humanized antibodies in vivo in animals The efficacy of humanized antibodies Hab21F10C10.12-DLE (containing a DLE mutation in the Fc region, the constant region sequence of which is shown in SEQ ID NO: 53) and 43H7C9.12-DLE (containing a DLE mutation in the Fc region, the constant region sequence of which is shown in SEQ ID NO: 53) was evaluated in a tumor xenograft model. NCG mice were allowed to acclimate for at least two days after arrival at the facility. Cryopreserved human PBMCs were resuscitated and counted in vitro and then co-cultured with mitomycin C-treated A375 melanoma cells. After co-culture, PBMCs were collected, mixed with fresh exponentially growing A375 melanoma cells, resuspended in PBS to the appropriate concentration, and subcutaneously inoculated into NCG mice. On the day of transplantation, mice were randomly divided into groups based on body weight and intraperitoneally injected with the above-mentioned anti-PVRIG monoclonal antibody at 20 mg / kg once every other day. Tumor volume was measured. In the control group, the mean tumor volume on day 13 of treatment was 201.9 ± 53.6 (mean ± standard error). Compared to the control group, the 43H7C9.12-DLE group suppressed tumor growth, with a tumor inhibition rate of 27.3% and a tumor volume of 146.8 ± 45.0. In the 43H7C9.12-DLE + SG-001 (PD-1 antibody, sequence shown below) group, the tumor inhibition rate was 61.5% and the tumor volume was 77.8 ± 52.6. In the 21F10C10.12-DLE + SG-001 group, the tumor inhibition rate was 74.2% and the tumor volume was 52.2 ± 10.1 (Figure 10).

[0095] SG-001 heavy chain QVQLVESGGGVVQPGRSLRLTCKASGLTFSSSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNNDYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 54) SG-001 light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYTASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 55)

[0096] Example 10: Pharmacokinetic properties of anti-PVRIG humanized antibodies The pharmacokinetic characteristics of the anti-PVRIG monoclonal antibody in cynomolgus monkeys were investigated by measuring serum drug concentrations after a single intravenous injection of 43H7C9.12-DLE humanized antibody (20 mg / kg) into cynomolgus monkeys. Two male cynomolgus monkeys were intravenously injected with 20 mg / kg of anti-PVRIG monoclonal antibody. Whole blood samples were collected before and after drug administration, and serum was separated. ELISA was used to detect the content of anti-PVRIG monoclonal antibody in the serum samples, and PK parameters were obtained. At this dose, the half-life was approximately 227 hours. The results are shown in Figure 11 and Table 15, with M1 and M2 representing two cynomolgus monkeys.

[0097] [Table 15]

Claims

1. (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and / or LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (2) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and / or LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 25, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26; or (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38, and / or LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 39, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 40, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 41 An anti-human PVRIG antibody or an antigen-binding fragment thereof, comprising:

2. (1) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (2) HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26; (3) HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 38, and LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 39, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 40, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 41 The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 1 , comprising:

3. comprising the sequence of a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16; (2) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32, and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35; or (3) The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47, and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO:

50. The anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 2.

4. comprising the sequence of a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 47, and / or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 50; (2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32, and / or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35, or (3) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10, and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

14. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 3.

5. comprising the sequence of a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 50; (2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 35; or (3) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

14. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 4.

6. comprising the sequence of a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16; (2) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:35; or (3) The heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50, An anti-human PVRIG antibody or an antigen-binding fragment thereof.

7. comprising the sequence of a heavy chain variable region and / or a light chain variable region, (1) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:47, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO:50; (2) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 32, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 35; or (3) the heavy chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 10, and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% homology to the amino acid sequence set forth in SEQ ID NO: 14; The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 6.

8. The anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is a humanized antibody or a human antibody or a portion thereof.

9. The anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is an IgG1 antibody or an IgG4 antibody or a portion thereof.

10. Fab, Fab', F(ab') ２ 10. The antigen-binding fragment of any one of claims 1 to 9, which is selected from: Fd, Fv, scFv, dAb or CDR.

11. An antigen-binding fragment according to any one of claims 1 to 10, wherein the Fc region of the antibody has mutations, preferably the Fc mutations include at least one of S239D, A330L, and I332E, and more preferably the Fc region mutations simultaneously include the three point mutations S239D, A330L, and I332E.

12. An isolated nucleic acid molecule encoding the anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

13. An expression vector comprising the nucleic acid molecule of claim 10.

14. A host cell comprising the expression vector of claim 13.

15. A method for preparing an anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising culturing the host cell of claim 14, thereby expressing the nucleic acid molecule.

16. The method of claim 15, further comprising recovering the anti-human PVRIG antibody or antigen-binding fragment thereof from the host cell culture.

17. An antibody-drug conjugate comprising the anti-human PVRIG antibody or antigen-binding fragment thereof of any one of claims 1 to 11 conjugated to a therapeutic agent.

18. A bispecific molecule comprising an anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, which targets another antigenic epitope or is conjugated to a peptide of another antigen.

19. A composition comprising the anti-human PVRIG antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or the antibody-drug conjugate of claim 17, or the bispecific molecule of claim 18.

20. 20. Use of an anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or an antibody-drug conjugate according to claim 17, or a bispecific molecule according to claim 18, or a composition according to claim 19, in the preparation of a medicament for modulating an immune response in a subject.

21. Use of an anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or an antibody-drug conjugate according to claim 17, or a bispecific molecule according to claim 18, or a composition according to claim 19, in the preparation of a drug for inhibiting the binding of PVRIG protein to CD112 protein.

22. 20. Use of an anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or an antibody-drug conjugate according to claim 17, or a bispecific molecule according to claim 18, or a composition according to claim 19, in the preparation of a medicament for inhibiting the growth of tumor cells in a subject.

23. The tumor cells are from bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute ...

23. The use of claim 22, wherein the tumor cells are selected from tumor cells of cancers including: myocardial infarction, ...

24. 20. Use of a therapeutically effective amount of the anti-human PVRIG antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or the antibody-drug conjugate of claim 17, or the bispecific molecule of claim 18, or the composition of claim 19, in the preparation of a medicament for treating a hyperproliferative disease.

25. The hyperproliferative disease is cancer, and preferably the cancer is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, 25. The use of claim 24, wherein the cancer is selected from the group consisting of hematologic malignancies, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and any combination of said cancers.

26. The use according to any one of claims 20 to 25, wherein the anti-human PVRIG antibody or antigen-binding fragment thereof is combined with an anti-PD-1 antibody or antigen-binding fragment thereof.

Images