Recombinant anti-human PVRIG antibody and its application

Abstract

The present invention discloses an anti-human PVRIG antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises antigenic determining regions VH CDR1, VH CDR2 and VH CDR3, and the VL comprises antigenic determining regions VL CDR1, VL CDR2 and VL CDR3. The humanized anti-human PVRIG antibody can specifically bind to PVRIG, showing good clinical anti-tumor application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a novel artificially designed PVRIG antibody. The present invention also relates to the therapeutic and diagnostic uses of these PVRIG-binding antibodies, especially in the treatment, prevention and / or diagnosis of PVRIG-related diseases, such as tumors.

[0002] Background Overview

[0003] At present, cancer antibody therapy has gradually been established and has become one of the most successful and important strategies for treating hematological malignancies and solid tumors. This therapy can work by changing the function of antigens or receptors (such as using agonists or antagonists) or coupling specific drugs to antibodies targeting specific antigens.

[0004] A poliovirus receptor-associated immunoglobulin domain-containing protein (PVRIG) that independently mediates inhibitory signals by binding to the CD112 ligand. It also competes with CD226 for ligands and blocks the stimulatory signals it transmits, thereby achieving negative regulation of T cell function, but does not bind to CD155. PVRIG has a high affinity for the ligand CD112 on the surface of tumor cells. After binding, it can inhibit the cellular action of lymphocytes and prevent their interaction with the CD112 receptor overexpressed on tumor cells, thereby driving the activation of NK cells and T cells and exerting immune effects. Experiments have shown that PVRIG and TIGIT are effective inhibitory receptors on CD8+T cells. Intervention targeting these two targets can effectively enhance anti-tumor responses in vitro.

[0005] However, existing antibody treatments have a low clinical efficacy. For most cancer types, if used indiscriminately, the efficacy is only 10%-20% on average. Therefore, more effective antibody drug molecules in the anti-tumor field need to be studied. Summary of the invention

[0006] The present invention aims to solve the problems of tumor treatment in the prior art, specifically, to solve the technical problem of low efficacy of tumor-targeting antibody molecules. To this end, one object of the present invention is to provide an antibody or antigen-binding fragment that specifically binds to PVRIG.

[0007] PVRIG has a high affinity for the ligand CD112 on the surface of tumor cells. After binding, it can inhibit the cellular action of lymphocytes and prevent their interaction with the CD112 receptor overexpressed on tumor cells, thereby driving the activation of NK cells and T cells and exerting immune effects.

[0008] Thus, according to one aspect of an embodiment of the present invention, an anti-human PVRIG antibody or an antigen-binding fragment thereof is provided, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises antigenic determining regions VH CDR1, VH CDR2 and VH CDR3, the VH CDR1 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 11, the VH CDR2 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 12, and the VH CDR3 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 13; the VL comprises antigenic determining regions VL CDR1, VL CDR2 and VL CDR3, the VL CDR1 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 14, the VL CDR2 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 15, and the VL CDR3 comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 16. NO: 16 amino acid sequence has an amino acid sequence with at least 80% identity.

[0009] The PVRIG antibody or antigen-binding fragment according to the embodiment of the present invention can bind to human PVRIG, and the binding activity of human PVRIG and the inhibition of the activity of hPVRL2 binding to human PVRIG are better than the control antibody, and have strong affinity and high specificity for human PVRIG, showing good clinical anti-tumor application prospects.

[0010] In addition, the antibodies or antigen-binding fragments according to the above embodiments of the present invention may also have the following additional technical features:

[0011] According to an embodiment of the present invention, the VH comprises an amino acid sequence having at least 80% identity with SEQ ID NO:3, and the VL comprises an amino acid sequence having at least 80% identity with SEQ ID NO:4.

[0012] According to an embodiment of the present invention, the VH comprises an amino acid sequence having at least 80% identity with SEQ ID NO:7, and the VL comprises an amino acid sequence having at least 80% identity with SEQ ID NO:8.

[0013] According to an embodiment of the present invention, the VH comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7, and the VL comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 10.

[0014] According to an embodiment of the present invention, the VH comprises an amino acid sequence having at least 80% identity with SEQ ID NO:9, and the VL comprises an amino acid sequence having at least 80% identity with SEQ ID NO:8.

[0015] According to an embodiment of the present invention, the VH comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 9, and the VL comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 10. It is further stated that the VH and VL sequences of the antibody or antigen-binding fragment may be obtained by inserting, deleting, mutating or modifying one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0016] According to an embodiment of the present invention, the anti-human PVRIG antibody or antigen-binding fragment thereof has cross-reactivity with monkey PVRIG, but has no binding activity with mouse PVRIG.

[0017] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is a monoclonal antibody. A monoclonal antibody is a highly uniform antibody produced by a single B cell clone and directed only against a specific antigen epitope. For example, the antibody of the embodiment of the present invention specifically binds only to PVRIG.

[0018] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody. Humanized antibodies mainly refer to antibodies that are re-expressed after the mouse monoclonal antibody is modified by gene cloning and DNA recombination technology, and binds to human targets after modification. Most of its amino acid sequences are replaced by human sequences, which basically retains the affinity and specificity of the parent mouse monoclonal antibody, and reduces its heterology, which is beneficial for application in the human body. For example, the antibody or antigen-binding fragment of the embodiment of the present invention can specifically bind to human PVRIG after humanization.

[0019] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds to human PVRIG and is selected from the group consisting of Fv, Fab, Fab', scFv and F(ab')2.

[0020] According to another aspect of the present invention, the present invention provides an isolated polynucleotide. According to an embodiment of the present invention, the isolated polynucleotide encodes the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the isolated polynucleotide encodes the PVRIG antibody or antigen-binding fragment described in the preceding item. Thus, the polypeptide encoded by the polynucleotide can specifically bind to PVRIG.

[0021] According to another aspect of the present invention, the present invention provides an isolated polynucleotide. According to an embodiment of the present invention, the isolated polynucleotide encodes the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof. According to an embodiment of the present invention, the isolated polynucleotide encodes the light chain variable region or heavy chain variable region, or the light chain or heavy chain of the anti-human PVRIG antibody or antigen-binding fragment thereof described in the preceding item.

[0022] According to another aspect of the present invention, the present invention provides a vector. According to an embodiment of the present invention, the vector comprises the aforementioned one or more polynucleotides.

[0023] According to an embodiment of the present invention, the vector comprises the aforementioned two polynucleotides, and the vector encodes a VL region and a VH region, and the VL region and the VH region together bind to PVRIG.

[0024] According to another aspect of the present invention, the present invention provides a vector pair. According to an embodiment of the present invention, each of the vector pairs comprises one of the aforementioned polynucleotides, wherein the vector pair jointly encodes a VL region and a VH region, and the VL region and the VH region together bind to PVRIG.

[0025] According to another aspect of the present invention, the present invention provides a host cell. According to an embodiment of the present invention, the host cell comprises the aforementioned vector or the aforementioned vector pair.

[0026] According to another aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof, the aforementioned polynucleotide, the aforementioned vector, the aforementioned vector pair, or the aforementioned host cell; and optional pharmaceutically acceptable excipients. Thus, the composition can specifically bind to PVRIG, and has a good tumor inhibition effect and good drug efficacy.

[0027] According to another aspect of the present invention, the present invention provides a method for preparing an anti-human PVRIG antibody or a binding fragment thereof. According to an embodiment of the present invention, the method comprises: culturing the aforementioned host cell under suitable conditions; isolating and recovering the anti-human PVRIG antibody or a binding fragment thereof. The present invention adopts a method for isolating and purifying the target protein after in vitro cell culture to obtain an antibody that specifically binds to PVRIG.

[0028] According to another aspect of the present invention, the present invention provides a use. According to an embodiment of the present invention, the use includes: use of the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof, the aforementioned polynucleotide, the aforementioned vector, the aforementioned vector pair, the aforementioned host cell or the aforementioned composition in the preparation of a cancer diagnostic reagent.

[0029] According to another aspect of the present invention, the present invention provides a use. According to an embodiment of the present invention, the use includes: use of the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof, the aforementioned polynucleotide, the aforementioned vector, the aforementioned vector pair, the aforementioned host cell or the aforementioned composition in the preparation of a drug for treating cancer.

[0030] According to an embodiment of the present invention, the cancer includes breast cancer, colorectal cancer, female reproductive system cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, liver cancer (HCC), endometrial cancer, pancreatic cancer, gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, high MSI cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, myeloma.

[0031] According to another aspect of the present invention, the present invention provides a method for preventing or treating a subject suffering from the disease. According to an embodiment of the present invention, the method comprises administering an effective amount of the aforementioned anti-PVRIG antibody or antigen-binding fragment thereof or the aforementioned pharmaceutical composition to the subject.

[0032] According to another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the aforementioned anti-PVRIG antibody or antigen-binding fragment thereof. Two or more (e.g., two, three or four) of any antibodies or antigen-binding fragments described herein may be present in any combination in a pharmaceutical kit, and the pharmaceutical kit may be prepared in any manner known in the art.

[0033] According to another aspect of the present invention, the present invention provides a method for detecting PVRIG in a sample. According to an embodiment of the present invention, the method comprises:

[0034] (a) contacting the sample with the aforementioned antibody or antigen-binding fragment thereof; and

[0035] (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and PVRIG;

[0036] Optionally, the antibody is detectably labeled.

[0037] According to the above embodiments of the present invention, the inventors found that the antibodies of the embodiments of the present invention can bind to and activate PVRIG with an affinity of less than 3.97E-10, which solves the defects of insufficient affinity and specificity and adverse reactions of anti-human PVRIG antibodies in the prior art, has good anti-tumor application prospects, high stability, strong affinity, higher biological activity, and shows good clinical anti-tumor application prospects.

[0038] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0040] Figure 1 The chimeric antibody according to one embodiment of the present invention is shown to bind to human PVRIG ELISA activity;

[0041] Figure 2 It shows that the chimeric antibody according to one embodiment of the present invention inhibits hPVRL2 binding to human PVRIGELISA activity;

[0042] Figure 3 The biological activity of the chimeric antibody reporter gene according to one embodiment of the present invention is shown;

[0043] Figure 4 It shows that the humanized antibody according to one embodiment of the present invention inhibits hPVRL2 binding to human PVRIGELISA activity;

[0044] Figure 5 It is shown that the anti-PVRIG humanized antibody according to one embodiment of the present invention enhances the activity of CEFT-activated PBMC in secreting IFN-γ. DETAILED DESCRIPTION

[0045] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0046] In order to better understand the present invention, some terms are first defined. Other definitions are listed throughout the detailed description.

[0047] The term "specificity" refers to the ability to determine the presence or absence of a protein in a heterogeneous population of proteins and / or other organisms. Thus, under specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0048] The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy chains and light chains being linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conservative framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0049] The term "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule preparation of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope.

[0050] The term "antigen-binding fragment" (or simply antibody portion) of an antibody herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) a Fd fragment consisting of VH and CH1; (iv) a Fv fragment consisting of a single-arm VL and VH of an antibody; (v) a dAb fragment consisting of VH (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity determining region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by recombinant methods via a synthetic linker that makes the two into a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (called single-chain Fc (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single-chain antibodies are also intended to be included in the meaning of the term. These antibody fragments can be obtained by common techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0051] Antigen binding fragments of the present invention include those that are capable of specifically binding to an antigen. Examples of antibody binding fragments include, for example, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv (scFv) fragments, and single domain fragments.

[0052] The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. The Fab' fragment is produced by cleaving the disulfide bond at the hinge cysteine ​​of the F(ab')2 pepsin digest. Additional chemical coupling of antibody fragments is known to those of ordinary skill in the art. Fab and F(ab')2 fragments lack the fragment crystallizable (Fc) region of the intact antibody, are cleared more rapidly from the circulation of the animal, and may have less nonspecific tissue binding than intact antibodies (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0053] As is generally understood in the art, an "Fc" region is a fragment crystallizable constant region of an antibody that does not contain an antigen-specific binding region. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments, derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

[0054] The term "binding affinity" is used herein as a measure of the strength of non-covalent interactions between two molecules (e.g., an antibody or its fragment, and an antigen). The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules (e.g., an antibody or its fragment, and an antigen) via a monovalent interaction can be quantitatively determined by measuring the dissociation constant (KD). Subsequently, KD can be determined by measuring the complex formation and dissociation kinetics, such as by the SPR method. The rate constants corresponding to the association and dissociation of the monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is associated with ka and kd by the equation KD=kd / ka. According to the above definition, the binding affinity associated with different molecular interactions, such as the comparison of the binding affinity of different antibodies for a given antigen, can be compared by comparing the KD values ​​of each antibody / antigen complex. Similarly, the specificity of the interaction can be evaluated by measuring and comparing the KD value of the interaction of interest (e.g., specific interaction between antibody and antigen) with the KD value of the interaction of no interest. The value of the dissociation constant can be directly determined by well-known methods, for example, standard assays for evaluating the binding ability of a ligand (e.g., antibody) to a target are known in the art, and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art, such as SPR. Competitive binding assays can be performed, in which the binding of an antibody to a target and the binding of another ligand (e.g., another antibody) to the target are compared.

[0055] Anti-human PVRIG antibodies and antigen-binding fragments thereof

[0056] The present invention provides antibodies and antigen-binding fragments thereof that specifically bind to PVRIG. The anti-human PVRIG antibodies or antigen-binding fragments thereof according to the embodiments of the present invention can heterosexually bind to human PVRIG or mouse PVRIG protein, and can promote the PVRIG-CD112 signal transduction pathway, preventing its interaction with the CD112 receptor overexpressed on tumor cells, thereby driving the activation of NK cells and T cells and exerting immune effects.

[0057] The embodiments of the present invention provide anti-PVRIG antibodies, including mouse antibody 1718-525, and humanized antibodies and chimeric antibodies thereof. These chimeric antibodies have VH and VL from mouse antibodies. However, the constant domains of these chimeric antibodies are from human antibodies (e.g., human IgG1, human IgG2, human IgG3, or human IgG4), and the chimeric antibodies are labeled as 1718-525HL. In addition, the embodiments of the present invention also provide humanized antibodies 1718-525H1L0 and 1718-525H1L1.

[0058] The CDR sequences (Kabat definition), heavy and light chain variable regions, and heavy and light chain constant region sequences of some published antibodies are shown in Table 1 below.

[0059] Table 1

[0060]

[0061]

[0062] The embodiments of the present invention also provide the amino acid sequences of the heavy chain variable region and the light variable region of the humanized antibody. Due to the different ways to humanize mouse antibodies (e.g., different amino acid substitutions can be used to modify the sequence), the heavy chain and light chain of the antibody can have more than one form of humanized sequence. The amino acid sequence of the heavy chain variable region of the humanized 1718-525 antibody is shown in SEQ ID NO: 5, 7 and 9. The amino acid sequence of the light chain variable region of the humanized 1718-525 antibody is shown in SEQ ID NO: 6, 8 and 10. Any of these heavy chain variable region sequences (SEQ ID NO: 5, 7 and 9) can be paired with any of these light chain variable region sequences (SEQ ID NO: 6, 8 and 10).

[0063] In some embodiments, the antibody may have a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises complementarity determining regions CDR1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence; and the VL comprises CDR 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL The CDR2 amino acid sequence has or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the selected VL CDR3 amino acid sequence.

[0064] "Percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific amino acid sequence shown in this specification, after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a considerable degree of identity, such as at least 80%, 85%, 90%, 95% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes.

[0065] In some embodiments, the antibodies or antigen-binding fragments described herein may contain heavy and light chain variable regions containing one, two or three CDRs selected from the group consisting of SEQ ID NOs: 11-16 with zero, one or two amino acid insertions, deletions or substitutions in one, two or three of the selected CDRs.

[0066] The present invention also provides an antibody or antigen-binding fragment thereof that binds to PVRIG, wherein the antibody or antigen-binding fragment has VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are determined based on various CDR definitions known in the art, such as the Kabat definition, the Chothia definition, or the IMGT definition. The sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are shown in Table 1.

[0067] Nucleic acids, vectors and cells

[0068] The embodiments of the present invention also provide a nucleic acid comprising a polynucleotide encoding a polypeptide, wherein the polypeptide comprises an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or the immunoglobulin light chain comprises a CDR as shown in Table 1, or has a sequence as shown in Table 1. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to PVRIG (e.g., human PVRIG).

[0069] Embodiments of the present invention also provide vectors comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cells contain the polynucleotides and / or vectors containing the polynucleotides), and recombinant antibody polypeptides or fragments thereof produced by recombinant technology.

[0070] As used herein, a "vector" is any construct capable of delivering one or more target polynucleotides to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more target polynucleotides as encoded polypeptides in a host cell into which the expression vector has been introduced. Therefore, in an expression vector, the target polynucleotide is positioned in the vector for expression by being operably connected to a regulatory element such as a promoter, an enhancer, and / or a polyadenylic acid tail, and the regulatory element is located at or near or on both sides of the integration site of the target polynucleotide in the vector or in the genome of the host cell, so that the target polynucleotide will be translated in the host cell into which the expression vector has been introduced.

[0071] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with a recombinant virus). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0072] The present invention provides host cells transformed with the vectors described above. The host cell can be a prokaryotic or eukaryotic cell. The preferred prokaryotic host cell is Escherichia coli. Preferably, the eukaryotic cell is selected from: protist cells, animal cells, plant cells and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS cells. The preferred fungal cell is Saccharomyces cerevisiae.

[0073] Method for preparing anti-PVRIG antibodies

[0074] Isolated fragments of human PVRIG can be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be generated in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected with at least one adjuvant. In some embodiments, the antigenic peptides or proteins can be conjugated to an agent that is immunogenic in the species to be immunized. The antigenic peptides or proteins can be injected into animals multiple times.

[0075] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, a recombinantly expressed polypeptide or a chemically synthesized polypeptide (e.g., a fragment of human PVRIG). The preparation may further comprise an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0076] Pharmaceutical compositions, uses and methods of treatment

[0077] The present invention also provides a pharmaceutical composition containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described in the present invention. Two or more (e.g., two, three, or four) of any antibody or antigen-binding fragment described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0078] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, etc. that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc. and combinations thereof. In many cases, it is preferred to include an isotonic agent, such as sugar, polyols such as mannitol, sorbitol or sodium chloride in the composition. A pharmaceutically acceptable carrier may further include a small amount of auxiliary substances, such as a wetting agent or emulsifier, a preservative or a buffer, which increases the shelf life or effectiveness of the antibody.

[0079] The present invention provides one or more antibodies or their antigen binding fragments that can be used for a variety of therapeutic purposes. In one aspect, the present disclosure provides a method for treating a subject's cancer, a method for reducing the rate at which a subject's tumor volume increases over time, a method for reducing the risk of metastasis, or a method for reducing the risk of another metastasis in a subject. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of a subject's cancer.

[0080] In one aspect, the disclosure features a method comprising administering to a subject in need thereof (e.g., a subject having, identified as having, or diagnosed as having a cancer) a therapeutically effective dose of an antibody or antigen-binding fragment thereof disclosed herein, wherein the cancer is, for example, breast cancer, colorectal cancer, female reproductive system cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, liver cancer (HCC), endometrial cancer, pancreatic cancer, gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia / lymphoma, myeloma.

[0081] In one aspect, the present invention provides the use of the aforementioned anti-human PVRIG antibody or its antigen-binding fragment in the preparation of a drug, the drug is used to treat cancer. According to an embodiment of the present invention, the cancer includes breast cancer, colorectal cancer, female reproductive system cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, liver cancer (HCC), endometrial cancer, pancreatic cancer, gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer, melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cell carcinoma (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, high MSI cancer, KRAS mutant tumor, adult T cell leukemia / lymphoma, myeloma.

[0082] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients with cancer can be identified using a variety of methods known in the art.

[0083] Reagent test kit

[0084] The present invention also provides a kit containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described in the present invention. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein may be present in the kit in any combination. The pharmaceutical kit may be prepared in any manner known in the art.

[0085] The kit may also contain: (1) a solid phase carrier coated with an antigen or antibody (immunosorbent); (2) an enzyme-labeled antigen or antibody (conjugate); (3) an enzyme substrate; (4) negative and positive controls (in qualitative determinations), reference standards and control serum (in quantitative determinations); (5) diluents for the conjugate and specimens; (6) a washing solution. In plate-based ELISA, a commonly used diluent is phosphate-buffered saline containing 0.05% Tween 20; and (7) an enzyme reaction stop solution. A commonly used HRP reaction stop solution is sulfuric acid.

[0086] The present invention provides the use of the aforementioned anti-human PVRIG antibody or antigen-binding fragment thereof kit in a detection reagent. According to an embodiment of the present invention, the detection includes RT-PCR, Northern blot, Dot blot, Real time RTPCR, chip analysis, polyA screening, in vitro translation, RNase protection analysis and molecular cloning and other detection experiments.

[0087] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0088] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in the art (e.g., reference to "Molecular Cloning Experiment Guide" by J. Sambrook, Huang Peitang, etc., the third edition, Science Press) or the product instructions are followed. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially, such as can be purchased from Sigma.

[0089] Example 1: Mouse Antibody Screening

[0090] (1) Animal immunization

[0091] Human PVRIG (NP_076975.2) (Met1-Leu150) protein with 6 histidine tags fused to the C-terminus was expressed in CHO-K1. Four BALB / c mice were immunized according to the conventional Freund's adjuvant immunization procedure, divided into two batches of 2 mice, with an interval of two weeks between the two batches. Each batch of animals was immunized 4 times, and Human PVRIG his was used for ELISA detection. If the animal serum titer was >1:100000, the animal was finally immunized, and the spleen was collected 3-4 days later.

[0092] (2) B cell selection and culture

[0093] Two days before the formal experiment, the feeder cells were spread in 4 10cm culture dishes (corning, cat.430167) using culture medium. One day before the formal experiment, the feeder cells were spread in 40 96-well plates (corning, cat.3599) at 10,000 cells / well, 100μL / well. At the same time, the collected immunized mouse B cells were selected using the coated antigen Human PVRIG his, and the selected B cells were spread in the above 96-well plates that had been laid with feeder cells. Cultured at 37°C and 5% CO2 for 10 to 14 days, the B cell culture supernatant was taken for ELISA and cell-based assay screening.

[0094] (3) Mouse anti-supernatant screening

[0095] Mouse antibody binding Human PVRIG his ELISA screening: The above immunogen Human PVRIG his was diluted to 1 μg / mL with coating buffer, 50 μL / well, and coated overnight at 4°C; the next day, the coated plate was taken out and washed 3 times with PBST, then incubated with blocking solution at room temperature for 1 hour, and then washed 3 times with PBST, and the supernatant in the B cell wells that had formed obvious clones was added to the ELISA plate; incubated at room temperature for 1 hour, washed three times with PBST, and added goat anti-mouse secondary antibody (1:10000), 50 μL / well; incubated at room temperature for 1 hour, washed three times with PBST, added TMB 50 μL / well, color developed in dark for 10 minutes, and terminated with 2M sulfuric acid 100 μL / well; read the OD450 value on the microplate reader. The OD value of the negative control was 10 times or more as the positive judgment standard, and the positive clone was picked for B cell sequencing.

[0096] Example 2: B cell sequencing

[0097] Follow PureLink TM RNA Mini Kit Instructions Extract B cell clone mRNA and store it in aliquots at -80℃; Use PrimeScript TM II 1st Strand cDNA Synthesis Kit protocol was used to reverse transcribe into cDNA and stored in aliquots at -80°C.

[0098] Using the heavy chain VH fishing primer and light chain VL fishing primer shown in Tables 2 and 3, the above cDNA was used as a template, and VH and VL were amplified using Ex Taq enzyme, and then ligated to the pMD18T vector for sequencing.

[0099] Table 2 Heavy chain VH fishing primers

[0100]

[0101]

[0102] Table 3 Light chain VL fishing primers

[0103]

[0104]

[0105] The antibody is named after the cell clone number from which the light and heavy chains (HL) are derived. In this experiment, 1718-525 cells were used for cloning to obtain the mouse antibody 1718-525, whose heavy chain variable region and light chain variable region are SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0106] Example 3: Recombinant expression and screening of chimeric antibodies

[0107] (1) Recombinant expression of chimeric antibodies

[0108] The light and heavy chains from the same clone amplified in Example 2 were transfected into CHO-K1 cells. 10 μg / mL MSX was added for pressure screening 24 hours after transfection. After the cell density and viability were restored, they were inoculated for feed-batch expression. The supernatant after expression was centrifuged and purified by protein A. The antibody concentration was quantified by the BCA method and used for quantitative screening. The obtained chimeric antibody was named "1718-525HL".

[0109] (2) Screening of chimeric antibody binding activity to human PVRIG

[0110] COM701 was used as a control antibody. COM701 was derived from patent CN110088132B, wherein the heavy chain of COM701 is shown in SEQ ID NO: 9 of the patent, and the light chain is shown in SEQ ID NO: 14 of the patent. Human PVRIG his was diluted to 1 μg / mL with coating buffer, added to a 96-well plate at 50 μL / well, and coated overnight at 4°C; the coated plate was removed on the second day and washed three times with PBST; then incubated with blocking solution at room temperature for 1 hour, and washed three more times with PBST. The chimeric antibody was diluted 3 times from 1000 ng / mL, and a total of 8 concentrations were diluted, and added to the 96-well plate at 50 μL / well; incubated at room temperature for 1 hour, washed three times with PBST, added with goat anti-human secondary antibody (1:10000), and added to the 96-well plate at 50 μL / well; incubated at room temperature for 1 hour, washed three times with PBST, added TMB to the 96-well plate at 50 μL / well, color developed in the dark for 10 minutes, and terminated with 2M sulfuric acid 100 μL / well; read the OD450 value on the microplate reader, and the results are shown in Table 4 and Figure 1 As shown in Table 4, the obtained chimeric antibody 1718-525HL has better binding activity to human PVRIG than the control antibody COM701.

[0111] Table 4 Chimeric antibody binding activity to human PVRIG and relative activity compared to COM701

[0112] Antibody <![CDATA[EC 50 (ng / mL)]]> Relative activity (%) 1718-525HL 90.14 110.71 COM701 99.79 -

[0113] (3) Screening of chimeric antibodies that inhibit hPVRL2 binding to human PVRIG

[0114] The human PVRIG his protein was diluted to 1 μg / mL using coating solution, coated into a 96-well plate at 50 μL / well, and incubated overnight in a 4°C refrigerator; washed three times with PBST, added with blocking solution at 100 μL / well, and incubated at room temperature for 1 hour; washed three times with PBST. Biotin-hPVRL2his was diluted to a concentration of 400 μg / mL using diluent, and the chimeric antibody was diluted to a starting concentration of 200 μg / ml using diluent, and then diluted 3 times, a total of 8 concentrations, the diluted biotin-hPVRL2his and the diluted chimeric antibody were mixed 1:1, added to a 96-well plate at 50 μL / well, and incubated at room temperature for 1 hour; washed three times with PBST, HRP-labeled streptavidin (1:10000) was added, 50 μL / well, and incubated at room temperature for 1 hour; washed three times with PBST, TMB was added to a 96-well plate at 50 μL / well, color was developed in the dark for 10 minutes, and 2M sulfuric acid 100 μL / well was used to terminate; the OD450 value was read on an enzyme reader, and the results are shown in Tables 5 and Figure 2 As shown in Table 5, the obtained chimeric antibody 1718-525HL has better activity in inhibiting hPVRL2 binding to human PVRIG than COM701.

[0115] Table 5 Chimeric antibodies inhibit hPVRL2 binding to human PVRIG and their relative activity compared to COM701

[0116] Antibody <![CDATA[IC 50 (ng / mL)]]> Relative activity (%) 1718-525HL 78.82 440.72 COM701 319.20 -

[0117] (4) Screening of biological activity of chimeric antibody reporter gene

[0118] Take logarithmically growing CD112 / TCR Activator / CHO cells, digest them with trypsin, and resuspend them in fresh F12K medium containing 10% FBS. Adjust the resuspended cell density to 4*10 5 / mL. The resuspended cells were inoculated into a 96-well cell culture plate with a white wall and a transparent bottom, with 100 μL / well of cell suspension, and cultured in a 37-degree incubator overnight. The next day, the F12K culture medium in the 96-well plate inoculated with CD112 / TCRActivator / CHO cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) was aspirated, and the sample was gradient-treated with 10% serum RPMI1640 culture medium. The gradient-diluted 2* concentration sample (50 μL / well) was added to the 96-well plate inoculated with cells. The sample started from the highest concentration of 40 μg / mL (2* concentration), and 11 concentration gradients were diluted 3 times, and a blank culture medium control well was set up. Take the logarithmic growth PVRIG (CD112R) Effector Reporter cells, centrifuge and discard the supernatant, resuspend in fresh 10% FBS RPMI1640 culture medium, and adjust the resuspended cell density to 4*105 / mL, then add the cells to the 96-well plate in step 3, 50μL per well, and continue to culture in a 37-degree incubator for 5-6 hours. Take the 96-well plate out of the incubator and add 100μL / well Bright-Glo TM The luciferase detection reagent (purchased from Promega) was placed for 3 to 5 minutes and then placed in a microplate reader to read the value. According to the reading corresponding to each gradient concentration well, the Prism Graphpad software was used to fit the gradient curve of the sample on cell activation and calculate the EC of the sample. 50 The results are shown in Table 6 and Figure 3 As shown in Table 6, the reporter gene biological activity of the obtained chimeric antibody 1718-525HL is comparable to that of COM701.

[0119] Table 6 Chimeric antibody reporter gene biological activity and relative activity compared to COM701

[0120] Antibody <![CDATA[EC 50 (ng / mL)]]> Relative activity (%) 1718-525HL 171.30 75.25 COM701 228.90 -

[0121] (5) Cross-binding activity of chimeric antibodies against mouse and monkey PVRIG

[0122] Mouse PVRIG his and cyno PVRIG his were diluted to 1 μg / mL with coating buffer, added to 96-well plates at 50 μL / well, and coated overnight at 4°C; the coated plates were taken out the next day and washed three times with PBST; then incubated with blocking solution at room temperature for 1 hour, and washed three times with PBST. The chimeric antibody was diluted 3 times from 2000 ng / mL, with a total of 8 concentrations, added to the 96-well plate at 50 μL / well; incubated at room temperature for 1 hour, washed three times with PBST, added goat anti-human secondary antibody (1:10000), added to the 96-well plate at 50 μL / well; incubated at room temperature for 1 hour, washed three times with PBST, added TMB to the 96-well plate at 50 μL / well, color developed in the dark for 10 minutes, and terminated with 2M sulfuric acid 100 μL / well; read the OD450 value on the microplate reader, and the results are shown in Table 7. It can be seen from Table 7 that the obtained chimeric antibodies all cross-bind to monkey PVRIG but not to mouse PVRIG.

[0123] Table 7 Cross-binding activity of chimeric antibodies against mouse and monkey PVRIG

[0124]

[0125] Example 4: Humanization of 1718-525HL antibody

[0126] The chimeric antibody 1718-525HL antibody was selected for humanization design, and the specific method is as follows:

[0127] The heavy chain and light chain variable region sequences of chimeric antibody 1718-525HL were compared with human germline sequences, which was a blast search performed on the IMGT database. Redundant genes and those genes with unpaired cysteine ​​were removed from the group of human germline genes. The remaining closest matching human germline genes were selected as the receptor human framework in both the framework and CDR regions. FR-4 was selected based on the sequence similarity of IGHJ / IGJK germline genes, and IGHV1-69*08 was used as the humanized heavy chain variable region template, and IGKV2-30*02 was used as the light chain variable region template to carry out the humanization design of antibody variable regions. Table 8 shows the humanized sequence form of chimeric antibody 1718-525HL, in which the HZ0 version represents only CDR transplantation, and the HZ1 version introduces back mutations. The specific sequence after humanization is shown in Table 8, in which the corresponding CDR (according to the definition of enhanced Chothia / AbMCDR) is shown underlined.

[0128] Table 8 Humanized sequence formats of the heavy and light chain variable regions of the chimeric antibody 1718-525HL

[0129]

[0130] Example 5: Recombinant expression and screening of humanized antibodies

[0131] (1) Recombinant expression of humanized antibodies

[0132] Humanized light and heavy chains derived from the same chimera were constructed using the heavy chain constant region shown in SEQ ID NO.1 and the light chain constant region shown in SEQ ID NO.2, matched and transfected into CHO-K1 cells, and 10 μg / mL MSX was added for pressure screening 24 hours after transfection. After the cell density and viability were restored, they were inoculated for feed-batch expression. The centrifugal supernatant after expression was purified by protein A, and the antibody concentration was quantified by the BCA method for quantitative screening.

[0133] The collocation of the heavy and light chain variable regions of the humanized antibodies is shown in Table 9, wherein the suffix "ix" indicates that the antibody is a corresponding chimeric antibody.

[0134] Table 9 The sequence matching of the light and heavy chain variable regions of the humanized antibody 1718-525

[0135] 1718-525L 1718-525L-HZ0 1718-525L-HZ1 1718-525H 1718-525ix - - 1718-525H-HZ0 - 1718-525H0L0 1718-525H0L1 1718-525H-HZ1 - 1718-525H1L0 1718-525H1L1

[0136] (2) Humanized antibody affinity determination

[0137] All experiments were performed at 25°C in 1×HBS-EP+ buffer. The above-mentioned 1718-525ix, 1718-525H1L0 and 1718-525H1L1, each humanized antibody concentration was 5μg / ml, captured on the Protein A chip surface, and the capture time was 60s. The human PVRIG his injection solution, with a starting concentration of 5nM, was diluted two-fold to 6 points, the association was monitored for 180s, the dissociation was monitored for 600s, and the sensor surface was regenerated by injecting a glycine solution at pH 1.5. The kinetic data were analyzed using a 1:1 binding model. The highest concentration point was removed based on the fitting. The specific results can be seen in Table 10. It can be seen from Table 10 that the affinity of the humanized antibody 1718-525H1L0 is comparable to that of COM701.

[0138] Table 10 Humanized antibody affinity determination

[0139] Antibody ka(1 / Ms) kd(1 / s) KD(M) 1718-525ix 1.98E+06 0.001248 6.30E-10 1718-525H1L0 1.40E+06 5.55E-04 3.97E-10 1718-525H1L1 9.13E+05 1.62E-03 1.77E-09 COM701 1.36E+06 4.70E-04 3.46E-10

[0140] (3) Screening of humanized antibodies that inhibit hPVRL2 binding to human PVRIG

[0141] The human PVRIG his protein was diluted to 1 μg / mL using coating solution, coated into a 96-well plate at 50 μL / well, and incubated overnight in a 4°C refrigerator; washed three times with PBST, added with blocking solution at 100 μL / well, and incubated at room temperature for 1 hour; washed three times with PBST. Biotin-hPVRL2his was diluted to a concentration of 400 μg / mL using diluent, and humanized antibody was diluted to a starting concentration of 200 μg / ml using diluent, and then diluted 3 times, a total of 8 concentrations, the diluted biotin-hPVRL2his and diluted humanized antibody were mixed 1:1, added to a 96-well plate at 50 μL / well, and incubated at room temperature for 1 hour; washed three times with PBST, HRP-labeled streptavidin (1:10000) was added, 50 μL / well, and incubated at room temperature for 1 hour; washed three times with PBST, TMB was added to a 96-well plate at 50 μL / well, color was developed in the dark for 10 minutes, and 2M sulfuric acid 100 μL / well was used to terminate; OD450 values ​​were read on an enzyme reader, and the results are shown in Tables 11 and Figure 4 As shown in Table 11, the obtained humanized antibody 1718-525H1L0 has a significantly better inhibitory effect on the binding activity of hPVRL2 to human PVRIG than COM701.

[0142] Table 11 Humanized antibodies inhibit hPVRL2 binding to human PVRIG activity and relative activity compared to COM701

[0143] Antibody <![CDATA[IC 50 (ng / mL)]]> Relative activity (%) 1718-525H1L0 258.30 189.54 1718-525H1L1 101.90 480.47 COM701 489.60 -

[0144] (4) pH-sensitive binding of antibodies

[0145] Therapeutic antibodies that bind to their targets with reduced affinity at low pH can have enhanced PK properties, which is attributed to the release of the antibody target at low pH in the lysosome, followed by target degradation and antibody recycling. Biosensor analysis of pH-sensitive binding of antibodies 1718-525H1L0 and COM701 was performed on a Biacore X100. The association rate at pH 7.4 and the dissociation rate at pH 5.5 and 7.4 were monitored at a flow rate of 10 μL / min. The kinetic data were analyzed using a 1:1 binding model. The specific results can be seen in Table 12. It can be seen from Table 12 that the dissociation rate of antibody 1718-525H1L0 is significantly faster at reduced pH values.

[0146] Table 12 Kd change fold of antibodies at pH 5.5 and pH 7.4

[0147]

[0148] (5) Functional experiment of anti-PVRIG humanized antibody to enhance IFN-γ secretion by CEFT-activated PBMC

[0149] This test is used to evaluate the effect of anti-PVRIG humanized antibody alone or in combination with anti-PD-1 antibody on the production of IFN-γ by PBMC stimulated with antigen peptide mixture CEFT (purchased from JPT Peptide Technologies, catalog number: PM-CEFT-4). PBMC (purchased from Miaotong (Shanghai) Biotechnology Co., Ltd.) was used and resuspended in RPMI 1640 (purchased from thermo) + 10% FBS (purchased from thermo) medium containing 100 IU / ml IL-2 and 4 μg / ml antigen peptide mixture CEFT, and the cell density was adjusted to 1╳10 6 100 μl / well; 20 μg / ml of COM701, 1718-525H1L0, isotype IgG4 control antibody (hIgG4) and anti-PD-1 antibody pembrolizumab (Nanjing Noaixin Biotechnology Co., Ltd.) were added to the 96-well plate at 100 μl / well, with duplicate wells; after culturing in a 37°C, 5% CO2 incubator for 6 days, the supernatant was taken and the IFN-γ supernatant concentration was determined using an IFN-γ ELISA kit (purchased from R&D Company). The results are as follows: Figure 5 shown.

[0150] Depend on Figure 5It can be seen that COM701 and 1718-525H1L0 can significantly promote IFN-γ secretion, and the combination effect with PD-1 antibody is also better than that of the single drug group.

[0151] The 1718-525HL antibody obtained in the embodiment of the present invention can bind to human PVRIG, and the activity of binding to human PVRIG and inhibiting the activity of hPVRL2 binding to human PVRIG are better than the control antibody COM701. The humanized antibody 1718-525H1L0 inhibits the activity of hPVRL2 binding to human PVRIG significantly better than COM701. The antibody obtained in the embodiment of the present invention has strong affinity and high activity, and has good clinical application prospects.

[0152] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0153] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. Sequence Listing <110> Nanjing Noaixin Biotechnology Co., Ltd. <120> Recombinant anti-human PVRIG antibody and its application <160> 94 <170> SIPOSequenceListing 1.0 <210> 1 <211> 326 <212> PRT <213> Artificial Sequence <400> 1 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly 325 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <400> 2 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 3 <211> 113 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Phe Tyr Pro Gly Ser Gly Asp Thr Phe Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Glu Leu Arg Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ala <210> 4 <211> 112 <212> PRT <213> Artificial Sequence <400> 4 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Gly Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 5 <211> 113 <212> PRT <213> Artificial Sequence <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 6 <211> 112 <212> PRT <213> Artificial Sequence <400> 6 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 7 <211> 113 <212> PRT <213> Artificial Sequence <400> 7 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Phe Tyr Pro Gly Ser Gly Asp Thr Phe Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Glu Leu Arg Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 8 <211> 112 <212> PRT <213> Artificial Sequence <400> 8 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Gly Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 9 <211> 113 <212> PRT <213> Artificial Sequence <400> 9 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Phe Tyr Pro Gly Ser Gly Asp Thr Phe Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Glu Leu Arg Phe Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 10 <211> 112 <212> PRT <213> Artificial Sequence <400> 10 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Gly Gly Lys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <400> 11 Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile Ser 1 5 10 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <400> 12 Glu Phe Tyr Pro Gly Ser Gly Asp Thr Phe Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <400> 13 Glu Leu Arg Phe Thr Tyr 1 5 <210> 14 <211> 13 <212> PRT <213> Artificial Sequence <400> 14 Gln Ser Leu Leu Asp Ser Gly Gly Lys Thr Tyr Leu Asn 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <400> 15 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Trp Gln Gly Thr His Phe Pro Gln Thr 1 5 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <400> 17 Gly Gly Thr Phe Ser Ser Tyr Thr Ile Ser 1 5 10 <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <400> 18 Arg Ile Ile Pro Ile Leu Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 19 <211> 6 <212> PRT <213> Artificial Sequence <400> 19 Ala Arg Tyr Phe Asp Tyr 1 5 <210> 20 <211> 13 <212> PRT <213> Artificial Sequence <400> 20 Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu Asn 1 5 10 <210> twenty one <211> 7 <212> PRT <213> Artificial Sequence <400> twenty one Lys Val Ser Asn Arg Asp Ser 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial Sequence <400> twenty two Met Gln Gly Thr His Trp Pro Leu Thr 1 5 <210> twenty three <211> twenty three <212> PRT <213> Artificial Sequence <400> twenty three Ser Ala Gly Gly Thr Cys Cys Ala Gly Cys Thr Gly Cys Ala Gly Cys 1 5 10 15 Ala Gly Tyr Tyr Thr Gly Gly 20 <210> 24 <211> 23 <212> PRT <213> Artificial Sequence <400> 24 Cys Ala Gly Gly Thr Arg Cys Ala Gly Cys Thr Gly Ala Ala Gly Ser 1 5 10 15 Ala Gly Thr Cys Ala Gly Gly 20 <210> 25 <211> 23 <212> PRT <213> Artificial Sequence <400> 25 Gly Ala Lys Gly Thr Gly Cys Ala Gly Cys Thr Thr Cys Ala Gly Cys 1 5 10 15 Ala Gly Thr Cys Arg Gly Gly 20 <210> 26 <211> 23 <212> PRT <213> Artificial Sequence <400> 26 Gly Ala Val Gly Thr Gly Ala Trp Gly Cys Thr Gly Gly Thr Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Arg 20 <210> 27 <211> 23 <212> PRT <213> Artificial Sequence <400> 27 Gly Ala Ala Gly Thr Gly Cys Ala Gly Cys Thr Gly Thr Thr Gly Gly 1 5 10 15 Ala Gly Ala Cys Thr Gly Gly 20 <210> 28 <211> 23 <212> PRT <213> Artificial Sequence <400> 28 Gly Ala Gly Gly Thr Thr Cys Ala Gly Cys Thr Gly Cys Ala Gly Cys 1 5 10 15 Ala Gly Thr Cys Thr Gly Lys 20 <210> 29 <211> 23 <212> PRT <213> Artificial Sequence <400> 29 Cys Ala Gly Gly Thr Thr Cys Ala Cys Cys Thr Ala Cys Ala Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 30 <211> 23 <212> PRT <213> Artificial Sequence <400> 30 Gly Ala Gly Gly Thr Gly Ala Ala Gly Cys Thr Thr Cys Thr Cys Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 31 <211> 23 <212> PRT <213> Artificial Sequence <400> 31 Gly Ala Gly Gly Thr Gly Ala Ala Gly Cys Thr Gly Arg Thr Gly Gly 1 5 10 15 Ala Arg Thr Cys Thr Gly Arg 20 <210> 32 <211> 23 <212> PRT <213> Artificial Sequence <400> 32 Cys Ala Gly Gly Thr Thr Ala Cys Thr Cys Thr Gly Ala Ala Ala Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 33 <211> 23 <212> PRT <213> Artificial Sequence <400> 33 Cys Ala Gly Ala Thr Cys Cys Ala Gly Thr Thr Gly Gly Tyr Gly Cys 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 34 <211> 23 <212> PRT <213> Artificial Sequence <400> 34 Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys Thr Thr Gly Thr Thr Gly 1 5 10 15 Ala Gly Trp Cys Thr Gly Gly 20 <210> 35 <211> 23 <212> PRT <213> Artificial Sequence <400> 35 Cys Ala Gly Ala Thr Gly Cys Ala Gly Cys Thr Thr Cys Ala Gly Gly 1 5 10 15 Ala Gly Thr Cys Ala Gly Gly 20 <210> 36 <211> 23 <212> PRT <213> Artificial Sequence <400> 36 Gly Ala Ala Gly Thr Gly Ala Ala Gly Cys Thr Thr Gly Ala Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 37 <211> 23 <212> PRT <213> Artificial Sequence <400> 37 Gly Ala Ala Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Cys 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 38 <211> 23 <212> PRT <213> Artificial Sequence <400> 38 Gly Ala Gly Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Cys 1 5 10 15 Thr Gly Thr Cys Thr Gly Gly 20 <210> 39 <211> 23 <212> PRT <213> Artificial Sequence <400> 39 Gly Ala Thr Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Cys 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 40 <211> 23 <212> PRT <213> Artificial Sequence <400> 40 Gly Ala Ala Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Cys 1 5 10 15 Thr Gly Thr Cys Thr Gly Gly 20 <210> 41 <211> 23 <212> PRT <213> Artificial Sequence <400> 41 Gly Ala Gly Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Cys 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 42 <211> 23 <212> PRT <213> Artificial Sequence <400> 42 Gly Ala Thr Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Cys 1 5 10 15 Thr Gly Thr Cys Thr Gly Gly 20 <210> 43 <211> 23 <212> PRT <213> Artificial Sequence <400> 43 Gly Ala Ala Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 44 <211> 23 <212> PRT <213> Artificial Sequence <400> 44 Gly Ala Gly Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Gly 1 5 10 15 Thr Gly Thr Cys Thr Gly Gly 20 <210> 45 <211> 23 <212> PRT <213> Artificial Sequence <400> 45 Gly Ala Thr Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 46 <211> 23 <212> PRT <213> Artificial Sequence <400> 46 Gly Ala Ala Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Gly 1 5 10 15 Thr Gly Thr Cys Thr Gly Gly 20 <210> 47 <211> 23 <212> PRT <213> Artificial Sequence <400> 47 Gly Ala Gly Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Gly 1 5 10 15 Ala Gly Thr Cys Thr Gly Gly 20 <210> 48 <211> 23 <212> PRT <213> Artificial Sequence <400> 48 Gly Ala Thr Gly Thr Gly Met Ala Met Thr Thr Lys Ser Trp Gly Gly 1 5 10 15 Thr Gly Thr Cys Thr Gly Gly 20 <210> 49 <211> 20 <212> PRT <213> Artificial Sequence <400> 49 Gly Ala Gly Gly Ala Ala Ala Cys Gly Gly Thr Gly Ala Cys Cys Gly 1 5 10 15 Thr Gly Gly Thr 20 <210> 50 <211> 20 <212> PRT <213> Artificial Sequence <400> 50 Gly Ala Gly Gly Ala Gly Ala Cys Thr Gly Thr Gly Ala Gly Ala Gly 1 5 10 15 Thr Gly Gly Thr 20 <210> 51 <211> 20 <212> PRT <213> Artificial Sequence <400> 51 Gly Cys Ala Gly Ala Gly Ala Cys Ala Gly Thr Gly Ala Cys Cys Ala 1 5 10 15 Gly Ala Gly Thr 20 <210> 52 <211> 20 <212> PRT <213> Artificial Sequence <400> 52 Gly Ala Gly Gly Ala Gly Ala Cys Gly Gly Thr Gly Ala Cys Thr Gly 1 5 10 15 Ala Gly Gly Thr 20 <210> 53 <211> 23 <212> PRT <213> Artificial Sequence <400> 53 Gly Ala Thr Gly Tyr Thr Lys Thr Lys Val Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Ala Ala Cys Thr Cys Cys 20 <210> 54 <211> 23 <212> PRT <213> Artificial Sequence <400> 54 Arg Ala Cys Ala Thr Thr Gly Thr Gly Cys Thr Gly Ala Cys Met Cys 1 5 10 15 Ala Ala Thr Cys Thr Cys Cys 20 <210> 55 <211> 23 <212> PRT <213> Artificial Sequence <400> 55 Ser Ala Ala Ala Trp Thr Gly Thr Lys Cys Thr Cys Trp Cys Cys Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 56 <211> 23 <212> PRT <213> Artificial Sequence <400> 56 Ser Ala Ala Ala Trp Thr Cys Thr Lys Cys Thr Cys Trp Cys Cys Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 57 <211> 23 <212> PRT <213> Artificial Sequence <400> 57 Ser Ala Ala Ala Trp Thr Thr Thr Lys Cys Thr Cys Trp Cys Cys Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 58 <211> 23 <212> PRT <213> Artificial Sequence <400> 58 Ala Arg Cys Ala Thr Thr Gly Thr Gly Ala Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Gly Trp Cys Thr Cys Ala 20 <210> 59 <211> 23 <212> PRT <213> Artificial Sequence <400> 59 Ala Arg Cys Ala Thr Thr Gly Thr Gly Ala Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Gly Trp Cys Thr Cys Cys 20 <210> 60 <211> 23 <212> PRT <213> Artificial Sequence <400> 60 Gly Arg Cys Ala Thr Thr Gly Thr Gly Ala Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Gly Trp Cys Thr Cys Ala 20 <210> 61 <211> 23 <212> PRT <213> Artificial Sequence <400> 61 Gly Arg Cys Ala Thr Thr Gly Thr Gly Ala Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Gly Trp Cys Thr Cys Cys 20 <210> 62 <211> 23 <212> PRT <213> Artificial Sequence <400> 62 Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Ala Cys 1 5 10 15 Ala Gly Ala Cys Thr Ala Cys 20 <210> 63 <211> 23 <212> PRT <213> Artificial Sequence <400> 63 Gly Ala Met Ala Thr Cys Met Trp Gly Ala Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Arg Thr Cys Thr Cys Cys 20 <210> 64 <211> 23 <212> PRT <213> Artificial Sequence <400> 64 Gly Ala Thr Ala Thr Thr Gly Thr Gly Ala Thr Arg Ala Cys Asx Cys 1 5 10 15 Ala Gly Gly Tyr Thr Gly Ala 20 <210> 65 <211> 23 <212> PRT <213> Artificial Sequence <400> 65 Gly Ala Thr Ala Thr Thr Gly Thr Gly Ala Thr Arg Ala Cys Asx Cys 1 5 10 15 Ala Gly Gly Tyr Thr Gly Cys 20 <210> 66 <211> 23 <212> PRT <213> Artificial Sequence <400> 66 Gly Ala Tyr Ala Thr Tyr Cys Thr Gly Ala Thr Arg Ala Cys Arg Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 67 <211> 23 <212> PRT <213> Artificial Sequence <400> 67 Gly Ala Tyr Ala Thr Tyr Gly Thr Gly Ala Thr Arg Ala Cys Arg Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 68 <211> 23 <212> PRT <213> Artificial Sequence <400> 68 Gly Ala Tyr Ala Thr Tyr Thr Thr Gly Ala Thr Arg Ala Cys Arg Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 69 <211> 23 <212> PRT <213> Artificial Sequence <400> 69 Gly Ala Tyr Ala Thr Tyr Cys Thr Gly Cys Thr Arg Ala Cys Arg Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 70 <211> 23 <212> PRT <213> Artificial Sequence <400> 70 Gly Ala Tyr Ala Thr Tyr Gly Thr Gly Cys Thr Arg Ala Cys Arg Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 71 <211> 23 <212> PRT <213> Artificial Sequence <400> 71 Gly Ala Tyr Ala Thr Tyr Thr Thr Gly Cys Thr Arg Ala Cys Arg Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 72 <211> 23 <212> PRT <213> Artificial Sequence <400> 72 Gly Ala Cys Ala Thr Thr Gly Thr Gly Ala Thr Gly Ala Cys Thr Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 73 <211> 23 <212> PRT <213> Artificial Sequence <400> 73 Gly Ala Cys Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Tyr Tyr Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 74 <211> 23 <212> PRT <213> Artificial Sequence <400> 74 Gly Ala Thr Gly Thr Cys Cys Ala Gly Ala Thr Arg Ala Tyr Tyr Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 75 <211> 23 <212> PRT <213> Artificial Sequence <400> 75 Gly Ala Cys Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Trp Cys 1 5 10 15 Ala Arg Thr Cys Thr Tyr Cys 20 <210> 76 <211> 23 <212> PRT <213> Artificial Sequence <400> 76 Gly Ala Cys Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Met Met Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 77 <211> 23 <212> PRT <213> Artificial Sequence <400> 77 Gly Ala Ala Ala Cys Ala Ala Cys Thr Gly Thr Gly Ala Cys Cys Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 78 <211> 23 <212> PRT <213> Artificial Sequence <400> 78 Ala Cys Thr Gly Gly Ala Gly Ala Ala Ala Cys Ala Ala Cys Ala Cys 1 5 10 15 Ala Gly Gly Cys Thr Cys Cys 20 <210> 79 <211> 23 <212> PRT <213> Artificial Sequence <400> 79 Arg Ala Met Ala Thr Thr Ala Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Ala Cys 20 <210> 80 <211> 23 <212> PRT <213> Artificial Sequence <400> 80 Arg Ala Met Ala Thr Thr Ala Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Ala Thr 20 <210> 81 <211> 23 <212> PRT <213> Artificial Sequence <400> 81 Arg Ala Met Ala Thr Thr Gly Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Ala Cys 20 <210> 82 <211> 23 <212> PRT <213> Artificial Sequence <400> 82 Arg Ala Met Ala Thr Thr Gly Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Ala Thr 20 <210> 83 <211> 23 <212> PRT <213> Artificial Sequence <400> 83 Arg Ala Met Ala Thr Thr Thr Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Ala Cys 20 <210> 84 <211> 23 <212> PRT <213> Artificial Sequence <400> 84 Arg Ala Met Ala Thr Thr Thr Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Ala Thr 20 <210> 85 <211> 23 <212> PRT <213> Artificial Sequence <400> 85 Arg Ala Met Ala Thr Thr Ala Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 86 <211> 23 <212> PRT <213> Artificial Sequence <400> 86 Arg Ala Met Ala Thr Thr Ala Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Thr 20 <210> 87 <211> 23 <212> PRT <213> Artificial Sequence <400> 87 Arg Ala Met Ala Thr Thr Gly Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 88 <211> 23 <212> PRT <213> Artificial Sequence <400> 88 Arg Ala Met Ala Thr Thr Gly Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Thr 20 <210> 89 <211> 23 <212> PRT <213> Artificial Sequence <400> 89 Arg Ala Met Ala Thr Thr Thr Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys 20 <210> 90 <211> 23 <212> PRT <213> Artificial Sequence <400> 90 Arg Ala Met Ala Thr Thr Thr Thr Gly Trp Thr Gly Trp Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Thr 20 <210> 91 <211> 20 <212> PRT <213> Artificial Sequence <400> 91 Ala Cys Gly Thr Thr Thr Gly Ala Thr Thr Thr Cys Cys Ala Gly Cys 1 5 10 15 Thr Thr Gly Gly 20 <210> 92 <211> 20 <212> PRT <213> Artificial Sequence <400> 92 Ala Cys Gly Thr Thr Thr Thr Ala Thr Thr Thr Cys Cys Ala Gly Cys 1 5 10 15 Thr Thr Gly Gly 20 <210> 93 <211> 20 <212> PRT <213> Artificial Sequence <400> 93 Ala Cys Gly Thr Thr Thr Thr Ala Thr Thr Thr Cys Cys Ala Ala Cys 1 5 10 15 Thr Thr Thr Gly 20 <210> 94 <211> 20 <212> PRT <213> Artificial Sequence <400> 94 Ala Cys Gly Thr Thr Thr Cys Ala Gly Cys Thr Cys Cys Ala Gly Cys 1 5 10 15 Thr Thr Gly Gly 20

Claims

1. An anti-human PVRIG antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The VH comprises antigenic determining regions VH CDR1, VH CDR2 and VH CDR3, wherein the VH CDR1 has an amino acid sequence as shown in SEQ ID NO: 11, the VH CDR2 has an amino acid sequence as shown in SEQ ID NO: 12, and the VH CDR3 has an amino acid sequence as shown in SEQ ID NO: 13; The VL comprises antigenic determining regions VL CDR1, VL CDR2 and VL CDR3, wherein the VL CDR1 has an amino acid sequence as shown in SEQ ID NO: 14, the VL CDR2 has an amino acid sequence as shown in SEQ ID NO: 15, and the VL CDR3 has an amino acid sequence as shown in SEQ ID NO:

16.

2. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The VH comprises an amino acid sequence at least 80% identical to SEQ ID NO:3, and the VL comprises an amino acid sequence at least 80% identical to SEQ ID NO:

4.

3. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The VH comprises an amino acid sequence at least 80% identical to SEQ ID NO:7, and the VL comprises an amino acid sequence at least 80% identical to SEQ ID NO:

8.

4. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The VH comprises an amino acid sequence at least 80% identical to SEQ ID NO:7, and the VL comprises an amino acid sequence at least 80% identical to SEQ ID NO:

10.

5. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The VH comprises an amino acid sequence at least 80% identical to SEQ ID NO:9, and the VL comprises an amino acid sequence at least 80% identical to SEQ ID NO:

8.

6. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The VH comprises an amino acid sequence at least 80% identical to SEQ ID NO:9, and the VL comprises an amino acid sequence at least 80% identical to SEQ ID NO:

10.

7. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The anti-human PVRIG antibody or antigen-binding fragment thereof has cross-reaction with monkey PVRIG, but has no binding activity with mouse PVRIG.

8. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The antibody or antigen-binding fragment thereof is a monoclonal antibody.

9. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 8, characterized in that: The antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody.

10. The anti-human PVRIG antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds to human PVRIG and is selected from the group consisting of Fv, Fab, Fab', scFv and F(ab')2.

11. An isolated polynucleotide encoding the anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. An isolated polynucleotide encoding the anti-human PVRIG antibody according to any one of claims 1 to 10.

13. A vector comprising one or more polynucleotides according to claim 11 or 12.

14. The carrier according to claim 13, characterized in that The vector comprises the two polynucleotides of claim 11 or 12, and the vector encodes a VL region and a VH region, and the VL region and the VH region together bind to PVRIG.

15. A carrier pair, characterized in that: Each vector pair comprises one of the polynucleotides of claim 11 or 12, wherein the vector pair jointly encodes a VL region and a VH region, which together bind to PVRIG.

16. A host cell comprising the polynucleotide of claim 11 or 12, the vector of claim 13 or 14, or the vector pair of claim 15, wherein: The host cell is a prokaryotic or eukaryotic cell, and the eukaryotic cell is a protist cell, an animal cell or a fungal cell.

17. A composition comprising: 1) the anti-human PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the polynucleotide according to claim 11 or 12, the vector according to claim 13 or 14, the vector pair according to claim 15, or the host cell according to claim 16; and 2) Optional pharmaceutically acceptable excipients.

18. A method for preparing an anti-human PVRIG antibody or an antigen-binding fragment thereof, comprising: 1) culturing the host cell according to claim 16 under suitable conditions; 2) Isolating and recovering the anti-human PVRIG antibody or its antigen-binding fragment.

19. A kit, characterized in that: An anti-PVRIG antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

20. A method for detecting PVRIG in a sample, the method comprising (a) contacting a sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10; and (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and PVRIG; Optionally, the antibody is detectably labeled.

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