Anti-PVRIG antibody and its use

Abstract

This disclosure provides an anti-PVRIG antibody, a nucleic acid molecule encoding the anti-PVRIG antibody, and an expression vector and host cells used for the expression of the anti-PVRIG antibody. This disclosure further provides a method for preventing and treating cancer and immunodeficiency by administering the anti-PVRIG antibody.

Description

[Technical Field]

[0001] <Cross reference> This application claims the interests of the international patent application PCT / CN2023 / 098601, filed on 6 June 2023. The entire contents of said application are incorporated herein by reference.

[0002] <Sequence Listing> This application includes a sequence listing, the entire contents of which are incorporated herein by reference.

[0003] <Technical field> This application generally relates to antibodies. More specifically, it relates to a monoclonal antibody against PVRIG, a method for preparing the same, and the use of the said antibody. [Background technology]

[0004] Cancer cells may evade or suppress the immune response by utilizing natural regulatory mechanisms that limit T cell activation in the body, thereby preventing uncontrolled T cell activity. The goal of immunotherapy is to restore the ability of immune effector cells, particularly T cells, to recognize and eliminate cancer. In the tumor microenvironment, persistent antigen stimulation can lead to T cell exhaustion (a state of T cell dysfunction), and co-inhibitory receptors such as PD-1, LAG-3, TIM3, and TIGIT may be overexpressed. Currently, numerous strategies are being investigated to reactivate exhausted T cells using approaches involving small molecules or therapeutic antibodies, either alone or in combination.

[0005] Poliovirus receptor-associated Ig domain-containing protein (PVRIG, also known as CD112R) is a co-suppressive immune checkpoint protein. PVRIG plays a crucial role in the recovery of T cells from exhaustion and the increased activation of NK cells. PVRIG belongs to the nectin and nectin-like family, which also includes TIGIT, DNAM-1 (CD226), and CD96. PVRIG is expressed in NK cells and T cells, and its expression increases further in T cells when activated. The interaction between PVRIG and its ligand, PVRL2 (CD112), which is expressed in APCs and several tumor cells, suppresses the activation of T cells and NK cells. PVRL2 is also a ligand for CD226, and CD226 activates human T cells and human NK cells through interaction with its ligand.

[0006] In multiple preclinical mouse tumor models, PVRIG blocking has demonstrated a promising strategy for cancer treatment. COM-701, developed by Compugen, is the first anti-PVRIG antagonist antibody to enter clinical development. Results from a Phase 1 clinical trial suggested that COM-701, both as monotherapy and in combination with nivolumab, is well-tolerated and has a manageable safety profile. Best-case responses, such as complete response (CR), partial response (PR), or stable disease (SD), were observed in 11 out of 21 patients (52%) with previously resistant disease and 13 out of 18 patients (52%) who had received prior treatment with immune checkpoint inhibitors. GSK4381562, co-developed by GSK and Surface Oncology, is another anti-PVRIG antibody currently in clinical trials. GSK4381562 binds to different epitopes on PVRIG and blocks the interaction between PVRIG and CD112, which may promote the activation of both NK cells and T cells, potentially inducing a potent antitumor response and enhancing immunological memory.

[0007] PVRIG may be a promising therapeutic target in tumor immunotherapy, either as a monotherapy or in combination with other immunomodulatory agents. [Overview of the Initiative]

Means for Solving the Problems

[0008] These objects and other objects are provided by the present disclosure, which in a broad sense relates to compounds, methods, compositions, and products that provide antibodies with improved efficacy. The advantages provided by the present disclosure are widely applicable in the fields of antibody therapy and diagnosis and can be used in combination with other antibodies that react with various targets.

[0009] The present disclosure provides antagonist antibodies against PVRIG. The antibodies disclosed herein can increase the activation of T cells and NK cells, among other things, by suppressing the signal transduction pathway triggered by the binding interaction between PVRIG and PRLR2. Furthermore, a method of treating a subject having cancer, an immune disorder, or a pathogen infection by administering the anti-PVRIG antibody disclosed herein is also provided. The present disclosure also provides methods for verifying the function of the antibody in vitro and in vivo.

[0010] In some aspects, the present disclosure a heavy-chain CDR (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; a light-chain CDR (LCDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 and provides an isolated antibody or an antigen-binding portion thereof.

[0011] In some embodiments, the isolated antibody or an antigen-binding portion thereof comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), wherein the VH is: (i) the amino acid sequence shown in SEQ ID NO: 7; (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 7; or (iii) Amino acid sequences having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 7 It includes or consists of and / or The above VL is: (i) The amino acid sequence shown in any of Sequence IDs 8-9; (ii) an amino acid sequence that is at least 85%, 90%, or 95% identical to any of sequence numbers 8-9; or (iii) Amino acid sequences having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid additions, deletions, and / or substitutions compared to any of sequence numbers 8-9 It includes or consists of.

[0012] In some embodiments, the isolated antibody or its antigen-binding moiety includes HCDR1, HCDR2, and HCDR3 of the VH region shown in SEQ ID NO: 7, and LCDR1, LCDR2, and LCDR3 of the VL region shown in any of SEQ ID NOs: 8 to 9.

[0013] In some embodiments, the isolated antibody or its antigen-binding portion includes: HCDR1 shown in SEQ ID NO: 1; HCDR2 shown in SEQ ID NO: 2; HCDR3 shown in SEQ ID NO: 3; LCDR1 shown in SEQ ID NO: 4; LCDR2 shown in SEQ ID NO: 5; and LCDR3 shown in SEQ ID NO: 6.

[0014] In some embodiments, the isolated antibody or its antigen-binding portion includes a VH region containing the amino acid sequence of SEQ ID NO: 7 and a VL region containing the amino acid sequence of SEQ ID NO: 8.

[0015] In some embodiments, the isolated antibody or its antigen-binding moiety further comprises a human IgG constant region, such as a human IgG1, IgG4, IgG2, or IgG3 constant region, which may be native or a variant thereof. Specifically, the antibody may comprise a human IgG1 Fc region or a human IgG4 Fc region.

[0016] In some embodiments, the isolated antibody or its antigen-binding moiety comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 12, 15, or 17 and a light chain containing the amino acid sequence of SEQ ID NO: 13, 16, or 18.

[0017] In some embodiments, the anti-PVRIG antibody disclosed herein is a human antibody or a humanized antibody. In some embodiments, the antibody herein is an anti-PVRIG antagonist antibody.

[0018] In some embodiments, the Disclosure provides isolated nucleic acid molecules comprising nucleic acid sequences encoding a heavy chain variable region and / or light chain variable region of an isolated antibody or its antigen-binding moiety disclosed herein. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO: 10 and / or the nucleic acid sequence shown in SEQ ID NO: 11.

[0019] In some embodiments, the present disclosure provides an expression vector comprising nucleic acid molecules disclosed herein.

[0020] In some embodiments, the present disclosure provides host cells comprising the expression vectors disclosed herein.

[0021] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody or an antigen-binding moiety thereof disclosed herein and a pharmaceutically acceptable carrier.

[0022] In some embodiments, the Disclosure provides a method for preparing the antibody or its antigen-binding moiety, comprising expressing the antibody or its antigen-binding moiety in a host cell and isolating the antibody or its antigen-binding moiety from the host cell. In some embodiments, the host cell is transfected or transformed with an expression vector encoding the heavy chain and light chain of the antibody disclosed herein. The nucleic acid sequences encoding the heavy chain and the nucleic acid sequences encoding the light chain may be in the same vector or in separate vectors.

[0023] In some embodiments, the Disclosure provides a method for modulating an immune response in a subject, comprising administering an antibody or an antigen-binding moiety thereof disclosed herein to the subject. The immune response may be PVRIG-related, T-cell-related, and / or NK-cell-related.

[0024] In some embodiments, the present disclosure provides a method for inhibiting the proliferation of tumor cells in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding moiety thereof or a pharmaceutical composition disclosed herein, either alone or in combination with another anticancer agent.

[0025] In some embodiments, the Disclosure provides a method for treating or preventing cancer or an immune-related disorder in a subject, comprising administering an effective amount of an antibody or its antigen-binding moiety disclosed herein to the subject. The method may further comprise the administration of additional anticancer agents, such as chemotherapeutic agents, monoclonal antibodies, or antibody-drug conjugates. In some embodiments, the anticancer agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody.

[0026] The cancers listed above can be selected from colon cancer, lung cancer (such as NSCLC), breast cancer, prostate cancer, bladder cancer, ovarian cancer, stomach cancer, colorectal cancer, esophageal cancer, head and neck cancer, cervical cancer, pancreatic cancer, testicular cancer, lymphoma, leukemia, malignant melanoma, and soft tissue cancer. The immune-related disorders listed above may be T-cell dysfunction, infections, or inflammatory diseases.

[0027] In some embodiments, the Disclosure provides the use of antibodies or their antigen-binding moieties disclosed herein, either alone or in combination with other anticancer agents, in the manufacture of pharmaceuticals for treating or preventing diseases such as cancer and immune-related disorders.

[0028] In some embodiments, the Disclosure provides the use of antibodies or antigen-binding moieties disclosed herein in the manufacture of diagnostic agents for diagnosing diseases associated with PVRIG overexpression.

[0029] In some embodiments, the Disclosure provides antibodies or antigen-binding moieties disclosed herein for use in the treatment or prevention of cancer and immune-related disorders.

[0030] In some embodiments, the Disclosure provides a method for detecting the presence of PVRIG antigen in a sample or for measuring the amount of PVRIG antigen, the method comprising contacting the sample with an anti-PVRIG antibody or its antigen-binding moiety disclosed herein.

[0031] In some embodiments, the present disclosure provides a kit or apparatus comprising an antibody or its antigen-binding portion disclosed herein in one or more containers.

[0032] The above is a summary and therefore naturally includes simplifications, generalizations, and omissions of details. Those skilled in the art will understand that this summary is illustrative and not intended to be limiting. This summary is not intended to identify the main or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. [Brief explanation of the drawing]

[0033] [Figure 1] Figure 1 shows antibody binding to human PVRIG engineered cells as determined by FACS. [Figure 2]Figure 2 shows the binding of antibodies to human CD8+ cells as determined by FACS. [Figure 3] Figure 3 shows antibody binding to cynomolgus monkey PVRIG engineered cells as determined by FACS. [Figure 4] Figure 4 shows the antibody binding results to mouse PVRIG as determined by ELISA. [Figure 5] Figure 5 shows the affinity of anti-PVRIG antibodies to human PVRIG in FACS affinity tests. [Figure 6] Figure 6 shows the binding of antibodies to the PVRIG paralog protein as determined by ELISA. [Figure 7] Figure 7 shows the results of ELISA, which determined that the antibody blocked the binding of PVRL2 to PVRIG. [Figure 8] Figure 8 shows the antibody results in the NFAT (Nuclear Factor for Activated T Cells) reporter gene assay. [Figure 9] Figure 9 shows the effect of antibodies on the stimulation of human CD8+ T cells. [Figure 10] Figure 10 shows the stability of antibodies in human serum. [Figure 11] Figure 11 shows the binding of IgG1 and IgG4 form antibodies to human PVRIG engineered cells as determined by FACS. [Figure 12] Figure 12 shows the activity of IgG1 and IgG4 type antibodies in the NFAT reporter gene assay. [Figure 13] Figure 13 shows the activity of IgG1 and IgG4 type antibodies in a T cell activation assay. [Figure 14] Figure 14 shows the pharmacokinetic results of anti-PVRIG antibodies in rats. [Figure 15] Figure 15 shows the binding of WT1175-1.158.12-m1-uIgG1L to human PVRIG engineered cells as determined by FACS. [Figure 16]Figure 16 shows the activity of WT1175-1.158.12-m1-uIgG1L in the NFAT reporter gene assay. [Modes for carrying out the invention]

[0034] While the present invention can be embodied in many different forms, this specification discloses specific exemplary embodiments illustrating the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated. Furthermore, the section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described.

[0035] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" shall include multiple references unless otherwise explicitly indicated in the context. Thus, for example, a reference to "a protein" includes multiple proteins, and a reference to "a cell" includes a mixture of cells. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "comprising," as well as other forms such as "comprises" and "comprised," is not limited. Also, the scopes indicated herein and in the appended claims include both endpoints and all points between those endpoints.

[0036] In general, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, as described herein are well known and commonly used in the art. The methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art, unless otherwise indicated, as described in the various general and more specific references cited and discussed throughout this specification. For example, Abbas et al., Cellular and Molecular Immunology, 6 th See also WBSaunders Company (2010), Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000), Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002), Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998), and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature, experimental procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well known and commonly used in the art.

[0037] definition To better understand this disclosure, the following definitions and explanations of relevant terms are provided.

[0038] As used herein, the term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains linked by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be classified into κ light chains and λ light chains. The heavy chains can be classified into μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1, C H 2, and C H 3). Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ). The V H region and the V L region can be further divided into hypervariable regions (called complementarity determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). Each V H and V L consists of three CDRs and four FRs in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (V H and V L ) of each heavy / light chain pair form an antigen binding site, respectively. The antibody can be a different antibody isotype, such as an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0039] In the context of this application, the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, which can be used interchangeably, refer to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to an antigen to which a full-length antibody specifically binds, and / or competes with the full-length antibody for binding to the same antigen. Generally, please refer to Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Under certain conditions, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides containing at least a portion of an antibody capable of conferring specific antigen-binding ability to the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., monoclonal anti-human PVRIG antibody as shown herein) by conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and their specificity can be screened in the same manner as for screening intact antibodies.

[0040] As used herein, the terms “monoclonal antibody” or “mAb” refer to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.

[0041] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species, for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody.

[0042] As used herein, the term “humanized antibody” refers to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as rat or mouse, is grafted onto a human framework sequence. Further framework region modifications can be performed within the human framework sequence. Humanized antibodies will also optionally include an immunoglobulin constant region (e.g., Fc), typically at least a portion of the human immunoglobulin constant region.

[0043] As used herein, the terms “human antibody” or “fully human antibody” are intended to include antibodies having variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody includes a constant region, that constant region is also derived from a human germline immunoglobulin sequence.

[0044] The terms “PVRIG” or “Poliovirus Receptor Related Immunoglobulin Domain Containing Protein” include known or wild-type PVRIG, as well as their variants, complexes, or fragments (in particular the ECD fragment). PVRIG is a transmembrane domain protein having a signal peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. PVRIG is expressed on the cell surface of NK cells and T cells and shares some similarities with other known immune checkpoints. The identification and methods used to demonstrate that PVRIG is a checkpoint receptor can be found in International Publication No. 2016 / 134333, incorporated herein by reference. An example of a human PVRIG amino acid sequence is shown in Sequence ID No. 14 (MRTEAQVPALQPPEPGLEGAMGHRTLVLPWVLLTLCVTAGTPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDPGLSAPPTPAPILRADLAGILGVSGVLLFGCVYLLHLLRRHKHRPAPRLQPSRTSPQAPRARAWAPSQASQAALHVPYATINTSCRPATLDTAHPHGGPSWWASLPTHAAHRPQGPAAWASTPIPARGSFVSVENGLYAQAGERPPHTGPGLTLFPDPRGPRAMEGPLGVR).

[0045] In this specification, the term "binding affinity" is used as a measure of the strength of a non-covalent interaction between two molecules, such as an antibody or its antigenic moiety and an antigen. The binding affinity between two molecules can be quantified by various assays, including surface plasmon resonance (SPR), flow cytometry (FACS), and kinetic exclusion assays (KinExA). Flow cytometry is a common technique for analyzing ligand binding to proteins presented on the cell surface. In flow cytometry affinity assays, the equilibrium binding constant (Ka, i.e., k) is used. on / k off ) and equilibrium dissociation constant (KD, i.e., K off / K on The binding rate and binding affinity of the antibody can be determined and evaluated. The binding reaction rate and binding affinity of the antibody can be evaluated by standard assays known in the art or by the methods described in Section 3.4 of the Examples below.

[0046] The term "EC" as used herein 50 The term "50% effective concentration," also known as the "50% effective concentration," refers to the concentration of a drug, antibody, or toxin that induces an intermediate response between baseline and maximum after a specific exposure period.

[0047] The term "IC" as used in this specification 50 The term "50% inhibitory concentration" (or "50% inhibitory concentration") refers to the 50% inhibitory concentration of a drug, antibody, or other substance. It is a measure of the potency of a substance in inhibiting a specific biological or biochemical function.

[0048] As used herein, the term “isolated” refers to a state obtained from its natural state by artificial means. Where a particular “isolated” substance or component exists in nature, this is made possible by alteration of its natural environment, or by isolation of the substance from its natural environment, or both. For example, certain non-isolated polynucleotides or polypeptides exist naturally within certain animal organisms, and a high-purity version of the same polynucleotide or polypeptide isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term “isolated” does not exclude any artificial or synthetic substances or other impurities that do not affect the activity of the isolated substance.

[0049] As used herein, the term “isolated antibody” is intended to refer to an antibody that substantially contains no other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to the PVRIG protein substantially contains no antibodies that specifically bind to antigens other than the PVRIG protein). However, an isolated antibody that specifically binds to the human PVRIG protein may cross-react to other antigens, such as PVRIG proteins from other species. Also, an isolated antibody may substantially contain no other cellular material and / or chemical substances.

[0050] As used herein, the term “vector” refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it is capable of expressing a protein encoded by the inserted polynucleotide. A vector can express its genetic material elements in a host cell by transformation, transduction, or transfection of the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but is not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may contain an origin of replication.

[0051] As used herein, the term “host cell” refers to a cell line that can be manipulated to produce the protein, protein fragment, or peptide of interest. Examples of host cells include, but are not limited to, cultured mammalian cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, and YB2 / 0; human tissue or hybridoma cells; yeast cells; insect cells; and cells contained within transgenic animals or cultured tissues. This term encompasses not only individual target cells but also their offspring. Such offspring may not be identical to their parent cells due to mutations or environmental influences, but they are still included within the scope of the term “host cell.”

[0052] As used herein, the term “identity” refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by aligning and comparing their sequences. “Identity percentage” means the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the smallest size of the compared molecules. For these calculations, any gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., “algorithm”). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48:1073.

[0053] As used herein, the term “immunogenicity” refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the properties of an antigen that stimulate specific immune cells to be activated, proliferate, and differentiate to ultimately produce immunological effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response in which antibodies or sensitized T lymphocytes are formed in the organism’s immune system after the organism has been stimulated by the antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the production of an immune response in a host depends on three factors: the properties of the antigen, the host’s reactivity, and the means of immunization.

[0054] As used herein, the term “transfection” refers to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods, such as electroporation. Several transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981, Gene 13:197. In a particular embodiment of the present invention, the human PVRIG gene was transfected into 293F cells.

[0055] As used herein, the terms “SPR” or “Surface Plasmon Resonance” refer to, and include, optical phenomena that enable real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix using systems such as the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, New Jersey). For further explanation, see Example 5 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26, Jonsson, U., et al. (1991) Biotechniques 11:620-627, Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131, and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0056] As used herein, the terms “fluorescence-activated cell sorting” or “FACS” refer to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of living cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence properties of each cell (FlowMetric. “Sorting Out Fluorescence Activated Cell Sorting”. Retrieved 2017-11-09). Instruments for performing FACS are known to those skilled in the art and are generally commercially available. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, California), the Epics C from Coulter Epics Division (Hyalia, Florida), and the MoFlo from Cytomation (Colorado Springs, Colorado).

[0057] As used herein, the terms “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refer to a cytotoxic form in which secreted immunoglobulins (Ig) bound to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-carrying target cells and subsequently kill them with cytotoxicity. Antibodies “arm” the cytotoxic cells and are absolutely necessary for such killing. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo, for example, in animal models, such as those disclosed in Clynes et al. PNAS (USA), 95:652-656 (1998).

[0058] The terms “subject” and “patient” are used interchangeably and include mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. This term does not necessarily indicate that the subject has been diagnosed with a specific disease and typically refers to an individual under medical supervision.

[0059] As used herein with reference to specific disease conditions in mammals, the terms “prevent,” “prevent,” or “preventing” mean preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or subclinical symptoms.

[0060] As used herein in the context of treating a condition, the terms “treatment,” “treating,” or “treated” generally refer to treatments and therapies, whether in humans or animals, that achieve any desired therapeutic effect, such as inhibiting the progression of a condition, including a reduction in the rate of progression, cessation of progression, regression of the condition, improvement of the condition, and cure of the condition. In the case of cancer, “treating” may mean weakening or slowing the growth, proliferation, or metastasis of a tumor or malignant cells, or any combination thereof.

[0061] As used herein, the term “effective dose” refers to the amount of an active compound or material, composition, or dosage form containing an active compound that is effective in producing some desired therapeutic effect commensurate with a reasonable benefit-to-risk ratio when administered according to a desired therapeutic regimen. For example, when used in connection with the treatment of a disease or condition, “effective dose” refers to an amount or concentration of antibody or its antigen-binding moiety that is effective in treating the disease or condition.

[0062] As used herein, the term “pharmaceutically acceptable” means that its vehicle, diluent, excipient, and / or salt are chemically and / or physically compatible with the other components of the formulation and physiologically compatible with the recipient.

[0063] As used herein, the term “pharmaceutically acceptable carriers and / or excipients” means carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and activator, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH modifiers, surfactants, adjuvants, and ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween®-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0064] As used herein, the term “adjuvant” refers to a nonspecific immunostimulant that, when delivered to an organism with an antigen or prior to the antigen, can enhance the immune response to an antigen or alter the type of immune response in the organism. Various adjuvants exist, including, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund’s adjuvants (e.g., complete Freund’s adjuvant and incomplete Freund’s adjuvant), Corynebacterium parvum, lipopolysaccharides, and cytokines. Freund’s adjuvants are currently the most commonly used adjuvants in animal studies. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0065] Anti-PVRIG antibody In some embodiments, the disclosure provides antibodies or antigen-binding moieties thereof against PVRIG (such as human, mouse, or cynomolgus monkey PVRIG). Preferably, the antibody binds to PVRIG with sufficient affinity to substantially or completely block the interaction and / or binding of PVRIG to PVRL2. When PVRIG binds to its ligand, PVRL2, it triggers an inhibitory signal that acts to attenuate the immune response of NK cells and T cells against target cells. By blocking the binding of PVRL2 to PVRIG, such inhibitory signals of PVRIG are blocked, and as a result, the immune response of NK cells and T cells is regulated.

[0066] In some embodiments, the anti-PVRIG antibodies disclosed herein are fully human antibodies. In some embodiments, the anti-PVRIG antibodies disclosed herein are humanized antibodies. The antigen-binding moiety of the antibody may be Fab, Fab', F(ab')2, a single-chain variable fragment (scFv), or a diabody. In some embodiments, the human antibody may include further modifications to CDR residues and framework residues to remove potential post-translational modifications or to improve antibody performance such as binding affinity.

[0067] The antibodies disclosed herein can bind with high affinity to at least one of human, mouse, and cynomolgus monkey PVRIG. The binding of the antibodies disclosed herein to PVRIG can be evaluated using one or more techniques well established in the art, such as ELISA. The binding specificity of the antibodies disclosed herein can also be determined by monitoring the binding of the antibodies to cells expressing the PVRIG protein, such as by flow cytometry. For example, the antibodies can be tested by a flow cytometry assay in which the antibodies react with human PVRIG-expressing cell lines, such as HEK293 cells transfected to express PVRIG on the cell surface. Additionally or alternatively, the binding of the antibodies, such as the binding reaction rate (e.g., K), can be evaluated. D The value can be tested using a BIAcore binding assay.

[0068] In some embodiments, the antibody or its antigen-binding moiety can specifically bind to cynomolgus monkey PVRIG in addition to human PVRIG. For example, the antibody or its antigen-binding moiety can bind to human PVRIG-expressing cells with an EC50 of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, or 0.08 nM or less, as measured by FACS; and can bind to cynomolgus monkey PVRIG-expressing cells with an EC50 of 0.5 nM or less, 0.3 nM or less, 0.1 nM or less, 0.08 nM or less, 0.06 nM or less, or 0.04 nM or less. In some embodiments, the antibody or its antigen-binding moiety of this disclosure can bind to cells expressing cynomolgus monkey PVRIG with a KD of 1 × 10⁻¹⁶ when measured by FACS affinity testing. -9 M or less, 5×10 -10 M or less, 1×10 -10 M or less, 5×10 -11 M or less, 4×10 -11 M or less, 3×10 -11 M or less, or 2 × 10 -11 It binds to human PVRIG at M or lower.

[0069] The anti-PVRIG antibodies disclosed herein can inhibit the interaction between PVRIG and PVRL2 (CD112). By blocking PVRL2-mediated signaling, the functional response of T cells to antigen stimulation (e.g., proliferation, cytokine production, target cell killing) can be restored from a dysfunctional state. The ability of an anti-PVRIG antibody to inhibit such interaction can be evaluated by measuring whether the physical interaction between PVRIG and CD112 is reduced in a binding assay. In many cases, the binding assay is a competitive binding assay. The assay may be carried out in various formats, including, but is not limited to, ELISA assays, flow cytometry, surface plasmon resonance (SPR) assays (e.g., Biacore®), or biolayer interferometry (e.g., ForteBio Octet®). In some embodiments, the anti-PVRIG antibodies herein can block the binding of human PVRL2 to PVRIG when measured by ELISA, with an IC50 of ≤0.5 nM, ≤0.4 nM, or ≤0.3 nM.

[0070] Anti-PVRIG antibody containing CDR In some embodiments, the present disclosure provides an isolated antibody or its antigen-binding moiety, the antibody or its antigen-binding moiety is A) One or more heavy chain CDRs (HCDRs) selected from the group consisting of: HCDR1 containing an amino acid sequence different from SEQ ID NO: 1, or SEQ ID NO: 1, due to the addition, deletion, and / or substitution of two or fewer amino acids; HCDR2 containing an amino acid sequence different from SEQ ID NO: 2, or SEQ ID NO: 2, due to the addition, deletion, and / or substitution of two or fewer amino acids; and HCDR3 containing an amino acid sequence different from SEQ ID NO: 3, or SEQ ID NO: 3, due to the addition, deletion, and / or substitution of two or fewer amino acids; B) One or more light chain CDRs (LCDRs) selected from the group consisting of: LCDR1 containing an amino acid sequence different from SEQ ID NO: 4, or SEQ ID NO: 4, due to the addition, deletion, and / or substitution of two or fewer amino acids; LCDR2 containing an amino acid sequence different from SEQ ID NO: 5, or SEQ ID NO: 5, due to the addition, deletion, and / or substitution of two or fewer amino acids; and LCDR3 containing an amino acid sequence different from SEQ ID NO: 6, or SEQ ID NO: 6, due to the addition, deletion, and / or substitution of two or fewer amino acids; or C) One or more HCDRs from A) and one or more LCDRs from B) Includes.

[0071] In some embodiments, CDR identification follows the IMGT / Kabat definition.

[0072] In some embodiments, the present disclosure provides an isolated antibody or its antigen-binding moiety comprising HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6.

[0073] The framework area and the scope of the CDR can be precisely identified using methods known in the art, for example, by definitions of Kabat, Chothia, AbM, Contact, IMGT (all well known in the art), and any combination thereof. For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877, Chothia, C. et al. al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Antibodies”, chapter 5, 2007. Furthermore, please refer to hgmp.mrc.ac.uk and bioinf.org.uk / abs. The correspondence or alignment between the numberings based on different definitions can be found, for example, at www.imgt.org / . (See also Giudicelli V et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206-11 and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55-77).

[0074] As those skilled in the art will understand, the exact numbering and arrangement of CDRs may differ depending on the numbering system. However, regardless of the numbering method or definition scheme adopted, it should be understood that the disclosure of a variable heavy chain sequence and / or variable light chain sequence includes the disclosure of the relevant (unique) CDRs. Thus, the disclosure of each variable region is a disclosure of the CDRs (e.g., HCDR1, HCDR2, and HCDR3). Two antibodies having the same VH and VL means that their CDRs are identical when determined by the same method (e.g., Kabat, AbM, Chothia, Contact, and IMGT definitions known in the art). Even the same antibody disclosed herein may have different CDR sets when determined by different definition schemes.

[0075] In some embodiments, the Disclosure provides an isolated antibody or its antigen-binding moiety comprising one, two, or all three HCDRs of the amino acid sequence shown in SEQ ID NO: 7 and one, two, or all three LCDRs of the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, an anti-PVRIG antibody comprising one, two, or all three HCDRs of the amino acid sequence shown in SEQ ID NO: 7 and one, two, or all three LCDRs of the amino acid sequence shown in SEQ ID NO: 9 is provided herein.

[0076] In some embodiments, the Disclosure provides an isolated antibody or its antigen-binding moiety comprising the VH sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 7 and the VL sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 8. In some embodiments, an anti-PVRIG antibody comprising the VH sequences HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 7 and the VL sequences LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 9 is provided herein.

[0077] Variable regions and CDRs in antibody sequences can be identified according to general rules developed in the art, or by aligning the sequence against a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described on the "Abysis" website (www.bioinf.org.uk / abs, maintained by AC Martin of the Department of Biochemistry and Molecular Biology, University College London, UK) and the VBASE2 website (www.vbase2.org, described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005)), and can be accessed through these websites. Sequences may be analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structure data from the PDB. See the chapter "Protein Sequence and Structure Analysis of Antibody Variable Domains" in the Antibody Engineering Lab Manual by Dr. Andrew CRMartin (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk / abs). The Abysis database website further includes general rules developed for identifying CDRs, which can be used in accordance with the teachings herein.

[0078] In some embodiments, the anti-PVRIG antibody disclosed herein comprises the following VH region and VL region: the VH region comprises FRW1-HCDR1-FRW2-HCDR2-FRW3-HCDR3-FRW4 (wherein HCDR1 has the amino acid sequence shown in SEQ ID NO: 1, HCDR2 has the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 has the amino acid sequence shown in SEQ ID NO: 3); and / or the VL region comprises FRW1-LCDR1-FRW2-LCDR2-FRW3-LCDR3-FRW4 (wherein LCDR1 has the amino acid sequence shown in SEQ ID NO: 4, LCDR2 has the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 6).

[0079] In some embodiments, the framework region is derived from human germline, such as human immunoglobulin. In some embodiments, specific residues in the framework region are mutated to improve antibody performance, such as stability, binding affinity, isomerization, and immunogenicity. In some embodiments, S7 in FRW1 of the VL region and / or T43 (according to Kabat numbering) in FRW2 of the VL region are mutated. In some specific embodiments, the VL region of the antibody contains an S7P / T43A substitution. In some embodiments, the FRW1 and FRW4 at the N-terminus and C-terminus of the VH region and / or VL region may be cleaved to contain only partial FRW1 and / or partial FRW4. In some embodiments, the CDR and FR regions are PTM removal optimized.

[0080] In some embodiments, anti-PVRIG antibodies are provided herein that comprise at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 7 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 8. In some embodiments, anti-PVRIG antibodies are provided herein that comprise at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 7 and at least one of the amino acid sequences FRW1, FRW2, FRW3, and FRW4 shown in SEQ ID NO: 9.

[0081] Anti-PVRIG antibody containing heavy chain variable region and light chain variable region In some embodiments, the isolated antibody or its antigen-binding portion is: (A) (i) Does it contain the amino acid sequence of SEQ ID NO: 7? (ii) Having the same CDR set as SEQ ID NO: 7 and containing an amino acid sequence having at least 85%, 90%, or 95% identity in the framework region; or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acid additions, deletions, and / or substitutions in the framework region compared to the amino acid sequence of SEQ ID NO: 7 Heavy chain variable region (VH), and / or (B) (i) Does it contain the amino acid sequence of SEQ ID NO: 8 or 9? (ii) Having the same CDR set as SEQ ID NO: 8 or 9 and containing an amino acid sequence having at least 85%, 90%, or 95% identity in the framework region; or (iii) an amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2) amino acid additions, deletions, and / or substitutions in the framework region compared to the amino acid sequence of SEQ ID NO: 8 or 9. Light chain variable region (VL) Includes.

[0082] The percentage of identity between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (Comput.Appl.Biosci.,4:11-17(1988)) incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J.Mol.Biol.48:444-453(1970)) incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either the Blossum62 matrix or the PAM250 matrix, gap weights 16, 14, 12, 10, 8, 6, or 4, and length weights 1, 2, 3, 4, 5, or 6.

[0083] Additionally or alternatively, the protein sequences of this disclosure can be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) described in Altschul, et al. (1990) J.MoI. Biol. 215:403-10. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. To obtain gapped alignments for comparative purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST program and the Gapped BLAST program, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0084] In some further embodiments, the isolated antibody or its antigen-binding moiety may include conservative amino acid substitutions or modifications in the variable regions of the heavy and / or light chains. In the art, it is understood that certain conservative sequence modifications can be made that do not remove antigen binding. For example, see Brummell et al. (1993) Biochem 32:1180-8, de Wildt et al. (1997) Prot.Eng.10:835-41, Komissarov et al. (1997) J.Biol.Chem.272:26864-26870, Hall et al. (1992) J.Immunol.149:1605-12, Kelley and O' Connell (1993) Biochem.32:6862-35, Adib-Conquy et al. (1998) Int.Immunol.10:341-6, and Beers et al. (2000) Clin.Can.Res.6:2835-43.

[0085] As used herein, the term “conservative substitution” refers to an amino acid substitution that will not adversely affect or alter the essential properties of a protein / polypeptide, including its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties (including the ability to form covalent or hydrogen bonds)). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with alkaline side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al., Protein Eng. 12(10):879-884 (1999), and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997) (these are incorporated herein by reference)). In some further embodiments, the antibodies or antigen-binding moieties disclosed herein have a conserved substitution at the S7 and / or T43 positions (by Kabat numbering) in the VL region.

[0086] In certain embodiments, the isolated antibody or its antigen-binding moiety includes a heavy chain variable region containing or comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region containing or comprising the amino acid sequence of SEQ ID NO: 8 or 9.

[0087] In other embodiments, the amino acid sequences of the heavy chain variable region and / or light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the respective sequences shown above.

[0088] Fc area The anti-PVRIG antibodies and antigen-binding moieties provided herein further comprise an immunoglobulin constant region, such as a human IgG1, IgG2, IgG3, or IgG4 immunoglobulin constant region (natural type or variant thereof), which includes an Fc region and optionally a hinge region. The anti-PVRIG antibodies herein may be IgG1 or IgG4 isotypes. In some embodiments, the Fc region is a natural Fc region or an Fc variant. The Fc variant may have at least about 80% homology or at least about 90% homology, e.g., at least about 95% homology, to the natural sequence Fc region. In some embodiments, the Fc region is a human IgG4 Fc region, such as a wild-type Fc region or an Fc variant containing an S228P substitution. In certain embodiments, the antibodies disclosed herein comprise a wild-type human IgG1 Fc region.

[0089] In some embodiments, the Fc region includes one or more amino acid changes (e.g., insertions, deletions, or substitutions) that modify the binding interaction between Fc and FcRn or FcγR.

[0090] In certain embodiments, the Fc region is an IgG4 Fc region containing the S228P mutation (according to EU numbering, as in Kabat et al.) which prevents Fab arm exchange and stabilizes the IgG4 molecule. In certain embodiments, the Fc region is an IgG1 Fc region containing the LALA mutation, i.e., the L234A and L235A mutations. The LALA mutation is perhaps the most commonly used mutation to interfere with antibody effector function, for example, by eliminating Fc binding to specific FcγR and reducing PBMC and monocyte-mediated ADCC activity. The “EU numbering system” or “EU index” is generally used to refer to residues in the constant domain of the immunoglobulin heavy chain (e.g., the EU index reported by Kabat et al. above). “Kabat-like EU numbering” or “Kabat-like EU index” refers to the residue numbering of a human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody mean residue numbering according to the EU numbering system.

[0091] In certain embodiments, the hinge region may be derived from the same human IgG immunoglobulin as the Fc region.

[0092] Anti-PVRIG antibodies with specific properties The antibodies of this disclosure are characterized by their specific functional features or properties. Based on the mechanism of action against the target, the in vitro functional properties and pharmacological activity of the antibodies were thoroughly evaluated at both the molecular and cellular levels. In some embodiments, the isolated antibody or its antigen-binding moiety has one or more of the following properties: (a) Specifically binds to at least one of the following: human PVRIG protein and cynomolgus monkey PVRIG protein; (b) Does not cross-bind with PVRIG paralog proteins; (c) Block the binding of PVRIG to its ligand PVRL2 (CD112); (d) Activate immune cells such as T cells and NK cells; (e) Good serum stability and thermal stability; (f) It shows significantly superior efficacy compared to benchmark antibodies in cancer treatment.

[0093] As shown in the examples, CTLs (cytotoxic T lymphocytes, e.g., CD8+ T cells) and NK cells can be used in functional assays to evaluate antibody function. CTLs express T cell receptors (TCRs) that recognize specific antigens (Ag) presented by MHC molecules. When TCRs bind to Ag, CTLs are activated, which manifests as cell proliferation, upregulation of activation markers (e.g., CD35, CD137), cytokine secretion, and cytotoxic activity. When PVRIG comes into contact with PVRL2 expressed on cancer cells or antigen-presenting cells, it mediates a negative signal to CTLs, thereby downregulating CTL activation. Therefore, contact with an anti-PVRIG antibody that exhibits binding to and / or receptor-ligand binding inhibition of CTLs disrupts the PVRIG-PVRL2 interaction, thereby releasing the negative signal mediated by PVRIG and enhancing antigen-specific CTL activation, which manifests as cell proliferation, upregulation of activation markers (e.g., CD25, CD137, etc.), and cytokine secretion (e.g., interferon-gamma, IL-2, TNF-alpha, etc.). In some embodiments, human CD8+ T cells treated with anti-PVRIG antibodies showed increased IFN-γ secretion compared to those observed using isotype controls and reference antibodies.

[0094] NK cells express various activating and inhibitory receptors. When PVRIG comes into contact with PVRL2 expressed on cancer target cells, it mediates a negative signal to NK cells, thereby downregulating NK cell activation. When an anti-PVRIG antibody comes into contact with NK cells, the PVRIG-PVRL2 interaction is disrupted, thereby releasing the negative signal mediated by PVRIG, enhancing NK cell activation, which manifests as cell proliferation, cytokine secretion (e.g., interferon-gamma, IL-2, TNFα, etc.), and / or cytotoxic activity.

[0095] Method for producing antibodies Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals (e.g., XenoMouse®), or any combination thereof. For example, monoclonal antibodies can be produced using hybridomas and biochemical and genetic engineering techniques recognized in the art, such as An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1 st ed.2009, Shire et.al.(eds.)Current Trends in Monoclonal Antibody Development and Manufacturing,Springer Science+Business Media LLC,1 st Further details are provided in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988, and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, 563-681 (Elsevier, NY, 1981), where each reference is incorporated herein by reference in its entirety. The selected binding sequences can be further modified, for example, to improve affinity for the target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, or produce multispecific antibodies, and it should be understood that antibodies containing modified target binding sequences are also antibodies of the present invention.

[0096] To obtain human antibodies with minimal immunogenicity, transgenic mouse technology has been developed that can produce fully human therapeutic mAbs. OmniRat (Open Monoclonal Technology Company) has developed a chimeric human / rat IgH gene locus (22 human VH , all humans D and J H The segment is naturally composed of rat C H This transgenic rat possesses both a locus linked to the endogenous Ig locus and a complete human IgL locus (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig locus was silenced using designer zinc finger nuclease. OmniRat rats can produce antibodies with the human idiotype with the same efficiency as wild-type rats produce rat antibodies.

[0097] Nucleic acid molecules encoding antibodies in this disclosure In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of isolated antibodies disclosed herein.

[0098] The nucleic acids of this disclosure can be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice possessing human immunoglobulin genes), the cDNA encoding the light and heavy chains of the antibodies produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0099] Isolated nucleic acids encoding the VH region can be converted into full-length heavy chain genes by operably ligating the VH-encoding nucleic acid to another DNA molecule encoding the heavy chain constant domains (CH1, CH2, and CH3). Sequences of human heavy chain constant domain genes are known in the art (see, for example, Kabat et al. (1991) cited above), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant domains may be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant domains, but are more preferably IgG1 or IgG4 constant domains.

[0100] Isolated nucleic acids encoding the VL region can be converted into full-length light chain genes (and Fab light chain genes) by operably ligating the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al. cited above), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa constant region or a lambda constant region.

[0101] Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques to convert, for example, a variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operably ligated to another DNA fragment encoding another protein, such as an antibody constant region or a mobile linker. The term "operably ligated" as used in this context is intended to mean that the two DNA fragments are ligated in such a way that the amino acid sequences encoded by these two DNA fragments remain in frame.

[0102] In some embodiments, this disclosure relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region of an isolated antibody disclosed herein. In some specific embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain variable region of an isolated antibody, selected from the group consisting of: (A) Nucleic acid sequence encoding the heavy chain variable region shown in Sequence ID No. 7; (B) Nucleic acid sequence shown in Sequence ID No. 10; or (C) A nucleic acid sequence hybridized to the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions.

[0103] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding the light chain variable region of an isolated antibody disclosed herein.

[0104] In some specific embodiments, the isolated nucleic acid molecule encodes the light chain variable region of the isolated antibody and includes a nucleic acid sequence selected from the group consisting of: (A) Nucleic acid sequences encoding the light chain variable region shown in Sequence ID No. 8 or 9; (B) Nucleic acid sequence shown in Sequence ID No. 11; or (C) A nucleic acid sequence hybridized to the complementary strand of the nucleic acid sequence of (A) or (B) under high stringency conditions.

[0105] For example, the nucleic acid molecule includes sequence numbers 10 and 11. In some other embodiments, the nucleic acid molecule shares at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with sequence number 10 or 11. In some specific embodiments, the identity percentage is based on the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0106] Exemplary high-stringency conditions include hybridization at 45°C in 5×SSPE and 45% formamide, and final washing at 65°C in 0.1×SSC. In the art, it is understood that equivalent stringency conditions can be achieved by varying the temperature and buffer or salt concentration, as described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3~6.4.10. Modification of hybridization conditions can be determined empirically or precisely calculated based on probe length and guanosine / cytosine (GC) base pairing ratio. Hybridization conditions can be calculated as described in Sambrook, et al. (Eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47~9.51.

[0107] host cell The host cells disclosed herein may be any cells suitable for expressing the antibodies disclosed herein, such as yeast, bacteria, fungi, plants, and animal cells, and preferably mammalian cells. Examples of mammalian host cells for expressing the antibodies disclosed herein include Chinese hamster ovary (CHO cells) (e.g., dhfr CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with a DHFR selection marker as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621, etc.), 293F cells, NSO myeloma cells, COS cells, and SP2 cells. In particular, when used in conjunction with NSO myeloma cells, other expression systems include the GS gene expression systems disclosed in International Publication No. 87 / 04462, International Publication No. 89 / 01036, and European Patent No. 338,841.Additionally, the following cells were used: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL1651), human fetal kidney cell line (293 or 293 cells subcloned for proliferation in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), and human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocytes (Hep G2, HB8065), mouse mammary tumor cells (MMT060562, ATCC CCL51), TRI cells (Mather et al., 1982, Annals NYAcad. Sci. 383:44-68), MRC5 cells, FS4 cells, mouse myeloma cells, e.g., NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC Examples include CRL1581), rat myeloma cells, e.g., YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL1662), PER.C6 cells, and human hepatocellular carcinoma cell lines (Hep G2). CHO cells are one of the cell lines that can be used herein, with CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555(1986)), and Lec13 being exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44, or CHO-DP12 host cells, the ability to fucosylate expressed proteins may be modified to be deficient.In some embodiments, the host cells herein are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NSO cells, or lymphocytes.

[0108] Suitable prokaryotes for this purpose include bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as the genera Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as Bacillus subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0109] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains, such as Schizosaccharomyces pombe, Clibellomyces hosts, such as K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC36,906), K. thermotlerans, and K. marxianus; Yarrowia (European Patent No. 402,226); Pichia pastris (European Patent No. 183,070); Candida; Trichoderma reesia (European Patent No. 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces Hosts of *Occidentalis* and filamentous fungi, such as *Nepeta crassa*, *Penicolus*, *Tripocladium*, and *Aspergillus*, such as *A. nidulans* and *A. niger*, are commonly available and useful herein.

[0110] When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to be expressed in the host cells or secreted into the culture medium in which the host cells are cultured. The antibody can be recovered from the culture medium using standard protein purification methods.

[0111] Pharmaceutical composition In some embodiments, this disclosure relates to a pharmaceutical composition comprising at least one antibody or antigen-binding moiety disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a pharmaceutical composition comprising a nucleic acid encoding an antibody or antigen-binding moiety disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a pharmaceutical composition comprising cells expressing an antibody or antigen-binding moiety disclosed herein and a pharmaceutically acceptable carrier.

[0112] Composition components The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of this disclosure may also be administered in combination therapy with another immunostimulant, anticancer agent, antiviral agent, or vaccine. Examples of pharmaceutically acceptable carriers include pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspension / dispersant agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, and various combinations thereof.

[0113] Suitable components include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propyl gallate. For example, a composition comprising an antibody or antigen-binding fragment of the present disclosure may contain one or more antioxidants (such as methionine) to suppress the oxidation of the antibody or its antigen-binding fragment. Suppression of oxidation can prevent or reduce a decrease in binding affinity, thereby increasing antibody stability and extending shelf life. Accordingly, in some embodiments, the present disclosure provides compositions comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants (such as methionine). The disclosure further provides various methods by which an antibody or its antigen-binding fragment is mixed with one or more antioxidants (such as methionine) to prevent oxidation of the antibody or its antigen-binding fragment, thereby extending their shelf life and / or increasing their activity.

[0114] Further examples of pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as plant-derived fixative oils such as cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents at bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate buffer or citrate buffer; antioxidants such as sodium bisulfate; and local anesthetics. Examples of pharmaceuticals include procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN®-80), metal ion sequestering agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers, including phenol or cresol, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride, may be added to the pharmaceutical composition in a multi-dose container. Suitable excipients include, for example, water, physiological saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or pharmaceuticals such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0115] Administration, formulation, and dosage The pharmaceutical compositions of this disclosure may be administered in vivo to the target subject as needed by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or otherwise by implantation or inhalation. The compositions may be formulated in the form of solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols, but not limited to these. The appropriate formulation and route of administration may be selected according to the intended use and treatment regimen.

[0116] Formulations suitable for intravenous administration include hard or soft gelatin capsules, pills, coated tablets, elixirs, suspensions, syrups, or inhalants, as well as controlled-release forms thereof.

[0117] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous isotonic pyrogen-free sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may further contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the recipient's blood (or other relevant body fluids). Examples of excipients include, for example, water, alcohol, polyols, glycerol, and vegetable oils. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosing regimens, including dosage, timing, and repetition, will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0118] The frequency of administration may be determined and adjusted throughout the course of treatment, based on reducing the number of proliferative or tumorigenic cells, maintaining a reduction in such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dose administered may be adjusted or attenuated to manage potential side effects and / or toxicity. Alternatively, a sustained-release formulation of the therapeutic composition in question may be appropriate.

[0119] Those skilled in the art will understand that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit with any risks or adverse side effects. The dosage level to be selected will depend on various factors, including, but are not limited to, the activity of the specific compound, the route of administration, the timing of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the patient's species, sex, age, weight, condition, overall health, and medical history. The dosage is generally selected to achieve a local concentration at the site of action that produces the desired effect without causing significant adverse side effects, but the amount of compound and the route of administration are ultimately at the discretion of the physician, veterinarian, or clinician.

[0120] In general, the antibody or its antigen-binding moiety of this disclosure can be administered in a variety of doses. These include approximately 5 μg / kg body weight to approximately 40 mg / kg body weight per dose, approximately 50 μg / kg body weight to approximately 5 mg / kg body weight per dose, and approximately 100 μg / kg body weight to approximately 10 mg / kg body weight per dose. Other ranges include approximately 100 μg / kg body weight to approximately 20 mg / kg body weight per dose, and approximately 0.5 mg / kg body weight to approximately 20 mg / kg body weight per dose. In certain embodiments, the dose is at least approximately 100 μg / kg body weight, at least approximately 250 μg / kg body weight, at least approximately 750 μg / kg body weight, at least approximately 3 mg / kg body weight, at least approximately 5 mg / kg body weight, and at least approximately 10 mg / kg body weight.

[0121] In any case, the antibody or its antigen-binding portion of this disclosure is preferably administered as needed to the target. The frequency of administration can be determined by a person skilled in the art, such as the attending physician, taking into consideration the condition being treated, the age of the target, the severity of the condition, and the general health status of the target.

[0122] In certain preferred embodiments, a therapeutic course comprising the antibody or its antigen-binding moiety of the Disclosure would involve multiple administrations of a selected formulation over a period of several weeks or months. More specifically, the antibody or its antigen-binding moiety of the Disclosure may be administered once daily, every two days, every four days, once a week, every ten days, every two weeks, every three weeks, once a month, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be understood that the dosage may be changed or the intervals adjusted based on the patient's response and clinical practice.

[0123] Dosages and regimens may also be empirically determined for the therapeutic compositions of the Disclosure in individuals receiving one or more doses. For example, an individual may be given an escalating dose of the therapeutic composition prepared as described herein. In selected embodiments, doses may be gradually increased, decreased, or attenuated based on empirically determined or observed side effects or toxicity. To evaluate the efficacy of the selected composition, markers of specific diseases, disorders, or conditions may be tracked as described above. In the case of cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques, improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples, measurement of indirect tumor markers (e.g., PSA in prostate cancer) or oncoplastic antigens identified according to the methods described herein, reduction of pain or paralysis, improvement of speech, vision, respiration, or other impairments related to the tumor, increased appetite, or improvement in quality of life as measured by approved tests, or extension of survival.

[0124] Suitable formulations for parenteral administration (e.g., intravenous injection) may contain the antibody or its antigen-binding moiety disclosed herein at concentrations ranging from approximately 10 μg / ml to approximately 100 mg / ml. It will be apparent to those skilled in the art that the dosage of the antibody or its antigen-binding moiety disclosed herein will vary depending on the individual, the type of neoplasm, the stage of the neoplasm, whether the neoplasm has begun to spread to other parts of the individual, past and concomitant treatments, and the dosage of any therapeutic agents used in combination with the antibody disclosed herein.

[0125] Uses of this disclosure The antibodies, antibody compositions, and methods of this disclosure have numerous in vitro and in vivo uses, including detection of PVRIG or enhancement of the immune response. For example, these molecules can be administered in vitro or ex vivo to cells in culture, or to human subjects, for example, in vivo, to enhance immunity in various situations. The immune response can be modulated, for example, enhanced, stimulated, or upregulated.

[0126] For example, the target population includes human patients who require enhancement of the immune response. This method is particularly suitable for treating human patients with disorders that can be treated by enhancing the immune response (e.g., T cell-mediated immune response). In certain embodiments, this method is particularly suitable for in vivo cancer treatment, such as cancer-induced immunosuppression.

[0127] The antibodies disclosed herein may be used in combination with additional therapeutic agents, such as anticancer agents including anticancer antibodies and chemotherapeutic agents. These additional therapeutic agents may be T-cell co-suppressive molecule antagonists or inhibitors, T-cell co-activating molecule agonists, or immunostimulatory cytokines. When an anti-PVRIG antibody is administered with another agent, such as an anti-PD-1 agent, the two agents may be administered in any order or simultaneously.

[0128] This disclosure further provides a method for detecting the presence of PVRIG antigen in a sample or for measuring the amount of PVRIG antigen, comprising contacting a sample and a control sample with an anti-PVRIG antibody or its antigen-binding portion under conditions that enable the antibody or portion to form a complex with PVRIG. The formation of the complex is then detected, and if there is a difference in complex formation between the sample and the control sample, it is determined that PVRIG antigen is present in the sample. Furthermore, PVRIG can be purified by immunoaffinity purification using the anti-PVRIG antibody of this disclosure.

[0129] Treatment of diseases such as cancer In some embodiments, the Disclosure provides a method for treating a disorder or disease in a mammal, comprising administering a therapeutically effective amount of the anti-PVRIG antibody disclosed herein or its antigen-binding moiety to a subject in need of treatment (e.g., a human). The disorder or disease includes, but is not limited to, proliferative disorders (such as cancer), immunodeficiencies, inflammatory diseases, or infections. For example, the disorder may be cancer.

[0130] In some embodiments, the cancer is characterized by high PRVL2 expression. In some embodiments, the cancer is characterized by an abundance of T cells or natural killer (NK) cells that express PVRIG.

[0131] Cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, lung cancer, such as non-small cell lung cancer (NSCLC) (NSCLC includes squamous cell NSCLC or non-squamous cell NSCLC, which include locally advanced unresectable NSCLC (e.g., stage IIIB NSCLC) or recurrent or metastatic NSCLC (e.g., stage IV) This includes lung adenocarcinoma or squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-resistant non-muscle-invasive bladder cancer (NMIBC)); urinary tract cancer; hepatocellular carcinoma; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer or renal cancer (e.g., renal cell carcinoma (RCC)); prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma including superficial spreading melanoma, lentigo malignant melanoma, acral lentigo melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma Tumors (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-incisional nuclear NHL; giant tumor NHL; mantle cell lymphoma; AIDS-associated lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorders (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal angiogenesis associated with nevus, edema (such as that associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and associated metastases.

[0132] As a co-inhibitory receptor on various immune cells, PVRIG is associated with a variety of cancers, whether malignant or benign, primary or secondary, which can be treated or prevented in the manner provided herein. The anti-PVRIG antibodies disclosed herein are preferably administered in combination with other anticancer agents. Cancers can be solid tumors or hematological malignancies. Examples of such cancers include lung cancers such as bronchogenic cancers (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondropathic hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyoma; osteochondroma, chondroma, chondroblastoma, chondromyxofibroma, osteoid, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, and Ewing's tumor. Bone cancers such as Ewing's sarcoma and reticular cell sarcoma; brain cancers such as gliomas (e.g., glioblastoma multiforme), undifferentiated astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannomas, ependymoma, meningioma, pituitary adenoma, pineal glandoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratoma, dermoid cystoma, and hemangioma; colon cancer, leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, and colon polyps Cancers of the digestive system, such as colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, hyperephroma, and transitional cell carcinoma of the renal pelvis; bladder cancer; skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancers; eye-related cancers such as retinoblastoma and intraocular melanoma; benign prostate cancer. Examples include cancers of the male reproductive system such as adenomatous hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonic carcinoma, and choriocarcinoma); breast cancer; cancers of the female reproductive system such as uterine cancer (endometrial cancer), cervical cancer, ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (papillary carcinoma, follicular carcinoma, undifferentiated carcinoma, or medullary carcinoma, etc.); pheochromocytoma (adrenal gland); non-cancerous proliferation of the parathyroid gland; and pancreatic cancer. In certain embodiments, the cancer is colon cancer.

[0133] In some other embodiments, the disorder or disease to be treated or prevented is an immune-related disorder. The immune-related disorder may be associated with T-cell dysfunction. In some embodiments, T-cell dysfunction is characterized by reduced responsiveness to antigen stimulation. In some embodiments, T-cell dysfunction is characterized by T-cell anergy, or by reduced cytokine secretion, reduced proliferation, or reduced cytolytic activity. In some embodiments, T-cell dysfunction is characterized by T-cell exhaustion. In some embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments, the immune-related disorder is selected from the group consisting of unresolved acute infections, chronic infections, and reduced tumor immunity.

[0134] Stimulation of the immune response In some embodiments, the Disclosure further provides a method for enhancing (e.g., stimulating) an immune response in a subject, comprising administering an antibody of the Disclosure or an antigen-binding moiety thereof to the subject so as to enhance the immune response in the subject. For example, the subject is a mammal. In certain embodiments, the subject is a human.

[0135] The term "enhance the immune response," or its grammatical variations, means stimulating, inducing, increasing, improving, or enhancing any response of the mammalian immune system. The immune response can be a cellular response (i.e., cell-mediated, e.g., cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated), and can be a primary or secondary immune response. An example of enhancing the immune response is CD4 +Enhancement of the immune response can be evaluated using many in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Typically, the methods of the disclosure enhance the immune response in mammals compared to the immune response in untreated mammals or mammals not treated with the methods disclosed herein. In one embodiment, an antibody or its antigen-binding moiety is used to enhance the human immune response to a microbial pathogen (such as a virus). In another embodiment, an antibody or its antigen-binding moiety is used to enhance the human immune response to a vaccine. In one embodiment, the method enhances the cellular immune response, particularly the cytotoxic T cell response. In another embodiment, the cellular immune response is a T helper cell response. In yet another embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-2 production. Antibodies or their antigen-binding portions may be used to enhance the human immune response to microbial pathogens (such as viruses) or vaccines.

[0136] The antibody or its antigen-binding portion may be used alone as monotherapy, or in combination with other antibodies (such as anti-PD-1 antibodies or anti-PD-L1 antibodies), chemotherapy, radiotherapy, targeted therapy, or cellular immunotherapy.

[0137] Combination with chemotherapy Antibodies or their antigen-binding portions may be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0138] The terms “anticancer agent” or “antiproliferative agent” mean any agent that can be used to treat cell proliferation disorders such as cancer, and include, but are not limited to, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, weight-reducing agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents, and immunotherapy agents. It will be understood that such anticancer agents may include conjugates and may be conjugated with the antibodies of this disclosure before administration. More specifically, in certain embodiments, an engineered conjugate is provided by linking a selected anticancer agent to an unpaired cysteine ​​of an engineered antibody. Thus, such an engineered conjugate is explicitly intended to be within the scope of this disclosure. In some other embodiments, the anticancer agent is given in combination with an antibody-drug conjugate containing a different therapeutic agent.

[0139] As used herein, the term “cytotoxic agent” means a substance that is toxic to cells, reduces or inhibits cellular function, and / or causes cell destruction. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-sarcin, restrictosin), plants (e.g., abrin, lysine, modesin, biscumin, pokeweed antiviral protein, saporin, geronin, momorizin, tricosanthin, barley toxin, Chinese tallow tree protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitors, curcin, crotin, soapwort inhibitors, geronin, mitejerin, restrictosin, phenomycin, neomycin, and trichothecene), or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase, DNase I (including its fragments and / or variants)).

[0140] For the purposes of this disclosure, “chemotherapeutic agents” include chemical compounds (e.g., cytotoxic agents or cell proliferation inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents often target intracellular processes necessary for cell growth or division and are therefore generally particularly effective against cancer cells that grow and divide rapidly. For example, vincristine depolymerizes microtubules and, consequently, inhibits cells from entering mitosis. Generally, chemotherapeutic agents may include any chemical agents that inhibit or are designed to inhibit cancer cells or cells that are likely to become cancerous or produce tumorigenic offspring (e.g., TICs). Such agents are often administered and are most effective in combination with regimens such as CHOP or FOLFIRI.

[0141] Examples of anticancer agents that may be used in combination with the antibodies of this disclosure (either as components of site-directed complexes or in an unbound state) include alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methylamelamines, acetogenins, camptothecin, bryostatin, calistatin, CC-1065, cryptophycin, dorastatin, duocalmycin, eleuterobin, pancratistatin, sarcodicin, spongistin, nitrogen mustard, antibiotics, enediyne antibiotics, dinemycin, bisphosphonates , Esperamycin, pigment protein enediin antibiotic chromophore, Acrasinomycin, Actinomycin, Ausramycin, Azaserin, Bleomycin, Cactinomycin, Carabicin, Carminomycin, Cardinophilin, Chromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazo-5-Oxo-L-Norleucine, ADRIAMYCIN®, Doxorubicin, Epirubicin, Esolubicin, Idarubicin, Marcelomycin, Mitomycin, Mycophenolate Nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin, antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folate analogs, purine analogs, androgens, anti-adrenal agents, folate supplements such as folic acid, acegraton, aldofosphamide glycoside, aminolevulinic acid, enyl Uracil, Amsacrin, Bestrabusil, Bisanthren, Edatraxate, Dehofamine, Demecolsin, Diadicone, Elhornitine, Erliptinium Acetate, Epothiron, Etoglucid, Gallium Nitrate, Hydroxyurea, Lentinan, Ronidinin, Mytansinoid, Mitoguazone, Mitoxanthrone, Mopidammol, Nitraerine, Pentostatin, Fenamet, Pirarubicin, Rosoxanthrone, Podophyllic Acid, 2-Ethylhydrazide, Procarbazine, PSK (Registered Trademark) Polysaccharide Complex (JHS Natural Products, Eugene, Oregon), Lazoxane, Rhizoxin, Schizophyllan, Spirogermanium, Tenuazonic Acid, Triadicone, 2,2',2"-Trichlorotriethylamine, Trichothecenes (especially T-2 toxin, Beraclin A, Loridine A, and Angidin), Urethane, Vindesine, Dacarbazine, Mannomustine, Mitobronitol, Mitractol, Pipobroman, Gacitosine, Arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Taxoid, Chlorambucil, GEMZAR® Gemcitabine, 6-Thiogunine, Mercaptopurine, Methotrexate, Platinum Analogue, Vinblastine, Platinum, Etoposide (VP-16), Ifosfamide, Mitoxantrone, Vincristine, NAVE Examples of pharmaceutically acceptable substances include, but are not limited to, LBINE® vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeroda, ibandronate, irinotecan (Camptosar, CPT-11), the topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoids, capecitabine, combretastatin, leucovorin, oxaliplatin, inhibitors of PKC-alpha, Raf, H-Ras, EGFR, and VEGF-A that reduce cell proliferation, and any pharmaceutically acceptable salts, acids, or derivatives of the above. This definition also includes anti-hormone agents that act to control or inhibit hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, an enzyme that controls estrogen production in the adrenal gland, and anti-androgens, as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog), antisense oligonucleotides, ribozymes, such as VEGF expression inhibitors, vaccines, PROLEUKIN, (R) rIL-2, LURTOTECAN (R) Topoisomerase 1 inhibitor, ABARELIX (R) This also includes rmRH, vinorelbine, and esperamycin, as well as any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0142] Combined use with radiation therapy This disclosure also provides combinations of antibodies or their antigen-binding moieties with radiotherapy (i.e., any mechanism for inducing localized DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combination therapies using directed delivery of radioisotopes to tumor cells are also envisioned, and the antibodies of this disclosure may be used in conjunction with targeted anticancer agents or other targeting means. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy may be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy may be administered as a single dose or as a series of doses.

[0143] Pharmaceutical packs and kits Pharmaceutical packs and kits are also provided, comprising one or more containers containing one or more doses of antibody or its antigen-binding moiety. In certain embodiments, a unit dosage form is provided, comprising a predetermined amount of a composition containing, for example, an antibody or its antigen-binding moiety, together with or without one or more additional agents. In other embodiments, such a unit dosage form is supplied as a single-use pre-filled syringe for injection. In yet another embodiment, the composition contained in the unit dosage form may include, and / or be formulated within a stable and effective pH range, including, physiological saline, sucrose, and buffering agents such as phosphates. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder that is reconstituted upon addition of a suitable liquid, such as sterile water or physiological saline. In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, for example, sucrose and arginine, but not limited to the following. Any label on or accompanying the container indicates that the encapsulated antibody is used to treat a selected neoplastic disease condition.

[0144] This disclosure also provides kits comprising single or multi-dose units of an antibody and, optionally, one or more anticancer agents. The kits comprise a container and labels or accompanying documents on or associated with the container. Suitable containers include, for example, bottles, vials, and syringes. Containers may be formed from a variety of materials, such as glass or plastic, and may contain a pharmaceutically effective amount of the antibody of this disclosure. In some embodiments, the container includes a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper puncturable by a subcutaneous needle). Such kits generally comprise a pharmaceutically acceptable formulation of the antibody in a suitable container and, optionally, one or more anticancer agents in the same or different containers. The kits may also comprise other pharmaceutically acceptable formulations for either diagnostic or combination therapy. For example, in addition to the antibody or its antigen-binding moiety of the present disclosure, such kit may include one or more of the following anticancer agents: chemotherapeutic agents or radiotherapy agents, anti-angiogenic agents, anti-metastatic agents, targeted anticancer agents, cytotoxic agents, and / or other anticancer agents.

[0145] More specifically, the kit may have a single container containing the antibody or its antigen-binding moiety with or without additional components, or it may have separate containers for each desired drug. If concomitant therapeutic agents are provided for binding, a single solution may be pre-mixed in molar equivalent combinations or such that one component outweighs the other. Alternatively, the antibody and optional anticancer agents of the kit may be maintained separately in separate containers before administration to the patient. The kit may also include second / third container means for sterile, pharmaceutically acceptable buffers or other diluents, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextrose solution.

[0146] If the components of this kit are provided in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, and sterile aqueous solutions or physiological saline are particularly preferred. However, the components of this kit may also be provided as dry powders. If the reagents or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent.

[0147] As briefly stated above, the kit may also include means for administering the antibody or its antigen-binding portion and optional components to a patient, for example, one or more needles, IV bags or syringes, or eyedroppers, pipettes or other such instruments that can inject or introduce the formulation into an animal or apply it to a diseased area of ​​the body. The kit of the disclosure will also typically include means for tightly sealing and housing vials and other components for commercial sale, for example, injection-molded or blow-molded plastic containers that hold the desired vials or other instruments.

[0148] Sequence List Overview A sequence listing containing several nucleic acid and amino acid sequences is attached to this application. Tables A, B, and C below provide an overview of the included sequences.

[0149] [Table 1]

[0150] [Table 2]

[0151] [Table 3] [Examples]

[0152] The disclosure, broadly described above, will be more readily understood by referring to the following examples, which are provided as illustrations but are not intended to limit the disclosure. The examples are not intended to represent all or only experiments that have been conducted.

[0153] Example 1 Preparation of antigens, benchmark antibodies, and cell lines 1.1 Antigen Preparation WT117-hPro1.ECD.His is the extracellular domain of human PVRIG (NP_076975.2) with a C-terminal polyhistidine tag. WT117-hPro1.ECD.hFc is the extracellular domain of human PVRIG (NP_076975.2) with a human IgG1 Fc region at its C-terminus. WT117-mPro1.ECD.His is the extracellular domain of mouse PVRIG (XP_011239268.1) with a C-terminal polyhistidine tag. WT117-mPro1.ECD.hFc is the extracellular domain of mouse PVRIG (XP_011239268.1) with a human IgG1 Fc region at its C-terminus. WT117-hPro1L1.ECD.mFc is the extracellular domain of human PVRL2 (NP_001036189.1) which has the mouse IgG2a Fc region at its C-terminus.

[0154] 1.2 Preparation of Benchmark Antibody (BMK) Anti-human PVRIG reference antibodies WT117-BMK1 and WT117-BMK3 were prepared according to the sequences disclosed in their respective patents. This information is summarized in Table 1.

[0155] [Table 4]

[0156] 1.3 Preparation of cell pools / cell lines The human PVRIG-expressing cell line WT117-293F.hPro1.G11 was created using 293F cells transfected with full-length human PVRIG (NP_076975.2). The cynomolgus monkey PVRIG-expressing cell pool WT117-Flpin293.cPro1.pool was created using Flpin293 cells transfected with full-length cynomolgus monkey PVRIG (XP_005549281.1).

[0157] Example 2 Production of human antibodies against PVRIG 2.1 Immunization Two female OmniRats (Open Monoclonal Technology Company), aged 8-13 weeks, were purchased from Charles River and reared in an IACUC-certified animal facility. These two animals were alternately immunized with the WT117-hPro1.ECD.hFc and WT117-mPro1.ECD.hFc proteins or plasmids. Immunization was repeated approximately weekly for a total of 111 days.

[0158] 2.2 Detection of serum titer Anti-human / mouse PVRIG antibody titers in serum samples were determined by ELISA. Microplates were coated with 0.5 μg / mL of WT117-hPro1.ECD.His in 100 μL of coating buffer (Na2CO3 / NaHCO3, pH 9.2) per well and incubated overnight at 4°C. On the day of the assay, diluted rat serum samples (first 1:100 dilution using 1×PBS / 2%BSA, then 3-fold dilution) and negative controls were added to plates blocked with 1×PBS / 2%BSA for 1 hour, and then incubated at room temperature for 1 hour. After washing three times with 1×PBST (PBS containing 0.05% Tween®-20), HRP-labeled goat anti-rat IgG Fc (Bethyl, cat#A110-236P) was added and incubated at room temperature for 1 hour. After removing unbound substances, the TMB (3,3',5,5'-tetramethylbenzidine) substrate was added, and the reaction was stopped with 2M HCl. Absorbance at 450 nm was detected using a microplate spectrophotometer.

[0159] Table 2 shows the serum titers of immunized OMT rats. Lymph nodes were collected from two animals and used for fusion.

[0160] [Table 5]

[0161] 2.3 Hybridoma generation, antibody screening, and subcloning Lymph nodes and spleens were harvested from OMT rats under sterile conditions, dissociated, and prepared as single-cell suspensions. These suspensions were then mixed with myeloma cells SP2 / 0 in a 1:1.2 ratio. Electrofusion was performed using a BTX 2001 Electro cell manipulator following an optimized electrofusion procedure. After fusion, the cells were transferred to 96-well plates containing DMEM medium supplemented with 20% FBS and 1% HAT selective reagent (1 × 10⁶). 4 Cells were cultured in a 96-well assay plate at 37°C and 5% CO2, and monitored regularly. When the clones reached approximately 80% confluence in the wells, 100 μL of the supernatant was transferred from the tissue culture plate to a 96-well assay plate for antibody screening.

[0162] Positive cell lines in the logarithmic growth phase were diluted to approximately 200 cells per 1.5 mL of semi-solid HAT medium. The cell suspension was gently mixed in a vortex shaker for 5–10 seconds, and then seeded into 6-well plates. The plates were kept at 37°C and 5% CO2 for 7–8 days. Once cell clusters had grown, visible single colonies were harvested and seeded into 96-well plates containing DMEM medium supplemented with 10% fetal bovine serum. After 2–3 days, the supernatant of each clone was collected and screened again to obtain positive hybridoma single clones.

[0163] 2.4 Sequencing of Hybridomas RNA was isolated from monoclonal hybridoma cells, and cDNA was amplified using the SuperScript® III First-Strand Synthesis SuperMix Kit according to the manufacturer's instructions. Subsequently, the obtained cDNA was used as a template for PCR amplification using primers specific to the target gene. The PCR product was inserted into a pMD18-T vector, and the ligation product and PCR product were sent for sequencing.

[0164] 2.5 Production of fully human antibodies The DNA sequences of the VH and VL domains were amplified by PCR and then subcloned into pcDNA expression vectors containing the constant region of human IgG1 or human IgG4. Plasmids containing the VH and VL genes were co-transfected into Expi293 cells, and the cells were cultured for approximately 5 days before the supernatant was collected. Antibodies were purified from the supernatant using a Protein A column.

[0165] Forty-five positive cell lines were selected and subcloned using primary and secondary binding screening, as well as PVRIG / PVRL2 block and TCR / NFAT luciferase activation assays. After confirming monoclonal antibody status, 20 hits were selected and sequenced, and four of these were converted to human IgG. Following further in vitro characterization (see below), one clone was identified (named WT1175-1.158.12-uIgG4LV1). Its sequence is shown in Tables A and B above.

[0166] Example 3 In vitro characterization 3.1 Human PVRIG binding assay WT117-293F.hPro1.G11(1×10 5 Cells / wells or activated human CD8 + T cells (1×10 5Cells (per well) were incubated with various concentrations of anti-PVRIG antibody at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, PE-labeled goat anti-human IgG (JacksonImmunoResearch, cat#109-115-098), was added, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. Anti-human PVRIG antibody WT117-BMK1 was used as a positive control. Human IgG4 isotype antibody was used as an isotype control. The cells were then washed and resuspended in 1×PBS / 1%BSA. Cellular filtration efficiency (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0167] WT117-293F.hPro1.G11 or Human CD8 + Figures 1 and 2 show the binding results of anti-PVRIG antibodies to T cells. These results indicate that WT1175-1.158.12-uIgG4LV1 can strongly bind to human PVRIG-expressing cells, and its binding affinity is significantly higher than that of the reference antibody. Table 3 below outlines the antibody binding process.

[0168] 3.2 PVRIG binding assay in cynomolgus monkeys WT117-Flpin293F.cPro1.pool(1×10 5 Cells (per well) were incubated with various concentrations of anti-PVRIG antibody at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, PE-labeled goat anti-human IgG (JacksonImmunoResearch, cat#109-115-098), was added, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. Anti-human PVRIG antibody WT117-BMK1 was used as a positive control. Human IgG4 isotype antibody was used as an isotype control. The cells were then washed and resuspended in 1×PBS / 1%BSA. Cellular filtration efficiency (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0169] Figure 3 shows the binding results of the anti-PVRIG antibody to WT117-Flpin293F.cPro1.pool cells. These results indicate that WT1175-1.158.12-uIgG4LV1 can strongly bind to cynomolgus monkey PVRIG-expressing cells, and its binding affinity is significantly higher than that of the reference antibody. Table 3 below outlines the antibody binding process.

[0170] 3.3 Mouse PVRIG binding assay Plates were pre-coated overnight at 4°C with 0.5 μg / mL of WT117-hPro1.ECD.His or WT117-mPro1.ECD.His in 100 μL of coating buffer per well. After blocking with 200 μL of 1×PBS / 2%BSA, 100 μL of anti-PVRIG antibody at a concentration of 6.67 nM was added to the plate and incubated at room temperature for 1 hour. After incubation, the plate was washed three times with 1×PBST. HRP-labeled goat anti-human IgG antibody (Bethyl, cat#A80-304P) diluted with 1×PBS / 2%BSA was added and incubated at room temperature for 1 hour. After washing with 1×PBST, 100 μL of TMB substrate was dispensed and allowed to develop color, then 100 μL of 2M HCl was added to stop the reaction. Absorbance was read at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0171] Figure 4 shows the binding results of the anti-PVRIG antibody to the extracellular domain of mouse PVRIG. From these results, it can be seen that WT1175-1.158.12-uIgG4LV1 does not bind to mouse PVRIG. Table 3 below summarizes the antibody binding process.

[0172] [Table 6]

[0173] 3.4 Human PVRIG affinity assay The binding affinity of the WT1175 antibody to human PVRIG on the cell surface was measured by FACS. WT117-293F.hPro1.G11 cells were measured in 5 × 10⁶ units.4 Cells were transferred to a 96-well round-bottom plate at a cell / well density. Anti-PVRIG antibody was serially diluted in 1×PBS / 1%BSA and incubated with the cells at 4°C for 1 hour. Secondary antibody, FITC-labeled goat anti-human IgG Fc (Jackson Immuno Research, cat#109-095-098), was added and incubated in the dark at 4°C for 0.5 hours. Subsequently, the cells were washed once, resuspended in 1×PBS / 1%BSA, and analyzed by flow cytometry (BD). Fluorescence intensity was converted to the number of bound molecules per cell based on quantitative beads (Bangs Laboratories, cat#555pB).

[0174] Figure 5 and Table 4 show the binding affinity results of anti-PVRIG antibodies against WT117-293F.hPro1.G11 cells. Bmax represents maximum specific binding, and KD is the ligand concentration required to achieve 50% of maximum binding at equilibrium. WT1175-1.158.12-uIgG4LV1 binds to human PVRIG on the cell surface with high affinity.

[0175] [Table 7]

[0176] 3.5 PVRIG Paralog Protein Binding Assay Plates were pre-coated overnight at 4°C with 1 μg / mL of WT117-hPro1.ECD.His, recombinant human TIGIT, CD226, CD96, or PD-1 extracellular domain in 100 μL of coating buffer per well. After blocking with 200 μL of 1×PBS / 2%BSA, 100 μL of test antibody at a concentration of 10 μg / mL was added to the plate and incubated at room temperature for 1 hour. After incubation, the plate was washed three times with 1×PBST. HRP-labeled goat anti-human IgG antibody (Bethyl, cat#A80-304P) diluted with 1×PBS / 2%BSA was added and incubated at room temperature for 1 hour. After washing six times with 1×PBST, 100 μL of TMB substrate was dispensed and allowed to develop color, then 100 μL of 2M HCl was added to stop the reaction. Absorbance was read at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0177] Figure 6 shows the binding results of the anti-PVRIG antibody to the PVRIG paralog protein. From these results, it can be seen that WT1175-1.158.12-uIgG4LV1 specifically binds to PVRIG without cross-reactivity with human TIGIT, CD226, CD96, or PD-1.

[0178] 3.6 Human PVRIG / PVRL2 Blocking Assay Plates were pre-coated overnight at 4°C with WT1175-hPro1.ECD.His at 2 μg / mL in 100 μL of coating buffer per well. 200 μL of 1×PBS / 2% BSA was added for blocking. Serially diluted anti-PVRIG antibody was mixed with a constant concentration of WT1175-hPro1L1.ECD.mFc (final concentration 10 μg / mL) in a 1:1 volume ratio. After blocking for 1 hour, the antibody / ligand mixture was added to the plate and incubated at room temperature for 2 hours. After washing three times with 1×PBST, HRP-labeled goat anti-mouse IgG (Bethyl, cat#A90-231P) was added to the plate and incubated at room temperature for 1 hour. After washing six times with 1×PBST, TMB substrate was added and the interaction was stopped with 2M HCl. Absorbance was read at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0179] Figure 7 shows the results of human PVRIG / PVRL2 binding blocking. These results indicate that WT1175-1.158.12-uIgG4LV1 can effectively block the binding of human PVRL2 to PVRIG. Table 5 below summarizes the antibody blocking activity. The maximum inhibition rate is given by (OD) = (%). max -OD bottom ) / OD max It was calculated as ×100%. In the formula, OD max This was defined as the (OD450-OD540) value in the absence of the antibody.

[0180] 3.7 Jurkat PVRIG / NFAT-luciferase reporter gene assay Jurkat cells overexpressing human PVRIG and NFAT-luciferase reporters were stimulated by co-culturing them with CHOK1 cells expressing human PVRL2 and TCR activator, thereby binding them to T cell receptors. 4 × 10⁶ CHOK1 / PVRL2 / TCR activator cells were placed in a 96-well plate. 4Cells were seeded at a density of cells / well and cultured overnight at 37°C and 5% CO2. On day 2, after removing the supernatant and non-adherent cells, serially diluted anti-PVRIG antibody and Jurkat / PVRIG / NFAT luciferase cells (2 × 10⁶ cells) were used. 4 The cells (per well) were added to the plate and co-cultured at 37°C and 5% CO2 for 5-6 hours. After incubation, the reconstituted luciferase substrate (Promega, cat#E6130) was added to each well and mixed thoroughly. Luciferase intensity was read using an Envision microplate reader (PerkinElmer).

[0181] Figure 8 shows the results of reversing the suppression of NFAT signaling induced by the PVRIG / PVRL2 interaction when an anti-PVRIG antibody was applied. This result indicates that WT1175-1.158.12-uIgG4LV1 can enhance TCR / NFAT activation. Table 5 summarizes the antibody RGA activity.

[0182] [Table 8]

[0183] 3.8 Human Primary T Cell Activation Assay Human first CD8 + T cells were stimulated by binding T cell receptors to human CD8 cells through co-culture with CHOK1 cells expressing human PVRL2 and TCR activator. + T cells were isolated from human peripheral blood mononuclear cells (PBMCs) by magnetic selection using human CD8 MicroBeads (Miltenyi Biotec, cat#130-045-201) according to the manufacturer's protocol. + T cells (1 x 10 per well) 5 Irradiated CHOK1 / PVRL2 / TCR activator cells (2 × 10 cells per well) in the presence of serially diluted anti-PVRIG antibodies. 4The cells were co-cultured with the other cells at 37°C and 5% CO2 for 5 days. After incubation, the supernatant was collected and IFN-γ was measured by ELISA (capture antibody Thermo cat#M700A, detection antibody Thermo cat#M701B). Absorbance was detected using an M5e microplate reader (Molecule Devices).

[0184] Human CD8 treated with anti-PVRIG antibody + T cells secreted more IFN-γ than when observed using isotype controls and reference antibodies. The results indicate that WT1175-1.158.12-uIgG4LV1 enhances CD8+ T cell activation. The data are shown in Figure 9.

[0185] 3.9 Antibody serum stability assay Fresh human serum was isolated from a healthy donor. Anti-PVRIG antibody was diluted in the serum. The sample was dispensed into five tubes and incubated at 37°C. Subsequently, samples were collected on days 0, 1, 4, 7, and 14, rapidly frozen, and stored in a freezer set to -70°C until ready for analysis. The binding activity of the samples was evaluated by FACS according to the method described in Section 3.1.

[0186] Figure 10 shows the binding of WT1175-1.158.12-uIgG4LV1 incubated with serum to WT117-293F.hPro1.G11. Antibodies incubated with serum for up to two weeks maintained binding activity and very similar EC50 values ​​to fresh antibodies. The results indicate that WT1175-1.158.12-uIgG4LV1 is stable in human serum at 37°C for at least two weeks.

[0187] 3.10 Antibody Thermal Stability Assay Conformational stability is a crucial property for promising antibodies. Conformational stability can be evaluated by measuring thermal stability using differential scanning fluorescence (DSF), a method that can detect changes in protein folding. DSF measures the unfolding transition temperature (Tm) of a protein based on changes in the fluorescence intensity of the environmentally sensitive dye SYPRO Orange.

[0188] DSF was performed using a Quant Studio 7 Flex real-time PCR instrument (Applied Biosystems) in each formulation buffer. SYPRO Orange dye (Invitrogen cat#S6651) was added to the antibody, and the mixture was transferred to a 96-well plate. The plate was placed in a Quant Studio® 7 Flex real-time PCR instrument, with the temperature range set to 26°C to 95°C and the heating rate set to 0.9°C / min. The first two temperatures during protein unfolding were recorded as Tm1 and Tm2. These two values ​​were calculated according to the melting curve using Quant Studio® real-time PCR software (v1.3).

[0189] The DSF thermogram of the WT1175-1.158.12-uIgG4LV1 antibody shows two transitions: the first with a low melting point (Tm1) and the second with a high melting point (Tm2), at 62.5°C and 64.9°C, respectively. The results are shown in Table 6.

[0190] [Table 9]

[0191] 3.11 Comparison of Human IgG1 and IgG4 Formats To compare the effects of different Fc values ​​on antibody activity, a corresponding IgG1 format antibody for WT1175-1.158.12-uIgG4LV1 was also prepared. This antibody was named WT1175-1.158.12-uIgG1L. Additionally, a reference antibody containing IgG1 Fc, WT117-BMK3, was prepared and used as a positive control.

[0192] The binding activity of WT1175-1.158.12-uIgG1L to WT117-293F.hPro1.G11 was evaluated according to the method described in Section 3.1. The PVRIG-mediated inhibitory signal reversal effect of WT1175-1.158.12-uIgG1L was evaluated according to the method described in Section 3.7. The CD8+ T cell activation enhancing activity of WT1175-1.158.12-uIgG1L was evaluated according to the method described in Section 3.8.

[0193] Binding (Figure 11), TCR / NFAT luciferase activation assay (Figure 12), and CD8 + The results of enhanced T cell activation (Figure 13) are shown. These results indicate that WT1175-1.158.12-uIgG4LV1 and WT1175-1.158.12-uIgG1L have equivalent binding and functional activity.

[0194] 3.12 Production and Characterization of Mutant PVRIG Antibodies In the antibody WT1175-1.158.12-uIgG1L, positions S7 and T43 (by Kabat numbering) in the VL were identified as potentially unstable residues. Stability was improved by introducing the S7P and T43A mutations, and this mutated mutant was renamed WT1175-1.158.12-m1-uIgG1L. The activity of these two antibodies was compared using a FACS binding assay (method 3.1) and an NFAT reporter gene assay (method 3.7). The thermal stability of WT1175-1.158.12-m1-uIgG1L was evaluated according to the method described in Section 3.10 to demonstrate its improvement.

[0195] The binding results are shown in Figure 15, and the reporter gene assay results are shown in Figure 16. These results show that WT1175-1.158.12-uIgG1L and WT1175-1.158.12-m1-uIgG1L have equivalent binding and functional activity.

[0196] The DSF results are shown in Table 7. Compared to WT1175-1.158.12-uIgG4LV1, WT1175-1.158.12-m1-uIgG1L showed improved thermal stability, with Tm1 increasing from 62.5°C to 68.3°C.

[0197] [Table 10]

[0198] Example 4 In vivo characteristic evaluation 4.1 Pharmacokinetic studies in rodents This study aimed to determine the pharmacokinetics of WT1175-1.158.12-uIgG1L and WT1175-1.158.12-uIgG4LV1 in untreated rats after a single intravenous bolus administration. Eight animals were randomly divided into two groups and treated with either WT1175-1.158.12-uIgG1L (10 mg / kg) or WT1175-1.158.12-uIgG4LV1 (10 mg / kg), respectively.

[0199] PK blood samples were collected before administration, at 0.5 hours, 4 hours, and on days 2, 3, 5, 7, 10, 12, 14, and 21. Serum concentrations of anti-PVRIG antibody were determined by ELISA. Briefly, goat anti-human IgG Fc was used as the capture reagent and biotinylated goat anti-human IgG Fc was used as the detection reagent. The samples were color-developed using streptavidin-HRP and TMB substrate, and the reaction was stopped with 2M HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. Serum concentrations of WT1175-1.158.12-uIgG1L and WT1175-1.158.12-uIgG4LV1 were subjected to non-compartmental pharmacokinetic analysis using Phoenix WinNonlin software. PK parameters were obtained by applying the linear / logarithmic trapezoid rule.

[0200] Since these two antibodies did not bind to rat PVRIG, no target-mediated pharmacokinetic effects were observed. Linear pharmacokinetics were observed at a single dose of 10 mg / kg (Figure 14). The PK parameter results are summarized in Table 8. WT1175-1.158.12-uIgG1L had a mean serum clearance of 4.89 mL / day / kg, a half-life of 429 hours, a volume of distribution of 123 mL / kg, and an AUC. 0-t The value was 25547 h*μg / mL. On the other hand, WT1175-1.158.12-uIgG4LV1 had a mean serum clearance of 5.03 mL / day / kg, a half-life of 368 hours, a volume of distribution of 108 mL / kg, and an AUC. 0-t The result was 29589 h*μg / mL.

[0201] [Table 11]

[0202] Those skilled in the art will further understand that this disclosure can be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of this disclosure discloses only exemplary embodiments, it should be understood that other modifications are also within the scope of this disclosure. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, the appended claims should be used to illustrate the scope and content of the invention.

Claims

1. Heavy chain CDR (HCDR) 1 containing the amino acid sequence of SEQ ID NO: 1; HCDR2 containing the amino acid sequence of SEQ ID NO: 2; HCDR3 containing the amino acid sequence of SEQ ID NO: 3; Light chain CDR (LCDR) 1 containing the amino acid sequence of SEQ ID NO: 4; LCDR2 containing the amino acid sequence of SEQ ID NO: 5; LCDR3 containing the amino acid sequence of SEQ ID NO: 6 An isolated antibody containing or its antigen-binding moiety.

2. (A) (i) containing the amino acid sequence shown in SEQ ID NO: 7; or (ii) Containing an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 7 Heavy chain variable region (VH); and / or (B) (i) containing the amino acid sequence shown in either SEQ ID NOs: 8 or 9; or (ii) Containing an amino acid sequence that is at least 85%, 90%, or 95% identical to either SEQ ID NOs: 8 or 9 Light chain variable region (VL) The isolated antibody or antigen-binding moiety according to claim 1, comprising:

3. An isolated antibody or antigen-binding moiety according to claim 1 or 2, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable region containing the amino acid sequence shown in SEQ ID NO:

8.

4. The isolated antibody or antigen-binding portion thereof according to any one of claims 1 to 3, wherein the isolated antibody further comprises a human IgG constant region.

5. The isolated antibody or antigen-binding moiety according to claim 4, wherein the above-mentioned human IgG constant region is the human IgG1, IgG4, IgG2, or IgG3 constant region or a variant thereof.

6. The isolated antibody or antigen-binding moiety thereof according to any one of claims 1 to 5, wherein the antibody optionally includes a human IgG4 Fc region or a human IgG1 Fc region containing the S228P mutation.

7. The isolated antibody or antigen-binding portion thereof according to any one of claims 1 to 6, wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.

8. An isolated nucleic acid molecule comprising an isolated antibody according to any one of claims 1 to 7, or a nucleic acid sequence encoding a heavy chain variable region and / or a light chain variable region of the antigen-binding portion thereof.

9. A vector comprising an isolated nucleic acid molecule as described in claim 8.

10. A host cell comprising the vector according to claim 9.

11. A pharmaceutical composition comprising an isolated antibody or an antigen-binding moiety thereof as defined in any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

12. A method for producing an antibody or its antigen-binding portion as defined in any one of claims 1 to 7, A step of culturing host cells containing the above-mentioned antibody or an expression vector encoding its antigen-binding portion under appropriate conditions; A step of collecting the above antibody or its antigen-binding portion from a cell culture. A method that includes this.

13. A method for inhibiting the proliferation of tumor cells in a subject, comprising administering an effective amount of an antibody or its antigen-binding portion as defined in any one of claims 1 to 7, or the pharmaceutical composition described in claim 11, to the subject.

14. A method for treating or preventing cancer or an immune-related disorder in a subject, comprising administering to the subject an effective amount of an antibody or its antigen-binding portion as defined in any one of claims 1 to 7, or the pharmaceutical composition described in claim 11.

15. The method according to claim 14, wherein the cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, central nervous system tumors, mesothelioma, myeloma, and sarcoma.

16. The method according to claim 14, wherein the above-mentioned immune-related disorder is T-cell dysfunction or infection.

17. The method according to any one of claims 14 to 16, further comprising administering an additional therapeutic agent.

18. Use of an antibody or antigen-binding moiety as defined in any one of claims 1 to 7 in the manufacture of a pharmaceutical product for treating or preventing cancer or an immune-related disorder.

19. An antibody or antigen-binding moiety as defined in any one of claims 1 to 7, for use in the treatment or prevention of cancer or immune-related disorders.

20. A kit comprising a container containing an antibody or an antigen-binding portion thereof as defined in any one of claims 1 to 7.

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