Methods and compositions for treating cancers or viral infections with PLA2g2d antagonists

Abstract

To provide methods and compositions for treating cancers or viral infections with PLA2G2D antagonists.SOLUTION: The present application provides methods of treating diseases (such as cancers or infectious diseases) comprising antagonists targeting the PLA2G2D signaling pathway (such as antagonists targeting PLA2G2D). The present application also provides PLA2G2D polypeptides that are non-naturally occurring. The application in one aspect provides a method of treating cancers or viral infections in an individual comprising administering to the individual an effective amount of an antagonist that targets the PLA2G2D signaling pathway.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 968,060 (filed January 30, 2020), which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Computer Readable Format (CRF) of Sequence Listing (Filename: 196882000140SEQLIST.TXT, Recording Date: January 29, 2021, Size: 34KB).

[0003] Field of the Application The present invention relates to methods and compositions for treating diseases or conditions involving antagonists that target the PLA2G2D signaling pathway. [Background technology]

[0004] background The mechanism by which immune system responds to infection or disease depends on the complex interaction between innate and adaptive immune elements.Unwanted suppression of immune response remains a major obstacle to promising treatments such as immunotherapy, which uses patient's own immune system to fight disease or infection.For example, the fundamental problem in the effort to treat patients with immunotherapy is that tumor-bearing state is associated with immunosuppressive mechanisms originating from both tumor and host's disturbed immune system, thereby preventing treatment from achieving ideal efficacy.

[0005] The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are incorporated by reference in their entirety for all purposes. Summary of the Invention [Means for solving the problem]

[0006] BRIEF SUMMARY OF THE APPLICATION The present application provides methods for treating a disease or condition, such as cancer or a viral infection.

[0007] In one aspect, the present application provides a method for treating cancer or viral infection in an individual, comprising administering to the individual an effective amount of an antagonist that targets the PLA2G2D signaling pathway. In some embodiments, the antagonist is an antagonist that targets PLA2G2D. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the antagonist reduces the enzymatic activity level of PLA2G2D. In some embodiments, the antagonist that targets the PLA2G2D signaling pathway blocks the catalytic site on PLA2G2D. In some embodiments, the antagonist targets the H67 catalytic site on human PLA2G2D according to SEQ ID NO: 1 or 5.

[0008] In some embodiments, the antagonist comprises an siRNA, miRNA, antisense RNA, or a gene editing system.

[0009] In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to an immune cell or an agent that inhibits the activity of PLA2G2D). In some embodiments, the immune cell is a T cell.

[0010] In some embodiments, the antagonist comprises an anti-PLA2G2D antibody. In some embodiments, the anti-PLA2G2D antibody is a monoclonal antibody. In some embodiments, the antagonist is a fusion protein or immunoconjugate comprising an anti-PLA2G2D antibody portion and a second portion. In some embodiments, the second portion comprises a cytokine.

[0011] In some embodiments, the antagonist comprises an inhibitory PLA2G2D polypeptide that blocks binding of PLA2G2D to immune cells.

[0012] In some embodiments, the inhibitory PLA2G2D polypeptide binds to immune cells with higher affinity than PLA2G2D. In some embodiments, the immune cells are T cells. In some embodiments, the inhibitory polypeptide further comprises a stabilization domain. In some embodiments, the stabilization domain is an Fc domain. In some embodiments, the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids. In some embodiments, the inhibitory PLA2G2D polypeptide has a mutation at a position corresponding to histidine 67 (H67) according to SEQ ID NO: 1 or 5. In some embodiments, the inhibitory PLA2G2D polypeptide comprises the amino acid sequence of SEQ ID NO: 3, 4, 7, or 8.

[0013] In some embodiments of any one of the above methods, the disease or symptom is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer has increased levels of PLA2G2D. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer, and melanoma. In some embodiments, the disease or symptom is a viral infection. In some embodiments, the site of infection has increased levels of PLA2G2D.

[0014] In some embodiments of any one of the above methods, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent. In some embodiments, the second agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises cells comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the cancerous tissue or site of infection.

[0015] In some embodiments according to any of the above methods, the antagonist is administered at a dose of about 0.001 μg / kg to about 100 mg / kg.

[0016] In some embodiments of any of the above methods, the individual experiences an increase in the number of immune cells in the cancerous tissue or site of infection after administration of the antagonist. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are activated T cells. In some embodiments, the number of immune cells in the cancerous tissue or site of infection increases by at least about 5% (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold) after administration of the antagonist.

[0017] In some embodiments of any of the above methods, immune cells in the cancer tissue or at the site of infection produce increased levels of cytokines after administration of the antagonist. In some embodiments, the cytokines are IFNγ and / or IL-2. In some embodiments, the cytokine levels are increased by at least about 5% (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold) after administration of the antagonist. [Brief explanation of the drawings]

[0018] [Figure 1A-1B] Figures 1A-1D show that PLA2G2D is highly differentially expressed in human (Figure 1A) lung adenocarcinoma, (Figure 1B) triple-negative breast cancer, (Figure 1C) hepatocellular carcinoma, and (Figure 1D) gastric adenocarcinoma. The relative expression and significance of PD-1, CTLA-4, and TIGIT are also shown. [Figure 1C-1D] Figures 1A-1D show that PLA2G2D is highly differentially expressed in human (Figure 1A) lung adenocarcinoma, (Figure 1B) triple-negative breast cancer, (Figure 1C) hepatocellular carcinoma, and (Figure 1D) gastric adenocarcinoma. The relative expression and significance of PD-1, CTLA-4, and TIGIT are also shown.

[0019] [Figure 2A] FIG. 2A shows that soluble human PLA2G2D-Fc protein dose-dependently suppresses PBMC-derived CD4+ and CD8+ T cell proliferation in the presence of anti-CD3 and anti-CD28 stimulation.

[0020] [Figure 2B] FIG. 2B shows a quantitative graph of the effect of human PLA2G2D-Fc protein on PBMC-derived CD4+ and CD8+ T cell proliferation.

[0021] [Figure 3A]Figures 3A-3C show that soluble PLA2G2D protein dose-dependently suppresses T cell proliferation in different PBMC donors in the presence of anti-CD3 and anti-CD28 stimulation. T cell CFSE proliferation analyzed by flow cytometry is shown on the left, and quantitative representation of the percentage of CD4+ and CD8+ T cell proliferation is shown on the right. [Figure 3B] Figures 3A-3C show that soluble PLA2G2D protein dose-dependently suppresses T cell proliferation in different PBMC donors in the presence of anti-CD3 and anti-CD28 stimulation. T cell CFSE proliferation analyzed by flow cytometry is shown on the left, and quantitative representation of the percentage of CD4+ and CD8+ T cell proliferation is shown on the right. [Figure 3C] Figures 3A-3C show that soluble PLA2G2D protein dose-dependently suppresses T cell proliferation in different PBMC donors in the presence of anti-CD3 and anti-CD28 stimulation. T cell CFSE proliferation analyzed by flow cytometry is shown on the left, and quantitative representation of the percentage of CD4+ and CD8+ T cell proliferation is shown on the right.

[0022] [Figure 4A] 4A-4B show that soluble PLA2G2D protein dose-dependently suppresses IFNγ and IL-2 levels, correlating with suppression of T cell proliferation across different donors. [Figure 4B] 4A-4B show that soluble PLA2G2D protein dose-dependently suppresses IFNγ and IL-2 levels, correlating with suppression of T cell proliferation across different donors.

[0023] [Figure 5] Figure 5 shows that immobilized PLA2G2D protein dose-dependently suppresses T cell proliferation in PBMC cultures in the presence of anti-CD3 and anti-CD28 stimulation. T cell CFSE proliferation analyzed by flow cytometry is shown on the left, and quantitative representation of the percentage of CD4+ and CD8+ T cell proliferation is shown on the right.

[0024] [Figure 6] Figure 6 shows that immobilized PLA2G2D protein dose-dependently suppresses proliferation of isolated T cell cultures in the presence of anti-CD3 and anti-CD28 stimulation. T cell CFSE proliferation analyzed by flow cytometry is shown on the left, and quantitative representation of the percentage of CD4+ and CD8+ T cell proliferation is shown on the right.

[0025] [Figure 7A] Figure 7A shows the desired structural and functional features of the human PLA2G2D protein (SEQ ID NO: 22), including its signal peptide (first 20 amino acids), calcium binding site, catalytic site, N-linked glycosylation site, and active site. An H67Q catalytic site mutant was generated to generate an enzyme-deficient PLA2G2D protein.

[0026] [Figure 7B] 7B-7C show that the H47Q-PLA2G2D catalytic mutant retains most of the immunosuppressive function on CD4+ (7B) and CD8+ (7C) T cells. [Figure 7C] 7B-7C show that the H47Q-PLA2G2D catalytic mutant retains most of the immunosuppressive function on CD4+ (7B) and CD8+ (7C) T cells.

[0027] [Figure 8] FIG. 8 shows that LY315920, a general inhibitor for various PLA2 small molecule inhibitors, does not rescue immunosuppression by PLA2G2D.

[0028] [Figure 9A] Figures 9A-9C show that human PLA2G2D-Fc preferentially binds to activated CD4+ and CD8+ T cells in different donor T cells compared to control-Fc proteins. PLA2G2D binds weakly to unstimulated T cells, but binding increases dramatically upon T cell stimulation. Figure 9C shows a quantitative representation of PLA2G2D-Fc binding to stimulated T cells. [Figure 9B] Figures 9A-9C show that human PLA2G2D-Fc preferentially binds to activated CD4+ and CD8+ T cells in different donor T cells compared to control-Fc proteins. PLA2G2D binds weakly to unstimulated T cells, but binding increases dramatically upon T cell stimulation. Figure 9C shows a quantitative representation of PLA2G2D-Fc binding to stimulated T cells. [Figure 9C] Figures 9A-9C show that human PLA2G2D-Fc preferentially binds to activated CD4+ and CD8+ T cells in different donor T cells compared to control-Fc proteins. PLA2G2D binds weakly to unstimulated T cells, but binding increases dramatically upon T cell stimulation. Figure 9C shows a quantitative representation of PLA2G2D-Fc binding to stimulated T cells.

[0029] [Figure 10A] FIG. 10A shows the predicted automated 3D structure based on the Swiss model.

[0030] [Figure 10B] Figure 10B shows the sequence homology to PLA2G2D of two family members from different PLA2 groups. The sequence numbers from top to bottom are SEQ ID NOS: 22-32.

[0031] [Figure 10C] Figure 10C shows the sequence homology of PLA2G2D from different species to human: human (SEQ ID NO: 33), mouse (SEQ ID NO: 34), rat (SEQ ID NO: 35), rhesus monkey (SEQ ID NO: 36) and chimpanzee (SEQ ID NO: 37).

[0032] [Figures 11A-11B] Figure 11A shows that the mean tumor volume of syngeneic subcutaneous MC38 colon adenocarcinoma tumors implanted in PLA2G2D knockout (KO) mice (n = 16) was significantly reduced compared to wild-type (WT) C57BL6 mice (n = 16). Figure 11B shows the tumor growth kinetics of individual animals in each group.

[0033] [Figures 11C-11D] Figure 11C shows that the mean tumor volume of syngeneic subcutaneous B16F10 melanoma tumors implanted in PLA2G2D knockout (KO) mice (n = 16) was significantly reduced compared to wild-type (WT) C57BL6 mice (n = 16). Figure 11D shows tumor growth kinetics for individual animals in each group.

[0034] [Figures 11E-11F] Figure 11E shows that the mean tumor volume of syngeneic subcutaneous E.G7-OVA T-cell lymphoma tumors implanted in PLA2G2D knockout (KO) mice (n = 16) was significantly reduced compared to wild-type (WT) C57BL6 mice (n = 16). Figure 11F shows tumor growth kinetics for individual animals in each group.

[0035] [Figure 12A] FIG. 12A shows that the mean fluorescence intensity (MFI) of PLA2G2D-Fc binding to activated T cells in PBMC cultures can be blocked by anti-PLA2G2D antibodies.

[0036] [Figures 12B-12C] 12B-12C show that PLA2G2D-Fc-mediated suppression of IL-2 and IFNγ levels in T cell-activated PBMC cultures can be reversed by the addition of a function-blocking anti-PLA2G2D antibody. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention In one aspect, the present application provides a method for treating a disease or condition (such as cancer or an infectious disease) comprising administering an antagonist that targets the PLA2G2D signaling pathway. In some embodiments, the antagonist comprises an agent that binds to PLA2G2D (such as an agent comprising an anti-PLA2G2D antibody moiety). In some embodiments, the antagonist comprises an inhibitory PLA2G2D polypeptide. In some embodiments, the antagonist comprises a nucleic acid agent that targets PLA2G2D (such as an siRNA or antisense RNA). In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D enzyme activity. In another aspect, the present application provides non-naturally occurring polypeptides, such as inhibitory PLA2G2D polypeptides, that can be used for treatment.

[0038] This application is based, at least in part, on the remarkable discovery that PLA2G2D plays an important role in suppressing T cells, a key player in the immune system. Specifically, PLA2G2D was found to be 56-fold more expressed in CD8+ high tumors than in CD8+ low tumors. As shown in more detail in the Examples, PLA2G2D can inhibit the proliferation, activation, and / or cytokine production of both CD4+ and CD8+ T cells both directly and indirectly (e.g., via cross-linking with antigen-presenting cells), which can result in a significant level of suppression of the immune response in diseases such as cancer, particularly in diseased tissues such as cancer tissues. It has also been found that the enzymatic activity of PLA2G2D only partially contributes to its role in suppressing the immune response, and that PLA2G2D can directly bind to T cells, particularly activated T cells. Without wishing to be bound by theory, it is believed that at least part of PLA2G2D's suppressive function on T cells is exerted by PLA2G2D binding to the cells. The present application presents for the first time a promising new method of treating diseases or conditions in which the immune response is suppressed, including, for example, cancer and infectious diseases (such as viral infectious diseases), using antagonists that target the PLA2G2D pathway (such as drugs comprising anti-PLA2G2D antibody moieties or inhibitory PLA2G2D polypeptides).

[0039] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Furthermore, any methods or materials similar or equivalent to those described herein can be used in the practice of this application. For purposes of this application, the following terms are defined:

[0040] It is understood that embodiments of the present application described herein with the term "comprising" include "consisting of" and / or "consisting essentially of."

[0041] As used herein, the term "wild type" is a term of the art understood by those skilled in the art and means the typical form of an organism, strain, gene or characteristic occurring in nature, as distinguished from mutant or variant forms.

[0042] As used herein, the term "variant" should be taken to mean a property whose expression has a pattern that deviates from that which occurs in nature.

[0043] The terms "non-naturally occurring" and "engineered" are used interchangeably and indicate the involvement of the hand of man. When referring to a nucleic acid molecule or polypeptide, this term means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is naturally associated and found in nature.

[0044] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide are sometimes collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0045] The terms "therapeutic agent," "therapeutic capable agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to an individual. Beneficial effects include enabling a diagnostic determination; ameliorating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or pathological condition.

[0046] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, chimeric antibodies, full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Antibodies and / or antibody fragments can be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camel antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0047] "Percent (%) amino acid sequence identity" or "homology" for the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004, each of which is incorporated by reference in its entirety for all purposes).

[0048] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences multiplied by 100, divided by the number of positions compared. For example, if 6 of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are performed when two sequences are aligned to maximize homology.

[0049] As used herein, the term "epitope" refers to the specific group of atoms or amino acids on an antigen to which an antibody or diabody binds. Two antibodies or antibody portions may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0050] The term "polypeptide" or "peptide" is used herein to encompass all types of natural and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including, but not limited to, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.).

[0051] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody (such as a diabody). In certain embodiments, specific binding determines the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules (e.g., cell surface receptors). For example, an antibody that specifically recognizes a target (which may be an epitope) is an antibody (such as a diabody) that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than its binding to other molecules. In some embodiments, the extent of binding of the antibody to unrelated molecules is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≤ 10 -12The antibody has a dissociation constant (KD) of M. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require, exclusive binding. The binding specificity of an antibody or antigen-binding domain can be experimentally determined by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE™, and peptide scan.

[0052] As used herein, "composition" or "compositions" includes and is applicable to the compositions of the present application. The present application also provides pharmaceutical compositions containing the components described herein.

[0053] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms attributable to the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the disease state, providing remission (partial or total) of the disease, reducing the dose of one or more other medications needed to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. The methods of the present application contemplate any one or more of these aspects of treatment. The benefit to the treated individual is statistically significant or at least perceptible to the patient or physician.

[0054] As used herein, the term "effective amount" refers to an amount of an agent or composition sufficient to treat a particular condition, disorder, symptom, or disease, e.g., ameliorate, alleviate, reduce, and / or delay one or more of its symptoms (e.g., clinical or subclinical symptoms). For therapeutic use, beneficial or desired results include, for example, reducing one or more symptoms (biochemical, histological, and / or behavioral) resulting from the disease, including its complications and intermediate pathological phenotypes manifested during the development of the disease; improving the quality of life of a person suffering from the disease; reducing the dose of other medications required to treat the disease; enhancing the effectiveness of another medication; slowing the progression of the disease; and / or extending patient survival. With respect to cancer, an effective amount includes an amount sufficient to shrink and / or reduce the growth rate of cancer tissue, or prevent or delay other undesirable cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay the onset of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. In the case of cancer, an effective amount of a drug or composition can: (i) reduce the number of tumor cells; (ii) reduce tumor size; (iii) inhibit, prevent, slow to some extent, and preferably stop tumor cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. It should be noted that when a combination of active ingredients is administered, an effective amount of the combination may or may not include the amount of each ingredient that would be effective when administered individually. The exact amount required will vary from individual to individual, depending on the individual's species, age, and general condition, the severity of the condition being treated, the specific drug(s) used, the mode of administration, etc.

[0055] As used herein, the term "co-administration" means that the first and second therapies in a combination therapy are administered at a time interval of about 15 minutes or less, e.g., about 10, 5, or 1 minute or less. When the first and second therapies are administered at the same time, the first and second therapies can be included in the same composition (e.g., a composition containing both the first and second therapies) or in separate compositions (e.g., the first therapy is included in one composition and the second therapy is included in another composition).

[0056] As used herein, the term "sequential administration" means that the first and second therapies in a combination therapy are administered at a time interval of more than about 15 minutes, for example, about 20, 30, 40, 50, 60 minutes, or more. Either the first or second therapy may be administered first. The first and second therapies are contained in separate compositions that may be contained in the same or different packages or kits.

[0057] As used herein, the term "co-administration" means that the administration of a first therapy and a second therapy in a combination therapy overlap with each other.

[0058] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable; e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically acceptable carrier or excipient preferably meets the required standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration or other state / federal government, or is listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, more particularly humans.

[0059] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water or aqueous solutions, such as saline and aqueous dextrose and glycerol solutions, are preferably used as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more of binders (for compressed pills), lubricants, encapsulating agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, which is incorporated by reference in its entirety for all purposes.

[0060] The term "tumor" refers to or describes the physiological condition in mammals that is typically characterized by uncontrolled cell growth and includes benign or malignant abnormal growths of tissue. The term "tumor" includes cancer.

[0061] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human. In preferred embodiments, the individual is a human.

[0062] Reference herein to "about" a value or parameter includes (and accounts for) a variation that is directed to the value or parameter itself. For example, a statement of "about X" includes the statement "X." In certain embodiments, a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The allowable variation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art.

[0063] Here, "about X to Y" is synonymous with "about X to about Y."

[0064] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the kind described herein and / or that will become apparent to those of ordinary skill in the art upon reading this disclosure. As will be apparent to one of ordinary skill in the art, the individual being evaluated, selected, and / or treated is an individual in need of such activity.

[0065] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al., eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al., eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al., eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al., eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al., eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al., eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are described, for example, in U.S. Patent No. 7,912,698 and U.S. Patent Application Publication Nos. 2011 / 0202322 and 2011 / 0307437, each of which is incorporated by reference in its entirety for all purposes.

[0066] The terms and expressions used are used as terms of description rather than of limitation, and the use of such terms and expressions does not exclude equivalents of the features shown and described or portions thereof, but rather allows various modifications within the scope of the claimed technology.

[0067] II. Treatment Methods In one aspect, the present application provides a method for treating a disease or condition (such as cancer or an infectious disease) in an individual, the method comprising administering to the individual an effective amount of an antagonist that targets the PLA2G2D signaling pathway. In some embodiments, the antagonist comprises an agent that binds to PLA2G2D (such as an agent comprising an anti-PLA2G2D antibody moiety). In some embodiments, the antagonist comprises an inhibitory PLA2G2D polypeptide. In some embodiments, the antagonist comprises a nucleic acid agent (such as an siRNA or antisense RNA) that targets PLA2G2D. In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D enzyme activity.

[0068] In some embodiments, methods are provided for treating cancer (such as solid tumors, colon cancer, melanoma, or T-cell lymphoma) in an individual, comprising administering to the individual an effective amount of an antagonist comprising an agent that inhibits PLA2G2D (e.g., an agent that blocks binding of PLA2G2D to immune cells or an agent that inhibits the activity of PLA2G2D). In some embodiments, the immune cell is a T cell (e.g., an activated T cell, such as an activated CD4+ T cell or CD8+ T cell). In some embodiments, the antagonist comprises an anti-PLA2G2D antibody. In some embodiments, the anti-PLA2G2D antibody is a monoclonal antibody. In some embodiments, the antagonist is a fusion protein or immunoconjugate comprising an anti-PLA2G2D antibody portion and a second portion, such as a second portion comprising a cytokine (e.g., a pro-inflammatory cytokine). In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer, and melanoma. In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent. In some embodiments, the second agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises cells comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently.In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0069] In some embodiments, methods of treating an infectious disease (e.g., a viral infectious disease) in an individual are provided, comprising administering to the individual an effective amount of an antagonist comprising an agent that inhibits PLA2G2D (e.g., an agent that blocks binding of PLA2G2D to immune cells or an agent that inhibits the activity of PLA2G2D). In some embodiments, the immune cell is a T cell (e.g., an activated T cell, e.g., an activated CD4+ T cell or CD8+ T cell). In some embodiments, the antagonist comprises an anti-PLA2G2D antibody. In some embodiments, the anti-PLA2G2D antibody is a monoclonal antibody. In some embodiments, the antagonist is a fusion protein or immunoconjugate comprising an anti-PLA2G2D antibody portion and a second portion. In some embodiments, the second portion comprises a cytokine (e.g., a pro-inflammatory cytokine). In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the site of infection has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent comprises an immunotherapy. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0070] In some embodiments, methods of treating cancer (e.g., solid tumor, colon cancer, melanoma, or T-cell lymphoma) in an individual are provided, comprising administering to the individual an effective amount of an antagonist comprising an inhibitory PLA2G2D polypeptide that inhibits PLA2G2D (e.g., an inhibitory peptide that blocks binding of PLA2G2D to immune cells). In some embodiments, the inhibitory PLA2G2D polypeptide binds to immune cells with higher affinity than PLA2G2D (e.g., wild-type PLA2G2D). In some embodiments, the immune cell is a T cell (e.g., an activated T cell, e.g., an activated CD4+ T cell or CD8+ T cell). In some embodiments, the inhibitory PLA2G2D polypeptide further comprises a stabilization domain. In some embodiments, the stabilization domain is an Fc domain. In some embodiments, the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids. In some embodiments, the inhibitory PLA2G2D polypeptide has a) a mutation at a position corresponding to histidine 67 (H67) according to SEQ ID NO: 1 or 5, or b) a mutation at a position corresponding to glycine 80 (G80) according to SEQ ID NO: 5. In some embodiments, the inhibitory PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12, or a variant thereof. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer, and melanoma. In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent. In some embodiments, the second agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor.In some embodiments, the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises cells comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0071] In some embodiments, methods are provided for treating an infectious disease (e.g., a viral infectious disease) in an individual, comprising administering to the individual an effective amount of an antagonist comprising an inhibitory PLA2G2D polypeptide that blocks binding of PLA2G2D to immune cells. In some embodiments, the inhibitory PLA2G2D polypeptide binds to immune cells with higher affinity than PLA2G2D (e.g., wild-type PLA2G2D). In some embodiments, the immune cell is a T cell (e.g., an activated T cell, e.g., an activated CD4+ T cell or CD8+ T cell). In some embodiments, the inhibitory PLA2G2D polypeptide further comprises a stabilization domain. In some embodiments, the stabilization domain is an Fc domain. In some embodiments, the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids. In some embodiments, the inhibitory PLA2G2D polypeptide has a) a mutation at a position corresponding to histidine 67 (H67) according to SEQ ID NO: 1 or 5, or b) a mutation at a position corresponding to glycine 80 (G80) according to SEQ ID NO: 5. In some embodiments, the inhibitory PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12, or a variant thereof. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the infected site has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent comprises immunotherapy. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0072] In some embodiments, a method for treating cancer in an individual (such as a solid tumor, colon cancer, melanoma, or T-cell lymphoma) is provided, comprising administering to the individual an effective amount of an antagonist comprising a nucleic acid agent that inhibits expression of PLA2G2D. In some embodiments, the nucleic acid agent comprises siRNA, miRNA, or antisense RNA. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer, and melanoma. In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent. In some embodiments, the second agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises cells comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0073] In some embodiments, a method of treating cancer in an individual (such as a solid tumor, colon cancer, melanoma, or T-cell lymphoma) is provided, comprising administering to the individual an effective amount of an antagonist comprising a nucleic acid agent that inhibits expression of PLA2G2D, wherein the individual has high T-cell infiltration in the cancer tissue. In some embodiments, high T-cell infiltration comprises a high number, percentage, or density of T cells (e.g., CD3 T cells, CD4 T cells, CD8 T cells, activated T cells, activated CD4 T cells, activated CD8 T cells) in the cancer tissue. In some embodiments, high T-cell infiltration exists when the number of T cells in the cancer is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% greater than the number of corresponding T cells in a reference tissue. In some embodiments, high T cell infiltration exists when the number of T cells in the cancer is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold the number of corresponding T cells in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the number of corresponding T cells in the reference tissue is the average number of corresponding T cells in the same tissue in a group of individuals (e.g., 10, 30, 50, 100 individuals) with the same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has cancer but has a less suppressed immune response in the cancer tissue, as indicated by biomarkers. Examples of biomarkers indicative of an immunosuppressive tumor microenvironment (TME) include: a) a high number, percentage, and / or density of M2 macrophages (e.g., CD68+CD163+ cells) in the tissue; and b) a high expression level of an immune checkpoint agent (e.g., PD-1 or PD-L1). Methods for assessing and evaluating these biomarkers are known. See, for example, Hensler et al., Journal for ImmunoTherapy of Cancer 2020;8:e000979; Chen et al., J Biomed Sci 26,78(2019); Teng et al., Cancer Res. 2015 Jun 1;75(11):2139-2145.In some embodiments, the nucleic acid agent comprises siRNA, miRNA, or antisense RNA. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer, and melanoma. In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent. In some embodiments, the second agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises cells comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0074] In some embodiments, provided herein is a method for treating cancer (such as solid tumor, colon cancer, melanoma or T-cell lymphoma) in an individual, comprising administering to the individual an effective amount of antagonist, comprising a nucleic acid drug that inhibits the expression of PLA2G2D, wherein the individual has a high expression level of PLA2G2D in cancer tissue.In some embodiments, when the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% higher than the expression level of PLA2G2D in reference tissue, the cancer tissue has a high expression level of PLA2G2D. In some embodiments, a cancer tissue has a high expression level of PLA2G2D if the expression level of PLA2G2D (e.g., as assessed by immunohistochemistry) is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, the reference tissue is a corresponding tissue in a healthy individual. In some embodiments, the expression level of PLA2G2D in the reference tissue is the average expression level of PLA2G2D in the same tissue in a group (e.g., 10, 30, 50, 100 individuals) of individuals with the same or similar cancer. In some embodiments, the reference tissue is a corresponding tissue in an individual who also has cancer but in whom the immune response in the cancer tissue is less suppressed, as indicated by biomarkers (e.g., high M2 macrophages or high expression of immune checkpoint agents such as PD-1 or PD-L1). In some embodiments, the nucleic acid agent comprises siRNA, miRNA, or antisense RNA. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer, and melanoma.In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent. In some embodiments, the second agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises cells comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0075] In some embodiments, a method for treating cancer (such as a solid tumor, colon cancer, melanoma, or T-cell lymphoma) in an individual is provided, comprising administering to the individual an effective amount of an antagonist comprising a nucleic acid drug that inhibits expression of PLA2G2D, wherein the individual has a) high T cell infiltration (e.g., CD3 T cells, e.g., CD4 T cells, e.g., CD8 T cells, e.g., activated CD3 or CD4 or CD8 T cells) within the cancer tissue, and / or b) high PLA2G2D expression within the cancer tissue.

[0076] In some embodiments, the methods described herein further include selecting an individual for treatment based on high T cell infiltration (e.g., high CD3 T cells, high CD8 T cells, high CD4 T cells, activated T cells, activated CD8 T cells, or activated CD4 T cells) within the cancer tissue. High T cell infiltration can be determined by a) assessing the number of T cells (e.g., CD3 T cells, CD4 T cells, CD8 T cells, activated T cells, activated CD4 T cells, activated CD8 T cells) within the tumor, and b) comparing that number to the number of corresponding T cells in a reference tissue. In some embodiments, high T cell infiltration is present when the number of T cells in the cancer is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% greater than the number of corresponding T cells in the reference tissue. In some embodiments, high T cell infiltration exists when the number of T cells in the cancer is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold the number of corresponding T cells in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the number of corresponding T cells in the reference tissue is the average number of corresponding T cells in the same tissue in a group of individuals (e.g., 10, 30, 50, 100 individuals) with the same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has cancer but has a less suppressed immune response in the cancer tissue, as indicated by biomarkers. Examples of biomarkers indicative of an immunosuppressive tumor microenvironment (TME) include: a) a high number, percentage, and / or density of M2 macrophages (e.g., CD68+CD163+ cells) in the tissue; and b) a high expression level of an immune checkpoint agent (e.g., PD-1 or PD-L1).

[0077] In some embodiments, the method further comprises selecting an individual for treatment based on a high expression level of PLA2G2D in cancer tissue. In some embodiments, a cancer tissue has a high expression level of PLA2G2D if the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, a cancer tissue has a high expression level of PLA2G2D if the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the expression level of PLA2G2D in a reference tissue is the average expression level of PLA2G2D in the same tissue in a group (e.g., 10, 30, 50, 100 individuals) of individuals with the same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has cancer but has a less suppressed immune response in the cancer tissue, as indicated by biomarkers (e.g., high M2 macrophages or high expression of immune checkpoint agents such as PD-1 or PD-L1).

[0078] In some embodiments, the methods described herein include selecting an individual for treatment, wherein the individual has a) high T cell infiltration (e.g., CD3 T cells, e.g., CD4 T cells, e.g., CD8 T cells, e.g., activated CD3 or CD4 or CD8 T cells) within the cancer tissue, and / or b) high PLA2G2D expression within the cancer tissue.

[0079] In some embodiments, methods are provided for treating a disease (such as a viral infectious disease) in an individual, comprising administering to the individual an effective amount of an antagonist, including a nucleic acid agent that inhibits expression of PLA2G2D. In some embodiments, the nucleic acid agent comprises siRNA, miRNA, or antisense RNA. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the site of infection has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent comprises immunotherapy. In some embodiments, the antagonist and the second agent are administered simultaneously or in parallel. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0080] In some embodiments, a method of treating cancer in an individual (such as a solid tumor, colon cancer, melanoma, or T-cell lymphoma) is provided, comprising administering to the individual an effective amount of an antagonist that reduces the enzymatic activity level of PLA2G2D. In some embodiments, the antagonist targeting the PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the antagonist comprises an agent that specifically inhibits the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: 1 or 5. In some embodiments, the antagonist targets the H67 catalytic site on human PLA2G2D according to SEQ ID NO: 1 or 5. In some embodiments, the agent interferes with calcium binding to PLA2G2D. In some embodiments, the agent blocks calcium binding to one or more residues among H47, G49, G51, and D68 according to SEQ ID NO: 1 or 5. In some embodiments, the antagonist comprises an agent that specifically reduces the enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D set forth in SEQ ID NO: 1 or 5 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the cancer tissue has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the cancer is an advanced or malignant tumor. In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer, and melanoma. In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent. In some embodiments, the second agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor.In some embodiments, the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent comprises cells comprising a chimeric antigen receptor that specifically binds to a tumor antigen. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0081] In some embodiments, a method for treating a disease (such as a viral infectious disease) in an individual is provided, comprising administering to the individual an effective amount of an antagonist that reduces the enzymatic activity level of PLA2G2D. In some embodiments, the PLA2G2D is human PLA2G2D. In some embodiments, the antagonist targeting the PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D. In some embodiments, the antagonist comprises an agent that specifically inhibits the catalytic His67-Asp68 Dyad of human PLA2G2D as set forth in SEQ ID NO: 1 or 5. In some embodiments, the antagonist targets the H67 catalytic site on human PLA2G2D according to SEQ ID NO: 1 or 5. In some embodiments, the agent interferes with calcium binding to PLA2G2D. In some embodiments, the agent blocks calcium binding to one or more residues among H47, G49, G51, and D68 according to SEQ ID NO: 1 or 5. In some embodiments, the antagonist comprises an agent that specifically reduces the enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D set forth in SEQ ID NO: 1 or 5 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the site of infection has an increased expression level of PLA2G2D compared to a reference tissue (e.g., a corresponding tissue in a healthy individual). In some embodiments, the method further comprises administering a second agent. In some embodiments, the second agent comprises immunotherapy. In some embodiments, the antagonist and the second agent are administered simultaneously or concurrently. In some embodiments, the antagonist and the second agent are administered sequentially. In some embodiments, the antagonist and / or the second agent are administered parenterally. In some embodiments, the antagonist is administered directly to the diseased tissue.

[0082] Administration of the antagonists described herein may also be useful for promoting local immune responses, promoting the proliferation and / or activation of immune cells (such as T cells), and promoting a favorable tumor microenvironment. In some embodiments, provided are methods for promoting local immune responses in cancer tissues of individuals with cancer (such as solid tumors), comprising administering any of the antagonists described herein. In some embodiments, provided are methods for promoting local immune responses at the site of infection in individuals with infection (such as viral infection), comprising administering any of the antagonists described herein.

[0083] In some embodiments, methods are provided for promoting T cell proliferation and / or activation in cancerous tissue of an individual with cancer (e.g., a solid tumor), comprising administering any of the antagonists described herein. In some embodiments, methods are provided for promoting T cell proliferation and / or activation at the site of infection in an individual with an infection (e.g., a viral infection), comprising administering any of the antagonists described herein. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells.

[0084] In some embodiments, there is provided a method of promoting a favorable tumor microenvironment in cancer tissue of an individual having cancer (e.g., a solid tumor), comprising administering any of the antagonists described herein. In some embodiments, there is provided a method of promoting a favorable tumor microenvironment at the site of infection in an individual having an infection (e.g., a viral infection), comprising administering any of the antagonists described herein. "Promoting a favorable tumor microenvironment" generally refers to or includes the conversion of tumor tissue that is resistant to cancer treatment (e.g., immunotherapy) into tumor tissue that is less resistant to cancer treatment.

[0085] Antagonists targeting the PLA2G2D signaling pathway The antagonist can be an antibody, polypeptide, peptide, polynucleotide, peptidomimetic, natural product, carbohydrate, aptamer, avimer, anticalin, spiegelmer, or small molecule that targets (i.e., inhibits or downregulates) the PLA2G2D signaling pathway. In some embodiments, the antagonist targets (i.e., inhibits or downregulates) PLA2G2D. Specific examples of potential agents are described below. In some embodiments, the antagonist is a fusion protein (e.g., a fusion protein including a half-life-extending domain (e.g., an Fc domain)).

[0086] PLA2G2D PLA2G2D (phospholipase A2 group IID, sPLA2-IID) is a secreted member of the phospholipase A2 family. Phospholipase A2 family members hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce lysophospholipids and free fatty acids. To date, 10 sPLA2 isoforms (IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII) have been identified in mammals. These isoforms, except for group III isoforms, share a highly conserved catalytic site, a Ca 2+ binding loop, and a common molecular weight of 14–19 kDa. Among these sPLA2 isoforms, sPLA2-IIA, sPLA2-IIC, sPLA2-IID, sPLA2-IIE, sPLA2-IIF, and sPLA2-V share the same chromosomal locus (1p34–p36), often referred to as group II subfamily sPLA2s. A biological feature of group II subfamily sPLA2 is that almost all isoforms, except for sPLA2-IIC (a pseudogene in humans), are associated with inflammatory and immune processes.

[0087] Human PLA2G2D is a basic protein (pI ∼8.7) with 14 cysteines at precisely conserved positions. Presumably due to its cationic nature, PLA2G2D binds to heparin or heparin sulfate on the cell surface in vitro when overexpressed in cultured cells.

[0088] In some embodiments, PLA2G2D comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, PLA2G2D comprises the amino acid sequence set forth in SEQ ID NO: 5 or 6.

[0089] Antagonists targeting PLA2G2D In some embodiments, the antagonist reduces the expression level of PLA2G2D. In some embodiments, the antagonist reduces the enzymatic activity level of PLA2G2D. In some embodiments, the anti-PLA2G2D antibody does not completely inhibit or block the catalytic activity of PLA2G2D (e.g., blocks less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the total catalytic activity). In some embodiments, the anti-PLA2G2D antibody does not inhibit or block the catalytic activity of PLA2G2D.

[0090] In some embodiments, the antagonist includes an agent that inhibits PLA2G2D (such as an agent that blocks the binding of PLA2G2D to immune cells or an agent that inhibits the activity of PLA2G2D) (such as T cells, activated T cells, activated CD4+ T cells, activated CD8+ T cells, etc.).

[0091] A. Drugs that bind to PLA2G2D In some embodiments, the antagonist is an agent that recognizes and specifically binds to PLA2G2D, hi some embodiments, the agent comprises an anti-PLA2G2D antibody moiety (such as an anti-PLA2G2D antibody).

[0092] In some embodiments, the anti-PLA2G2D antibody portion blocks or reduces the binding of PLA2G2D to immune cells. In some embodiments, the anti-PLA2G2D antibody portion reduces the binding of PLA2G2D to immune cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the binding of PLA2G2D to immune cells is independent of binding via heparin sulfate on the cell surface.

[0093] In some embodiments, the PLA2G2D recognized by the anti-PLA2G2D antibody is human PLA2G2D. In some embodiments, the human PLA2G2D comprises or has the amino acid sequence of SEQ ID NO: 1 or a natural variant of human PLA2G2D. In some embodiments, the natural variant of human PLA2G2D is derived from tumor tissue. In some embodiments, the natural variant of human PLA2G2D is derived from a viral infection site.

[0094] The predicted 3D structure of PLA2G2D based on the Swiss model is shown in Figure 10A. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, or 5) of Q65, H73, S80, H96, and R121 according to SEQ ID NO: 1. (Q65, H73, S80, H96, and R121 are sites for varying natural variants.) In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) of amino acids from R121 to C145 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids from V32 to A59 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids from T60 to T76. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids from Q77 to Y85 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids from G21 to Q31 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids from Y86 to W103 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids from C104 to R121 according to SEQ ID NO: 1. In some embodiments, the epitope is a discontinuous epitope.In some embodiments, the epitope is a continuous epitope.

[0095] The sequence homology of human PLA2G2D to two family members of different PLA2 groups was analyzed and is shown in Figure 10B. The sequence homology of human PLA2G2D to PLA2G2D of different species was analyzed and is shown in Figure 10C. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes one or more residues a) at a position that differs from the corresponding residue in two family members of the other PLA2 groups and / or b) at the same position as PLA2G2D in other species. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids at positions 22, 23, 25, 26, 27, or 31 according to SEQ ID NO:1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) of the amino acids at positions 36, 37, 38, 42, 43, 55, or 59 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) of the amino acids at positions 62, 65, 66, 72, 73, or 76 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) of the amino acids at positions 77, 80, 81, 83, 84, or 85 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) of the amino acids at positions 87, 89, 90, 92, 93, 94, 96, 98, 99, 100, 101, 102, or 103 according to SEQ ID NO:1.In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) of the amino acids at positions 105, 106, 107, 108, 110, 114, 115, 117, 119, or 120 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) of the amino acids at positions 123, 124, 127, 129, 130, 131, 132, 134, 135, 136, 137, 139, 141, 144, or 145 according to SEQ ID NO: 1. In some embodiments, the anti-PLA2G2D antibody portion binds to an epitope on PLA2G2D that includes any one or more (e.g., 1, 2, 3, 4, 5, or more) of the amino acids at positions 22, 26, 31, 36, 42, 43, 72, 73, 76, 77, 80, 81, 83, 85, 87, 89, 90, 92, 94, 96, 100, 101, 102, 103, 106, 110, 114, 115, 117, 120, 134, 135, 136, 141, or 144 according to SEQ ID NO: 1. In some embodiments, the epitope is a discontinuous epitope. In some embodiments, the epitope is a continuous epitope.

[0096] In some embodiments, the agent comprises an anti-PLA2G2D antibody. In some embodiments, the anti-PLA2G2D antibody is a polyclonal antibody. In some embodiments, the anti-PLA2G2D antibody is a monoclonal antibody.

[0097] In some embodiments, the anti-PLA2G2D antibody is an anti-human PLA2G2D antibody.

[0098] In some embodiments, the anti-PLA2G2D antibody is humanized or chimeric.

[0099] In some embodiments, the anti-PLA2G2D antibody is a full-length antibody or an immunoglobulin derivative. In some embodiments, the anti-PLA2G2D antibody is an antigen-binding fragment, such as a single chain Fv (scFv), Fab, Fab', F(ab'), Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv), V H The anti-PLA2G2D antibody is an antigen-binding fragment selected from the group consisting of H, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, and tetrabody. In some embodiments, the anti-PLA2G2D antibody is an scFv. In some embodiments, the anti-PLA2G2D antibody is Fab or Fab'. In some embodiments, the anti-PLA2G2D antibody is chimeric, human, partially humanized, fully humanized, or semi-synthetic. The antibody and / or antibody fragment may be derived from a mouse antibody, a rabbit antibody, a human antibody, a fully humanized antibody, a camel antibody variable domain and humanized version, a shark antibody variable domain and humanized version, and a camel antibody variable domain.

[0100] In some embodiments, the anti-PLA2G2D antibody comprises an Fc fragment (such as any of the Fc fragments described herein). In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is derived from human IgG. In some embodiments, the Fc fragment comprises the Fc region of human IgG1, IgG2, IgG3, IgG4, or a combination or hybrid IgG.

[0101] In some embodiments, the anti-PLA2G2D antibody does not completely inhibit or block the catalytic activity of PLA2G2D (e.g., blocks less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the total catalytic activity). In some embodiments, the anti-PLA2G2D antibody does not inhibit or block the catalytic activity of PLA2G2D.

[0102] In some embodiments, the anti-PLA2G2D antibody blocks PLA2G2D binding to T cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0103] In some embodiments, anti-PLA2G2D antibodies can restore T cell activation to at least about 50%, 60%, 70%, 80%, 90%, 95% or 100%. T cell activation can be indicated, for example, by its cytokine secretion level. Exemplary cytokines include IL-2 and IFN-γ.

[0104] Epitope mapping Determining whether an antibody moiety binds within an epitope region can be performed by methods known to those skilled in the art. As an example of such a mapping / characterization method, the epitope region of an anti-PLA2G2D antibody can be determined by epitope "footprinting" using chemical modification of exposed amines / carboxyls in the PLA2G2D protein. One specific example of such a footprinting technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry), in which hydrogen / deuterium exchange, binding, and back-exchange of receptor and ligand protein amide protons occurs, leaving the backbone amide groups involved in protein binding protected from back-exchange and therefore deuterated. At this point, the relevant regions can be identified by digestive proteolysis, rapid microbore high-performance liquid chromatography separation, and / or electrospray ionization mass spectrometry. See, for example, Ehring H, Analytical Biochemistry, Vol. 267(2) pp. 252-259 (1999); Engen, JR and Smith, DL (2001) Anal. Chem. 73, 256A-265A. Each is incorporated herein by reference in its entirety for all purposes. Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), which typically compares the positions of signals in two-dimensional NMR spectra of free antigen and antigen complexed with an antigen-binding peptide such as an antibody. The antigen is typically selectively isotopically labeled with 15N so that only signals corresponding to the antigen are observed in the NMR spectrum, and no signals from the antigen-binding peptide are observed. Antigen signals derived from amino acids involved in interaction with the antigen-binding peptide typically shift in position in the spectrum of the complex compared to the spectrum of the free antigen, allowing the amino acids involved in binding to be identified in this manner.See, e.g., Ernst Schering Res Found Workshop. 2004;(44):149-67; Huang et al., Journal of Molecular Biology, Vol. 281(1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 Jun;9(3):516-24, each of which is incorporated herein by reference in its entirety for all purposes.

[0105] Epitope mapping / characterization can also be performed using mass spectrometry. See, for example, Downard, J Mass Spectrom. 2000 Apr;35(4):493-503 and Kiselar and Downard, Anal Chem. 1999 May 1;71(9):1792-1801, each of which is incorporated herein by reference in its entirety for all purposes. Protease digestion techniques can also be useful in the context of epitope mapping and identification. Antigenic determinant-related regions / sequences can be determined by protease digestion, for example, trypsin at a ratio of approximately 1:50 for PLA2G2D or overnight digestion at pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. Peptides protected from trypsin cleavage by the anti-PLA2G2D binder can then be identified by comparing individualized samples to trypsin digestion with samples incubated with the antibody and then digested, such as with trypsin, thereby revealing the footprint of the binder. Other enzymes, such as chymotrypsin and pepsin, can also or alternatively be used in similar epitope characterization methods. Furthermore, enzyme digestion can provide a rapid method for analyzing whether potential antigenic determinant sequences lie within regions of a PLA2G2D polypeptide (such as the polypeptide set forth in SEQ ID NO: 1) that are not surface-exposed and therefore of little relevance in terms of immunogenicity / antigenicity.

[0106] Site-directed mutagenesis is another technique useful for elucidating binding epitopes. For example, in "alanine scanning," each residue in a protein segment is replaced with an alanine residue and the resulting effect on binding affinity is measured. If the mutation results in a significant decrease in binding affinity, it is most likely involved in binding. Monoclonal antibodies specific for structural epitopes (i.e., antibodies that do not bind to unfolded proteins) can be used to verify that alanine substitutions do not affect the overall folding of the protein. See, for example, Clackson and Wells, Science 1995;267:383-386; and Wells, Proc Natl Acad Sci USA 1996;93:1-6.

[0107] Electron microscopy can also be used for epitope "footprinting." For example, Wang et al., Nature 1992;355:275-278, used the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of Fab fragments on the capsid surface of native cowpea mosaic virus.

[0108] Other forms of " label-free " assays for epitope evaluation include surface plasmon resonance (SPR, BIACORE) and reflectance interference spectroscopy (RifS).See, for example, Fagerstam et al., Journal of Molecular Recognition 1990;3:208-14; Nice et al., J.Chroma-togr.1993;646:159-168; Leipert et al., Angew.Chem.Int.Ed.1998;37:3308-3311; Kroger et al., Biosensors and Bioelectronics 2002;17:937-944.

[0109] Immunoconjugates In some embodiments, the agents that bind to PLA2G2D described herein further comprise a second moiety. In some embodiments, the second moiety comprises a therapeutic agent. In some embodiments, the second moiety comprises a label. In some embodiments, the anti-PLA2G2D antibody moiety and the second moiety are linked via a linker (such as any of the linkers described in the "Linkers" section).

[0110] In some embodiments, the second agent is a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. In some embodiments, the cytotoxic agent is a growth inhibitory agent. In some embodiments, the cytotoxic agent is a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof). In some embodiments, the cytotoxic agent is a radioisotype (i.e., a radioconjugate).

[0111] Immunoconjugates allow for targeted delivery of, and in some embodiments, intracellular accumulation of, drug moieties to tissues (such as tumors), where systemic administration of unconjugated drugs can result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

[0112] Antibody-drug conjugates (ADCs) are targeted chemotherapy molecules that combine the properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (Teicher, BA (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, PJ and Senter PD (2008) The Cancer Jour:14(3):154-169; Chari, RV (2008) ACC. Chen. Res. 41.98-107).

[0113] In the context of cancer treatment, ADC compounds of the present application include those with anti-cancer activity. In some embodiments, the ADC compounds comprise an antibody conjugated, i.e., covalently attached, to a drug moiety. In some embodiments, the antibody is covalently attached to the drug moiety via a linker. In some embodiments, a second agent is linked to the anti-PLA2G2D antibody moiety via a linker (such as a linker described herein). In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable.

[0114] The antibody-drug conjugates (ADCs) of the present application selectively deliver an effective dose of drug to tumor tissue, thereby enabling greater selectivity, i.e., lower effective doses, to be achieved while increasing the therapeutic index ("therapeutic window"). The drug moiety of the antibody-drug conjugates (ADCs) may include any compound, moiety, or group that has a cytotoxic or cytostatic effect. The drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including, but not limited to, tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drug moieties include, but are not limited to, maytansinoids, dolastatins, auristatins, calicheamicins, pyrrolobenzodiazepines (PBDs), nemobicin and its derivatives, PNU-159682, anthracyclines, duocarmycins, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecin, elinafide, and their stereoisomers, isosteres, analogs, and derivatives that have cytotoxic activity.

[0115] The production of the immunoconjugates described herein can be found, for example, in US Pat. No. 9,562,099 and US Pat. No. 7,541,034, which are incorporated herein by reference in their entireties.

[0116] fusion proteins In some embodiments, the agent binds to PLA2G2D comprising a fusion protein comprising an anti-PLA2G2D antibody portion and a second portion.

[0117] In some embodiments, the second portion comprises an Fc fragment (such as any of the Fc fragments described herein). In some embodiments, the half-life extending moiety is an albumin binding moiety (e.g., an albumin binding antibody moiety).

[0118] In some embodiments, the second moiety comprises a cytokine, hi some embodiments, the cytokine is a pro-inflammatory cytokine (e.g., TNF-α, IL-1B, IL-6, or IL-10).

[0119] In some embodiments, the anti-PLA2G2D antibody portion and the second portion are linked via a linker (such as any of the linkers described in the "Linkers" section).

[0120] 1. Fc fragment The terms "Fc region," "Fc domain," or "Fc" refer to the C-terminal non-antigen-binding region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may be present or absent without affecting the structure or stability of the Fc region. Unless otherwise specified herein, the numbering of amino acid residues in an IgG or Fc region is in accordance with Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. According to the EU numbering system for antibodies, also called the EU index, as described in Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0121] In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof, hi some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

[0122] In some embodiments, the Fc fragment has reduced effector function compared to the corresponding wild-type Fc fragment (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% reduced effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC)).

[0123] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises an L234A mutation and / or an L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising an S228P, F234A, and / or L235A mutation. In some embodiments, the Fc fragment comprises an N297A mutation. In some embodiments, the Fc fragment comprises an N297G mutation.

[0124] 2. Linker In some embodiments, an anti-PLA2G2D immunoconjugate or fusion protein described herein comprises an anti-PLA2G2D antibody described herein fused to a second moiety via a linker.

[0125] The length, degree of flexibility, and / or other properties of the linker used in an anti-PLA2G2D immunoconjugate or fusion protein can have several effects on properties including, but not limited to, the affinity, specificity, or avidity of the anti-PLA2G2D and / or the affinity, specificity, or avidity for one or more specific antigens or epitopes present on the PLA2G2D. For example, a longer linker can be selected to ensure that two adjacent antibody moieties do not sterically interfere with each other. In some embodiments, the linker (such as a peptide linker) contains flexible residues (such as glycine and serine) to allow adjacent antibody moieties to move freely relative to each other. For example, a glycine-serine duplex can be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.

[0126] Other linker considerations include their effect on the physical or pharmacokinetic properties of the resulting anti-PLA2G2D immunoconjugate or fusion protein, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and programmed degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, ability to be incorporated into micelles or liposomes, etc.

[0127] Peptide Linker Any one or all of the linkers described herein can be peptide linkers. The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody can be used as a linker. See, for example, International Publication No. 1996 / 34103, the entire contents of which are incorporated by reference for all purposes. In some embodiments, the peptide linker comprises the amino acid sequence of CPPCP, a sequence found in the natural IgG1 hinge region.

[0128] The peptide linker may be of any suitable length, in some embodiments, the length of the peptide linker is any of about 1 aa to about 10 aa, about 1 aa to about 20 aa, about 1 aa to about 30 aa, about 5 aa to about 15 aa, about 10 aa to about 25 aa, about 5 aa to about 30 aa, about 10 aa to about 30 aa, about 30 aa to about 50 aa, about 50 aa to about 100 aa, or about 1 aa to about 100 aa.

[0129] The essential technical feature of such a peptide linker is that it does not contain polymerization activity. The characteristics of peptide linkers, including the absence of secondary structure promotion, are known in the art, and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80, each of which is incorporated by reference in its entirety for all purposes). A particularly preferred amino acid in the context of a "peptide linker" is Gly. Furthermore, peptide linkers that do not promote secondary structure are preferred. The linkage of molecules to each other can be provided, for example, by genetic engineering. Methods for preparing fused, operably linked antibody constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994; or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001, each of which is incorporated by reference in its entirety for all purposes).

[0130] In some embodiments, the peptide linker is a stable linker that is not cleaved by proteases, such as matrix metalloproteinases (MMPs).

[0131] In some embodiments, the peptide linker does not adopt a rigid three-dimensional structure, but rather tends to provide flexibility to the polypeptide (e.g., the first and / or second moieties), such as providing flexibility between the anti-PLA2G2D and the second moiety. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n (SEQ ID NO: 13), glycine-serine polymers (e.g., (GS) n (SEQ ID NO: 14), (GSGGS) n (SEQ ID NO: 15), (GGGGS) n (SEQ ID NO: 16), and (GGGS) n (SEQ ID NO: 17), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can function as neutral tethers between components. Glycine has access to much more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Those skilled in the art will recognize that the design of anti-PLA2G2D can include linkers that are wholly or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that provide a less flexible structure to provide the desired immunoconjugate or fusion protein structure.

[0132] Additionally, exemplary linkers also include (GGGGS) n(SEQ ID NO: 16), where n is an integer from 1 to 8, such as (GGGGS)3 (SEQ ID NO: 18; hereinafter referred to as "(G4S)3" or "GS3") or (GGGGS)6 (SEQ ID NO: 19; hereinafter referred to as "(G4S)6" or "GS6"). In some embodiments, the peptide linker is (GSTSGSGKPGSGEGS) n (SEQ ID NO: 20), where n is an integer of 1 to 3.

[0133] Natural linkers adopt various secondary conformations, such as helices, beta strands, coils / bends, and turns, to perform their functions. An α-helical linker can function as a rigid spacer to effectively separate protein domains, thereby reducing their unfavorable interactions. A non-helical linker with a Pro-rich sequence can increase the rigidity of the linker and reduce inter-domain interference. In some embodiments, the anti-PLA2G2D antibody portion and the second portion are linked to each other by an α-helical linker having the amino acid sequence A(EAAAK)4A (SEQ ID NO: 21).

[0134] Non-peptide linkers Any one or all of the linkers described herein can be achieved by any chemical reaction that bonds two molecules together, so long as the components or fragments retain their respective activities, such as binding to the target PLA2G2D or the function of the second moiety (e.g., binding to an FcR or cytokine receptor). This bond can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complexation. In some embodiments, the bond is covalent. Covalent bonding can be achieved either by direct condensation of existing side chains or by incorporation of an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful for coupling protein molecules, such as second moieties, to the anti-PLA2G2D antibodies of the present invention. For example, representative coupling agents include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of coupling agents known in the art, but rather is exemplary of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987), which are incorporated by reference in their entireties for all purposes).

[0135] Linkers that can be applied in the present application have been described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester), which is incorporated by reference in its entirety for all purposes). In some embodiments, non-peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat. #21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co., Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide) conjugated to EDC (Pierce Chem. Co., Cat. #2165-G). Chem. Co., Cat. #24510).

[0136] The above linkers contain components with different properties, resulting in PLA2G2D-binding agents (e.g., anti-PLA2G2D immunoconjugates or fusion proteins) with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. The SMPT linker also contains a sterically hindered disulfide bond, allowing for the formation of highly stable fusion proteins. Disulfide bonds are generally less stable than other linkages because they are cleaved in vitro, resulting in less available fusion protein. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. Carbodiimide coupling (e.g., EDC) when used in combination with sulfo-NHS forms esters that are more resistant to hydrolysis than carbodiimide coupling alone.

[0137] B. Inhibitory PLA2G2D Polypeptides or Variants Thereof In some embodiments, the methods described herein involve the use of inhibitory PLA2G2D polypeptides that completely or partially block the binding between PLA2G2D (e.g., wild-type PLA2G2D) and immune cells (e.g., block the binding between PLA2G2D and immune cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 95%). In one aspect, the present application provides novel, non-naturally occurring polypeptides, including inhibitory PLA2G2D polypeptides that block the binding of PLA2G2D to immune cells. In some embodiments, the inhibitory PLA2G2D polypeptide is a soluble polypeptide.

[0138] In some embodiments, the inhibitory PLA2G2D polypeptide is membrane-bound.In some embodiments, the membrane-bound inhibitory PLA2G2D polypeptide is bound to immune cells, but does not induce the PLA2G2D signal transduction pathway in immune cells.In some embodiments, the membrane-bound inhibitory PLA2G2D polypeptide is bound to immune cells, and attenuates the PLA2G2D signal transduction pathway in immune cells.In some embodiments, the membrane-bound inhibitory PLA2G2D polypeptide is introduced by gene editing system or mRNA delivery vehicle.

[0139] In some embodiments, the inhibitory PLA2G2D polypeptide comprises a naturally occurring PLA2G2D polypeptide. In some embodiments, the naturally occurring PLA2G2D polypeptide is derived from a human with an autoimmune disease or inflammatory disease (such as chronic obstructive pulmonary disease (COPD)). In some embodiments, the inhibitory PLA2G2D polypeptide has a mutation at a position corresponding to the polymorphism described in Takabatake et al. (Am J Respir Crit Care Med. 2005 Nov 1;172(9):1097-104) or Igarashi et al. (Respiration. 2009;78(3):312-21).

[0140] In some embodiments, the inhibitory PLA2G2D polypeptide comprises a mutation at a position corresponding to histidine 67 (H67) according to SEQ ID NO: 1 or 5. In some embodiments, the inhibitory PLA2G2D polypeptide comprises the amino acid sequence of SEQ ID NO: 3, 4, 7, or 8, or a variant thereof. In some embodiments, the variant has at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3, 4, 7, or 8.

[0141] In some embodiments, the inhibitory PLA2G2D polypeptide comprises a mutation at a position corresponding to G80 according to SEQ ID NO: 5. In some embodiments, the inhibitory PLA2G2D polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or 10, or a variant thereof. In some embodiments, the variant has at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 9 or 10.

[0142] In some embodiments, the inhibitory PLA2G2D polypeptide comprises a) a mutation at a position corresponding to histidine at position 67 (H67), and b) a mutation at a position corresponding to G80, according to SEQ ID NO: 5. In some embodiments, the inhibitory PLA2G2D polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12, or a variant thereof. In some embodiments, the variant has at least about 80% (e.g., about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 11 or 12.

[0143] In some embodiments, the inhibitory PLA2G2D polypeptide is selected from the group consisting of 22, 23, 25, 26, 27, 31, 36, 37, 38, 42, 43, 55, 59, 62, 65, 66, 72, 73, 76, 77, 80, 81, 83, 84, 85, 87, 89, 90, 92, 93, 94, 96, 98, 99, 100, 101, 102, 103, 105, 106, 107, 108, 110, 114 , 115, 117, 119, 120, 123, 124, 127, 129, 130, 131, 132, 134, 135, 136, 137, 139, 141, 144, and 145, where amino acid numbering is based on SEQ ID NO:1.

[0144] In some embodiments, the inhibitory PLA2G2D polypeptide further comprises at least one or more (e.g., about at least 10, 15, 20, 25, 30, or all) of the residues at positions 22, 26, 31, 36, 42, 43, 72, 73, 76, 77, 80, 81, 83, 85, 87, 89, 90, 92, 94, 96, 100, 101, 102, 103, 106, 110, 114, 115, 117, 120, 134, 135, 136, 141, or 144, where amino acid numbering is based on SEQ ID NO:1.

[0145] In some embodiments, the variants described herein are naturally occurring variants. In some embodiments, the variants do not contain non-conservative substitutions. In some embodiments, the variants contain only one or more conservative substitutions. In some embodiments, the one or more conservative substitutions comprise or consist of the substitutions shown in Table 1 below under the heading "Preferred Substitutions." [Table 1]

[0146] In some embodiments, the inhibitory PLA2G2D polypeptide binds to immune cells with higher affinity than wild-type PLA2G2D. In some embodiments, the inhibitory PLA2G2D polypeptide binds to immune cells with higher affinity than wild-type PLA2G2D. In some embodiments, the inhibitory PLA2G2D polypeptide binds to immune cells with higher affinity than wild-type PLA2G2D. D binds to immune cells at up to half, 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or 1 / 1000 of the original amount.

[0147] In some embodiments, the inhibitory PLA2G2D polypeptide further comprises a stabilization domain. The stabilization domain can be any domain that stabilizes the inhibitory PLA2G2D polypeptide (e.g., increases the half-life of the inhibitory PLA2G2D polypeptide in vivo). In some embodiments, the stabilization domain comprises an Fc fragment. Exemplary Fc fragments include those described in the "Fc Fragment" section.

[0148] In some embodiments, the inhibitory PLA2G2D polypeptide is about 50 to about 1000 amino acids in length, e.g., about 50 to 800, 50 to 500, 50 to 400, 50 to 300, or 50 to 200 amino acids in length. In some embodiments, the inhibitory polypeptide is about 50 to about 100 amino acids, about 100 to about 150 amino acids, or about 150 to about 200 amino acids in length.

[0149] C. Nucleic acid drugs targeting PLA2G2D In some embodiments, antagonists that target PLA2G2D include nucleic acid agents (eg, siRNA, shRNA, miRNA, or antisense RNA) that target PLA2G2D (such as human PLA2G2D).

[0150] In some embodiments, the antagonist comprises an siRNA or RNAi. In some embodiments, the antagonist comprises an antisense RNA. In some embodiments, the antagonist comprises a short hairpin ribonucleic acid (shRNA). In some embodiments, the antagonist comprises a microRNA (miRNA).

[0151] Those skilled in the art can select interfering RNA (RNAi) or siRNA specifically targeting PLA2G2D. The selected nucleic acid may be RNAi or siRNA, or a nucleic acid encoding such a product. As used herein, the term "RNAi" refers to double-stranded RNA (dsRNA), which can be used to mediate the degradation of specific mRNA and reduce or eliminate gene expression. As used herein, the terms "small interfering nucleic acid," "siNA," ​​"small interfering RNA," "siRNA," "small interfering nucleic acid molecule," "small interfering oligonucleotide molecule," or "chemically modified small interfering nucleic acid molecule" refer to any nucleic acid molecule directed against a gene. For example, siRNA can inhibit or downregulate gene expression or viral replication, for example, by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. See, for example, Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., International Publication No. WO 00 / 44895; Zernicka-Goetz et al., International Publication No. WO 01 / 36646; Fire, International Publication No. WO 99 / 32619; Plaetinck et al., International Publication No. WO 00 / 01846; Mello and Fire, International Publication No. WO 01 / 29058; Deschamps-Depaillette, International Publication No. WO 99 / 07409; and Li et al., International Publication No. WO 00 / 44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner See, for example, Reinhart et al., 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). The length of siRNA is not particularly limited as long as it does not exhibit toxicity. Examples of modified RNAi and siRNA include STEALTH™ forms (Invitrogen Corp., Carlsbad, Calif.), U.S. Patent Application Publication No. 2004 / 0014956 (Application No. 10 / 357,529) and U.S. Patent Application Publication No. 11 / 049,636, filed February 2, 2005), and other forms described below.

[0152] siRNA can be a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a part thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. siRNA can be assembled from two separate oligonucleotides, one of which is a sense strand and the other is an antisense strand, and the antisense strand and the sense strand are self-complementary (i.e., each strand comprises a nucleotide sequence complementary to the nucleotide sequence of the other strand; for example, when the antisense strand and the sense strand form a duplex or double-stranded structure, the double-stranded region is, for example, about 19 base pairs); the antisense strand comprises a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a part thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof. Alternatively, siRNA can be assembled from a single oligonucleotide, and the self-complementary sense region and the antisense region of siRNA are linked by a nucleic acid-based or non-nucleic acid-based linker(s). siRNA can be the polynucleotide of double stranded, asymmetric double stranded, hairpin or asymmetric hairpin secondary structure, with self-complementary sense region and antisense region, antisense region comprises the nucleotide sequence complementary to the nucleotide sequence in separate target nucleic acid molecule or a part thereof, and sense region has the nucleotide sequence corresponding to target nucleic acid sequence or a part thereof.siRNA can be the circular single-stranded polynucleotide of two or more loop structures and the stem comprises self-complementary sense region and antisense region, antisense region comprises the nucleotide sequence complementary to the nucleotide sequence in target nucleic acid molecule or a part thereof, and sense region has the nucleotide sequence corresponding to target nucleic acid sequence or a part thereof, and circular polynucleotide can be processed in vivo or in vitro to produce the active siRNA molecule that can mediate RNAi.siRNA can also comprise a single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., when such an siRNA molecule does not require the presence of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof within the siRNA molecule), and the single-stranded polynucleotide can further comprise a terminal phosphate group such as a 5'-phosphate (see, for example, Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or a 5',3'-diphosphate. In certain embodiments, the siRNA molecule of the present invention comprises separate sense and antisense sequences or regions, and the sense and antisense regions are covalently linked by a nucleotide or non-nucleotide linker molecule known in the art, or non-covalently linked by ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, and / or stacking interactions. In certain embodiments, the siRNA molecule of the present invention comprises a nucleotide sequence complementary to the nucleotide sequence of a target gene. In another embodiment, the siRNA molecule of the present invention interacts with the nucleotide sequence of the target gene in a manner that causes inhibition of expression of the target gene.

[0153] The double-stranded RNA portion of an siRNA in which two RNA strands are paired is not limited to a perfectly paired form and may contain unpaired portions due to mismatches (corresponding nucleotides are not complementary) or bulges (one strand lacks a corresponding complementary nucleotide). Unpaired portions may be present to the extent that they do not interfere with siRNA formation. As used herein, a "bulge" often contains one to two unpaired nucleotides, while the double-stranded RNA region of an siRNA in which two RNA strands are paired may contain one to seven bulges, or sometimes one to five bulges. Furthermore, as used herein, a "mismatch" is sometimes present in the double-stranded RNA region of an siRNA in which two RNA strands are paired, sometimes in the number of one to seven, and occasionally one to five. In a commonly used mismatch, one nucleotide is guanine and the other is uracil. Such mismatches may result from, but are not limited to, mutations in the DNA encoding the sense RNA from C to T, G to A, or a mixture thereof. Furthermore, in the present invention, the double-stranded RNA region of siRNA where the two RNA strands pair may contain both bulges and mismatches, the total number of which may sometimes be 1 to 7, and occasionally 1 to 5. The terminal structure of siRNA may be blunt or sticky (overhanging), as long as it allows siRNA to silence target gene expression due to its RNAi effect.

[0154] As used herein, siRNA molecule does not need to be limited to the molecule that contains only RNA, and also includes chemically modified nucleotide and non-nucleotide.In addition, as used herein, the term RNAi means to be equivalent to other terms that are used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition or epigenetic.For example, the siRNA molecule of the present invention can be used to epigenetically silence gene at both post-transcriptional level and pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by the siRNA molecules of the present invention may result from siRNA-mediated modification of chromatin structure to alter gene expression (see, e.g., Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

[0155] RNAi can be designed by methods known to those skilled in the art. In one example, siRNA can be designed by classifying RNAi sequences, for example, 1,000 sequences, based on functionality, with functional groups being classified as having knockdown activity of more than 85%, and non-functional groups being classified as having knockdown activity of less than 85%. The distribution of base composition is calculated for all RNAi target sequences for both functional and non-functional groups. Then, the ratio of the base distribution of functional and non-functional groups can be used to construct a score matrix for each position of the RNAi sequence. For a given target sequence, the bases at each position are scored, and then the logarithmic ratio of the multiplication of all positions is taken as the final score. Using this scoring system, a very strong correlation can be found between functional knockdown activity and the logarithmic ratio score. Once the target sequence is selected, it can be filtered through both the fast NCBI blast and slow Smith Waterman algorithm search against the Unigene database to identify gene-specific RNAi or siRNA. Sequences with at least one mismatch in the last 12 bases can be selected.

[0156] Antisense nucleic acids can be designed, prepared and / or utilized by those skilled in the art to inhibit nucleic acids encoding PLA2G2D. "Antisense" nucleic acid refers to a nucleotide sequence that is complementary to a "sense" nucleic acid encoding PLA2G2D or a fragment (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). Antisense nucleic acids can be complementary to the entire coding strand, or a portion thereof, or a substantially identical sequence thereof. In another embodiment, antisense nucleic acid molecules are antisense to the "non-coding region" of the coding strand of a nucleotide sequence.

[0157] Antisense nucleic acid can be complementary to the entire coding region of mRNA encoded by PLA2G2D nucleotide sequence, and in many cases, antisense nucleic acid is an oligonucleotide antisense to only a part of the coding region or non-coding region of mRNA.For example, antisense oligonucleotide can be complementary to the region surrounding the translation start site of mRNA, for example, the region between the -10 region and the +10 region of the nucleotide sequence of the target gene of interest.Antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more nucleotides in length.Antisense nucleic acid can be constructed by chemical synthesis or enzyme ligation reaction using standard procedures. For example, antisense nucleic acids (e.g., antisense oligonucleotides) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine-substituted nucleotides can be used). Antisense nucleic acids can also be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., the RNA transcribed from the inserted nucleic acid is in an antisense orientation to the target nucleic acid of interest, as further described in the following subsection).

[0158] When used in a subject, antisense nucleic acids are typically administered to the subject (e.g., by direct injection into a tissue site) or generated in situ to hybridize or bind to cellular mRNA and / or genomic DNA encoding the polypeptide, thereby inhibiting polypeptide expression, for example, by inhibiting transcription and / or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified to specifically bind to receptors or antigens expressed on the surface of selected cells, for example, by linking the antisense nucleic acid molecule to a peptide or antibody that binds to the cell surface receptor or antigen. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. Sufficient intracellular concentrations of antisense molecules can be achieved by incorporating a strong promoter, such as a pol II or pol III promoter, into the vector construct. Antisense nucleic acid molecules can sometimes be alpha-anomeric nucleic acid molecules. Alpha-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15:6625-6641 (1987)). Antisense nucleic acid molecules can also contain 2'-o-methylribonucleotides (Inoue et al., Nucleic Acids Res. 15:6131-6148 (1987)) or chimeric RNA-DNA analogs (Inoue et al., FEBS Lett. 215:327-330 (1987)). Antisense nucleic acids can be composed of DNA or PNA or any other nucleic acid derivatives mentioned above.

[0159] In some embodiments, the antisense nucleic acid is a ribozyme. Ribozymes with specificity for the Aid nucleotide sequence can contain one or more sequences complementary to such nucleotide sequences and sequences with known catalytic regions responsible for mRNA cleavage (e.g., U.S. Patent No. 5,093,246 or Haselhoff and Gerlach, Nature 334:585-591 (1988)). For example, derivatives of Tetrahymena L-19 IVS RNA can be used when the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in mRNA (e.g., Cech et al., U.S. Patent No. 4,987,071; and Cech et al., U.S. Patent No. 5,116,742). PLA2G2D mRNA sequences can also be used to select catalytic RNAs with specific ribonuclease activity from a pool of RNA molecules (e.g., Bartel & Szostak, Science 261:1411-1418 (1993)).

[0160] In some embodiments, the nucleic acid agent that targets PLA2G2D is a nucleic acid that can form a triple helix structure with an Aid nucleic acid. PLA2G2D expression can be inhibited by targeting nucleotide sequences complementary to the regulatory regions (e.g., promoters and / or enhancers) of the nucleotide sequences referred to herein or substantially identical sequences to form triple helix structures that prevent transcription of the gene in the target cell (see, e.g., Helene, Anticancer Drug Des. 6(6):569-84 (1991); Helene et al., Ann. NY Acad. Sci. 660:27-36 (1992); and Maher, Bioassays 14(12):807-15 (1992)). Triple helix formation can be enhanced by generating "switchback" nucleic acid molecules. Switchback molecules are synthesized in an alternating 5'-3', 3'-5' fashion so that they base pair first with one strand of the duplex, then with the other, eliminating the need for a significant stretch of purines or pyrimidines in one strand of the duplex.

[0161] D. Genome editing system targeting PLA2G2D In some embodiments, the PLA2G2D-targeting antagonist comprises a genome editing system targeting PLA2G2D. In some embodiments, the genome editing system comprises a DNA nuclease, such as an engineered (e.g., programmable or targetable) DNA nuclease, to induce genome editing of the PLA2G2D target DNA sequence. Any suitable DNA nuclease can be used, including, but not limited to, CRISPR-associated protein (Cas) nuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), meganuclease, other endonucleases or exonucleases, their mutants, fragments thereof, and combinations thereof. In some embodiments, the genome editing comprises modifying PLA2G2D so that the modified PLA2G2D no longer suppresses immune cells (e.g., T cells, e.g., activated T cells, e.g., activated CD4+ T cells, e.g., activated CD8+ T cells), or suppresses immune cells to a lesser extent than wild-type PLA2G2D. In some embodiments, the genome editing comprises modifying PLA2G2D such that the modified PLA2G2D no longer binds to immune cells (e.g., T cells, e.g., activated T cells, e.g., activated CD4+ T cells, e.g., activated CD8+ T cells) or binds to immune cells less than wild-type PLA2G2D. In some embodiments, the modification comprises inserting a transgene comprising a variant of PLA2G2D (such as any of the variants of PLA2G2D described herein). In some embodiments, the mutant PLA2G2D has a mutation at H67 based on SEQ ID NO:1. In some embodiments, the mutant PLA2G2D has a H67A mutation based on SEQ ID NO:1.

[0162] E. Drugs that inhibit PLA2G2D enzyme activity In some embodiments, the antagonist comprises an agent that inhibits PLA2G2D enzymatic activity (i.e., hydrolyzes fatty acids). In some embodiments, the antagonist comprises an agent that specifically inhibits the enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D set forth in SEQ ID NO: 1 or 5. In some embodiments, the antagonist comprises an agent that specifically reduces the enzymatic activity of the catalytic His67-Asp68 Dyad of human PLA2G2D set forth in SEQ ID NO: 1 or 5 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0163] In some embodiments, the agent interferes with calcium binding to PLA2G2D. In some embodiments, the agent blocks calcium binding to one or more of residues H47, G49, G51, and D68 according to SEQ ID NO: 1 or 5.

[0164] Disease or condition The methods described herein are applicable to diseases and conditions in which the body has a suppressed immune response, which at least in part contributes to less effective treatment of the disease. Exemplary diseases include cancer or infectious diseases (such as viral infectious diseases).

[0165] cancer In some embodiments, the disease or condition described herein is cancer.Cancers that can be treated using any of the methods described herein include any type of cancer.The types of cancers that can be treated with the agents described in this application include, but are not limited to, carcinomas, blastomas, sarcomas, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included.

[0166] In various embodiments, the cancer is an early stage cancer, a non-metastatic cancer, a primary cancer, an advanced cancer, a locally advanced cancer, a metastatic cancer, a cancer in remission, a recurrent cancer, a cancer in an adjuvant setting, a cancer in a neoadjuvant setting, or a cancer that is substantially resistant to treatment.

[0167] In some embodiments, the cancer is a solid tumor.

[0168] In some embodiments, the cancer is a liquid tumor.

[0169] In some embodiments, a cancer tissue has a high PLA2G2D expression level if the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, a cancer tissue has a high PLA2G2D expression level if the expression level of PLA2G2D (e.g., assessed by immunohistochemistry) is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold higher than the expression level of PLA2G2D in a reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the expression level of PLA2G2D in a reference tissue is the average expression level of PLA2G2D in the same tissue in a group (e.g., 10, 30, 50, 100 individuals) of individuals with the same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has cancer but has a less suppressed immune response in the cancer tissue, as indicated by biomarkers (e.g., high M2 macrophages or high expression of immune checkpoint agents such as PD-1 or PD-L1).

[0170] In some embodiments, the cancer tissue has high T cell infiltration (e.g., high CD3 T cells, high CD8 T cells, high CD4 T cells, activated T cells, activated CD8 T cells, or activated CD4 T cells) in the cancer tissue. In some embodiments, the cancer tissue has high T cell infiltration if the number of T cells in the cancer is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% higher than the number of corresponding T cells in the reference tissue. In some embodiments, high T cell infiltration exists if the number of T cells in the cancer is at least about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold the number of corresponding T cells in the reference tissue. In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the number of corresponding T cells in a reference tissue is the average number of corresponding T cells in the same tissue in a group (e.g., 10, 30, 50, 100 individuals) of individuals with the same or similar cancer. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has cancer but has a less suppressed immune response in the cancer tissue, as indicated by biomarkers (e.g., high M2 macrophages, high expression of immune checkpoint agents such as PD-1 or PD-L1, high expression levels of PLA2G2D).

[0171] In some embodiments, the cancer has a reduced number (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of immune cells (e.g., activated T cells, activated CD4+ T cells, or activated CD8+ T cells) in the cancer tissue compared to the reference tissue. In some embodiments, the cancer has a reduced number (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of activated immune cells (e.g., activated T cells, activated CD4+ T cells, or activated CD8+ T cells) in the cancer tissue compared to the reference tissue. In some embodiments, the cancer tissue has reduced levels (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of a cytokine (e.g., a pro-inflammatory cytokine, e.g., IFNγ or IL-2) compared to a reference tissue.

[0172] In some embodiments, the reference tissue is the corresponding tissue in a healthy individual. In some embodiments, the reference tissue is the corresponding tissue in an individual who also has cancer but has a less suppressed immune response in the cancer tissue. The suppression of the immune response can be assessed by measuring a) the number of immune cells (e.g., CD3+ cells); b) the proliferation / expansion state of the immune cells; c) the activation state of the immune cells; and / or d) cytokine levels. In some embodiments, any one or more of a)-d) are measured in the cancer tissue. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are CD8+ T cells (such as activated CD8+ T cells). In some embodiments, the immune cells are CD4+ T cells (such as activated CD4+ T cells).

[0173] Examples of cancers that can be treated by the methods of the present application include, but are not limited to, anal cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bladder carcinoma, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., astrocytoma, malignant glioma, medulloblastoma, and glioblastoma)), breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., uterine cancer), esophageal cancer, eye cancer (e.g., intraocular melanoma and retinoblastoma), gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hepatocellular carcinoma (HCC), and hepatocellular carcinoma (HCC). Alveolar (liver) cancer (e.g., hepatocarcinoma and hepatocytoma), liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), medulloblastoma, melanoma, mesothelioma, myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid cancer, cancer of the peritoneum, pituitary tumors, rectal cancer, kidney cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, and tuberous sclerosis. Further examples of cancers include The Merck Manual of Diagnosis and Therapy,19th Edition,§on Hematology and Oncology,published by Merck Sharp&Dohme Corp.,2011(ISBN 978-0-911910-19-3);The Merck Manual of Diagnosis and Therapy, 20th Edition, on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online version on the Merck Manuals internet website); and SEER Program Coding and Staging Manual 2016, each of which is incorporated by reference in its entirety for all purposes.

[0174] In some embodiments, the disease or condition is colon cancer.

[0175] In some embodiments, the disease or condition is melanoma.

[0176] In some embodiments, the disease or condition is T-cell lymphoma.

[0177] infectious disease In some embodiments, the disease or condition is an infectious disease, hi some embodiments, the infectious disease is a viral infectious disease.

[0178] In some embodiments, the viral infectious disease is characterized by infection with a hepatitis virus, human immunodeficiency virus (HIV), picornavirus, poliovirus, enterovirus, human coxsackievirus, influenza virus, rhinovirus, echovirus, rubella virus, encephalitis virus, rabies virus, herpes virus, papillomavirus, polyomavirus, RSV, adenovirus, yellow fever virus, dengue virus, parainfluenza virus, hemorrhagic virus, varicella virus, varicella-zoster virus, parainfluenza virus, reovirus, orbivirus, rotavirus, parvovirus, African swine fever virus, measles, coronavirus (e.g., SAR-CoV, MERS-CoV, 2019-nCoV), Ebola virus, mumps, or Norwalk virus. In some embodiments, the viral infectious disease is characterized by infection with an oncogenic virus such as CMV, EBV, HBV, KSHV, HPV, MCV, HTLV-1, HIV-1, or HCV.In some embodiments, the one or more genes encoding proteins involved in the development and / or progression of a viral infectious disease include RSV nucleocapsid, Pre-gen / Pre-C, Pre-S1, Pre-S2 / S, X, HBV conserved sequence, HIV Gag polyprotein (p55), HIV Pol polyprotein, HIV Gag-Pol precursor (p160), HIV matrix protein (MA, p17), HIV capsid protein (CA, p24), HIV spacer peptide 1 (SP1, p2), HIV nucleocapsid protein (NC, p9), HIV spacer peptide 2 (SP2, p1), HIV P6 protein, HIV reverse transcriptase (RT, p50), HIV RNase H (p15), HIV integrase (IN, p31), HIV protease (PR, p10), HIV These include, but are not limited to, genes encoding Env (gp160), gp120, gp41, HIV transactivator (Tat), HIV regulator of virion protein expression (Rev), HIV lentiviral protein R (Vpr), HIV Vif, HIV negative factor (Nef), HIV viral protein U (Vpu), human CCR5, miR-122, EBOV polymerase L, VP24, VP40, GP / sGP, VP30, VP35, NPC1, and TIM-1 (including mutants thereof).

[0179] In some embodiments, the viral infectious disease is characterized by infection with a coronavirus, hi some embodiments, the viral infectious disease is characterized by infection with an influenza virus.

[0180] An infection site refers to a tissue in the body where the virus appears in significant numbers and / or causes significant damage. In some embodiments, the infection site has an increased expression level of PLA2G2D compared to a reference tissue. In some embodiments, the PLA2G2D expression level at the infection site is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the reference tissue. In some embodiments, the PLA2G2D expression level at the infection site is increased by at least about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to the reference tissue.

[0181] In some embodiments, the site of infection has a reduced number (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of immune cells (e.g., activated T cells, activated CD4+ T cells, or activated CD8+ T cells) in the site of infection compared to a reference tissue. In some embodiments, the site of infection has a reduced number (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of activated immune cells (e.g., activated T cells, activated CD4+ T cells, or activated CD8+ T cells) in the site of infection compared to a reference tissue. In some embodiments, the site of infection has reduced levels (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of cytokines (e.g., pro-inflammatory cytokines, e.g., IFNγ or IL-2) compared to a reference tissue.

[0182] In some embodiments, the reference tissue is a corresponding tissue in a healthy individual. In some embodiments, the reference tissue is a corresponding tissue in an individual who also has a viral infection (e.g., the same type of viral infection) but has a less suppressed immune response at the site of infection. The suppression of the immune response can be assessed by measuring a) the number of immune cells; b) the proliferation / expansion state of the immune cells; c) the activation state of the immune cells; and / or d) cytokine levels. In some embodiments, circulating immune cells are assessed. In some embodiments, immune cells in diseased tissue are assessed. In some embodiments, immune cells in lymphoid tissue (e.g., lymph nodes) are assessed. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are CD8+ T cells (e.g., activated CD8+ T cells). In some embodiments, the immune cells are CD4+ T cells (e.g., activated CD4+ T cells).

[0183] individual In some embodiments, the individual is a mammal (such as a human).

[0184] In some embodiments, individuals are selected for treatment based on high expression of PLA2G2D in diseased tissue. In some embodiments, the tissue is cancerous tissue. In some embodiments, the tissue is an infection site.

[0185] In some embodiments, the PLA2G2D expression level at the site of infection is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% compared to the reference tissue, hi some embodiments, the PLA2G2D expression level at the site of infection is increased by at least about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to the reference tissue.

[0186] In some embodiments, the individual is selected for treatment based on an indication of a suppressed immune response. In some embodiments, the individual has a suppressed immune response in diseased tissue. In some embodiments, the tissue is cancerous tissue. In some embodiments, the tissue is a site of infection.

[0187] As described above, suppression of the immune response can be assessed by measuring a) the number of immune cells; b) the proliferation / expansion state of immune cells; c) the activation state of immune cells; and / or d) cytokine levels. In some embodiments, circulating immune cells are assessed. In some embodiments, immune cells in diseased tissue are assessed. In some embodiments, immune cells in lymphoid tissue (such as lymph nodes) are assessed. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are CD8+ T cells (such as activated CD8+ T cells). In some embodiments, the immune cells are CD4+ T cells (such as activated CD4+ T cells).

[0188] In some embodiments, the individual has a reduced number (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of immune cells (e.g., activated T cells, activated CD4+ T cells, or activated CD8+ T cells) in tissue (e.g., cancerous tissue or site of infection) compared to a reference tissue. In some embodiments, the individual has a reduced number (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of activated immune cells (e.g., activated T cells, activated CD4+ T cells, or activated CD8+ T cells) in tissue (e.g., cancerous tissue or site of infection) compared to a reference tissue. In some embodiments, the individual has a reduced level (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction) of a cytokine (e.g., a pro-inflammatory cytokine, e.g., IFNγ or IL-2) in a tissue (e.g., a cancerous tissue or a site of infection) compared to a reference tissue.

[0189] In some embodiments, the reference tissue is the corresponding tissue in a healthy individual, hi some embodiments, the reference tissue is the corresponding tissue in an individual who also has the same or a similar disease or condition but has a less suppressed immune response in the cancerous tissue.

[0190] In some embodiments, the individual has a compromised immune system.

[0191] In some embodiments, the individual is at least about 60, 65, 70, 75, 80, 85, or 90 years old.

[0192] In some embodiments, the individual has had at least one previous treatment. In some embodiments, the previous treatment includes radiation therapy, chemotherapy, and / or immunotherapy. In some embodiments, the individual is resistant, refractory, or relapsed to the previous treatment.

[0193] Combination therapy The present application also provides a method for administering an effective amount of an antagonist that targets the PLA2G2D signaling pathway to an individual to treat a disease or condition (such as cancer or an infectious disease), and further comprising administering a second drug or treatment. In some embodiments, the second drug or treatment is a standard or commonly used drug or treatment for treating the disease or condition.

[0194] In some embodiments, the antagonist is administered simultaneously with a second agent or therapy, hi some embodiments, the antagonist is administered together with a second agent or therapy, hi some embodiments, the antagonist is administered sequentially with a second agent or therapy.

[0195] Exemplary Combination Therapies for Cancer In some embodiments, the second agent or treatment comprises a chemotherapeutic agent. In some embodiments, the second agent or treatment comprises surgery. In some embodiments, the second agent or treatment comprises radiation therapy. In some embodiments, the second agent or treatment comprises immunotherapy. In some embodiments, the second agent or treatment comprises cell therapy (e.g., cell therapy comprising immune cells (e.g., CAR T cells)). In some embodiments, the second agent or treatment comprises an angiogenesis inhibitor.

[0196] In some embodiments, the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent.

[0197] In some embodiments, the second agent is a chemotherapeutic agent. In some embodiments, the second agent is an antimetabolite. In some embodiments, the antimetabolite is 5-FU.

[0198] In some embodiments, the second agent is an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. In some embodiments, the second agent is an anti-PD-1 antibody or fragment thereof. In some embodiments, the second agent is an anti-PD-L1 antibody or fragment thereof.

[0199] In some embodiments, the second agent comprises a cell (e.g., an immune cell, e.g., a T cell) that comprises a chimeric antigen receptor that specifically binds to a tumor antigen.

[0200] Exemplary combination therapies for infectious diseases (such as viral infectious diseases). In some embodiments, the second agent or treatment comprises a nucleotide analog.

[0201] In some embodiments, the second agent or treatment comprises a nucleoside analog.

[0202] In some embodiments, the second agent or treatment comprises a protease inhibitor. In some embodiments, the second agent or treatment comprises lopinavir. In some embodiments, the second agent or treatment comprises ritonavir.

[0203] In some embodiments, the second agent or treatment comprises a neuraminidase inhibitor. In some embodiments, the second agent or treatment comprises zanamivir. In some embodiments, the second agent or treatment comprises oseltamivir. In some embodiments, the second agent or treatment comprises peramivir.

[0204] In some embodiments, the second agent or treatment comprises a Cap-dependent endonuclease inhibitor. In some embodiments, the second agent or treatment comprises baloxavir.

[0205] In some embodiments, the second agent or treatment comprises a sialidase.

[0206] The second agent and the antagonist can be administered sequentially, together, or simultaneously. In some embodiments, the second agent is administered before the antagonist. In some embodiments, the second agent is administered after the antagonist.

[0207] Dosage regimen and route of administration The dose of the antagonist described herein and, in some embodiments, the second agent administered to an individual (such as a human) can vary depending on the particular composition, the method of administration, and the particular type and stage of the disease or condition being treated. The amount should be sufficient to bring about a desired response, such as a therapeutic response to the disease or condition. In some embodiments, the amount of the antagonist and / or the second agent is a therapeutically effective amount.

[0208] In some embodiments, the amount of antagonist is sufficient to reduce the suppression of an immune response in an individual. The presence or absence of a reduced suppression of an immune response and the degree of reduced suppression can be indicated by any of the following:

[0209] In some embodiments, the amount of antagonist is sufficient to increase the number of immune cells (e.g., T cells, e.g., CD4+ and / or CD8+ T cells) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of the antagonist. In some embodiments, circulating immune cells are assessed. In some embodiments, immune cells in diseased tissue are assessed. In some embodiments, immune cells in lymphoid tissue (e.g., lymph nodes) are assessed. In some embodiments, the immune cells comprise myeloid cells (e.g., dendritic cells). In some embodiments, the immune cells comprise NK cells. In some embodiments, the immune cells comprise T cells, such as CD4+ and / or CD8+ T cells. In some embodiments, the number of immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days after administration of the antagonist.

[0210] In some embodiments, the amount of antagonist is sufficient to increase the number of activated immune cells (e.g., activated CD4+ and / or CD8+ T cells) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of the antagonist. In some embodiments, activated immune cells in the circulation are assessed. In some embodiments, activated immune cells in diseased tissue are assessed. In some embodiments, activated immune cells in lymphoid tissue (e.g., lymph nodes) are assessed. In some embodiments, the immune cells comprise myeloid cells (e.g., dendritic cells). In some embodiments, the immune cells comprise NK cells. In some embodiments, the immune cells comprise T cells, such as CD4+ and / or CD8+ T cells. In some embodiments, the number of activated immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days after administration of the antagonist.

[0211] In some embodiments, the amount of antagonist is sufficient to increase the proliferation of immune cells or activated immune cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of the antagonist. In some embodiments, circulating immune cells or activated immune cells are assessed. In some embodiments, immune cells or activated immune cells in diseased tissue are assessed. In some embodiments, immune cells or activated immune cells in lymphoid tissue (such as lymph nodes) are assessed. In some embodiments, the immune cells comprise myeloid cells (such as dendritic cells). In some embodiments, the immune cells comprise NK cells. In some embodiments, the immune cells comprise T cells, such as CD4+ and / or CD8+ T cells. In some embodiments, the proliferation of immune cells or activated immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days after administration of the antagonist.

[0212] In some embodiments, the amount of antagonist is sufficient to increase cytokine levels (e.g., pro-inflammatory cytokines, e.g., IFNγ or IL-2) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of the antagonist. In some embodiments, cytokine levels in diseased tissue are assessed. In some embodiments, cytokine levels are assessed about 1, 2, 3, 4, 5, 6, or 7 days after administration of the antagonist.

[0213] In some embodiments, the amount of antagonist is sufficient to reduce inhibitory immune cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% after administration. In some embodiments, the inhibitory immune cells comprise regulatory T cells. In some embodiments, the inhibitory immune cells comprise myeloid-derived suppressor cells. In some embodiments, circulating inhibitory immune cells are assessed. In some embodiments, inhibitory immune cells are assessed in diseased tissue. In some embodiments, inhibitory immune cells are assessed in lymphoid tissue (such as lymph nodes). In some embodiments, the number of inhibitory immune cells is assessed about 1, 2, 3, 4, 5, 6, or 7 days after administration of the antagonist.

[0214] In some embodiments, the amount of antagonist is sufficient to increase the humoral immune response in an individual by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after administration of the antagonist. The humoral immune response can be assessed by measuring antibodies (such as IgG antibodies) targeting disease-associated antigens or plasmablasts producing such antibodies in the circulation. In some embodiments, the humoral immune response is assessed about 7 to 28 days (e.g., about 7 to 14 days) after administration of the antagonist.

[0215] In some embodiments, the amount of antagonist is sufficient to result in a reduction in tumor size, decrease the number of cancer cells, or decrease the growth rate of a tumor by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same individual before treatment, or compared to the corresponding activity in another individual not receiving treatment.

[0216] In some embodiments, the antagonist is administered at a dose of about 0.001 μg / kg to about 100 mg / kg of total body weight, e.g., about 0.005 μg / kg to about 50 mg / kg, about 0.01 μg / kg to about 10 mg / kg, or about 0.01 μg / kg to about 1 mg / kg.

[0217] In some embodiments of any one of the methods described herein, the antagonist and / or second agent composition is administered intravenously, intraarterially, intraperitoneally, intravesically, subcutaneously, intrathecally, intrapulmonary, intramuscularly, intratracheally, intraocularly, topically, transdermally, orally, or by inhalation. In some embodiments, the antagonist and / or second agent is administered intravenously.

[0218] In some embodiments, the antagonist is administered directly to the diseased tissue.

[0219] III. Diagnostic and Prognostic Methods Also provided herein are methods of diagnosing or prognosing an individual, comprising determining the individual's suitability for a treatment described in Section II or a different treatment, including immunotherapy, and determining the individual's likelihood of responsiveness to a method described in Section II or a different treatment.

[0220] In some embodiments, provided herein is a method for determining the suitability of an individual for treatment, comprising measuring the level of PLA2G2D expression in the diseased tissue of the individual.In some embodiments, the individual has cancer, and the tissue is tumor tissue.In some embodiments, the individual has infectious disease (such as viral infectious disease), and the tissue is the site of infection.

[0221] In some embodiments, a method of prognosis in an individual with cancer (such as a solid tumor) is provided, comprising measuring PLA2G2D expression levels in a tumor sample in vitro or in vivo, wherein a higher PLA2G2D expression level compared to a reference level indicates a higher likelihood of not responding or responding poorly to treatment (such as immunotherapy). In some embodiments, the reference level is the level of PLA2G2D expression (such as average PLA2G2D expression) in a non-tumor sample in the individual or in a corresponding tissue in a different individual (or group) without cancer.

[0222] In some embodiments, a method of prognosis in an individual with an infectious disease (such as a viral infectious disease) is provided, comprising measuring PLA2G2D expression levels in a sample from the site of infection in vitro or in vivo, wherein a higher PLA2G2D expression level compared to a reference level indicates a higher likelihood of no or poor response to treatment (such as immunotherapy). In some embodiments, the reference level is the level of PLA2G2D expression (such as average PLA2G2D expression) in a non-infected site sample in the individual or in a corresponding tissue in a different individual (or group) without the infectious disease.

[0223] In some embodiments, the therapy comprises cell therapy (such as CAR-T cell therapy).

[0224] In some embodiments, treating further comprises assessing the suppression of an immune response in the individual. Exemplary methods for assessing the suppression of an immune response are discussed above.

[0225] IV. Methods of Preparation, Nucleic Acids, Vectors, Host Cells and Culture Media In some embodiments, methods for preparing antagonists (e.g., siRNAs targeting PLA2G2D described herein, anti-PLA2G2D agents, inhibitory PLA2G2D polypeptides, agents that inhibit PLA2G2D enzyme activity), and compositions comprising agents, nucleic acid constructs, vectors, host cells, or culture media produced during preparation of the agents are provided.

[0226] Polypeptide Expression and Production Agents targeting PLA2G2D described herein (e.g., polypeptides comprising anti-PLA2G2D antibody moieties described in Chapter II) and agents targeting inhibitory PLA2G2D polypeptides can be prepared using any method known in the art, including those described below.

[0227] Polypeptides containing anti-PLA2G2D antibody moieties Monoclonal antibodies targeting PLA2G2D can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may exist in small amounts.Therefore, the modifier "monoclonal" indicates the characteristic of the antibody that it is not a mixture of individual antibodies.For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be produced by recombinant DNA methods (U.S. Patent No. 4,816,567).In the hybridoma method, mice or other suitable host animals, such as hamsters or llamas, are immunized as described above to induce lymphocytes that produce or can produce antibodies that specifically bind to the protein used for immunization.Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986).

[0228] The immunizing agent typically includes an antigen protein or a fusion variant thereof. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103, is incorporated by reference in its entirety for all purposes.

[0229] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells thus prepared are preferably seeded and grown in an appropriate culture medium containing one or more substances that inhibit the growth or survival of unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine, which inhibit the growth of HGPRT-deficient cells (HAT medium).

[0230] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, murine myeloma lines, such as MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA), and SP-2 cells (and their derivatives, such as X63-Ag8-653) (available from the American Type Culture Collection, Manassas, Va., USA), are preferred. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each of which is incorporated by reference in its entirety for all purposes).

[0231] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0232] The culture medium in which hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies against the desired antigen.Preferably, the binding affinity and specificity of monoclonal antibodies can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).Such techniques and assays are known in the art.For example, binding affinity can be determined by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0233] After hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, hybridoma cells can be grown in vivo as tumors in mammals.

[0234] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0235] Monoclonal antibodies can also be produced as described above by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells that do not produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to synthesize monoclonal antibodies in such recombinant host cells. Review articles on the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

[0236] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), each of which is incorporated by reference in its entirety for all purposes, describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications have described the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and combinatorial infection and in vivo recombination (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)) as strategies for constructing very large phage libraries. Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0237] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such a non-immunoglobulin polypeptide is substituted for the constant domains of an antibody or for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0238] The monoclonal antibodies described herein may be monovalent, and their preparation is well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at some point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residues may be substituted with other amino acid residues or deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments, particularly Fab fragments, can be accomplished using routine techniques known in the art.

[0239] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0240] Nucleic acid molecules encoding polypeptides In some embodiments, a polynucleotide encoding any one of the antibodies (such as an anti-PLA2G2D antibody) or polypeptides (such as an inhibitory PLA2G2D polypeptide) described herein is provided. In some embodiments, a polynucleotide prepared using any one of the methods as described herein is provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding a heavy chain or a light chain of an antibody (e.g., an anti-PLA2G2D antibody). In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding an inhibitory PLA2G2D polypeptide. In some embodiments, a nucleic acid molecule comprises both a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain of an antibody (e.g., an anti-PLA2G2D antibody). In some embodiments, a first nucleic acid molecule comprises a first polynucleotide encoding the heavy chain and a second nucleic acid molecule comprises a second polynucleotide encoding the light chain. In some embodiments, a nucleic acid molecule encoding an scFv (e.g., an anti-PLA2G2D scFv) is provided. In some embodiments, a nucleic acid molecule comprises a polynucleotide encoding an inhibitory PLA2G2D polypeptide.

[0241] In some such embodiments, the heavy and light chains of an antibody (e.g., an anti-PLA2G2D antibody) are expressed as two separate polypeptides from one nucleic acid molecule or from two separate nucleic acid molecules. In some embodiments, for example, when the antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both the heavy and light chains linked together.

[0242] In some embodiments, a polynucleotide encoding a heavy or light chain of an antibody (e.g., an anti-PLA2G2D antibody) comprises a nucleotide sequence encoding a leader sequence located at the N-terminus of the heavy or light chain upon translation. As noted above, the leader sequence may be the native heavy or light chain leader sequence or may be another heterologous leader sequence.

[0243] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the RNA is mRNA.

[0244] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0245] nucleic acid construct In some embodiments, a nucleic acid construct is provided that includes any one of the polynucleotides described herein. In some embodiments, a nucleic acid construct is provided that is prepared using any of the methods described herein.

[0246] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the polynucleotide, hi some embodiments, the polynucleotide corresponds to a gene and the promoter is the wild-type promoter of the gene.

[0247] vector The terms "vector," "cloning vector," and "expression vector" refer to a vehicle capable of introducing DNA or RNA sequences (e.g., foreign genes) into a host cell in order to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequences. Vectors include plasmids, synthetic RNA and DNA molecules, phages, viruses, and the like. In certain embodiments, the vector is a viral vector, for example, but not limited to, an adenovirus, adeno-associated, alphavirus, herpes, lentivirus, retrovirus, or vaccinia virus.

[0248] In some embodiments, vectors are provided comprising any polynucleotide encoding the heavy and / or light chain of any one of the antibodies (e.g., anti-PLA2G2D antibodies) described herein. In some embodiments, vectors are provided comprising any polynucleotide encoding a polypeptide (e.g., an inhibitory PLA2G2D polypeptide) described herein. In some embodiments, vectors are provided comprising any nucleic acid construct described herein. In some embodiments, vectors prepared using any method described herein are provided. Also provided are vectors comprising a polynucleotide encoding any polypeptide (such as an anti-PLA2G2D antibody or an inhibitory PLA2G2D polypeptide). Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, the vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy and light chains are expressed from the vector as two separate polypeptides.

[0249] In some embodiments, a first vector comprises a polynucleotide encoding a heavy chain of an antibody (e.g., an anti-PLA2G2D antibody), and a second vector comprises a polynucleotide encoding a light chain of an antibody (e.g., an anti-PLA2G2D antibody). In some embodiments, the first vector and the second vector are transfected into host cells in similar amounts (e.g., similar molar amounts or similar masses). In some embodiments, a molar or mass ratio of 5:1 to 1:5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of 1:1 to 1:5 is used for the vector encoding the heavy chain and the vector encoding the light chain. In some embodiments, a mass ratio of 1:2 is used for the vector encoding the heavy chain and the vector encoding the light chain.

[0250] In some embodiments, a vector is selected that is optimized for expression of a polypeptide in CHO or CHO-derived cells or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0251] In certain embodiments, the vector is a viral vector.In certain embodiments, the viral vector can be, but is not limited to, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, an alphavirus vector, a herpesvirus vector, and a vaccinia virus vector.In some embodiments, the viral vector is a lentivirus vector.

[0252] In some embodiments, the vector is a non-viral vector. Viral vectors can be plasmids or transposons (such as PiggyBac- or Sleeping Beauty transposons).

[0253] host cell In some embodiments, a host cell is provided that comprises any of the polypeptides, nucleic acid constructs, and / or vectors described herein. In some embodiments, a host cell is provided that has been prepared using any of the methods described herein. In some embodiments, the host cell is capable of producing, under fermentation conditions, any of the polypeptides (such as antibodies or inhibitory polypeptides) described herein.

[0254] In some embodiments, the polypeptides described herein (e.g., anti-PLA2G2D antibodies or inhibitory PLA2G2D polypeptides) can be expressed in prokaryotic cells, such as bacterial cells; or eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells (including CHO-S, DG44Lec13 CHO cells, and FUT8 CHO cells); PER.C6® cells (Crucell); and NSO cells. In some embodiments, the polypeptides described herein (e.g., anti-PLA2G2D antibodies or inhibitory PLA2G2D polypeptides) can be expressed in yeast. See, for example, U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy and / or light chains of the desired antibody. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0255] Introduction of one or more nucleic acids into desired host cells can be achieved by any method, including, but not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001), incorporated by reference in its entirety for all purposes. Nucleic acids can be transiently or stably transfected into desired host cells according to any suitable method.

[0256] The present invention also provides host cells containing any of the polynucleotides or vectors described herein. In some embodiments, the present invention provides host cells containing an anti-PLA2G2D antibody. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding antibodies, polypeptides, or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT Publication No. WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or B. subtillis) and yeast (e.g., S. cerevisae, S. pombe; or K. lactis).

[0257] In some embodiments, the polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

[0258] Polypeptide purification Polypeptides (e.g., anti-PLA2G2D antibodies, e.g., inhibitory PLA2G2D polypeptides) can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include ligands that bind to the ROR1 ECD and antibody constant regions. In some embodiments, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to bind to the constant region and purify antibodies containing Fc fragments. Hydrophobic interaction chromatography, such as butyl or phenyl columns, can also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) can also be suitable for purifying some polypeptides, such as antibodies. Mixed-mode chromatography (e.g., reversed-phase / anion exchange, reversed-phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) can also be suitable for purifying some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0259] V. COMPOSITIONS, KITS AND ARTICLES OF MANUFACTURE The present application also provides compositions, kits, medicaments, and unit dosage forms for use in any of the methods described herein.

[0260] composition Any of the antagonists described herein can be present in a composition (such as a formulation) that includes other agents, excipients, or stabilizers.

[0261] In some embodiments, the composition further comprises a targeting agent or carrier that facilitates delivery of the antagonist to diseased tissue. Exemplary carriers include liposomes, micelles, nanodispersed albumin and modifications thereof, polymeric nanoparticles, dendrimers, and inorganic nanoparticles of different compositions.

[0262] In some embodiments, the antagonist is packaged in a nanocarrier. In some embodiments, the nanocarrier has an average diameter of about 20 nm to about 200 nm. In some embodiments, the nanocarrier has an average diameter of about 50 nm.

[0263] In some embodiments, the antagonists described herein are coated with a serum protein (such as albumin). In some embodiments, the antagonists are coated with an opsonin.

[0264] In some embodiments, the antagonist comprises or is linked to a moiety that facilitates delivery of the antagonist to diseased tissue (such as cancer tissue or an infection site, as described above). In some embodiments, the moiety binds to an antigen expressed (e.g., overexpressed) or clustered on diseased tissue (such as cancer tissue) or cells within the diseased tissue. In some embodiments, the antigen is a tumor-associated antigen (e.g., Her2, folate receptor, CD44). See, e.g., Rosenblum et al., Nat Commun. 2018 Apr 12;9(1):1410.

[0265] In some embodiments, the compositions are suitable for administration to humans. In some embodiments, the compositions are suitable for administration to mammals, such as domestic pets and agricultural animals, in veterinary settings. A wide variety of suitable formulations of compositions containing antagonists exist. The following formulations and methods are merely exemplary and are in no way limiting. Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent such as water, saline, or orange juice; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient as a solid or granules; (c) suspensions in a suitable liquid; and (d) suitable emulsions. Tablet forms may contain one or more of lactose, mannitol, cornstarch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can contain the active ingredient in a flavoring, usually sucrose and acacia or tragacanth, and troches contain the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia, while emulsions, gels, and the like contain excipients as known in the art in addition to the active ingredient.

[0266] Examples of suitable carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, methyl- and propylhydroxybenzoate, talc, magnesium stearate and mineral oil.In some embodiments, the composition comprising the antagonist and the carrier described herein is present in a dried formulation (such as a lyophilized composition).The formulation can further comprise a lubricant, a wetting agent, an emulsifying and suspending agent, a preservative, a sweetener or a flavoring agent.

[0267] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation compatible with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.The preparations may be provided in unit-dose or multi-dose sealed containers such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid excipient for injection, such as water, immediately before use.Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type described above.Injectable preparations are preferred.

[0268] In some embodiments, the composition is formulated to have a pH range of about 4.5 to about 9.0 (e.g., including any of the pH ranges of about 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0). In some embodiments, the pH of the composition is formulated to be about 6 or higher, e.g., about 6.5, 7, or 8 or higher (e.g., about 8). The composition can also be made isotonic with blood by the addition of a suitable tonicity adjusting agent, such as glycerol.

[0269] kit The kits provided herein include one or more containers containing an antagonist or a pharmaceutical composition containing the antagonist and / or other agent(s) described herein, and in some embodiments, further include instructions for use according to any of the methods described herein. The kits may further include instructions for selecting individuals suitable for treatment. The instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions embodied on a magnetic or optical storage disk) are also acceptable.

[0270] In some embodiments, the kit comprises: a) a composition comprising an antagonist targeting the PLA2G2D signaling pathway, comprising an agent comprising an anti-PLA2G2D antibody moiety or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and optionally, b) instructions for administering the agent for the treatment of a disease or condition. In some embodiments, the agent is an anti-PLA2G2D antibody. In some embodiments, the agent is an anti-PLA2G2D fusion protein. In some embodiments, the agent is an anti-PLA2G2D immunoconjugate.

[0271] In some embodiments, the kit includes: a) a composition comprising an antagonist targeting the PLA2G2D signaling pathway, the antagonist comprising an inhibitory PLA2G2D polypeptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and, optionally, b) instructions for administering the agent for treating a disease or condition. In some embodiments, the inhibitory PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7-12.

[0272] In some embodiments, the kit includes: a) a composition comprising an antagonist targeting the PLA2G2D signaling pathway, including a nucleic acid drug (e.g., siRNA, shRNA, miRNA, or antisense RNA) targeting PLA2G2D or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, and, optionally, b) instructions for administering the drug for the treatment of a disease or condition.

[0273] In some embodiments, the kit includes: a) a composition comprising an antagonist targeting the PLA2G2D signaling pathway, including a genome editing system targeting PLA2G2D or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, and, optionally, b) instructions for administering the agent for the treatment of a disease or condition.

[0274] In some embodiments, the kit includes: a) a composition comprising an antagonist targeting the PLA2G2D signaling pathway, including an agent that inhibits PLA2G2D enzyme activity or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, and, optionally, b) instructions for administering the agent for the treatment of a disease or condition.

[0275] The kits of the present invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The kits may optionally provide additional components such as buffers and interpretive information. Thus, the present application also provides articles of manufacture that include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0276] In some embodiments, the kit includes one or more components that facilitate delivery of the composition comprising the antagonist, or agent, and / or additional therapeutic agent to an individual. In some embodiments, the kit includes, for example, a syringe and needle suitable for delivery of cells to an individual. In such embodiments, the composition comprising the antagonist or agent may be included in the kit in a bag or one or more vials. In some embodiments, the kit includes components that facilitate intravenous or intra-arterial delivery of the composition comprising the antagonist or agent to an individual. In some embodiments, the composition comprising the antagonist or agent may be included in, for example, a bottle or bag (e.g., a blood bag or similar bag that can hold up to about 1.5 L of solution containing cells), and the kit further includes tubing and needles suitable for delivery of the composition comprising the antagonist or agent to an individual.

[0277] The instructions for use of the composition generally include information on the dosage, administration schedule, and administration route for the intended treatment.The container can be a unit dose, bulk package (e.g., a multi-dose package), or sub-unit dose.For example, a kit can be provided that contains a sufficient dose of zinc disclosed herein to provide effective treatment for an individual for a long period of time, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or more.The kit can also include multiple unit doses of the pharmaceutical composition and instructions for use, and can be packaged in an amount sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies.

[0278] Illustrative Embodiments Embodiment 1. A method of treating cancer or a viral infection in an individual, comprising administering to said individual an effective amount of an antagonist that targets the PLA2G2D signaling pathway.

[0279] Embodiment 2. The method of embodiment 1, wherein the antagonist is an antagonist that targets PLA2G2D.

[0280] Embodiment 3. The method of embodiment 2, wherein said PLA2G2D is human PLA2G2D.

[0281] Embodiment 4. The method of embodiment 2 or embodiment 3, wherein the antagonist reduces the level of enzymatic activity of PLA2G2D.

[0282] Embodiment 5. The method of embodiment 4, wherein said antagonist that targets the PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D.

[0283] Embodiment 6. The method of embodiment 5, wherein said antagonist targets the H67 catalytic site on human PLA2G2D according to SEQ ID NO: 1 or 5.

[0284] Embodiment 7. The method of any one of embodiments 1-3, wherein the antagonist comprises an siRNA, miRNA, antisense RNA, or a gene editing system.

[0285] Embodiment 8. The method of any one of embodiments 1-3, wherein the antagonist comprises an agent that inhibits PLA2G2D (such as an agent that blocks binding of PLA2G2D to immune cells or an agent that inhibits the activity of PLA2G2D).

[0286] Embodiment 9. The method of embodiment 8, wherein the immune cells are T cells.

[0287] Embodiment 10. The method of any one of embodiments 1-3, wherein the antagonist comprises an anti-PLA2G2D antibody.

[0288] Embodiment 11. The method of embodiment 10, wherein the anti-PLA2G2D antibody is a monoclonal antibody.

[0289] Embodiment 12. The method of embodiment 10, wherein the antagonist is a fusion protein further comprising a second moiety.

[0290] Embodiment 13. The method of embodiment 12, wherein the second moiety comprises a cytokine.

[0291] Embodiment 14. The method of any one of embodiments 1-3, wherein the antagonist comprises an inhibitory PLA2G2D polypeptide that blocks binding of PLA2G2D to immune cells.

[0292] Embodiment 15. The method of embodiment 14, wherein said inhibitory PLA2G2D polypeptide binds to said immune cells with a higher affinity than to PLA2G2D.

[0293] Embodiment 16. The method of embodiment 15, wherein the immune cells are T cells.

[0294] Embodiment 17. The method of any one of embodiments 14-16, wherein the inhibitory PLA2G2D polypeptide further comprises a stabilization domain.

[0295] Embodiment 18. The method of embodiment 17, wherein the stabilization domain is an Fc domain.

[0296] Embodiment 19. The method of any one of embodiments 14-18, wherein the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids.

[0297] Embodiment 20. The method of any one of embodiments 14-19, wherein the inhibitory PLA2G2D polypeptide has a mutation at a position corresponding to histidine at position 67 (H67) according to SEQ ID NO: 1 or 5.

[0298] Embodiment 21. The method of embodiment 20, wherein the inhibitory PLA2G2D polypeptide comprises the amino acid sequence of SEQ ID NO: 3, 4, 7, or 8, or a variant thereof.

[0299] Embodiment 22. The method of any one of embodiments 1-21, wherein the disease or condition is cancer.

[0300] Embodiment 23. The method of embodiment 22, wherein the cancer is a solid tumor.

[0301] Embodiment 24. The method of embodiment 22 or embodiment 23, wherein the cancer is an aggressive or malignant tumor.

[0302] Embodiment 25. The method of any one of embodiments 22-24, wherein the cancer has an increased expression level of PLA2G2D.

[0303] Embodiment 26. The method of any one of embodiments 22-25, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer, and melanoma.

[0304] Embodiment 27. The method of any one of embodiments 1-21, wherein the disease or condition is a viral infection.

[0305] Embodiment 28. The method of embodiment 27, wherein the site of infection has increased levels of PLA2G2D.

[0306] Embodiment 29. The method of any one of embodiments 1-28, further comprising administering a second agent.

[0307] Embodiment 30. The method of embodiment 29, wherein the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent.

[0308] Embodiment 31. The method of embodiment 30, wherein the second agent is an immunomodulatory agent.

[0309] Embodiment 32. The method of embodiment 31, wherein the immunomodulatory agent is an immune checkpoint inhibitor.

[0310] Embodiment 33. The method of embodiment 28, wherein the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4.

[0311] Embodiment 34. The method of embodiment 33, wherein the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen.

[0312] Embodiment 35. The method of any one of embodiments 29-34, wherein the antagonist and the second agent are administered simultaneously or concurrently.

[0313] Embodiment 36. The method of any one of embodiments 29-34, wherein the antagonist and the second agent are administered sequentially.

[0314] Embodiment 37. The method of any one of embodiments 1-36, wherein the antagonist and / or the second agent is administered parenterally.

[0315] Embodiment 38. The method of any one of embodiments 22-37, wherein the antagonist is administered directly to the cancerous tissue or site of infection.

[0316] Embodiment 39. The method of any one of embodiments 1-38, wherein the antagonist is administered at a dose of from about 0.001 μg / kg to about 100 mg / kg.

[0317] Embodiment 40. The method of any one of embodiments 22-39, wherein the individual has an increased number of immune cells in the cancer tissue or the site of infection following administration of the antagonist.

[0318] Embodiment 41. The method of embodiment 40, wherein the immune cells are T cells.

[0319] Embodiment 42. The method of embodiment 40 or embodiment 41, wherein the T cells are activated T cells.

[0320] Embodiment 43. The method of any one of embodiments 40-42, wherein the number of immune cells in the cancer tissue or the site of infection is increased by at least about 5% after administration of the antagonist.

[0321] Embodiment 44. The method of any one of embodiments 22-43, wherein immune cells in the cancer tissue or the infected site produce increased levels of cytokines following administration of the antagonist.

[0322] Embodiment 45. The method of embodiment 44, wherein the cytokine is IFNγ and / or IL-2.

[0323] Embodiment 46. The method of embodiment 39 or embodiment 40, wherein the level of the cytokine is increased by at least about 5% after administration of the antagonist. [Example]

[0324] The following examples are intended to be purely illustrative of the present application and therefore should not be construed as limiting the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.

[0325] Example 1. Identification of the PLA2G2D signaling pathway Gene expression profiles were studied to identify new signaling pathways involved in cancer.

[0326] Specifically, batch-corrected RNA-seq datasets from the Cancer Genome Atlas (TCGA) were downloaded from the National Cancer Institute's Genomic Data Commons PanCanAtlas website (https: / / gdc.cancer.gov / about-data / publications / pancanatlas). The whole cancer dataset was separated into the following tumor types: bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), COAD (colon adenocarcinoma), ESCA (esophageal carcinoma), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), KIRC (renal clear cell carcinoma), KIRP (renal papillary cell carcinoma), LIHC (hepatocellular carcinoma), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), ovarian serous adenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), prostate adenocarcinoma (PRAD), and rectal adenocarcinoma (REA). D), prostate adenocarcinoma (SARC), cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), testicular germ cell tumor (TGCT), thyroid carcinoma (THCA), thymoma (THYM), triple-negative breast cancer (TN-BRCA), and uterine endometrial carcinoma (UCEC). For each tumor type, the following analysis was performed: genes with consistently low expression (e.g., genes with no counts in more than 80% of samples) were removed, and the data were log-transformed using log2(x+1). Then, a gene co-expression network was constructed using the weighted gene co-expression network analysis (WGCNA) R package to identify clusters of highly correlated genes. See Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, (2008).

[0327] Clusters containing T signature genes were extracted, and gene ontology analysis was performed using the R package clusterProfiler to confirm that the clusters were enriched for T cell-related pathways. Yu G, Wang L, Han Y, He Q (2012). "clusterProfiler: an R package for comparing biological themes among gene clusters." OMICS: A Journal of Integrative Biology, 16(5), 284-287. doi:10.1089 / omi.2011.0118. The expression dataset was then subset to genes within this T signature gene cluster, and the samples were clustered using the R package ConsensusClusterPlus, using the clustering algorithm k-means clustering, the distance measure Euclidean, and a maximum number of clusters k=20. Wilkerson, DM, Hayes, Neil D (2010). "ConsensusClusterPlus: a class discovery tool with confidence assessments and item tracking." Bioinformatics, 26(12), 1572-1573. Cumulative distribution function (CDF) plots and delta area plots showing the relative change in area under the CDF curve comparing k and k-1 were used to identify the optimal number of clusters. Expression values ​​of genes within each cluster were then summed to generate a single summary value describing each cluster. Tumors in the highest gene expression cluster were labeled "hot," and tumors in the lowest gene expression cluster were labeled "cold." The tumor RNA-seq read count matrix was then compiled into a database on Google Cloud, containing RNA-seq data from TCGA processed using Kallisto. Data were downloaded from the Pilot RNA-Sequencing for CCLE and TCGA project data reportory (https: / / osf.io / gqrz9 / ). See Tatlow, P. & Piccolo, S.R.A. cloud-based workflow to quantify transcript expression levels in public cancer compendia. Scientific Reports 6, (2016). Transcript level data were summed to the gene level, and a subset was summed to protein-coding genes. Differential expression analysis was then performed using the R package limma to compare "hot" samples with "cold" samples. See Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015). "limma powers differential expression analyses for RNA-sequencing and microarray studies." Nucleic Acids Research, 43(7), e47. Differential expression results were visualized using the R package EnhancedVolcano. See Blighe K, Rana S, Lewis M (2019). EnhancedVolcano: Publication-ready volcano plots with enhanced coloring and labeling. R package version 1.4.0, https: / / github.com / kevinblighe / EnhancedVolcano.

[0328] The results suggest that PLA2G2D is highly differentially expressed in the four cancer types (see Figures 1A-1D). Specifically and significantly, PLA2G2D was 56-fold more highly expressed in CD8+ high tumors compared with CD8+ low tumors.

[0329] Example 2. Role of PLA2G2D in suppressing T cell activation The effect of human PLA2G2D-Fc on T cell activation was evaluated in peripheral blood mononuclear cells (PBMCs) or isolated T cell cultures. PBMCs were isolated from leukocyte reduction system (LRS) chambers from healthy human donors by centrifugation on Ficoll-Paque Plus (GE Life Sciences), labeled with 5 μM CFSE solution (Molecular Probes) for 12 min at 37°C, and washed. 2 × 10 cells were then plated per well of a 96-well round-bottom plate. 5 Labeled PBMCs were stimulated with 1 μg / ml anti-CD3 (OKT3, Invitrogen) and 0.2 μg / ml anti-CD28 (CD28.2, Invitrogen) in the presence of soluble human PLA2G2D-Fc (0–20 μg / ml) or control human IgG1-Fc protein (Sino Biological) in a final volume of 200 μl of RPMI (Corning), as indicated. PBMC cultures were incubated at 37°C for 72 hours, after which supernatants were collected and measured for IFNγ and IL-2 levels using the MSD V-plex assay (Meso Scale Discovery). T cell proliferation within PBMC cultures was simultaneously assessed by staining cells with fluorophore-conjugated anti-CD3, anti-CD4, and anti-CD8 antibodies (Biolegend) and Live / Dead Fixable dead cell stain (Molecular Probes) and running them on a BD LSRFortessa X-20 flow cytometer (Becton Dickinson). FACS data were analyzed using FlowJo software.

[0330] Addition of soluble PLA2G2D-Fc dose-dependently suppressed CD4+ and CD8+ T cell proliferation in stimulated PBMC cultures, as measured by CFSE production tracing. Figures 2A-2B show CFSE histograms and quantification of T cell proliferation for one PBMC donor using increasing concentrations of PLA2G2D-Fc. Figures 3A-3C show CFSE histograms and quantification of proliferation for three additional independent PBMC donors, demonstrating that increasing concentrations of soluble PLA2G2D-Fc protein dose-dependently suppressed CD4+ and CD8+ T cell proliferation, while equivalent concentrations of control-Fc protein had no significant effect. Figures 4A-4B show that, consistent with T cell proliferation, levels of IFNγ and IL-2 in the different PBMC cultures were similarly dose-dependently and significantly reduced by increasing concentrations of soluble PLA2G2D-Fc protein, but not by control-Fc.

[0331] To assess the effect of immobilized PLA2G2D protein on T cell proliferation in PBMC cultures, the same assay was utilized, except that 100 μl of 0–10 μg / ml human PLA2G2D-Fc or control human IgG1-Fc protein was coated onto 96-well flat-bottom plates overnight at 4°C in PBS the day before PBMCs were prepared and added with anti-CD3 and anti-CD28 antibodies. Figure 5 shows that immobilized human PLA2G2D-Fc protein coated onto the plate surface also suppressed CD4+ and CD8+ T cell proliferation in stimulated PBMC cultures.

[0332] To evaluate the effect of PLA2G2D protein on isolated T cells, 96-well flat-bottom plates were coated overnight at 4°C with 0–10 μg / ml human PLA2G2D-Fc or control human IgG1-Fc protein in 100 μl of PBS, along with 1 μg / ml anti-CD3 (OKT3) and 0.2 μg / ml anti-CD28 (CD28.2). The next day, the plates were washed twice with PBS before adding T cells. T cells were isolated from PBMCs using the Pan T Cell Isolation Kit (Miltenyi) and labeled with CFSE as described above. 1x10 5 Purified and labeled T cells were added to each well of the coated plate in a final volume of 200 μl of RPMI. T cells were grown at 37°C for 72 hours, after which supernatants were collected for IFNγ and IL-2 analysis by MSD, and proliferation was analyzed by FACS.

[0333] Figure 6 shows that immobilized human PLA2G2D-Fc protein dose-dependently suppresses the proliferation of isolated T cell cultures in the presence of anti-CD3 and anti-CD28 stimulation. However, when soluble PLA2G2D-Fc protein was added to isolated T cell cultures instead of coating it on plates, there was no suppressive effect on T cells (data not shown). Collectively, these results suggest that PLA2G2D requires cross-linking by antigen-presenting cells or by immobilization on the plate surface to induce functional suppression of T cells.

[0334] Example 3. T cell suppression by PLA2G2D Human PLA2G2D contains an N-terminal 20-residue signal peptide, a highly conserved Ca 2+ It is a 145-amino acid secreted protein consisting of a binding site and a catalytic His-Asp dyad. In addition to these elements, human PLA2G2D is characterized by seven disulfide bonds that contribute to its high stability. Figure 7A shows the general structural and functional features of the human PLA2G2D protein.

[0335] To assess whether PLA2G2D enzymatic activity is required for its immunosuppressive function, we introduced an H67Q mutation into the highly conserved catalytic His67-Asp68 dyad of human PLA2G2D. Briefly, human PLA2G2D cDNA fused in-frame to human IgG1-Fc cDNA at the C-terminus was synthesized with a CAC→CAG point mutation corresponding to a His→Gln substitution at residue 67. The construct was cloned into a high-expression mammalian vector and transfected into HEK293 cells. Secreted human PLA2G2D-H67Q-Fc protein contained in the supernatant was purified by Protein A affinity chromatography. The purified PLA2G2D-H67Q-Fc protein was used in PBMC cultures as described above and compared with wild-type PLA2G2D-Fc and control human IgG1-Fc for T cell suppressive activity.

[0336] Figures 7B-7C show that the PLA2G2D-H67Q-Fc catalytic mutant inhibits CD4 + and CD8 + These results show that it retained most of the immunosuppressive function on T cells, with a dose of 10 μg / ml showing significantly reduced suppression.

[0337] Alternatively, 0-25 µM of the sPLA2 inhibitor LY315920 or DMSO control was added to assess T cell proliferation in PBMC cultures in the presence of wild-type PLA2G2D-Fc protein. Figure 8 shows that LY315920 does not reverse the immunosuppression induced by PLA2G2D.

[0338] Example 4. Binding of PLA2G2D to activated T cells To determine whether PLA2G2D induces immunosuppression by directly binding to T cells in vitro, the level of PLA2G2D binding was assessed in resting and activated primary human T cells. T cells were isolated from PBMCs using a Pan T Cell Isolation Kit (Miltenyi) and cultured in RPMI at 37°C for 48 hours in the presence or absence of beads loaded with anti-human CD2, CD3, and CD28 antibodies (Human T Cell Activation / Expansion Kit, Miltenyi). Stimulated or unstimulated T cells were then harvested, washed, and incubated with 0–10 μg / ml human PLA2G2D-Fc protein or human IgG1-Fc protein for 30 minutes at 4°C. Cells were washed three times with PBS, stained with Alexa 488-conjugated goat anti-human IgG1 antibody (Invitrogen) for 30 minutes at 4°C, washed, and then analyzed by FACS.

[0339] Figures 9A-9C show that human PLA2G2D-Fc preferentially binds to activated CD4+ and CD8+ T cells in two different donor T cells compared with control human IgG1-Fc protein. PLA2G2D binds weakly to unstimulated T cells, but binding increases dramatically upon T cell stimulation. Figure 9C shows a quantitative representation of PLA2G2D-Fc binding to stimulated T cells. Addition of heparin sulfate proteoglycan (HSPG) partially reduced PLA2G2D-Fc binding to T cells but did not alter the suppression of CD4+ and CD8+ T cell proliferation (data not shown). This suggests that the immunosuppression potentially associated with PLA2G2D binding to T cells is independent of binding via heparin sulfate on the cell surface.

[0340] Example 5. Syngeneic tumor growth in PLA2G2D-deficient mice PLA2G2D knockout mice were generated by deleting exon 2 of the mouse Pla2g2d gene from C57BL6 mice using CRISPR / Cas9-mediated gene editing. To confirm the absence of a functional Pla2g2d gene in these mice, spleens were collected from wild-type and knockout mice, and total RNA was isolated using TRIZol (Invitrogen). Total RNA was subjected to real-time RT-PCR to detect Pla2g2d mRNA. Hprt1 (hypoxanthine-guanine phosphoribosyltransferase) mRNA levels were also measured as a control. The results demonstrated that the knockout mice lacked Pla2g2d expression.

[0341] To assess the impact of Pla2G2d deficiency on tumor growth, mouse syngeneic tumor cell lines MC38 (colon adenocarcinoma), B16F10 (melanoma), and E.G7-OVA (T-cell lymphoma) were implanted into age-matched wild-type C57BL6 (WT) or PLA2G2D knockout mice. 1 x 10 cells suspended in 100 μl of PBS were used. 6 MC38 or E.G7-OVA cells, or 5x10 5 B16F10 cells were subcutaneously injected into WT (n=16) or PLA2G2D knockout mice (n=16), and tumor growth was monitored every 2 or 3 days. Tumor volume was calculated using the formula: tumor volume = 0.5 × length × width. 2 Body weight was also monitored weekly. Mice were sacrificed after 3–4 weeks or when they reached the designated endpoints.

[0342] As shown in Figures 11A-11F, tumor growth of all three syngeneic tumor cell lines was significantly reduced in PLA2G2D knockout mice compared to wild-type mice, indicating a role for PLA2G2D in tumor progression and supporting targeted inhibition of PLA2G2D as a potential immunotherapy.

[0343] Example 6. Disruption of PLA2G2D immunosuppressive function by anti-PLA2G2D monoclonal antibodies To demonstrate whether PLA2G2D immunosuppression can be neutralized and reversed by anti-PLA2G2D antibodies, we developed PLA2G2D-binding monoclonal antibodies by immunizing mice and generating hybridomas.

[0344] Blocking PLA2G2D binding to activated T cells As shown in the above examples (Figures 9A-9C), we found that PLA2G2D preferentially binds to activated T cells but only minimally to resting T cells, suggesting that PLA2G2D may confer inhibitory signaling by directly binding to T cells upon T cell activation. Therefore, we devised an assay to determine whether anti-PLA2G2D antibodies could interfere with PLA2G2D binding to activated T cells and potentially prevent immunosuppression. T cells in human PBMC cultures were stimulated with anti-CD3 and anti-CD28 antibodies (Invitrogen) for 24 hours at 37°C. PBMC cultures were then harvested, washed, and incubated with 2 μg / ml of human PLA2G2D-Fc protein or control human IgG1-Fc protein in the presence of 10 μg / ml of PLA2G2D antibody (developed in-house) or mouse IgG2a isotype control antibody (Invitrogen) for 30 minutes at 4°C. The cells were washed three times with PBS and then stained with fluorophore-conjugated anti-CD3, anti-CD4, and anti-CD8 antibodies (Biolegend) and PE-conjugated goat anti-human IgG1 antibody (Invitrogen) together with Live / Dead Fixable dead cell stain (Molecular Probes) for 30 min at 4°C, washed, and then analyzed by FACS.

[0345] Figure 12A shows that two representative PLA2G2D-binding antibodies can reduce PLA2G2D binding to activated T cells, as measured by the mean fluorescence intensity (MFI) of PLA2G2D staining on gated T cells. In contrast, a control mouse IgG2a isotype control had no effect on PLA2G2D binding.

[0346] Reversal of PLA2G2D-dependent T cell suppression in PBMC cultures To determine whether PLA2G2D antibodies could neutralize PLA2G2D-dependent suppression of T cell function, we used a similar approach to that we took to demonstrate PLA2G2D suppressive activity above (Figures 4A-4B). We then cultured 2 x 10 cells per well of a 96-well round-bottom plate. 5 Triplicate wells containing PBMCs were stimulated with 1 μg / ml anti-CD3 (OKT3, Invitrogen) and 0.2 μg / ml anti-CD28 (CD28.2, Invitrogen) in the presence of 1 μg / ml soluble human PLA2G2D-Fc protein (Sino Biological) and 10 μg / ml PLA2GD antibody or control mIgG2a isotype control antibody (Invitrogen) in a final volume of 200 μl of RPMI (Corning), as indicated. PBMC cultures were incubated at 37°C for 48 hours, after which supernatants were collected and measured for IL-2 and IFNγ levels using the MSD V-plex assay (Meso Scale Discovery).

[0347] Figures 12B-12C show that two representative function-blocking PLA2G2D antibodies can rescue PLA2G2D-mediated suppression of IL-2 and IFNγ secretion levels in this assay.

[0348] Example 7. Use of PLA2G2D antibodies to treat tumors To assess the effect of PLA2G2D antibodies on tumor growth, mouse syngeneic tumor cell lines MC38 (colon adenocarcinoma), B16F10 (melanoma), and E.G7-OVA (T-cell lymphoma) are implanted into age-matched wild-type C57BL6 mice at 1 x 10 6 MC38 or E.G7-OVA cells, or 5x10 cells suspended in 100ul of PBS 5 B16F10 cells were subcutaneously injected into C57BL6 mice. Tumor volumes were 50-150 mm. 3When tumor volume is within the range of 100 mg / kg, the mice are randomized into control and treatment groups. PLA2G2D antibody is administered at a dose of 10 mg / kg by IP injection on days 1, 4, 7, and 11 after randomization. Tumor growth is monitored every 2 or 3 days. Tumor volume is calculated using the formula: tumor volume = 0.5 x length x width. 2 Calculate using the . Body weight is also monitored weekly. Mice are sacrificed after 3-4 weeks or when they reach the designated endpoint. [Table 2-1] [Table 2-2] [Table 2-3] The present invention provides, for example, the following items. (Item 1) A method of treating cancer or a viral infection in an individual, comprising administering to said individual an effective amount of an antagonist that targets the PLA2G2D signaling pathway. (Item 2) 2. The method of claim 1, wherein the antagonist is an antagonist that inhibits or downregulates PLA2G2D. (Item 3) 3. The method of claim 2, wherein the PLA2G2D is human PLA2G2D. (Item 4) The method of item 2 or item 3, wherein the antagonist reduces the enzymatic activity level of PLA2G2D. (Item 5) 5. The method of claim 4, wherein the antagonist that targets the PLA2G2D signaling pathway blocks a catalytic site on PLA2G2D. (Item 6) 6. The method of claim 5, wherein the antagonist targets the H67 catalytic site on human PLA2G2D according to SEQ ID NO: 1 or 5. (Item 7) 4. The method of any one of items 1 to 3, wherein the antagonist comprises siRNA, miRNA, antisense RNA, or a gene editing system. (Item 8) 4. The method of any one of items 1 to 3, wherein the antagonist blocks binding of PLA2G2D to immune cells. (Item 9) 9. The method of claim 8, wherein the immune cells are T cells. (Item 10) 4. The method of any one of items 1 to 3, wherein the antagonist comprises an anti-PLA2G2D antibody. (Item 11) 11. The method of claim 10, wherein the anti-PLA2G2D antibody is a monoclonal antibody. (Item 12) 11. The method of claim 10, wherein the antagonist is a fusion protein or immunoconjugate further comprising a second moiety. (Item 13) 13. The method of claim 12, wherein the second moiety comprises a cytokine. (Item 14) 4. The method of any one of items 1 to 3, wherein the antagonist comprises an inhibitory PLA2G2D polypeptide that blocks binding of PLA2G2D to immune cells. (Item 15) 15. The method of claim 14, wherein the inhibitory PLA2G2D polypeptide binds to the immune cells with higher affinity than wild-type PLA2G2D. (Item 16) 16. The method of item 15, wherein the immune cells are T cells. (Item 17) 17. The method of any one of items 14 to 16, wherein the inhibitory PLA2G2D polypeptide further comprises a stabilization domain. (Item 18) 18. The method of claim 17, wherein the stabilization domain is an Fc domain. (Item 19) 19. The method according to any one of items 14 to 18, wherein the inhibitory PLA2G2D polypeptide has a length of about 50 to about 200 amino acids. (Item 20) 20. The method of any one of items 14 to 19, wherein the inhibitory PLA2G2D polypeptide has a) a mutation at a position corresponding to histidine 67 (H67) according to SEQ ID NO: 1 or 5, or b) a mutation at a position corresponding to glycine 80 (G80) according to SEQ ID NO: 5. (Item 21) 21. The method of item 20, wherein the inhibitory PLA2G2D polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, and 7 to 12, or a variant thereof. (Item 22) 22. The method of any one of items 1 to 21, wherein the disease or condition is cancer. (Item 23) 23. The method of claim 22, wherein the cancer is a solid tumor. (Item 24) 24. The method of claim 22 or 23, wherein the cancer is an advanced or malignant tumor. (Item 25) 25. The method of any one of items 22 to 24, wherein the cancer has an increased expression level of PLA2G2D. (Item 26) 26. The method according to any one of items 22 to 25, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, liver cancer, gastric cancer, cervical cancer, endometrial cancer, thyroid cancer, colorectal cancer, head and neck cancer, pancreatic cancer, kidney cancer, prostate cancer, urothelial cancer, testicular cancer, ovarian cancer and melanoma. (Item 27) 22. The method of any one of items 1 to 21, wherein the disease or condition is a viral infection. (Item 28) 28. The method according to item 27, wherein the expression level of PLA2G2D in an infected site is higher than the expression level in a non-infected site. (Item 29) 29. The method of any one of items 1 to 28, further comprising administering a second agent. (Item 30) 30. The method of claim 29, wherein the second agent is selected from the group consisting of a chemotherapeutic agent, an immunomodulatory agent, an anti-angiogenic agent, a growth inhibitory agent, and an anti-neoplastic agent. (Item 31) 31. The method of claim 30, wherein the second agent is an immunomodulatory agent. (Item 32) 32. The method of item 31, wherein the immunomodulatory agent is an immune checkpoint inhibitor. (Item 33) 29. The method of item 28, wherein the immune checkpoint inhibitor specifically targets PD-L1, PD-L2, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, or B7H4. (Item 34) 34. The method of claim 33, wherein the second agent comprises a cell comprising a chimeric antigen receptor that specifically binds to a tumor antigen. (Item 35) 35. The method of any one of items 29 to 34, wherein the antagonist and the second agent are administered simultaneously or concurrently. (Item 36) 35. The method of any one of items 29 to 34, wherein the antagonist and the second agent are administered sequentially. (Item 37) 37. The method of any one of items 1 to 36, wherein the antagonist and / or the second agent are administered parenterally. (Item 38) 38. The method of any one of items 22 to 37, wherein the antagonist is administered directly to the cancer tissue or the site of infection. (Item 39) The antagonist is administered at a dose of about 0.001 μg / kg to about 100 mg / kg. 39. The method according to any one of items 1 to 38, (Item 40) 40. The method according to any one of items 22 to 39, wherein the individual has an increased number of immune cells in the cancer tissue or the infected site after administration of the antagonist. (Item 41) 41. The method of claim 40, wherein the immune cells are T cells. (Item 42) 42. The method of claim 40 or 41, wherein the T cells are activated T cells. (Item 43) 43. The method of any one of items 40 to 42, wherein the number of immune cells in the cancer tissue or the infected site is increased by at least about 5% after administration of the antagonist. (Item 44) 44. The method of any one of items 22 to 43, wherein immune cells in the cancer tissue or the infected site produce increased levels of cytokines after administration of the antagonist. (Item 45) 45. The method of item 44, wherein the cytokine is IFNγ and / or IL-2. (Item 46) 41. The method of claim 39 or 40, wherein the level of the cytokine is increased by at least about 5% after administration of the antagonist.

Claims

[Claim 1] The invention described in the specification.

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