A Composition Immune Checkpoint Inhibition Comprising a WNK3 Inhibitor as an Active Ingredient

Abstract

The present invention relates to a composition for inhibiting immune checkpoints comprising a WNK3 inhibitor, and a composition for the prevention or treatment of cancer or a composition for immune enhancement comprising the same. The composition of the present invention exhibits multifaceted anticancer activity by increasing the sensitivity of tumors to immune cells by inhibiting PD-L1 expression, while simultaneously promoting the secretion of GRANZYME B and Perforin by T cells. Furthermore, the screening method of the present invention can rapidly and reliably identify candidate substances for fundamental anticancer therapeutic agents that efficiently inhibit the expression of PD-L1, which plays a key role in the immune evasion mechanism of tumor cells, and significantly improve the sensitivity of cancer cells to immune responses.

Description

Technology Field

[0001] The present invention relates to a method for treating cancer by discovering WNK3 as a novel target for immune checkpoint inhibition and blocking tumor immune evasion using the same. Background Technology

[0003] The immune system plays a role in recognizing and eliminating malignant tumors, but tumor cells can evade the immune system through genetic modifications acquired during evolution. 1 PD-L1, expressed in both cancer cells and cancer-associated immune cells, binds to its immune checkpoint receptor PD-1, inducing inhibitory signals toward activated T cells and causing T cell death, deficiency, and functional exhaustion. 2 Therefore, blocking the PD-L1 / PD-1 axis restores T-cell function and induces sustained tumor remission in cancer patients, particularly those with melanoma, non-small cell lung cancer, and renal cell adenocarcinoma. 3 Recent therapeutic strategies to block the PD-L1 / PD-1 axis involve the use of various monoclonal antibodies against PD-L1 or PD-1. However, more in-depth research is needed on the genetic regulators involved in tumor-specific PD-L1 increase, and in particular, discovering new regulators of the PD-L1 / PD-1 axis and establishing inhibition strategies is an important issue in immune checkpoint therapy.

[0004] IFN-γ, an inflammatory cytokine secreted by infiltrating T cells, is the most well-known PD-L1 inducer in the tumor microenvironment. IFN-γ binds to its cognate receptors and promotes PD-L1 expression through the activation of the JAK-STAT pathway. 4Furthermore, intrinsic tumor signals increase PD-L1 expression, protecting cancer cells from immune cell attacks. Oncogenes such as EGFR, ALK, MAPK, MET, AKT, MYC, STAT3, CDK5, and YAP increase PD-L1 expression. Recently, whole-genome screening has been performed to identify PD-L1 regulators, and a study using CRISPR-Cas9-mediated loss-of-function screening in pancreatic cancer cells 5 and research using haploid gene screening in HAP1 cells 6 CMTM6 was identified as a PD-L1 promoter. More recently, the transcription initiators eIF5B and MLLT6 were identified as novel regulators of PD-L1 through CRISPR-Cas9-based screening in lung and colorectal cancer cell lines. 7,8 However, the identified genes are difficult to apply pharmacologically, so these studies have limitations in the clinical application of the results.

[0006] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention. Prior art literature

[0008] Non-patent literature 1. Proc Natl Acad Sci USA 15;102(46):16783-16788(2005) The problem to be solved

[0009] The inventors have made diligent research efforts to develop an efficient immune checkpoint inhibitor that enhances the sensitivity of cancer cells to immune responses by significantly blocking the immune evasion response of tumor cells. As a result, WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , PAN3 , CTRL, SMAD4 and OSGEPL1 of The present invention was completed by discovering that 13 genes not only show a high correlation with the expression of PD-L1, a ligand of immune checkpoint receptors, but also that when the expression of these genes is suppressed, the PD-L1 level in cancer cells is significantly reduced, thereby significantly restoring the activity of T cells suppressed by the PD-L1 / PD-1 axis and inducing apoptosis in cancer cells.

[0010] Therefore, the objective of the present invention is to provide a composition for inhibiting immune checkpoints and a composition for preventing or treating cancer containing the same as an active ingredient.

[0011] Another objective of the present invention is to provide a screening method for compositions for immune checkpoint inhibition.

[0013] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0015] According to one aspect of the present invention, the present invention WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , PAN3 , CTRL, SMAD4 and OSGEPL1 A composition for inhibiting immune checkpoints is provided, comprising as an active ingredient an inhibitor for one or more genes selected from the group consisting of

[0016] The inventors have made diligent research efforts to develop an efficient immune checkpoint inhibitor that improves the sensitivity of cancer cells to immune responses by significantly blocking the immune evasion response of tumor cells. As a result, they discovered that the 13 genes listed above not only show a high correlation with the expression pattern identical to PD-L1, a ligand of the immune checkpoint receptor, but also that when the expression of these genes is inhibited, the PD-L1 level in cancer cells is significantly reduced, thereby significantly restoring the activity of T cells suppressed by the PD-L1 / PD-1 axis and inducing apoptosis of cancer cells.

[0017] In this specification, the term “immune checkpoint” refers to an intracellular signaling system that maintains self-tolerance and protects tissues from excessive immune responses that cause damage. Immune checkpoint proteins are cell membrane proteins that regulate immune checkpoints and can inhibit the differentiation, proliferation, and activity of immune cells; specifically, they are expressed in activated T cells to reduce T cell proliferation, cytokine secretion, and cytotoxicity, and to suppress excessive T cell activity. Some immune checkpoints act as one of the major mechanisms by which tumor cells evade a patient’s immune response. Accordingly, the terms “a composition for immune checkpoint inhibition” or “immune checkpoint inhibitor” refer to an anticancer component or an anticancer adjuvant component that promotes an anti-tumor immune response by enhancing T cell activity through blocking the expression or activity of immune checkpoint proteins.

[0018] In this specification, the term “inhibitor” means a substance that causes a decrease in the activity or expression of the 11 genes listed above, and means a substance that reduces the activity or expression of these genes to such an extent that not only is the activity or expression of these genes undetectable or present at a meaningless level, but the increase in PD-L1 expression by these genes and the resulting inhibition of immune cell activity can be significantly improved.

[0019] In this specification, the term “decrease in expression” may mean a state in which the expression level of these genes at the gene level (e.g., DNA or mRNA) or at the level of the protein they encode is reduced by, for example, 20% or more compared to the control group, more specifically a state in which it is reduced by 30% or more, and more specifically a state in which it is reduced by 40% or more.

[0020] In this specification, the term “reduction in activity” refers to a measurably significant decrease in the intrinsic in vivo function of these genes or the proteins they encode compared to a control group; specifically, it means a decrease in activity to the extent that immune cell activity within the subject can be significantly improved or restored. A reduction in activity includes not only a simple decrease in function but also ultimate inhibition of activity resulting from a decrease in stability.

[0021] The above inhibitor is, for example, one for which the nucleotide sequence and the amino acid sequence of the encoding protein are already known in the art. WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , CTRL or OSGEPL1 A CRISPR system comprising shRNA, siRNA, miRNA, ribozyme, PNA (peptide nucleic acids), antisense oligonucleotide, and guide RNA that recognizes a target gene, which inhibits the expression of these at the gene level; as well as antibodies or aptamers that inhibit their expression at the protein level, and small molecule compounds, peptides, and natural products that inhibit their activity, but not limited thereto, all possible means of inhibition at the gene and protein levels may be used.

[0023] According to a specific embodiment of the present invention, the inhibitor of the present invention is a nucleic acid molecule that inhibits the expression of the gene; or an antibody that specifically binds to a protein encoded by the gene, an antigen-binding fragment thereof, or an aptamer.

[0024] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman; Chemical Reviews , 90:543-584(1990)).

[0025] In this specification, the term “nucleic acid molecule that inhibits expression” refers to a nucleic acid molecule that specifically recognizes a target gene by including a complementary nucleic acid sequence that can hybridize with the target gene and causes a modification on the nucleotide structure that causes a decrease in its function, and includes, for example, the shRNA, siRNA, miRNA, ribozymes, PNA, antisense oligonucleotides, and gRNA included in the CRISPR system described above.

[0026] In this specification, the term “complementary” means that the nucleic acid molecule for expression inhibition is sufficiently complementary to selectively hybridize to the target nucleic acid sequence under specific annealing or hybridization conditions, and encompasses both substantially complementary and perfectly complementary, preferably meaning perfectly complementary. In this specification, the term “substantially complementary sequence” means that not only a perfectly matching sequence but also a sequence that is partially mismatched with the sequence to the extent that sequence-specific hybridization can occur upon annealing to a specific sequence.

[0027] In this specification, the term “shRNA (small hairpin RNA)” is 인 비보 It refers to an RNA sequence consisting of 50 to 70 nucleotides forming a single strand that forms a stem-loop structure, creating a tight hairpin structure to suppress the expression of a target gene through RNA interference. Typically, a long RNA of 19 to 29 nucleotides forms base pairs complementarily on both sides of a loop region of 5 to 10 nucleotides to form a double-stranded stem, and it is transduced into a cell via a vector containing a U6 promoter to ensure constant expression, and is usually transferred to daughter cells to inherit the suppression of the target gene's expression.

[0028] In this specification, the term “siRNA” refers to a short double-stranded RNA capable of inducing RNAi (RNA interference) phenomena through the cleavage of specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and both blunt and cohesive ends are possible as long as the expression of the target gene can be suppressed by the RNAi effect. The cohesive end structure can be either a structure with a protruding 3-terminal end or a structure with a protruding 5-terminal end.

[0029] In this specification, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is not expressed within a cell, has a short stem-loop structure, and inhibits target gene expression through complementary binding to the mRNA of the target gene.

[0030] In this specification, the term “ribozyme” refers to a type of RNA molecule that functions like an enzyme by recognizing a specific RNA base sequence and cleaving it. A ribozyme consists of a region that specifically binds to the base sequence complementary to the target mRNA strand and a region that cleaves the target RNA.

[0031] In this specification, the term “PNA (Peptide nucleic acid)” refers to a molecule that possesses the properties of both nucleic acids and proteins and is capable of binding complementarily to DNA or RNA. PNA is not found in nature but is artificially synthesized through chemical methods; it regulates the expression of target genes by forming a double strand through hybridization with natural nucleic acids of complementary base sequences.

[0032] In this specification, the term “antisense oligonucleotide” refers to a nucleic acid molecule that is a nucleotide sequence complementary to the sequence of a specific mRNA, which binds to the complementary sequence within the target mRNA to inhibit essential activities regarding its translation into protein, translocation into the cytoplasm, maturation, or any other overall biological function. Antisense oligonucleotides may be modified at one or more bases, sugars, or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995). The oligonucleotide backbone can be modified into phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatomics, heterocyclic glycosholfonates, etc.

[0033] In this specification, the term “gRNA (guideRNA)” refers to an RNA molecule used in a gene editing system that recognizes a target gene and specifically cleaves the recognized site by inducing a nuclease. A representative example of such a gene editing system is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0034] The nucleic acid molecule of the present invention described above is expressed within a subject (e.g., a cancer patient) by WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , CTRL or OSGEPL1 The expression of can be suppressed at the gene level.

[0035] In this specification, the term “to express” means that a gene becomes replicable within a subject’s cell as an extrachromosomal factor or through the completion of chromosomal integration by artificially introducing it using a gene carrier to cause the subject to express an exogenous gene or to increase the natural expression level of an endogenous gene. Accordingly, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.”

[0036] In this specification, the term “gene delivery system” refers to any means of transporting a gene into a cell, and gene delivery has the same meaning as the transduction of a gene. At the tissue level, the term gene delivery has the same meaning as the spread of a gene. Accordingly, the gene delivery system of the present invention may be described as a gene transduction system and a gene spread system.

[0038] According to the present invention, the inhibitor of the present invention may be a specific antibody that inhibits the activity of the 13 genes listed above at the protein level. The antibody that specifically recognizes the protein encoded by these genes is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

[0039] The antibodies of the present invention are produced by methods commonly practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), recombinant DNA method (U.S. Patent No. 4,816,567) or phage antibody library method (Clackson et al, Nature , 352:624-628 (1991) and Marks et al. J. Mol. Biol. It can be manufactured according to , 222:58, 1-597(1991)). The general process for antibody production is Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; and Zola, H.; Monoclonal Antibodies: A Manual of Techniques It is described in detail in CRC Press, Inc., Boca Raton, Florida, 1984.

[0040] In this specification, the term “antigen binding fragment” refers to a portion of the entire immunoglobulin structure to which an antigen can bind, including, but not limited to, F(ab')2, Fab', Fab, Fv, and scFv.

[0041] In this specification, the term “specifically binding” has the same meaning as “specifically recognizing,” and means that an antigen and an antibody (or a fragment thereof) interact specifically through an immunological reaction.

[0042] The present invention may inhibit the activity of a target protein by using an aptamer that specifically binds to the target protein instead of an antibody. In this specification, the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. For general information on aptamers, see Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R. “An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. This is disclosed in detail in 95(24):14272-7(1998).

[0043] In addition, the inhibitors used in the present invention also include small molecule compound inhibitors for the 13 genes mentioned above. Small molecule inhibitors known in the art are listed exemplarily in Table 6 below, but are not limited thereto, and any small molecule compound that reduces the expression or activity of the genes of the present invention to a measurable level may be used.

[0044] According to a specific embodiment of the present invention, the composition of the present invention is WNK3 or SIK3 It contains an inhibitor of as an active ingredient.

[0045] More specifically, the above inhibitor is WNK3 or SIK3 A nucleic acid molecule that inhibits the expression of; WNK3 or SIK3 It is an antibody that specifically binds to the protein encoded by, its antigen-binding fragment or aptamer; one or more inhibitors selected from the group consisting of WNK463; WNK Inhibitor 11; dasatinib; YKL-06-062; and YKL-05-099.

[0046] In the present invention, the term “WNK Inhibitor 11” is WNK3 It is a substance with inhibitory ability, with CAS number 2123489-30-3.

[0047] In the present invention, the term “WNK463” is WNK3 It is a substance with inhibitory ability, with CAS number 2012607-27-9.

[0048] In the present invention, the term “dasatinib” is SIK3 It is a substance with inhibitory ability, with CAS number 302962-49-8.

[0049] In the present invention, the term “YKL-06-062” is SIK3 It is a substance with inhibitory ability, with CAS number 2172617-16-0.

[0050] In the present invention, the term “YKL-05-099” is SIK3 It is a substance with inhibitory ability, with CAS number 1936529-65-5.

[0052] According to a specific embodiment of the present invention, the composition of the present invention reduces the expression or activity of PD-L1 (Programmed Death-Ligand 1).

[0054] According to another aspect of the present invention, the present invention provides a composition for the prevention or treatment of cancer comprising the composition of the present invention described above as an active ingredient.

[0055] In this specification, the term “prevention” means suppressing the occurrence of a disease or illness in subjects who have not been diagnosed with having such a disease or illness but are at risk of developing such a disease or illness.

[0056] In this specification, the term “treatment” means (a) inhibition of the progression of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. When the composition of the present invention is administered to a subject, anticancer immune activity is significantly restored by inhibiting the expression of PD-L1, thereby serving to inhibit, eliminate, or alleviate the progression of symptoms caused by tumors. Accordingly, the composition of the present invention may serve as a therapeutic composition for cancer itself, or it may be applied as an adjuvant for cancer treatment when administered together with other anticancer pharmacological components. Accordingly, in this specification, the terms “treatment” or “therapeutic agent” include the meaning of “therapeutic aid” or “therapeutic adjuvant.”

[0057] In this specification, the terms “administration” or “to administer” refer to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that an equal amount is formed within the subject’s body.

[0058] In the present invention, the term “therapeutic effective amount” refers to the content of a composition in which the pharmacological component within the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and includes the meaning of “preventive effective amount.”

[0059] In this specification, the term “object” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the object of the present invention is a human.

[0061] The composition of the present invention can prevent or treat all tumors having immune response evasion mechanisms without limitation by blocking immune cell suppression by the PD-L1 / PD-1 axis. For example, cancers that can be prevented or treated by the composition of the present invention include, but are not limited to, colorectal cancer, lung cancer, gastric cancer, glioma, liver cancer, melanoma, renal cell adenocarcinoma, urothelial carcinoma, head and neck cancer, Merkel-cell carcinoma, prostate cancer, multiple myeloma, breast cancer, colon cancer, rectal cancer, pancreatic cancer, brain cancer, glioblastoma among brain cancers, ovarian cancer, bladder cancer, bronchial cancer, skin cancer, cervical cancer, endometrial cancer, esophageal cancer, thyroid cancer, and bone cancer.

[0062] According to a specific embodiment of the present invention, the cancer prevented or treated by the composition of the present invention is selected from the group consisting of lung cancer, melanoma, brain cancer, renal cell adenocarcinoma, and colorectal cancer.

[0063] According to a specific embodiment of the present invention, the brain cancer of the present invention is a glioblastoma.

[0065] According to another aspect of the present invention, the present invention provides a screening method for a composition for immune checkpoint inhibitors comprising the following steps:

[0066] (a) WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , PAN3 , CTRL, SMAD4 and OSGEPL1 A step of contacting a candidate substance with a biological sample comprising one or more genes selected from a group consisting of, proteins encoded by them, or cells expressing them;

[0067] (b) a step of measuring the activity or expression amount of the gene or protein in the sample,

[0068] If the activity or expression level of the above gene or protein is reduced, the above candidate substance is determined to be a composition for inhibiting immune checkpoints.

[0069] In the present invention, the term “biological sample” refers to a sample obtained from mammals, including humans. WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , PAN3 , CTRL, SMAD4 or OSGEPL1 Any sample containing a gene, its encoding protein, or a cell expressing them, including but not limited to tissues, organs, cells, or cell culture media.

[0070] According to a specific embodiment of the present invention, the biological sample is a biological sample derived from cancer tissue. More specifically, the cancer may include, without limitation, any tumor having a mechanism to evade an immune response by the PD-L1 / PD-1 axis, and may be selected from the group consisting of, for example, lung cancer, melanoma, brain cancer, renal cell adenocarcinoma, and colorectal cancer.

[0071] The term “candidate substance” used in reference to the screening method of the present invention refers to an unknown substance used in screening to test whether it affects the activity or expression level of the aforementioned 13 genes by adding it to a sample containing cells expressing one or more of the aforementioned 13 genes. The test substance includes, but is not limited to, compounds, nucleotides, peptides, and natural extracts. The step of measuring the expression level or activity of the said genes in a biological sample treated with the test substance may be performed by various expression level and activity measurement methods known in the art. If, as a result of the measurement, the expression level or activity of the said genes is reduced, the test substance may be determined to be a composition for inhibiting immune checkpoints.

[0072] According to a specific embodiment of the present invention, the brain cancer of the present invention is a glioblastoma.

[0074] According to another aspect of the present invention, the present invention WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , PAN3 , CTRL , SMAD4 and OSGEPL1 A composition for measuring PD-L1 expression or activity is provided, comprising as an active ingredient a preparation for measuring the expression level of one or more genes selected from a group consisting of said genes or a protein encoded by said gene.

[0075] As described above, the inventors of the present invention WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , PAN3 , CTRL , SMAD4 and OSGEPL1 of It was first identified that 13 genes show a high correlation with the expression of PD-L1, a ligand of immune checkpoint receptors. Therefore, the expression levels of these genes or proteins can serve as reliable indicators for evaluating the expression or activity of PD-L1, and furthermore, the degree of immune evasion response and T cell suppression caused by PD-L1. Accordingly, the term “composition for measuring PD-L1 expression or activity” has the same meaning as “composition for diagnosing diseases (e.g., cancer) caused by PD-L1 overexpression or overactivation.”

[0076] In this specification, the term “diagnosis” includes the determination of an individual’s susceptibility to a specific disease, the determination of whether an individual currently possesses a specific disease, and the determination of the prognosis of an individual afflicted with a specific disease.

[0077] In this specification, the term “diagnostic composition” refers to an integrated mixture or device comprising means for measuring the genes listed above or proteins encoded therein to evaluate the PD-L1 expression (activity) level of a subject and to predict the resulting immune evasion response and the degree of T cell suppression, and may also be expressed as a “diagnostic kit.”

[0078] According to a specific embodiment of the present invention, a preparation for measuring the expression level of the gene is a primer or probe that specifically binds to a nucleic acid molecule of the gene.

[0079] As used herein, the term “primer” refers to an oligonucleotide that acts as an initiator for synthesis under conditions in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, namely, in the presence of a polymerizing agent such as a nucleotide and DNA polymerase, and under suitable temperature and pH conditions. Specifically, the primer is a single strand of deoxyribonucleotide. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides, and may also include ribonucleotides.

[0080] The primer of the present invention may be an extension primer that is annealed to a target nucleic acid and forms a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, and extends to the location where the immobilized probe is annealed and occupies the site where the probe is annealed.

[0081] The extension primer used in the present invention is a target nucleic acid, for example WNK3 It includes a hybridized nucleotide sequence that is complementary to a specific base sequence, such as a gene. The term “complementary” means that it is sufficiently complementary to the extent that a primer or probe selectively hybridizes to a target nucleic acid sequence under specific annealing or hybridization conditions, and encompasses both cases of being substantially complementary and perfectly complementary, specifically meaning the case of being perfectly complementary. In this specification, the term “substantially complementary sequence” includes not only sequences that match perfectly, but also sequences that are partially mismatched with the sequence to the extent that they can serve as a primer by annealing to a specific sequence.

[0082] Primers must be long enough to prime the synthesis of the extension product in the presence of a polymer. The suitable length of the primer is determined by a number of factors, such as temperature, pH, and the source of the primer, but is typically 15 to 30 nucleotides. Shorter primer molecules generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The design of such primers can be easily carried out by a person skilled in the art by referring to the target nucleotide sequence, for example, using a primer design program (e.g., PRIMER 3 program).

[0083] In this specification, the term “probe” refers to a natural or modified monomer or a linear oligomer having bonds comprising deoxyribonucleotides and ribonucleotides capable of hybridizing to a specific nucleotide sequence. Specifically, the probe is single-stranded for maximum efficiency in hybridization, and more specifically, is a deoxyribonucleotide. As a probe used in the present invention, WNK3 A sequence that is perfectly complementary to a specific nucleotide sequence of the gene of the present invention may be used, but a sequence that is substantially complementary may also be used within a range that does not interfere with specific hybridization. Generally, since the stability of the duplex formed by hybridization tends to be determined by the matching of the terminal sequences, it is preferable to use a probe that is complementary to the 3'-terminus or 5'-terminus of the target sequence.

[0084] Conditions suitable for hybridization are Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach This can be determined by referring to the information disclosed in , IRL Press, Washington, DC (1985).

[0086] According to a specific embodiment of the present invention, the agent for measuring the expression amount of the protein is an antibody that specifically binds to the protein, its antigen-binding fragment, or an aptamer. Since the antibody, its antigen-binding fragment, and aptamer used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0088] According to one aspect of the present invention, the present invention WNK3 An immune-enhancing composition is provided that includes a gene inhibitor as an active ingredient.

[0089] According to a specific embodiment of the present invention, the composition of the present invention enhances the activity of T cells. More specifically, the T cells whose activity is enhanced by the composition of the present invention include, but are not limited to, CD4+ T cells; CD8+ T cells; and CD4+CD8+ T cells. The present invention may also be used as a therapeutic adjuvant to further enhance the anticancer activity of an immunotherapy by activating CAR-T cells (Chimeric antigen receptor T cells) designed to target tumor antigens.

[0090] In this specification, the term "CD4+ T cell" also referred to as "CD4+ T lymphocyte" refers to a T cell that expresses CD4 on its surface. CD4+ T cells include naive CD4+ T cells (CD4+ TN), helper T cells (CD4+ TH), memory stem CD4+ T cells (CD4+ TMSc), central memory CD4+ T cells (CD4+ TCM), effector memory CD4+ T cells (CD4+ TEM), effector CD4+ T cells (CD4+ TE), or any combination thereof.

[0091] In this specification, the term “CD8+ T cell” refers to a T cell that expresses CD8 on its surface, also referred to as “CD8+ T lymphocyte.” CD8+ T cells include naive CD8+ T cells, cytotoxic T lymphocytes (CTL), memory stem CD8+ T cells (CD8+ TMsc), central memory CD8+ T cells (CD8+ TCM), effector memory CD8+ T cells (CD8+ TEM), effector CD8+ T cells (TE), or any combination thereof.

[0092] In this specification, the term “CD4+CD8+ T cell” refers to a double-positive T cell that expresses both CD4 and CD8.

[0093] In this specification, the term “CAR-T cell” refers to a T cell expressing a chimeric antigen receptor. In this specification, the term “chimeric antigen receptor” refers to a recombinant fusion protein having an antigen-specific extracellular domain coupled to an intracellular domain that directs the cell to perform a specific function upon antigen binding, and may also be referred to as an “extracellular domain,” “artificial T cell receptor,” “chimeric T cell receptor,” and “chimeric immune receptor.” The chimeric antigen receptor is distinguished from other antigen binders by its ability to bind to MHC-independent antigens and transmit activation signals through its intracellular domain.

[0094] In the present invention, immune enhancement refers to the function of enhancing biological defense capabilities by regulating immunity.

[0096] According to a specific embodiment of the present invention, the inhibitor is a nucleic acid molecule that inhibits the expression of the gene; or an antibody, its antigen-binding fragment, or an aptamer that specifically binds to a protein encoded by the gene; or a small molecule compound capable of inhibiting the activity of a protein encoded by the gene. Since the antibody, its antigen-binding fragment, and the aptamer used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0097] In the present invention, the term “small molecule compound” is also referred to as “small molecule inhibitor,” and generally refers to a compound having a small molecular weight of 500 Da or less that has the effect of inhibiting the expression or activity of a target protein. Specifically, it may be a compound listed in Table 6 of the specification of the present invention.

[0099] According to one aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of an immunodeficiency disease or an infectious disease comprising the immune-enhancing composition of the present invention as an active ingredient.

[0100] Infectious diseases that can be prevented or treated using a pharmaceutical composition for the prevention or treatment of infectious diseases comprising the immune-enhancing composition of the present invention as an active ingredient are diseases caused by infection with viruses or pathogens, and the concept includes all diseases that can be transmitted and contracted through the respiratory tract, blood, skin contact, etc. Such infectious diseases include, for example, hepatitis B and C, human papilloma virus (HPV) infection, cytomegalovirus infection, viral respiratory diseases, and influenza, but are not limited thereto.

[0102] According to a specific embodiment of the present invention, the immunodeficiency disease is selected from the group consisting of congenital immunodeficiency, cancer, chronic viral infection, immunodeficiency after bone marrow transplantation, bacterial sepsis, immunodeficiency after anticancer treatment, and immunodeficiency caused by stroke.

[0103] According to one aspect of the present invention, the present invention provides a screening method for an immune-enhancing composition comprising the following steps:

[0104] (a) WNK3 A step of contacting a candidate substance with a biological sample comprising a gene, a protein encoded by the said gene, or a cell expressing the same;

[0105] (b) a step of measuring the activity or expression amount of the gene or protein in the sample,

[0106] If the activity or expression level of the above gene or protein is reduced, the above candidate substance is determined to be an immune-enhancing composition.

[0107] In the present invention, the term “biological sample” refers to a sample obtained from mammals, including humans. WNK3 All samples containing cells expressing, including but not limited to tissues, organs, cells, or cell culture media.

[0108] The term “candidate substance” used when referring to the screening method of the present invention has already been described above, so its description is omitted to avoid excessive duplication.

[0109] According to a specific embodiment of the present invention, the biological sample is a biological sample derived from human blood.

[0111] According to one aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer for co-administration with an immune checkpoint inhibitor.

[0112] According to a specific embodiment of the present invention, the WNK3 inhibitor of the present invention is WNK463, and the immune checkpoint inhibitor of the present invention is a PD-1 inhibitor. More specifically, the PD-1 inhibitor is an anti-PD-1 antibody. Effects of the invention

[0114] The features and advantages of the present invention are summarized as follows:

[0115] (a) The present invention WNK3 The present invention provides a composition for inhibiting immune checkpoints comprising an inhibitor, and a composition for preventing or treating cancer or for enhancing immunity comprising the same.

[0116] (b) The composition of the present invention inhibits PD-L1 expression by It exhibits multifaceted anticancer activity by increasing the sensitivity of tumors to immune cells while promoting the secretion of GRANZYME B and Perforin by T cells.

[0117] (c) In addition, the screening method of the present invention can rapidly and reliably identify candidate substances for fundamental anticancer therapeutic agents that efficiently suppress the expression of PD-L1, which plays a key role in the immune evasion mechanism of tumor cells, and significantly improve the sensitivity of cancer cells to immune responses. Brief explanation of the drawing

[0119] Figure 1 shows the results of shRNA screening for pharmacological and oncogeneic genes regulating PD-L1 expression. Figures 1a and 1b show the membranes in NSCLC cells 24 hours after exposure to 10 ng / mL IFN-γ. This figure shows the results of measuring PD-L1 levels by flow cytometry (Fig. 1a) and immunoblotting (Fig. 1b). Fig. 1c shows a schematic diagram of the shRNA screening process. FACS was performed on H2009 cells transfected with lentiviral vectors expressing shRNA libraries at three time points (t0, t1, and t2), representing 3, 13, and 20 days after the initiation of puromycin screening, respectively, to classify them into PD-L1-high, -intermediate, and -low cells. The shRNA sequences inserted into the genomic DNA were amplified by PCR and subjected to next-generation sequencing (NGS). Fig. 1d shows the results of analyzing cells expressing shRNAs for core essential or non-essential genes. The left side shows the cumulative distribution function (CDF) for the RIGER rank distribution of the CCE (constitutive core essential, red) and non-essential (NE, blue) gene groups (Hart T et al.). 9 The graph on the right shows the variance of genes exhibiting a log2-fold change (t2 / t0). Figure 1e shows the results of gene-set amplification analysis for hit genes. Significantly increased gene sets (p<0.001) are represented as enrichment maps along with baseline parameters, where circles represent each gene set and connecting lines between circles represent the overlap between gene sets. The size of the circles is proportional to the number of genes within the gene set, and the color of the circles indicates the statistical significance of the gene set. Figure 2 is a figure showing the results of genetic and chemical verification for PD-L1 regulatory genes. Figure 2a is PD-L1 positive regulator genes are shown. The relative median fluorescence intensity (MFI) of PD-L1 in shRNA-expressing H2009 cells is shown on the left compared to the negative control (shNC; black) (N=1). Repression of target genes by the corresponding shRNAs was measured by qRT-PCR and is shown on the right. The most effective (light blue) shRNA and the next most effective (blue) shRNA in the screening for each gene are indicated, respectively. Error bars represent ± standard deviation (N=2). * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, not significant (ns) p >= 0.05; Statistical significance is two-sided independent student t- It was calculated by testing. Figures 2b and 2d are PD-L1 positive regulator genes are shown. The relative median fluorescence intensity (MFI) of PD-L1 in shRNA-expressing H2009 and H460 cells is shown on the left compared to the negative control (shNC; black) (N=3). Repression of target genes by the corresponding shRNAs was measured by qRT-PCR and is shown on the right. Figure 2b shows the best (blue) and next best (light blue) shRNAs in the screening for each gene, respectively. Error bars represent ± standard deviation (N=3). * p <0.05, ** p <0.01, *** p <0.001; statistical significance is two-sided independent student t-It was calculated by testing. Figure 2c shows the results of flow cytometry on PD-L1 in H460 cells transfected with shRNA of the 13 genes that best inhibit PD-L1. Figures 2e and 2f show the results of chemical verification for hit genes using H460 cells. In Figure 2f, flow cytometry (left) and cell viability analysis (right) for PD-L1 in H460 cells were performed after treating the cells for 5 days with two different concentrations of small molecule inhibitors of each gene or with DMSO (vehicle control) at the same concentration as the inhibitor. Unstained controls are shown at the top (black). Error bars represent ± standard deviation (N=3). * p <0.05, ** p <0.01, *** p <0.001, no significance (ns) p >= 0.05; statistical significance was calculated by a two-sided independent Student's t-test. Fig. 3 is 인 비트로 This figure shows the immunomodulatory effect of the candidate gene under co-culture conditions. Fig. 3a is sh PD-L1 Alternatively, this is the result of flow cytometry for PD-L1 in H460 cells transduced as a negative control (shNC). Figure 3b shows sh cells co-cultured for 72 hours with unstimulated PBMCs (1:0) or IL-2 and anti-CD3-activated PBMCs (1:5, 1:10). PD-L1 Alternatively, this is a figure showing the relative viability of shNC-expressing H460 cells. Error bars represent ± standard deviation (N=3). ** p <0.01, not significant(ns) p >= 0.05; Statistical significance is two-sided independent student t- Calculated by assay. Relative cell viability (%, sh) under each stimulation condition. PD-L1 / shNC) is indicated in parentheses. Figure 3c shows the relative viability of shPD-L1 or shNC-expressing H460 cells co-cultured with IL-2 and anti-CD3-activated PBMCs (1:5, 1:10) for 72 hours compared to co-culture conditions with unstimulated PBMCs. Error bars represent ± standard deviation (N=3). *** p <0.001; statistical significance is two-sided independent student t- It was calculated by the assay. Figure 3d shows sh co-cultured for 72 hours with PBMC not co-cultured (1:0) or IL-2 and anti-CD3-activated PBMC (1:5, 1:10). WNK3 Alternatively, this is a figure showing the relative viability of shNC-expressing H2009 cells. Error bars represent ± standard deviation (N=3). ** p <0.01, *** p <0.001; Two-sided independent Student's T-test. Relative cell viability (%, sh) under each stimulation condition WNK3 / shNC) is indicated in parentheses. Figure 3e shows sh co-cultured with IL-2 and anti-CD3-activated PBMC (1:5, 1:10) for 72 hours. WNK3 Alternatively, this figure shows the relative survival rate of shNC-expressing H2009 cells against the condition not co-cultured with PBMC (1:0). Error bars represent ± standard deviation (N=3). p <0.05; two-sided independent Student's T-test. Figure 3f shows the results of adding activated T cells to H2009 cells after treating them with 1 μM WNK463 or DMSO vehicle controls for 9 days. Co-culture was performed for 48 hours in the presence or absence of the inhibitor. Relative viability of H2009 cells pretreated with WNK463 or DMSO vehicles for 9 days and then co-cultured with PBMCs (1:0) or IL-2 and anti-CD3-activated PBMCs (1:5, 1:10) for 48 hours is shown, and error bars represent ± standard deviation (N=3).p <0.05, *** p <0.001; two-sided independent student t -Test. Relative cell viability (%, WNK463 / DMSO) under each stimulation condition is indicated in parentheses. Figure 3g shows the results of adding activated T cells to H2009 cells after treating them with 1 μM WNK463 or DMSO vehicle controls for 9 days. Co-culture was performed for 48 hours in the presence or absence of the inhibitor. Relative viability of H2009 cells co-cultured for 48 hours with IL-2 and anti-CD3-activated PBMCs (1:5, 1:10) after 9 days of pretreatment with WNK463 or DMSO vehicles versus the non-co-culture condition (1:0) is shown, with error bars representing ± standard deviation (N=3). ** p <0.01; two-sided independent student t -Test. Figure 3h shows the cell viability (left) and secreted GRANZYME B (right) measurements of PBMCs 72 hours after treatment with WNK463 or the DMSO vehicle control. Error bars represent ± standard deviation (N=2). * p <0.05; one-way independent student t- Analysis. Figure 3i shows the results of cell viability (left) (N=2) and secreted IFN-γ, TNF-α, and IL-10 (right) (N=3) of PBMCs 72 hours after treatment with WNK463 or the DMSO vehicle control. Error bars indicate ± standard deviation. ** p <0.01, *** p <0.001; one independent student t- Test. Fig. 3j shows sh stimulated with anti-CD3 / CD28 beads at different bead-to-cell ratios (0:1, 5:1, 10:1) (N=3). WNK3 Or, this is the result of measuring IL-2 (right) secreted from Jurkat T cells expressing shNC. In Jurkat T cells WNK3 Deficiency was measured by qRT-PCR (left) (N=2). Error bars represent ± standard deviation. * p <0.05, ** p <0.01; Two-sided independent Student's T-test. Relative IL-2 concentration (%, sh) under each stimulus condition WNK3 / shNC) is indicated in parentheses. Figure 3k shows the relative viability of H2009 cells not co-cultured with PBMCs in YKL-06-062 or DMSO vehicles, or co-cultured for 96 hours with IL-2 and anti-CD3-activated PBMCs (1:5, 1:10). Error bars represent ± standard deviation (N=3). * p <0.05, *** p <0.001; two-sided independent student t- Test. Relative cell viability (%, YKL-06-062 / DMSO) under each stimulation condition is indicated in parentheses. Figure 31 shows the relative viability of H2009 cells co-cultured for 96 hours with IL-2 and anti-CD3-activated PBMCs (1:5, 1:10) in YKL-06-062 or DMSO vehicles compared to the non-co-culture condition (1:0). Error bars represent ± standard deviation (N=3). * p <0.05; two-sided independent student t- Analysis. Figure 3m shows the results of measuring cell viability (left) and secreted GRANZYME B (right) of PBMCs 72 hours after treatment with the pan-SIK inhibitor or DMSO vehicle control. Error bars represent ± standard deviation (N=2). * p <0.05; one-way independent student t -Test. Figure 3n shows the results of measuring cell viability (left) and secreted GRANZYME B (right) of PBMCs 72 hours after treatment with the pan-SIK inhibitor or DMSO vehicle control. Error bars represent ± standard deviation (N=2). * p <0.05, ** p <0.01, ***p <0.001; One-way ANOVA and Tukey's multiple comparison test. Figure 4 is a figure showing the results confirming that WNK3 is a transcription regulator of PD-L1. Figure 4a is WNK3 In knockdown cells PD-L1 Shows mRNA levels in H2009 and H460 cells transfected with specified shRNA. WNK3 (Left) and PD-L1 (Right) mRNA levels were measured by qRT-PCR. Error bars represent ± standard deviation (N=2). * p <0.05, ** p <0.01; statistical significance is two-sided independent student t- It was calculated through testing. Figure 4b is WNK3 In knockdown cells PD-L1 Shows mRNA levels in H2009 and H460 cells transfected with specified shRNA. WNK3 (Left) and PD-L1 (Right) mRNA levels were measured by qRT-PCR. Error bars represent ± standard deviation (N=3). ** p <0.01, *** p <0.001; statistical significance is two-sided independent student t- It was calculated through testing. Figure 4c is WNK3 This shows bar graphs quantitatively illustrating the results of immunoblotting on whole-cell lysates of H2009 and H460 cell lines to measure the effect of knockdown on PD-L1 protein levels, and the results of image-based quantitative analysis for PD-L1, respectively. Fig. 4d is WNK3 In overexpressing cells PD-L1 This is a figure showing mRNA levels. By qRT-PCR WNK3 In H2009 and H460 cells transfected with cDNA plasmid or control plasmid WNK3 (Left) and PD-L1mRNA levels were measured on the (right). Error bars represent ± standard deviation (N=2). * p <0.05, ** p <0.01; two-sided independent student t- Test. Fig. 4e is WNK3 This bar graph quantitatively shows the results of immunoblotting on whole-cell lysates of H2009 and H460 cell lines to measure the effect of overexpression on the expression of each protein, and the results of image-based quantitative analysis for PD-L1, respectively. Error bars represent ± standard deviation (N=3). p <0.05; two-sided independent Student's t-test. Figure 4f shows the results after treatment with a specified concentration of WNK463 in H460 cells. PD-L1 This figure shows the results of measuring mRNA (left) and protein (right) levels using qRT-PCR and immunoblotting, respectively. Error bars represent ± standard deviation (N=2). p <0.05; not significant(ns) p >= 0.05; two-sided independent Student's T-test. Fig. 4g after treatment with specified concentrations of WNK463 in H460 cells PD-L1 This shows bar graphs quantitatively illustrating mRNA (left) and protein (right) levels measured by qRT-PCR and immunoblotting, respectively, as well as the results of image-based quantitative analysis for PD-L1. Error bars represent ± standard deviation (N=3). p <0.05, ** p <0.01, *** p <0.001; Two-sided independent Student's T-test. Figure 4h is sh WNK3 Alternatively, it shows the results of flow cytometry for MHC class I in H460 and H2009 cells expressing shNC (left) and H460 cells 72 hours after WNK463 treatment (right). Figure 5 illustrates that WNK3 maintains PD-L1 expression through the JNK / c-Jun pathway. Figures 5a and 5c are These are the results of measuring membrane PD-L1 levels by flow cytometry in specified cell lines treated with IFN-γ (20 ng / mL) or medium alone. Cells were transfected with specified shRNA (Fig. 5a) or treated with WNK463 (1 μM) (Fig. 5c). Figs. 5b and 5d are These are the results of flow cytometry measurements of membrane PD-L1 levels in designated cell lines treated with IFN-γ (10 ng / mL) or medium alone. Cells were transfected with designated shRNA (Fig. 5b) or treated with WNK463 (1 μM) (Fig. 5d). Error bars represent ± standard deviation (N=3). ** p <0.01, *** p <0.001; two-sided independent Student's t-test. Figure 5e in H2009 and H460 cell lines WNK3 This is the result of investigating, via immunoblotting, the effect of knockdown on the phosphorylation levels of known PD-L1 transcription regulators (JNK, c-Jun, ERK, P38, AKT, p65, and STAT3). Differently expressed proteins are indicated by arrowheads. Figure 5f shows the result of investigating, via immunoblotting, the effect of JNK inhibition using JNK-IN-8 on PD-L1 levels in H2009 cells. H2009 cells were treated with a specified concentration of JNK-IN-8 for 48 hours. Figure 5g is WNK3 This is the result of investigating the effect of JNK inhibition using JNK-IN-8 on PD-L1 levels in H2009 cells transfected with a cDNA plasmid or a control plasmid by immunoblotting. H2009 cells were treated with specified concentrations of JNK-IN-8 for 48 hours. Figure 5h is sh WNK3Alternatively, this figure illustrates the effect of AEBSF-induced JNK activation on PD-L1 levels in H2009 and H460 cells expressing control shRNA. H2009 cells were treated with 100 μM AEBSF for 24 hours, and H460 cells were treated with 250 μM AEBSF for 8 hours. Figure 5i shows a schematic diagram of the WNK3 protein domain. Conserved catalytic lysine (Lys 159) within the kinase domain is indicated in black. Figures 5j and 5k for H2009 cells WNK3 Wild type, WNK3 This is the result of evaluating the effect of K159M and negative control plasmid transfection on each protein expression by immunoblotting. Figure 6 illustrates that genetic and chemical inhibition of WNK3 increases the sensitivity of PD-L1-dependent synonymous tumors to anti-PD1 therapy. Figure 6a is Wnk3 - In deficient MC38 cells Pd-l1 Shows the expression level. si WNK In MC38 cells transfected with 3 or control siRNA Wnk 3 (left) and Pd-l1 mRNA levels (on the right) were measured by qRT-PCR. Error bars represent ± standard deviation (N=2). * p <0.05, ** p <0.01; statistical significance is two-sided independent student t- It was calculated by testing. Figure 6b is Wnk3 - In deficient MC38 cells Pd-l1 Shows the expression level. si WNK In MC38 cells transfected with 3 or control siRNA Wnk 3 (left) and Pd-l1 mRNA levels (on the right) were measured by qRT-PCR. Error bars represent ± standard deviation (N=3). ** p <0.01, *** p <0.001; statistical significance is two-sided independent student t-It was calculated by testing. Figure 6c is Wnk3 This shows the results of investigating the effect of knockdown on PD-L1 protein levels through immunoblotting on MC38 cells. Figure 6d shows the results of measuring PD-L1 membrane levels by flow cytometry 72 hours after treatment with WNK463 at specified concentrations. Figure 6e shows the results of performing immunoblotting with specified antibodies after treating MC38 cell lines with different concentrations of WNK463. Figure 6f is In Vibo A schematic diagram of the experimental protocol is shown. Figure 6g illustrates the effect of WNK463 treatment on MC38 tumor growth with or without concomitant treatment with CD8 antibodies (right). Tumor volume was measured at designated times. The arrowhead indicates Day 1 after WNK463 injection. Error bars represent the mean ± standard error. * p <0.05, ** p <0.01, not significant(ns) p >= 0.05 ; Two-sided independent student T- Test. n = 5 mice per group. Fig. 6h is CD8 + This figure shows the weight of MC38 tumors harvested from T-cell competent mice (Fig. 6g, left). * p <0.05, ** p <0.01; Significance was calculated by performing standard one-way ANOVA and Tukey's multiple comparison test using single pooled variance. Figure 6i shows the results of measuring apoptosis (Figure 6g, left) in extracted MC38 tumor cells via Annexin-V staining. p <0.05, ** p Standard one-way ANOVA and Tukey's multiple comparison test were performed using single pooled variance. Figure 6j shows the results of measuring PD-L1 levels in extracted MC38 tumor cells (Figure 6g, left) by flow cytometry. ** p <0.01, *** p<0.001; Standard one-way ANOVA and Tukey's multiple comparison test were performed using single pooled variance. Figure 6k shows tumor-infiltrating CD4 + and CD8 + This figure shows the results of flow cytometry analysis of IFN-γ and TNF-α expressed by T cells (Fig. 6g, left). p <0.05, ns, no significance, p >= 0.05; Standard one-way ANOVA and Tukey multiple comparison tests were performed using single pooled variance. Figure 61 shows the results of Kaplan-Meier survival analysis on the overall survival of lung cancer patients with high and low WNK3 expression in the TCGA (left) and E-MTAB-923 (right) cohorts. Patients had gene expression thresholds 10 They were classified based on [the criteria], and the difference in survival rates between each group was evaluated using the log-rank method. Figure 7 is a schematic diagram illustrating the synergistic anti-tumor mechanism by WNK3 inhibition. WNK3 activates the JNK / c-Jun downstream signaling cascade and promotes PD-L1 gene transcription in tumor cells, whereas CD4 + and CD8 + It inhibits the secretion of cytotoxic enzymes and cytokines by T cells. Accordingly, WNK463 treatment exerts a synergistic anti-tumor effect by suppressing tumor immune evasion and activating immune cells. Figure 8 illustrates the shRNA screening results for pharmacological and oncogenic genes regulating PD-L1 expression in relation to Figure 1. Figure 8a is a histogram showing the amounts of shRNA targeting pharmacological genes (N=5,069; left) and oncogenic genes (N=800; right). Figure 8b is a bar graph quantitatively representing the flow cytometry results of Figure 1a. The error bars represent ± standard deviation (N=4). ** p <0.01; statistical significance is two-sided independent student t-Calculation was performed using assays. Figure 8c is a bar graph quantitatively representing the quantitative values ​​of the immunoblotting densitometric analysis results in Figure 1b. Figure 8d shows the cell distribution before and after FACS sorting at t1 based on PD-L1 expression levels. The PD-L1-low cell group was defined based on unstained cells expressing control shRNA. The PD-L1-intermediate cell group (13-15% of the total), exhibiting intermediate PD-L1 expression levels, was defined based on cells expressing control shRNA, while the PD-L1-high cell group represents the top 1-2% of cells showing the highest PD-L1 expression. Figure 8e is a schematic diagram summarizing the NGS-based shRNA deconvolution process. Figure 8f shows the identification results of hit genes derived from RIGER analysis. The gene scatter plot displays the shRNAs with high enrichment scores as the first (y-axis) and second (x-axis). The enrichment of PD-L1-low and PD-L1-high cell populations is shown at the top and bottom, respectively. Hit genes (FDR < 0.05) identified by RIGER analysis are indicated by red dots. Figure 9 illustrates the results of genetic and chemical verification of PD-L1 regulatory genes in relation to Figure 2. Figure 9a shows the membrane expression levels of PD-L1 and cell viability following drug treatment. The relative amount of PD-L1 MFI (left; refer to the flow cytometry histogram in Figure 2e) and the viability of H460 cells (right) are shown 5 days after treatment with small molecule inhibitors compared to the DMSO vehicle control group. Error bars represent ± standard deviation (N=3). p <0.05, ** p <0.01, ns, no statistical significance. p >= 0.05; Statistical significance is two-sided independent student t-Calculated by assay. Figures 9b and 9c show the results of additional chemical validation using a different cell line, H2009. Flow cytometry for PD-L1 (Figure 9b) and cell viability analysis (Figure 9c) were performed after treating H2009 cells with WNK463, dasatinib, YKL-06-062, YKL-05-099, or with equal concentrations of DMSO (vehicle control). Error bars represent ± standard deviation (N=3). Figure 9d shows the results of additional chemical validation using a different cell line, H2009. Flow cytometry for PD-L1 (left) and cell viability analysis (right) were performed after treating H2009 cells with small molecule inhibitors for each gene or with equal concentrations of DMSO (vehicle control). Error bars represent ± standard deviation (N=3). * p <0.05, ** p <0.01, *** p <0.001, ns, no statistical significance. p >= 0.05; Statistical significance was calculated using a two-sided independent Student's t-test. Figure 10 is a figure showing the results confirming that WNK3 is a transcription regulator of PD-L1 in relation to Figure 4 above. Figures 10a and 10b are bar graphs quantitatively showing the image-based quantitative analysis results for PD-L1 in Figures 4c and 4e, respectively. Figure 10c is sh WNK3 It shows the effect of ectopic overexpression of WNK3, reduced by [it], on PD-L1 levels. Human WNK3 sh targeting the 3'UTR region of a gene WNK3 In H2009 cells expressing WNK3cDNA plasmids were treated. PD-L1 protein levels were measured by immunoblotting. Figure 10d is a bar graph quantitatively showing the image-based quantitative analysis results for PD-L1 in Figure 4f. Figure 10e shows the results of flow cytometry measurements of PD-L1 levels on the membranes of H2009 cell lines expressing each shRNA targeting different WNK family proteins. The knockdown efficiency of the target genes is shown in Figure 10f. Error bars represent ± standard deviation (N=2). * p <0.05, ** p <0.01; statistical significance is two-sided independent student t- It was calculated by assay. Figure 10g shows the effect of ectopic overexpression of other WNK family proteins on PD-L1 levels in H2009 cell line. Figure 11 is a figure showing that genetic and chemical inhibition of WNK3 in relation to Figure 6 increases the sensitivity of PD-L1-dependent synonymous tumors to anti-PD1 therapy. Figure 11a is sh WNK3 or HCT116 cells expressing control shRNA WNK3 (Left) and PD-L1 This is the result of measuring mRNA levels (on the right) by qRT-PCR. Error bars represent ± standard deviation (N=3). * p <0.05, ** p <0.01; statistical significance is two-sided independent student t- It was calculated using a test. Figure 11b is sh WNK3 Alternatively, this is the result of measuring membrane PD-L1 levels in HCT116 cells expressing control shRNA by flow cytometry. Fig. 11c shows CD4 extracted from untreated (Fig. 6g, left) or anti-Cd8 antibody-treated mice (Fig. 6g, right). + and CD8 + This is the result of measuring T cells by flow cytometry. *** p <0.001, ns, no statistical significance p>= 0.05; Statistical significance was calculated using standard one-way ANOVA and Tukey's multiple comparison test. Figure 11d shows the results of measuring PD-L1 levels in MC38 tumor cells (Figure 6g, right) by flow cytometry. p <0.05; statistical significance is two-sided independent student t- It was calculated using a test. Figure 12 illustrates that genetic or chemical inhibition of WNK3 enhances the production of cytokines and cytotoxic enzymes in CD4+ and CD8+ T cells. Figure 12a shows the results of flow cytometry measurements of PERRFORIN and GRANZYME B production in mouse CD4+ T cells (left) or CD8+ T cells (right) 36 hours after TCR stimulation following 6 hours of treatment with 1 μM WNK463 or DMSO (vehicle). Error bars represent ± standard error (N=3). ** p <0.01, *** p <0.001; two-sided independent Student's t-test. Fig. 12b shows sg 36 hours after TCR stimulation. Wnk3 This is the result of measuring PERFORIN and GRANZYME B levels by flow cytometry in mouse CD4+ T cells (left) or CD8+ T cells (right) transfected with a ribonucleoprotein complex composed of non-targeted sgRNA (Ctrl) and Cas9 protein. Wnk3 Depletion was measured by qRT-PCR. Error bars represent ± standard error (N=3). ** p <0.01, *** p <0.001; two-sided independent Student's t-test. Fig. 12c shows sh 36 hours after TCR stimulation. WNK3 Alternatively, this is the result of measuring the production levels of PERFORIN and GRANZYME B in human CD4+ T cells (upper) or CD8+ T cells (lower) expressing shNC by flow cytometry. WNK3 Depletion was measured by qRT-PCR. Error bars represent ± standard error (N=3). * p<0.05, ** p <0.01, *** p <0.001, ns, no statistical significance. p >= 0.05; statistical significance was calculated using a two-sided independent Student's t-test. Figure 12d shows the results after 48 hours of stimulation with anti-CD3 / CD28 beads at various bead-to-cell ratios (5:1, 10:1) (right). WNK3 This figure illustrates the IL-2 concentration by ELISA in the supernatant obtained from Jurkat T cells transfected with a lentiviral vector or an empty vector expressing a transgene. In Jurkat T cells WNK3 Overexpression was measured by qRT-PCR (left). Error bars represent ± standard deviation (N=3). * p <0.05, *** p <0.001; statistical significance was calculated using a two-sided independent Student's t-test. Figure 12e shows sg after 5 minutes of TCR stimulation. Wnk3 This is the result of measuring phospho-AKT and phospho-S6 levels by flow cytometry in mouse CD4+ T cells (left) or CD8+ T cells (right) transfected with a ribonucleoprotein complex composed of non-targeted sgRNA (Ctrl) and Cas9 protein. Wnk3 Depletion was measured by qRT-PCR. Error bars represent ± standard error (N=3). ** p <0.01, *** p <0.001, ns, no statistical significance. p >= 0.05; Statistical significance was calculated using a two-sided independent Student's t-test. Figure 12f plots phospho-AKT and phospho-S6 levels in mouse CD8+ T cells treated with 1 μM WNK463 or DMSO (vehicle) for 6 hours followed by TCR stimulation for 5 minutes. Error bars represent ± standard error (N=3). ** p <0.01, *** p<0.001; Statistical significance was calculated using a two-sided independent Student's t-test. Figure 13 illustrates the synergistic effect on tumor growth inhibition by the combination of WNK463 and PD-1 blockade. Figure 13a is in vivo This figure illustrates a schematic diagram of the experimental protocol. Figure 13b shows the results confirming the therapeutic effect of the combination of WNK463 and anti-PD-1 antibodies on MC38 tumor growth, where tumor volume was measured at the indicated time points. Error bars represent ± standard error. *** p <0.001; Statistical significance was calculated using two-way ANOVA. n = 5 mice per group. Figure 13c illustrates the results of weighing MC38 tumors obtained from mice at the endpoint. Error bars represent ± standard deviation. * p <0.05, *** p <0.001, ns, no statistical significance. p >= 0.05; Standard one-way ANOVA and Tukey's multiple comparison test were performed using single pooled variance. Figure 13d illustrates the results of evaluating PD-L1 levels in extracted MC38 tumor cells (Figure 13b) via flow cytometry. Error bars represent ± standard deviation. ** p <0.01, ns, no statistical significance. p Standard one-way ANOVA and Tukey's multiple comparison test were performed using single pooled variance. Figure 13e illustrates the results of evaluating the proportion of CD8+, PD-1+, TIM3+, and Tox+ T cells among the total CD8+ T cells in the tumor using flow cytometry. Error bars represent ± standard deviation. ** p <0.01, ns, no statistical significance. p>= 0.05; Standard one-way ANOVA and Tukey's multiple comparison test were performed using single pooled variance. Figure 13f shows the results of flow cytometry analysis of IFN-γ and GRANZYME B produced by tumor-infiltrating CD8+ T cells. Error bars represent ± standard deviation. * p <0.05, ** p <0.01, *** p <0.001, ns, no statistical significance. p >= 0.05; Standard one-way ANOVA and Tukey's multiple comparison test were performed using single pooled variance. Figure 13g illustrates the results of Kaplan-Meier survival analysis on the overall survival of colorectal (left) and gastric (right) cancer patients in the indicated cohorts with high or low WNK3 expression. Patients had a gene expression threshold 10 They were classified based on [the criteria], and the difference in survival rates between each group was evaluated using the log-rank method. Fig. 14a is sh PD-L1 Alternatively, this is the result of measuring PD-L1 deficiency in H2009 cells transduced as a negative control (shNC) by flow cytometry. Error bars represent ± standard deviation (N=3). *** p <0.001; statistical significance is two-sided independent student t- It was calculated by the assay. Figure 14b shows sh co-cultured with IL-2 and anti-CD3-activated PBMC (1:5, 1:10) for 72 hours. PD-L1 Alternatively, this figure shows the relative survival rate of shNC-expressing H2009 cells against the condition not co-cultured with PBMC (1:0). Error bars represent ± standard deviation (N=3). p <0.05; two-sided independent student t- Verification. Figure 14c is by qRT-PCR PD-L1This figure illustrates the results of flow cytometry analysis of PD-L1 levels in H2009 cells transfected with cDNA plasmids or control plasmids. Error bars represent ± standard deviation (N=3). *** p <0.001; two-sided independent student t- Verification. Fig. 14d shows IL-2 and anti-CD3-activated PBMCs (1:10) co-cultured for 48 hours in the presence or absence of an inhibitor after being pretreated with WNK463 or DMSO vehicle for 9 days. PD-L1 This figure shows the relative survival rates of H2009 cells transfected with cDNA plasmid or control plasmid against the condition without co-culture with PBMC (1:0). Error bars represent ± standard deviation (N=3). ** p <0.01; two-sided independent student t- Verification. Fig. 14e in the 7 NSCLC cell lines of Figs. 1a and 1b WNK3 (x-axis) and PD-L1 (y-axis) This is a figure showing the correlation of expression levels using Pearson correlation coefficients. Fig. 14f is In vitro These are the results of measuring the kinase activity of WNK3 wild-type and WNK3 K159M. After reacting the substance obtained by immunoprecipitation, substrate, ATP, and cofactor with HEK293 cells transfected with WNK3-Myc-Flag (wild-type), WNK3-K159M-Myc-Flag (K159M), or control plasmids, an ADP-Glo ​​kinase assay was performed (right). The WNK3 protein levels in the substance obtained by immunoprecipitation were measured by immunoblotting (left). Error bars represent ± standard deviation (N=3). p <0.05, ** p <0.01; two-sided independent student t- Black. 14g is In vitroThis shows the results of measuring the kinase activity of wild-type WNK3 following treatment with WNK463 and WNK inhibitor 11. HEK293 cells transfected with WNK3-Myc-Flag (wild type) or control plasmids were treated with 1 μM WNK463, 5 μM WNK inhibitor, or DMSO (vehicle) for 48 hours. Subsequently, the substance obtained by immunoprecipitation, substrate, ATP, and cofactor were reacted, followed by an ADP-Glo ​​kinase assay (right). The WNK3 protein level in the substance obtained by immunoprecipitation was measured by immunoblotting (left). Error bars represent ± standard deviation (N=3). *** p <0.001; One-way ANOVA and Tukey's multiple comparison test. Figure 14h shows the human glioblastoma cell line (human GMB cell lines) model, WNK3 This figure illustrates the results confirming that the level of PD-L1 decreases when the gene is knocked down with siRNA. Specific details for implementing the invention

[0120] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0122] Examples

[0123] Experimental method

[0124] cell line

[0125] Human NSCLC cell lines (H2009, H460, HCC44, HCC461, H647, H2122, A549) were provided by Michael A. White (UT Southwestern Medical Center, TX, USA). The cell lines were maintained in RPMI-1640 medium supplemented with 5% fetal bovine serum (Gibco / Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Jurkat T cell lines were purchased from ATCC (Manassas, VA, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Mouse MC38 cell lines were purchased from Kerafast (Boston, MA, USA). MC38 cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco / Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0127] Construction of an shRNA library

[0128] The list of drug-induced genes and cancer promoters is the drug-gene interaction database. 11 (Table 3) and pan-cancer genome atlas research paper 12-19 They were obtained from (Table 4), each containing 32,643 shRNAs for 5,069 drug-promoting genes and 5,611 shRNAs for 800 cancer-promoting genes. These were integrated into a single library containing 35,949 shRNAs for 5,592 genes.

[0129] To construct the shRNA pool, bacterial stock was cherry-picked from the MISSION shRNA library (Sigma-Aldrich, St. Louis, MO, USA) and plated on LB agar plates using a BioMek FXII (Beckman Coulter, Brea, CA, USA). After overnight incubation at 37°C, DNA was purified from the stirred bacterial clones using a DNA-mag plasmid DNA purification kit (#17255, iNtRON Biotechnology, Seongnam-si, Korea). HEK 293FT cells (1 x 10⁶ 7 The plasmid DNA pool was adsorbed onto a 150 mm dish overnight. Lentiviruses were prepared by co-transfecting HEK293FT cells with 7.2 μg of plasmid DNA pool, 5.4 μg of psPAX2 (provided by Didier Trono, Addgene plasmid # 12260 ; http: / / n2t.net / addgene:12260 ; RRID:Addgene_12260), and 1.8 μg of pMD2.G (provided by Didier Trono, Addgene plasmid # 12259 ; http: / / n2t.net / addgene:12259 ; RRID:Addgene_12259). The culture medium was replaced 24 hours after transfection, and the viral supernatant was collected twice, at 48 and 72 hours after transfection. Lentivirus titers were measured using the HIV Type 1 p24 Antigen ELISA kit (#0801111, Zeptometrix, City Road, London, UK) according to the manufacturer's instructions.

[0131] Lentivirus production and shRNA screening

[0132] H2009 cells (4.8 x 10⁶ 7Lentiviruses containing shRNA in ) were transduced at 0.3 MOI, and selection was performed with 1 μg / mL puromycin for 72 hours starting 48 hours after transduction. The selected cells were BD FACSAria III Classification was performed based on PD-L1 expression levels on the cell surface at day 3 [t0], day 13 [t1], and day 20 [t2] using (BD Biosciences, Franklin Lakes, NJ, USA). For classification, 1.8 x 10 7 Cells were collected using 10 mM EDTA and stained with 5 μg / mL PE anti-human PD-L1 (#12-5983-42, eBioscience, San Diego, CA, USA) in PBS containing 0.5% BSA. PD-L1-low cells were a group of cells showing PD-L1 expression equivalent to that of unstained control cells, while PD-L1-high and PD-L1-intermediate cells were groups showing high to intermediate PD-L1 expression, accounting for 1–2% and 13–15% of the total cells, respectively. 1.8 x 10⁶ cells before sorting 7 Cells were collected at [t0] and [t2], and the remaining cells were subcultured for FACS sorting.

[0133] Genomic DNA was isolated from FACS-sorted or unsorted cells using the QIAamp DNA Blood Mini Kit (#51104, QIAGEN, Germantown, MD, USA). The inserted shRNA was amplified by two rounds of PCR (20 cycles per round) using GoTaq Hot Start Polymerase (#M5001, Promega, Madison, WI, USA). The annealing temperatures for the first and second rounds were set to 52°C and 56°C, respectively. The primer sequences for the first round were Cowley GS et al. 20Primers identical to the sequences used were used. The PCR primers for the second round were redesigned to maintain base diversity during the first 16 cycles of the sequencing process by including Illumina P5 and P7 adapters and various sequences with stagger regions. Additionally, barcoded reverse primers were used to ensure that all samples could be multiplexed in a single lane. The primer sequences are shown in Table 1.

[0134] PCR products were purified using an AMPure XP purification system (#A63880, Beckman Coulter) and sequenced on a Hiseq2000 with paired-end 100 bp reads. The average coverage was 1000 times that of the shRNA pool.

[0135] Cutadapt 21 Sense and antisense sequences of shRNA were extracted by removing residual sequences from raw reads using [tool / method]. Sequences were aligned against a reference shRNA sequence within the limit allowing up to 3 nt of mismatch, and the aligned reads were [using Bedtools] 22 Reads were counted. Only reads in which sense and antisense sequences mapped to a single shRNA sequence were included in the count, and reads with a total number of less than 100 in the PD-L1-low, -intermediate, and -high cell groups were excluded. Read counts were normalized by the total number of reads and the number of cells in each sample. Subsequently, the enrichment score of the PD-L1-low cell group was calculated using the following formula: log2 low / (intermediate + high) (low, intermediate, and high are normalized read counts extracted from samples showing low, intermediate, and high expression of PD-L1, respectively). The enrichment score of the PD-L1-high cell group was calculated using the same method. The enrichment score is calculated using the RIGER algorithm, which converts shRNA-level scores into gene-level scores. 23 Analysis was performed using [the method], and target genes of high-expression shRNAs in classified cell populations were ranked. This ranking algorithm calculates gene scores based on the second-best shRNA.

[0136] Primer sequences for shRNA amplification primer order 1 st PCR Forward direction 5′-AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG-3′ 20 1 st PCR reverse direction 5′-CTTTAGTTTGTATGTCTGTTGCTATTATGTCTACTATTCTTTCCC-3′ 20 2 nd PCR Forward direction 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNTCTTGTGGAAAGGACGA-3′5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNGATCTTGTGGAAAGGACGA-3′5′-AATGA TACGGCGACCACCGAGATCTACACTCTTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGATCTTGTGGAAAGGACGA-3′5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNACGATCTTGTGGAAAGGACGA-3′5′-AATGATACG GCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCTAGAATCTTGTGGAAAGGACGA-3′5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNTGGACACATCTTTGTGAAAGGACGA-3′5′-AATGA TACGGCGACCACCGAGATCTACACTCTTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNGTCGGCACATCTTGTGGAAAGGACGA-3′5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNAACCAGCACATCTTGTGGAAAGGACGA-3′ 2 nd PCR reverse direction 5′-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGAACGTGTGCTCTTCCGATCTTCTACTATTCTTTTCCCCTGCACTGT-3′

[0138] Gene set amplification analysis and visualization

[0139] Metascape webtool on 73 screened hit genes (46 PD-L1 enhancers and 27 PD-L1 repressors) 24 Integrated gene-set amplification analysis was performed using Gene Ontology, MSigDB, KEGG, Reactome, Hallmark gene sets (https: / / metascape.org, v.3.5). The entire set of genes targeted by the collected shRNAs was used as the background for the baseline parameters. p -value cutoff, 0.01; minimum overlap, 3; minimum amplification factor, 1.5). Significantly amplified gene set (hypergeometric p <0.001) is Cytoscape(v.3.8.2)'s Enrichment Map 25 It was visualized as.

[0141] T cell-mediated tumor cell death assay

[0142] Tumor cells were adsorbed onto 96-well plates overnight and co-cultured with human peripheral blood mononuclear cells (PBMC; #70025, StemCell Technologies, Vancouver, BC, Canada) at different target-to-operator cell ratios (1:0, 1:5, 1:10). To activate T cells, 100 ng / mL anti-CD3 antibody and 10 ng / mL IL-2 (#317325, #589102, BioLegend, San Diego, CA, USA) were added to the wells. After co-culture for 48–96 hours, PBMCs were removed and washed with PBS, and tumor cell viability was measured using a CellTiter-Glo assay kit (Promega).

[0144] Cytokine assay

[0145] PBMCs (2 x 10⁶) activated with 100 ng / mL anti-CD3 antibody and 10 ng / mL IL-2 or Dynabeads™ Human T-Activator CD3 / CD28 (#11161D, Gibco). 5 ) were cultured with the test substance for 3 days. GRANZYME B levels in the collected supernatant were measured using the LEGEND MAX™ Human Granzyme B ELISA Kit (#439207, Biolegend) according to the manufacturer's instructions. Jurkat T cells (5 x 10⁶ 4) were activated by culturing with Dynabeads™ Human T-Activator CD3 / CD28 (#11161D, Gibco) at various bead-to-cell ratios (0:1, 5:1, 10:1) for 48 hours. IL-2 levels in the collected supernatant were measured using the LEGEND MAX™ Human IL-2 ELISA Kit (#431807, Biolegend) according to the manufacturer's instructions. Twelve cytokines, including IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 22, IFN-γ, and TNF-α, were measured using the Luminex platform (Human T Cytokine Panel, BioLegend) according to the manufacturer's instructions.

[0147] Genetic and chemical perturbation

[0148] Transduction: The shRNA plasmids for lentivirus packaging were purchased from Sigma-Aldrich and are as follows: WNK1(TRCN0000219719); WNK2(TRCN0000194773); WNK3 (TRCN0000219674, TRCN0000001531, TRCN0000195276, TRCN0000196566); HSP90AA1 (TRCN0000315007, TRCN0000001028); CA3(TRCN0000151024, TRCN0000184026); CAST(TRCN0000073638, TRCN0000073642); PFKFB4(TRCN0000037764, TRCN0000199612); SLC7A7 (TRCN0000043033, TRCN0000043037); HDAC3 (TRCN0000004824, TRCN0000196925); HSP90B1 (TRCN0000029427, TRCN0000276250); SIK3 (TRCN0000194845, TRCN0000037450); PAN3(TRCN0000049805, TRCN0000049807); CTRL(TRCN0000003652, TRCN0000003651); SMAD4 (TRCN0000010321, TRCN0000040031); OSGEPL1 (TRCN0000047052, TRCN0000047050); PD-L1 (TRCN0000056913, used on H460). pGIPZ-shPD-L1 (#RHS4430-200253051, used on H2009) was purchased from Horizon Discovery (Waterbeach, UK), and pGIPZ-PD-L1 WT 26It was provided by Mien-Chie Hung (Addgene plasmid # 121486 ; http: / / n2t.net / addgene:121486 ; RRID:Addgene_121486). To construct a lentiviral plasmid containing human WNK3 cDNA, WNK3-Myc-DDK (#RC220755) was subcloned into the pLVX vector (Clontech Laboratories, Mountain View, CA, USA). The lentivirus was 1.3 x 10⁶ 6 It was constructed by co-transfecting HEK293FT cells with 1 μg of plasmid DNA, 0.75 μg of psPAX2, and 0.25 μg of pMD2.G (Addgene), and 5 x 10⁴ cells seeded in 60 mm dishes 5 It was added to target cells. Afterwards, transfected cells were selected using 1 μg / mL (H2009) or 2 μg / mL (H460) puromycin.

[0149] cDNA transfection: 4 x 10 5 H2009 cells or 8 x 10 5H460 cells were seeded into 6-well plates and transfected with 2 μg (H2009) or 4 μg (H460) of plasmid using Lipofectamine 2000 (Invitrogen), and collected after 72 hours. Myc-DDK-tagged plasmids encoding human WNK1 (#RC218208), WNK2 (#RC212364), WNK3 (#RC220755L3), and WNK4 (#RC223269) were purchased from Origene (Rockville, MD, USA). The WNK3 kinase-loss mutant construct (WNK3 K159M) was constructed using the Q5 positioning mutant kit (#E0554S, New England Biolabs, Ipswich, MA, USA) according to the manufacturer's guidelines. The primer sequences used are as follows: WNK3 K159M forward, 5′-AGGAGCATTTATGACAGTATATAAAGG-3′, reverse, 5′-CTTCCTAGTTCTATGTCAAATTTC-3′.

[0150] siRNA transfection: cells (2 x 10 5 siRNA mixed with RNAiMAX (Invitrogen) was added to each well of a 6-well plate at a dose of 50 nM, and each experiment was conducted 72 hours after transfection. Each siRNA oligonucleotide with the following sequences was synthesized (Genolution, Seoul, Korea): Human WNK4: 5′-GAUUGCAGCUGCCAUGGUA-3′, Mouse WNK3: 5′-GCCTCACGTTTGTCAGTAT-3′, 5′-ATACTGACAAACGTGAGGC-3′, Negative control: 5′-GCAGGACCAGGCCAUAUGA-3′.

[0151] Compounds: WNK463 (#CD00005886, Crysdot, Bel Air, MD, USA), dasatinib (#S1021, Selleckchem, Houston, TX, USA), YKL-06-062 (#AOB37242, AOBIOUS, Gloucester, MA, USA), YKL-05-099 (#HY-101147, MedChemExpress, Monmouth Junction, NJ, USA), PU-H71 (#NSC 750424, Selleckchem), CH5138303 (#S7340, Selleckchem), RGFP966 (#S7229, Selleckchem), acetazolamide (#A6011, Sigma -Aldrich), 5MPN (#S656801, Sigma-Aldrich), JNK-IN-8 (#18096, Cayman Chemical, Ann Arbor, MI, USA) and AEBSF HCl (#A8456, Sigma-Aldrich) were used.

[0153] qRT-PCR assay

[0154] The cDNA template synthesized by RT-PCR was mixed with probes and TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and applied to the StepOne Plus real-time PCR system (Applied Biosystems). For the SYBR Green assay, the reaction was performed using the designed primers and the QuantiNova SYBR Green PCR Kit (Qiagen). The probes used in the Taqman assay are as follows: WNK3(Hs00908643_m1), HSP90AA1(Hs00743767_sH), CA3(Hs00193123_m1), CAST(Hs00156280_m1), PFKFB4 (Hs00894603_m1), SLC7A7(Hs00909952_m1), HDAC3(Hs00187320_m1), HSP90B1 (Hs00427665_g1), SIK3(Hs00228549_m1), PAN3(Hs01107000_m1), CTRL (Hs00157187_m1), SMAD4(Hs00929647_m1), OSGEPL1 (Hs01088658_g1), PD-L1 (Hs00204257_m1), and 18S (Hs99999901_s1). The primer sequences for the SYBR Green assay are This is summarized in Table 2. Human 18S ribosomal RNA or mouse β-actin was used as an internal control.

[0155] Primer sequences for shRNA amplification gene Primer sequence Human PD-L1 Forward, 5′-CAATGTGACCAGCACACTGAGAA-3′ Reverse, 5′-GGCATAATAAGATGGCTCCCAGAA-3′ Human WNK1 Forward, 5′- GCCGTCAGATCCTTAAAGGTC-3′ Reverse, 5′-CCAGTAGGGCCGGTGATAA-3′ Human WNK2 Forward, 5′-CGCTTCCTCAAGTTCGACATC-3′ Reverse, 5′-TGGACTCCCAGAAGTCGTAGA-3′ Human WNK3 Forward, 5′-ACTTCTCCTAGTGGCAGATTCC-3′ Reverse, 5′-GCAGCTCACACCAAGCAAC-3′ Human WNK4 Forward, 5′-CGATGGCCGATACCTCAAGTT-3′ Reverse, 5′-GTCGGTGTCTAGCCCTCGAT-3′ Human 18S Forward, 5′-ACTCAACACGGGAAACCTCA-3′ Reverse, 5′-AACCAGACAAATCGCTCCAC-3′ Mouse PD-L1 Forward, 5'-TGCGGACTACAAGCGAATCACG-3' Reverse, 5′- CTCAGCTTCTGGATAACCCTCG-3′ Mouse WNK3 Forward, 5′-GGTGGTCAGTCTTCAAACACAA-3′ Forward, 5′-ACTTCTCCTAGTGGCAGATTCC-3′ (Fig. 12b) Reverse direction, 5′-GTGAACATCCCCTTCTTACTGG-3′ Reverse direction, 5′-GCAGCTCACACCAAGCAAC-3′ (Fig. 12b) Mouse β-actin Forward, 5′-GTGACGTTGACATCCGTAAAGA-3′ Reverse, 5′-GCCGGACTCATCGTACTCC-3′

[0157] Flow cytometry

[0158] Cells were isolated and washed, then resuspended in PBS containing 0.5% BSA with the following fluorescent antibodies: 5 μg / mL PE anti-human PD-L1 (#12-5983-42, eBioscience), 2.5 μg / mL PE anti-mouse PD-L1 (#124308, Biolegend), and 3.2 μg / mL APC anti-human MHC class I (#311409, Biolegend). After incubation on ice for 20 minutes, cells were washed with PBS containing 0.5% BSA and processed on a BD FACSVerse flow cytometer (BD Biosciences). Results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). For the flow cytometry of IFN-γ-induced PD-L1, cells were treated with 10 or 20 ng / mL human IFN-γ (#PHC4031, Gibco) for 24 hours.

[0160] Flow cytometry of WNK3 knockdown human CD4+ and CD8+ T cells

[0161] To induce WNK3 knockdown in human CD4+ and CD8+ T cells, live CD4+ and CD8+ T cells from human PBMCs were sorted using an MA900 sorter (SONY, San Jose, CA, USA) and stimulated for 24 hours with plate-conjugated anti-human CD3 (#BE0001-2, BioXCell, Lebanon, NH, USA) / anti-human CD28 (#BE0248, BioXCell) and 50 IU IL-2. These cells were then treated with lentiviral supernatant (shNC and sh WNK3 Spin-infected with ) (900g, 90 min, 37°C). Four days after lentivirus infection, cells were selected with 1 µg / ml puromycin for 7 days. Producing PERRFORIN and GRANZYME B WNK3 To quantify knockdown human CD4+ and CD8+ T cells, shNC and sh WNK3CD4+ and CD8+ T cells were stimulated with Dynabeads Human T-Activator CD3 / CD28 (Gibco) for 36 hours. During the stimulation process, BD GolgiStop (#554724, BD Biosciences) was added during the last 6 hours. After stimulation, cells were washed and surface molecules were stained with anti-CD4 (OKT4, Biolegend) and anti-CD8 (SK1, Biolegend). Then, cells were fixed with eBioscience / Invitrogen Intracellular (IC) fixation buffer (#00-8222-49, Invitrogen), washed with 1x Permeabilization Buffer (#00-8333-56, Invitrogen), and stained with the following antibodies: anti-PERFORIN (B-D48, Biolegend) and anti-GRANZYME B (QA16A02, Biolegend).

[0163] Flow cytometry of WNK463-treated or Wnk3 knockout mouse CD4+ and CD8+ T cells

[0164] Splenectomy was performed on mice to prepare single-cell suspensions. CD4+ and CD8+ T cells were sterilized with biotinylated antibodies (Biolegend) against CD19 (6D5), B220 (RA3-6B2), CD11b (M1 / 70), CD11c (N418), and NK.1 (PK136) and EasySep Mouse Na The cells were enriched using the ve CD4+ T cell separation kit (#19765, STEMCELL Technologies) and sorted into CD4+ and CD8+ T naive cells using the MA900 sorter (SONY). Cells were stimulated with plate-bound anti-mouse CD3 (#BE0001-1, BioXcell) / anti-mouse CD28 (#BE0015-1, BioXcell) for 24 hours. Pre-designed sgRNAs targeting the mouse Wnk3 gene were synthesized at Integrated DNA Technologies (IDT; Coralville, IA, USA).

[0165] The sequence of the pre-designed sgRNA used in the experiment is as follows. Negative control: 5'-mA*mC*mG* rArUrU rCrCrU rArArG rArUrG rCrUrU rGrCrG rUrUrU rUrArG rArGrC rUrArG rArArA rUrArG rCrArA rGrUrU rArArA rArUrA rArGrG rCrUrA rGrUrC rCrGrU rUrArU rCrArA rCrUrU rGrArA rArArA rGrUrG rGrCrA rCrCrG rArGrU rCrGrG rUrGrC mU*mU*mU* rU-3', Wnk3 : 5'- mU*mA*mG* rUrUrC rGrArU rUrCrU rArUrG rArUrU rCrArG rUrUrU rUrArG rArGrC rUrArG rArArA rUrArG rCrArA rGrUrU rArArA rArUrA rArGrG rCrUrA rGrUrC rCrGrU rUrArU rCrArA rCrUrU rGrArA rArArA rGrUrG rGrCrA rCrCrG rArGrU rCrGrG rUrGrC mU*mU*mU* rU-3'.

[0166] To form ribonucleoproteins, Alt-R CRISPR-Cas9 guide RNA and Alt-R Sp HiFi Cas9 Nuclease V3 (#1081061, IDT) were mixed, the DN-100 electroporation program was applied, and activated CD4+ and CD8+ T cells were transfected using the Amaxa P3 primary cell 4D-nucleofector X kit (Lonza, Basel, Switzerland). The recovered cells were cultured for 72 hours prior to flow cytometry analysis.

[0167] To quantify the production of PERFORN and GRANZYME B, CD4+ and CD8+ T cells (2x10 5 ) were treated with 1 μM WNK463 or DMSO for 6 hours and stimulated with Dynabeads Mouse T-Activator CD3 / CD28 (#11452D, Invitrogen) for 36 hours. BD GolgiStop (BD Biosciences) was added during the last 6 hours of stimulation. Wnk3 Knockout CD4+ and CD8+ T cells (2x10 5 ) were stimulated with Dynabeads Mouse T-Activator CD3 / CD28 (Invitrogen) for 36 hours. After stimulation, WNK463 treatment and Wnk3Knockout CD4+ and CD8+ T cells were washed, and surface molecules were stained with anti-CD4 (GK1.5, Biolegend) and anti-CD8 (53-6.7, Biolegend). Then, cells were fixed with eBioscience / Invitrogen Intracellular (IC) Fixation Buffer (Invitrogen), washed with 1x Permeabilization Buffer (Invitrogen), and stained with anti-PERFORIN (S16009A, Biolegend) and anti-GRANZYME B (QA16A02, Biolegend). To quantify phospho-AKT and phospho-S6 proteins, WNK463 treatment or Wnk3 Knockout mouse CD4+ and CD8+ T cells (2x10 5 The cells were stimulated with Dynabeads Mouse T-Activator CD3 / CD28 (Invitrogen) for 5 minutes. After stimulation, 20 volumes of preheated 1x BD Phosflow lysis / fixation buffer (#558049, BD Biosciences) were added to immediately fix the cells, and they were permeated with BD Phosflow Perm / Wash buffer (#557885, BD Biosciences). The cells were stained with anti-phospho-AKT (Ser473) (#9271, Cell Signaling Technology, Danvers, MA, USA) and anti-phospho-S6 ribosomal protein (Ser240 / 244) (#5364, Cell Signaling Technology) antibodies, followed by staining with PE donkey anti-rabbit IgG (minimal X-reactivity) antibody (#406421, Biolegend). Cell acquisition was performed on a BD FACSCelesta cell analyzer (BD Bioscience) and the data were analyzed using FlowJo software.

[0169] Immunoblotting

[0170] Cells were washed with PBS and lysed in RIPA lysis buffer (Sigma-Aldrich) containing a protease and phosphatase inhibitor cocktail (GenDEPOT, Katy, TX, USA). Protein concentration was measured using Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on 4-15% Mini-PROTEAN TGX Precast Gels (Bio-Rad). Anti-PD-L1 (#13684S), anti-p-JNK (#4668S), anti-JNK (#9252S), anti-pc-Jun (#3270S), anti-p-ERK1 / 2 (#4370S), anti-ERK1 / 2 (#4348S), anti-p-p38 (#4631S), anti-p38 (#9212S), anti-p-AKT (#9271S), anti-AKT (#4691S), anti-p-P65 (#3033S), anti-P65 (#8242S), anti-p-STAT3 (#9145S), anti-STAT3 (#9139S), anti-HSP90 (#4877), and anti-flag (DYKDDDDK) (#2368S) antibodies were developed by Cell Signaling Technology (Danvers, MA). Anti-PD-L1 (mouse-specific, #ab213480, Abcam, Cambridge, MA, USA) and anti-β-actin (#sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were purchased from the respective manufacturers listed. Peroxidase AffiniPure Goat anti-Rabbit IgG (#111-035-144) and anti-Mouse IgG (#115-035-003, Jackson ImmunoResearch, West Grove, RA, USA) were used as secondary antibodies. The primary and secondary antibodies were diluted to 1:1000 and 1:5000, respectively.

[0172] Survival analysis

[0173] Download TCGA-lung cancer gene expression data using the TCGAbiolinks package (version 2.6.12), and TCGA pan-cancer clinical data resources 27 I downloaded the clinical data processed at. E-MTAB-923 Colot 28 The gene expression and clinical data of Arrayexpress(https: / / www.ebi.ac.uk / arrayexpress / experiments / E-MTAB-923 The data was downloaded from ). Gene expression and clinical data for colorectal (GSE39582) and gastric (GSE62254) cancer patients were downloaded from GEO (https: / / www.ncbi.nlm.nih.gov / geo / ). Gene expression levels from multiple samples of the same patient were averaged to eliminate duplicates. Next, patients were separated into high-expression and low-expression groups for each gene. The optimal expression cutoff was determined according to previously reported methods. 10 The two-sided log-ranking method was performed using the Survival package (version 2.42-6).

[0175] Immunoprecipitation and in vitro kinase activity analysis

[0176] HEK293FT cells (1.5 x 10⁶) in each well of a 6-well plate 5Cells were harvested 72 hours after forward-transfecting with 2.5 μg of WNK3 wild-type or WNK3-K159M using Lipfectamine 2000. Alternatively, 24 hours after WNK3 wild-type transfection, cells were treated with WNK463, WNK3 inhibitor 11, or DMSO for 48 hours. Cells were lysed on ice in a lysis buffer consisting of 50 mM Tris-HCl, pH 7.5, 1% NP-40, 130 mM 233 NaCl, 10% glycerol, 10 mM MgCl2, and a protease and phosphatase inhibitor cocktail (GenDEPOT). 600 μg of cell lysate was gently shaken with 2 μl of anti-Myc antibody (#2276S, Cell Signaling Technology) at 4°C for 1 hour, and then SureBeads Protein A and Protein G Magnetic Beads (#1614013, #1614023, Bio-Rad) mixed in a 1:1 ratio were added and reacted for 1 hour. The precipitate obtained in this way was washed three times with cold lysis buffer, and for an in vitro kinase assay, beads containing anti-Myc-precipitated kinase were diluted in 15 μl of kinase dilution buffer X (#K20-09, SignalChem, British Columbia, Canada) and 50 μM DTT (#D86-09), and then reacted with 10 μl of substrate / ATP mixture (1 μl of 10 mM ATP, 79 μl of kinase assay buffer III (#K03-09, SignalChem, DTT added to a concentration of 250 μM before use), 80 μl of 0.5 mg / ml MBP (#M42-51N, SignalChem), and 1 μl of 1 M MnCl2 (#M40-09, SignalChem)) at room temperature for 50 minutes. The ADP generated in the kinase reaction was measured using the ADP-Glo ​​kinase assay (#V6930, Promega).

[0178] animal testing

[0179] All animal experiments were conducted under the approval of the Animal Ethics Committee of Yonsei University College of Medicine (Seoul, Korea; 2019-0333). Prior to the experiment, animals were acclimatized to a 12-hour light / dark cycle for 7 days. C57BL / 6 mice were purchased from SLC, Inc. (Shizuoka, Japan). On day 0, 5 x 10⁶ were placed on the right flank of 6 to 8-week-old mice. 5 MC38 cells were injected subcutaneously. The average tumor volume was 50-200 mm³. 3 At the time of reaching [date], 5 mice per group were orally administered 0, 5, or 10 mg / kg WNK463 (#CD00005886, Crysdot, BelAir, MD, USA) daily. CD8 + To induce T-cell apoptosis, 300 μg of an anti-CD8 depletion antibody (clone 2.43, #BP0061, BioXcell, Lebanon, NH, USA) was administered intraperitoneally to mice on days -1, 3, 6, 10, and 13. For the combined treatment of WNK463 and a PD-1 blocking antibody, 10 μg of either the anti-PD-1 antibody or the IgG2a control antibody was administered intraperitoneally to mice 8 days after tumor injection, while WNK463 was administered orally at 0 or 10 mg / kg daily starting from day 8 of tumor injection. Mice were randomly assigned to treatment and control (vehicle) groups and maintained under standard conditions. Tumor size (length x width) was measured with calipers starting from the first day of drug injection. Tumor volume was calculated using the following formula: 0.5 x long diameter x short diameter. 2 The drug was administered to mice daily for 9 days, and the maximum tumor volume was 2 cm² 3 When it reached, sacrificed it.

[0181] Flow cytometry of tumor cells

[0182] The excised tumor was digested with type IV collagenase and DNase at 37°C for 20 minutes, and the digested tissue was filtered through a 100 μm filter to prepare a cell suspension. Subsequently, tumor cells and tumor-infiltrating lymphocytes (TILs) were purified using centrifugation (2000 rpm, 40 min, RT, break-off) with discontinuous concentration gradients of 40% and 80% using Percoll (GE Healthcare, Chicago, IL, USA).

[0183] Single-cell suspensions of TILs were prepared, dead cells were labeled by staining with Fixable Viability Dye (Invitrogen), and reacted with fluorescent chrome-conjugated antibodies. For surface staining, cells were washed with PBS and treated with the following antibodies diluted 1:400 in PBS: anti-CD4 (GK1.5, Biolegend), anti-CD8 (53-6.7, Biolegend), anti-CD45 (30-F11; BD Pharmingen, San Diego, CA, USA), anti-TCRβ (H57-597, Biolegend), anti-PD-L1 (10F.9G2, Biolegend), anti-PD1 (29F.1A12, BioLegend), and anti-TIM3 (RMT3-23, BioLegend). For intracellular transcription factor staining, cells were washed with eBioscience / Invitrogen Perm Buffer and treated with anti-TOX (REA473, Miltenyi Biotech). For intracellular cytokine staining, cells were stimulated with a cell stimulation cocktail and a protein transport inhibitor (00-4980-03, eBioscience / Invitrogen) for 6 hours, after which the cells were washed and surface molecules were stained. Subsequently, cells were fixed with eBioscience / Invitrogen Intracellular (IC) fixation buffer, washed with 1 x permeability buffer (Invitrogen), and stained with the following antibodies diluted 1:200 in PBS: anti-TNF (MP6-XT22, BD Pharmingen), anti-IFN-γ (XMG1.2, Biolegend), and anti-GRANZYME B (QA16A02, BioLegend). Cell apoptosis was detected using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) according to the manufacturer's instructions. Cell images were acquired using FACSCelesta (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). In ViboThe experimental data was analyzed using GraphPad Prism version 9 (San Diego, CA, USA).

[0185] glioblastoma

[0186] - Cell line

[0187] U87MG was purchased from ATCC (Manassas, VA, USA) and SF295 from AddexBio (San Diego, CA, USA) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0189] - siRNA transfection

[0190] Cells (4 x 10 5 siRNA mixed with RNAiMAX (Invitrogen) was added to each well of a 6-well plate at a dose of 100 nM, and each experiment was conducted 72 hours after transfection. Each siRNA oligonucleotide having the following sequences was used (Genolution, Seoul, Korea): Human WNK3: 5′-GCCTCACGTTTGTCAGTAT-3′, 5′-ATACTGACAAACGTGAGGC-3′, Negative control: 5′-GCAGGACCAGGCCAUAUGA-3′.

[0192] - Immunoblotting

[0193] Cells were washed with PBS and lysed in RIPA lysis buffer (Sigma-Aldrich) containing a protease and phosphatase inhibitor cocktail (GenDEPOT, Katy, TX, USA). Protein concentration was measured using Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on 4-15% Mini-PROTEAN TGX Precast Gels (Bio-Rad). Antibodies purchased from Bethyl Laboratories (Montgomery, TX, USA) for anti-WNK3 (#A301-877A), Cell Signaling Technology (Danvers, MA, USA) for anti-PD-L1 (#13684S), and Cell Signaling Technology (Danvers, MA, USA) for anti-HSP90 (#4877) were used. Peroxidase AffiniPure goat anti-rabbit IgG (#111-035-144) and anti-mouse IgG (#115-035-003, Jackson ImmunoResearch, West Grove, RA, USA) were used as secondary antibodies. The primary and secondary antibodies were diluted to 1:1000 and 1:5000, respectively.

[0195] Experimental results

[0196] shRNA screening results identified drug-induced genes and oncogenes that regulate PD-L1 expression.

[0197] The inventors generated and combined a concentrated shRNA library to identify drug-associated genes and oncogenes that regulate PD-L1 expression (N = 35,949). The set of drug-associated genes included 5,069 genes (32,643 shRNAs), which were related to drugs or kinases, receptors, and metabolic enzymes. 11 As shown, they belonged to the category of drug targets (Table 3). The oncogenic gene set included genes (5,611 shRNAs) exhibiting 800 of the most frequent mutations across 13 tumor types. 12-19(Table 4). The amounts of shRNA in both libraries were close to a normal distribution (Fig. 8a). To select an appropriate cell line for screening, basal and IFN-γ-induced PD-L1 expression levels were measured in seven non-small cell lung cancer (NSCLC) cell lines (H2009, HCC44, HCC461, H460, H647, H2122, A549), and H2009 was selected as it exhibited the highest basal and IFN-γ-induced PD-L1 levels (Figs. 1a, 1b, 8b, and 8c). To investigate tumor-specific PD-L1 regulatory mechanisms, screening was performed under conditions without IFN-γ treatment. H2009 cells were transduced with lentiviral shRNA (N = 35,949) and 32 non-targeted negative control shRNAs (Fig. 1c). FACS was performed 3 days (t0), 13 days (t1), and 20 days (t2) after puromycin screening to classify each cell into the following groups: PD-L1 low, PD-L1 intermediate, and PD-L1 high subpopulations (Figs. 1c and 8d). Next, amplicon sequencing was performed on the inserted shRNA in each cell population (Fig. 1c). Finally, the RIGER (RNAi Gene Enrichment Ranking) algorithm 23 Gene-level enrichment scores were calculated using the second-best method shown in Fig. 8e. Since shRNAs exhibit varying on-target efficiencies and off-target effects, this method was developed to observe phenotypic changes caused by at least two shRNAs. First, using unsorted t0 and t2 cells, the CCE (Constitutive core essential) gene 9 The quality of the screening data was evaluated by measuring the time-dependent deficiency of. While the CCE gene was significantly deficient at t2 compared to t0 (Kolmogorov-Smirnov test, p =2.2E-16), non-essential (NE) genes9 This was not the case (Fig. 1d), indicating that the screening data of the present invention is of high quality. As a result of the screening, 46 PD-L1 enhancers and 27 PD-L1 inhibitors were identified in the PD-L1 low and PD-L1 high populations, respectively, at t1 and t2 (Fig. 8f, Table 5). Among the identified targets, 40 pharmacological PD-L1 enhancers, 9 oncogenic agents that promote PD-L1 expression, and 7 oncogenic agents that inhibit PD-L1 expression were identified, respectively (Table 5).

[0198] Gene-set amplification analysis of 73 hit genes (46 screened PD-L1 enhancers and 27 PD-L1 repressors) revealed that PD-L1 regulators were significantly involved in “nucleotide deletion repair” and the “BMP / SMAD signaling pathway” ( p <0.001) (Fig. 1e). The “nucleotide deletion repair” gene set includes 4 PD-L1 repressors ( POLR2C , PARP1 , COPS5 , POLE4 ...was included (Fig. 1e, left), which is consistent with the observation that deficiency in the DNA repair signaling pathway increases PD-L1 expression through the neoantigen-T cell activation-IFN-γ pathway and ATR-CHK1-dependent DNA damage signaling. 29 The BMP / SMAD signaling gene set includes two PD-L1 enhancers ( BMPR2 , SMAD4 ) and one PD-L1 inhibitor( UBE2D3 ) was included (Fig. 1e, right). Ubiquitin conjugate E2D3( UBE2D3 Since ) suppresses the SMAD signal triggered by BMP-BMPR 30,31 This suggests the possibility that BMP signaling is involved in PD-L1 increase. As described above, the inventors have identified a PD-L1 enhancer that can serve as a direct therapeutic target.

[0199] The drug-like gene used in the present invention Data type literature [ref] gene Drug-induced genes GO 32 Russ & Lampel 33 Hopkins & Groom 34 dGene 35 BaderLabGenes 36 FoundationOneGenes 37 5815302626682256292229 drug-gene interaction DrugBank 38 PharmGKB 39 TTD 40 TEND 41 MyCancerGenome 42 TALC 43 ClearityFoundationClinicalTrial*MyCancerGenomeClinicalTrial 44 CancerCommons 45 ClearityFoundationBiomarkers* 204959447743016915393784834 Totals (unique) 5069

[0200] * https: / / www.clearityfoundation.org (Data currently inaccessible)

[0202] The drug-like gene used in the present invention literature Tumor morphology Cancer-causing genes Pan-cancer studies -Somatic mutations Ciriello G. et al. Nat Genet 45, 1127-33 (2013) 12 13 199 Kandoth C. et al. Nature 502, 333-9 (2013) 13 13 127 Vogelstein, B. et al. Science 339, 1546-58 (2013) 14 26 138 Chang M. T. et al. Nature Biotechnology 34, 155-163 (2016) 15 41 275 -Somatic copy number alterations Zack TI. et al. Nat Genet 45, 1134-40 (2013) 16 12 131 Beroukhim R. et al. Nature 463, 899-905 (2010) 17 26 196 Gastric cancer studies -Somatic mutations Cancer Genome Atlas Research Network. Nature 513, 202-9 (2014) 18 1 57 Cristescu R. et al. Nat Med 21, 449-56 (2015) 19 1 17 Totals (unique) 800

[0204] 73 candidate genes that promote or inhibit PD-L1 expression Category gene number PD-L1 Enhancer Drug-induced genes AKR1C1, BMPR2, CA3, CAST, CTRL, EPHB1, GGT5, GLRX, GSTT2, HCK, HDAC3, HSP90AA1, HSP90B1, KCNB2, KSR1, METAP1, NDUFS3, OAZ2, OR4Q3, OR52E2, OR9K2, OSGEPL1, PFKFB4, PLAUR, PSKH1, SDHC, SERPINA10, SIK3, SLC25A17, SLC7A7, SLC9A3, TAS2R13, TAS2R40, TIGD1, UQCRC1, USP17, WNK3 37 Cancer-causing genes ARIH1, BRD1, CDKN2C, MYH3, NDRG2, SMAD4 6 Drug-induced cancer-causing genes ADAMTS20, CCDC105, PAN3 3 PD-L1 inhibitors drug-induced genes EWSR1, PSMD9, NT5C2, RIOK3, POLE4, APOE, POLR2C, COPS5, CD70, RPS6KA6, CPE, CRYBB1, FCGR3B, LDHC, CDKL3, ACBD4, BTNL3, TGM6, OR52L1, TRIM49L1 20 Cancer-causing genes SMC3, SRSF2, UBE2D3, CHL1, IGFBP1, MYO18A 6 Drug-induced cancer-causing genes PARP1 1

[0206] WNK3 and SIK3 are positive regulators of PD-L1.

[0207] Next, the inventors investigated the PD-L1 regulatory function of 46 PD-L1 enhancers using cell lines (H2009 and H460) engineered to stably express first and second efficient shRNAs that inhibit candidate genes. Among these, 13 genes ( WNK3 , HSP90AA1 , CA3 , CAST , PFKFB4 , SLC7A7 , HDAC3 , HSP90B1 , SIK3 , PAN3 , CTRL , SMAD4 , OSGEPL1 It was confirmed through flow cytometry and qPCR analysis that PD-L1 levels significantly decrease when ) is knocked down (Figs. 2a-2d). Among these 13 PD-L1 enhancers, 2 oncologists ( PAN3 and SMAD4 Eleven of them, excluding ), have pharmacological properties. In particular, 7 of the pharmacological genes ( WNK3 , SIK3 , HSP90AA1 , HSP90B1 , HDAC3 , CA3 and PFKB4Commercially available small molecule inhibitors exist for ) (Table 6). Among these, WNK3, SIK3, HSP90AA1, and HSP90B1 inhibitors reduced membrane PD-L1 levels in H460 and H2009 cells (Figs. 2e-2f, Figs. 9a, 9b, 9d), and only WNK3 (WNK463) and SIK3 inhibitors (dasatinib, YKL-06-062, and YKL-05-099) reduced membrane PD-L1 levels without affecting cell viability (Figs. 2e-2f, Figs. 9a-9d). Surprisingly, the HDAC3 inhibitor RGFP966 increased PD-L1 levels (Figs. 2e-2f, Figs. 9a, 9d), which is sh HDAC3 Or it could be an off-target effect of RGFP966.

[0208] When these results are taken together, it can be seen that WNK3 and SIK3 are positive regulators of PD-L1 in lung cancer cells, and that inhibition of these genes inhibits membrane PD-L1 levels without inducing direct cytotoxicity in H460 (Figs. 2e-2f, Fig. 9a) and H2009 (Figs. 9b-9d).

[0209] Compounds that inhibit genes promoting PD-L1 expression PD-L1 Enhancer compound Target specificity WNK3 WNK463 Pan-WNK SIK3 Dasatinib Pan-SIK YKL-06-062 Pan-SIK YKL-05-099 Pan-SIK HSP90AA1, HSP90B1 PU-H71 Pan-Hsp90 HSP90AA1 CH5138303 Hsp90a HDAC3 RGFP966 HDAC3 CA3 Acetazolamide Pan-CA PFKFB4 5MPN PFKFB4

[0211] WNK3 exerts immunomodulatory effects under co-culture conditions.

[0212] The immune cell-dependent anticancer effects of WNK3 and SIK3-targeting shRNAs and their chemoinhibitors were evaluated using a cancer cell-immune cell co-culture method. Before evaluation, sh PD-L1 It was confirmed that monoclonal H460 cells (Fig. 3a) and H2009 cells (Fig. 14a) stably expressing [the substance] showed increased sensitivity to IL-2 and anti-CD3-activated peripheral blood mononuclear cells (PBMCs) (Figs. 3b, 3c, 14b).

[0213] Next, shRNA-mediated WNK3We investigated whether cancer cells could be sensitized to immune cell attacks through removal. As a result, compared to the condition in the absence of PBMCs, sh in the presence of activated PBMCs WNK3 An increase in cell death was observed (Figs. 3d, 3e). Similarly, under co-culture conditions, the pan-WNK inhibitor WNK463 significantly increased cancer cell death triggered by activated PBMCs compared to conditions in the absence of PBMCs (Figs. 3f, 3g). This effect of cancer cell death triggered by activated PBMCs was partially reversed by PD-L1 overexpression in cancer cells (Fig. 14c) (Fig. 14d), demonstrating that PD-L1 deficiency in cancer cells contributes to the enhancement of anti-cancer immunity. In particular, WNK463 not only inhibited PD-L1 but also directly activated immune cells by increasing Granzyme B secretion and the secretion of immuno-activating cytokines IFN-γ and TNF-α by PBMCs at concentrations that did not affect PBMC survival, and by inhibiting the immunosuppressive cytokine IL-10 (Figs. 3h, 3i). Through this, it was demonstrated that WNK463 possesses a dual anticancer mechanism by inhibiting PD-L1 expression in cancer cells and enhancing the activation of immune cells.

[0214] To further investigate how WNK463 promotes immune cell activation, CD4+ or CD8+ T cells were isolated from mouse spleens and stimulated in the presence or absence of WNK463. WNK463 was sufficient to increase the production of PERFORN and GRANZYME B in both CD4+ and CD8+ T cells compared to the vehicle (Fig. 12a). These results imply that WNK performs an intrinsic function in T cell activation.

[0215] The inventors found that the production of PERRFORIN and GRANZYME B in mouse CD4+ and CD8+ T cells Wnk3It was further demonstrated that they were significantly increased by the effective knockout of (Fig. 12b). Human CD4+ T cells and CD8+ T cells WNK3 [Unclear] consistently produced significantly more PERRFORIN and / or GRANZYME B when knocked down by shRNA compared to the control shRNA (Fig. 12c). In addition, sh WNK3 or stably express WNK3 Jurkat T cells overexpressing [it] were also shown to have significantly enhanced or inhibited IL-2 secretion, respectively (Fig. 3j, Fig. 12d). Based on these findings, it was found that WNK3 possesses an intrinsic T cell function that inhibits T cell activation.

[0216] Next, we examined how WNK3 inhibits T cell activation. WNK3 possesses kinase activity, and PI3K-AKT signaling GRANZYME B 46 Given its importance in generation, the inventors hypothesized that WNK3 could primarily affect proximal TCR signaling cascades by regulating the AKT pathway. Indeed, compared to control sgRNA and vehicle, Crispr / Cas9-mediated Wnk3 Knockout and WNK463 treatment were sufficient to increase the phosphorylation of AKT and pS6, particularly in CD8+ T cells, or potentially in CD4+ T cells as well (Fig. 12e, Fig. 12f).

[0218] On the other hand, SIK3 inhibition showed the opposite effect on immune cells. SIK3Although SIK3 inhibitors reduce PD-L1 levels in cancer cells (Figs. 2a-2f, 9a, 9b, and 9d), the SIK3 inhibitor YKL-06-062 confers resistance to immune cells to cancer cells under co-culture conditions (Figs. 3k, 3l). Consistent with these results, SIK3 inhibitors (dasatinib and YKL-06-062) significantly reduced GRANZYME B secretion by PBMCs (Figs. 3m, 3n), indicating that SIK3 inhibition has a cancer cell-independent anti-immunity effect.

[0219] These data reveal the dual function of WNK3 inhibition under co-culture conditions. WNK3 inhibition inhibits PD-L1 expression in cancer cells, thereby inhibiting CD8 + While it increases sensitivity to T cells, in immune cells, CD8 + It promotes the secretion of anti-tumor cytokines by T cells.

[0221] WNK3 is a transcription regulator of PD-L1.

[0222] Next, sh WNK3 expressing or WNK3 Using H2009 and H460 cell lines overexpressing, we investigated whether PD-L1 regulation by WNK3 occurs at the mRNA level or the protein level. Two sh WNK3 Both the mRNA and protein levels of PD-L1 were decreased in the expression cell lines (Figs. 4a, 4b, 4c, and 10a); furthermore, WNK3 - Both PD-L1 mRNA and protein levels were increased in overexpression cell lines (Figs. 4d, 4e, and 10b). WNK3 In cells expressing WNK3 Since the ectopic expression of was sufficient to restore the reduced PD-L1 levels, the inventors sh WNK3It was hypothesized that the mediated reduction of PD-L1 was an on-target effect (Fig. 10c). Furthermore, the expression of WNK3 and PD-L1 in seven NSCLC cell lines showed a positive correlation (Fig. 14e). Taken together, these results indicate that WNK3 transcriptionally regulates PD-L1 expression. Consistent with the genetic data, the pan-WNK inhibitor WNK463 also reduced the mRNA and protein levels of PD-L1 (Figs. 2e, 2f, 4f, 4g, 9a, 9b, 9d, and 10d). Since WNK463 is a pan-WNK inhibitor, other WNK family ( WNK1 , WNK2 , WNK4 We intended to investigate the PD-L1 regulatory ability of ). WNK3 Unlike WNK1 and WNK2 shRNA-mediated knockdown and WNK4 siRNA-mediated knockdown did not affect PD-L1 expression (Figs. 10e, 10f). Furthermore, in H2009 cells WNK1 , WNK2 or WNK4 Overexpression of did not affect PD-L1 levels (Fig. 10g). The fact that other WNK family members did not affect PD-L1 expression suggests that WNK463 regulates PD-L1 through WNK3 inhibition. In particular, since WNK3 inhibition did not affect type I MHC protein expression, CD8 in cancer cells + It can be seen that it does not impair antigen presentation to T cells (Fig. 4h). These results indicate that WNK3 is an essential factor regulating PD-L1 mRNA expression, and that WNK463 WNK3 - It can be seen that it mimics the phenotype of knockdown.

[0224] WNK3 promotes PD-L1 expression through the JNK / c-Jun pathway.

[0225] To investigate whether WNK3-dependent PD-L1 transcriptional activation is coupled with the IFN-γ pathway, in the presence or absence of IFN-γ WNK3Membrane PD-L1 levels were measured in deficient or suppressor cells. If WNK3 acts downstream of the IFN-γ signaling pathway, the addition of IFN-γ WNK3 It was hypothesized that PD-L1 expression reduced by deletion or inhibition would not be restored. However, the reduction in PD-L1 levels was restored by recombinant human IFN-γ, indicating that WNK3 acts independently of the IFN-γ signaling pathway (Figs. 5a-5d). To identify the signaling pathways involved in WNK3-mediated PD-L1 regulation, the inventors investigated several key signaling pathways regulating PD-L1 transcription in cancer cells, including JNK / c-Jun, ERK, p38, AKT, NF-κB, and JAK / STAT. 47 . As a result WNK3 By observing that only phospho-JNK and phospho-c-Jun were reduced by knockdown (Fig. 5e), it was determined that WNK3 is an upstream regulator of the JNK signaling pathway. Similarly, inhibition of JNK by JNK-IN-8 (Fig. 5f) in H2009 cells WNK3 While PD-L1 expression increased by overexpression was inhibited (Fig. 5g); JNK activation by 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride (AEBSF) WNK3 -PD-L1 expression was restored in knockdown cells (Fig. 5h). Next, to investigate whether the regulation of WNK3-mediated PD-L1 levels depends on the kinase activity of WNK3, a WNK3 kinase-deficient mutant (WNK3-K159M) 48,49The ability to regulate PD-L1 was evaluated (Fig. 5i). The WNK3-K159M mutant obtained by immunoprecipitation showed no kinase activity, whereas the wild-type WNK3 phosphorylated the substrate MBP (Fig. 14f). Since the WNK3-K159M mutant failed to increase PD-L1 expression levels like the wild-type WNK3 (Figs. 5j and 5k), PD-L1 regulation appears to be mediated at least partially by WNK3 kinase activity. Phosphorylated JNK levels were also associated with WNK3 kinase activity (Figs. 5j and 5k). Furthermore, WNK3 The kinase activity of WNK3 was inhibited by the pan-WNK inhibitor, WNK463, and WNK inhibitor 11 when treated in HEK293FT cells overexpressing [the substance] (Fig. 14g). These data show that WNK3 promotes PD-L1 expression through the JNK / c-Jun pathway.

[0227] WNK463, a pan-WNK inhibitor, inhibits tumor PD-L1 and CD8 + It inhibits MC38 tumor growth by activating T cells.

[0228] The WNK3 / PD-L1 relationship was also replicated in the human colorectal cancer cell line HCT116 (Figs. 11a, 11b), indicating that this axis operates in cancers other than NSCLC. To investigate the functional role of the WNK3 / PD-L1 axis, an MC38 synonymous colorectal adenocarcinoma mouse model responsive to anti-PD1 or anti-PD-L1 monotherapy was used. 50 Similar to human NSCLC cell lines, si Wnk3As indicated by mediated Pd-L1 deficiency (Figs. 6a, 6b, 6c), it was confirmed that MC38 possesses an intact WNK3 / PD-L1 axis. Furthermore, the pan-WNK3 inhibitor WNK463 reduced PD-L1 expression in MC38 cells (Figs. 6d, 6e). When WNK463 (0, 5, or 10 mg / kg; once daily) was administered orally via a nasogastric tube to C57BL / 6 mice with normal immune function carrying MC38 tumors (Fig. 6f), tumor growth was inhibited in a dose-dependent manner (Fig. 6g left and Fig. 6h). Similarly, WNK463 treatment promoted apoptosis in neoplasm cells or tumor cells (Fig. 6i). However, CD8 + In T-cell-deficient mice, PD-L1 expression was reduced by WNK463 treatment, but no significant tumor regression was observed (Figs. 11c, 11d and right of 6g), suggesting that the antitumor effect of WNK463 is primarily CD8 + It was found that it is primarily dependent on T cell-mediated immune responses. In particular, a dose-dependent decrease in PD-L1 membrane levels (Fig. 6j) in the CD45-negative tumor cell population (Fig. 6g, left) 9 days after WNK463 treatment and CD4 of tumor-infiltrating lymphocytes + and CD8 + A dose-dependent increase in IFN-γ and TNF-α secreted by T cells was also observed (Fig. 6k). These results suggest that WNK3 inhibition leads to CD8 through tumor PD-L1 inhibition and immune cell activation. + It can be seen that it enhances T cell-mediated anti-tumor immune responses.

[0230] The synergistic effect of WNK463 and PD-1 blockade combination therapy depends on the cytotoxicity of CD8+ T cells in vivo.

[0231] Since in vivo WNK463 treatment inhibits tumor growth by reducing PD-L1 expression, it was hypothesized that WNK463 could enhance the therapeutic efficacy of low-dose PD-1 blockade. Mice with subcutaneous MC38 tumors were administered WNK463 (10 mg / kg), an anti-PD-1 antibody (10 μg), or a combination (Fig. 13a). In contrast to monotherapy using a low-dose anti-PD-1 antibody (10 μg), which does not directly affect tumor growth, combination therapy with WNK463 completely blocked tumor growth (Fig. 13b) and showed a significant reduction in tumor weight (Fig. 13c). As expected, PD-L1 levels in the tumor cell population decreased in the WNK463 treatment group (Fig. 13d). Consistent with these observations, the combination therapy group showed the most significant reduction in CD8+, PD-1+, TIM3+, and TOX+ exhausted T cells (Fig. 13e), indicating that the activity of tumor-infiltrating CD8+ T cells was enhanced. Additionally, cytotoxic CD8+ T cells producing IFN-γ and GRANZYME B were significantly increased in tumors co-treated with WNK463 and anti-PD-1 (Fig. 13f). Overall, these results demonstrated that WNK463 and low-dose anti-PD-1 therapy have a synergistic effect on the activation of tumor-infiltrating CD8+ T cells and the inhibition of tumor growth. Finally, to evaluate the clinical significance of WNK3 in tumor immune resistance, the inventors compared the prognosis of lung cancer patients with WNK3-high expression and WNK3-low expression tumors. In both the TCGA and E-MTAB-923 cohorts, 3.6% to 10% of patients with the highest WNK3 expression showed a worse prognosis and shorter overall survival than patients with low WNK3 expression (Fig. 6l).Consistently, WNK3 expression levels were significantly associated with poor overall survival in colorectal (GSE39582) and gastric (GSE62254) cancer cohorts (Fig. 13g). These observations suggest that the worse prognosis of WNK3-high expression tumors may be due to the suppression of anti-tumor immunity induced by WNK3.

[0233] It was confirmed that PD-L1 is reduced by WNK3 inhibition in a glioblastoma cell line model.

[0234] Results of siRNA transfection and PD-L1 immunoblotting experiments on glioblastoma (GBM) cell line models U87MG and SF295 showed that even in glioblastoma cell lines WNK3 It was confirmed that PD-L1 decreased due to inhibition (Fig. 14h).

[0236] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

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Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A screening method for a composition for inhibiting the expression or activity of PD-L1 (Programmed Death-Ligand 1), comprising the following steps: (a) WNK3 (b) a step of contacting a candidate substance with a biological sample comprising a gene or a protein encoded by said gene; or a cell expressing them; (b) a step of measuring the activity or expression level of said gene or protein in said sample, and if the activity or expression level of said gene or protein is reduced, said candidate substance is determined to be a composition for inhibiting the expression or activity of PD-L1. Claim 9 A method according to claim 8, characterized in that the biological sample is a biological sample derived from cancer tissue. Claim 10 A method according to claim 9, characterized in that the cancer is selected from the group consisting of lung cancer, melanoma, brain cancer, renal cell adenocarcinoma, and colorectal cancer. Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 WNK3 An immune-enhancing composition comprising a gene inhibitor as an active ingredient, wherein the inhibitor is one or more inhibitors selected from the group consisting of a nucleic acid molecule that inhibits the expression of the gene; an antibody that specifically binds to a protein encoded by the gene, an antigen-binding fragment thereof; and WNK463, and wherein the composition is characterized by enhancing the activity of one or more cells selected from the group consisting of CD4+ T cells; CD8+ T cells; and CD4+CD8+T cells. Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 A pharmaceutical composition for the prevention or treatment of cancer for co-administration with an immune checkpoint inhibitor comprising a WNK3 inhibitor, wherein the WNK3 inhibitor is WNK463 and the immune checkpoint inhibitor is an anti-PD-1 antibody. Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 A method according to claim 10, characterized in that the brain cancer is a glioblastoma.

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