Pharmaceutical composition for preventing or treating gastric cancer

Abstract

The present specification provides a pharmaceutical composition for preventing or treating gastric cancer, comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, and DHFR, wherein the protein activity inhibitor is at least one of a compound, a peptide, a peptidomimetic, a substrate analog, an aptamer, and an antibody that specifically bind to a protein, and the gene expression inhibitor is at least one of an antisense nucleotide, RNAi, siRNA, miRNA, shRNA, and a ribozyme that complementarily bind to mRNA of a gene.

Description

Pharmaceutical composition for the prevention or treatment of gastric cancer

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of gastric cancer effective against gastric cancer containing intratumoral heterogeneity.

[0002] Gastric cancer is the most common malignant tumor in Korea and can be histologically classified into intestinal-type and diffuse-type gastric cancers. More specifically, intestinal-type gastric cancer is known to develop primarily from chronic gastritis induced by Helicobacter pylori (H. pylori), progressing through atrophic gastritis and precancerous mucosal changes such as intestinal metaplasia.

[0003] On the other hand, diffuse gastric cancer is known to develop from active inflammation of the gastric mucosa caused by H. pylori without undergoing precancerous mucosal changes. Furthermore, diffuse gastric cancer is known to occur at a younger age compared to intestinal gastric cancer, show no difference in the male-female ratio or tend to occur more frequently in women, and exhibit a more aggressive tendency than intestinal gastric cancer. Meanwhile, intestinal gastric cancer occurs predominantly in East Asia, such as Korea and Japan, whereas diffuse gastric cancer is geographically more evenly distributed compared to intestinal gastric cancer. For example, intestinal gastric cancer occurs predominantly in East Asia, such as Korea and Japan, while diffuse gastric cancer is geographically more evenly distributed compared to intestinal gastric cancer.

[0004] As such, diffuse gastric cancer has different carcinogenesis processes and biological behaviors compared to intestinal gastric cancer, requiring a differentiated approach to diagnosis and treatment; however, its pathophysiological identification has not been clear to date, and there is a need for a method to accurately diagnose and confirm it.

[0005] The background description of the invention is provided to facilitate a better understanding of the present invention. The matters described in the background description should not be construed as an acknowledgment that they exist as prior art.

[0006] Cancer is a monoclonal disease originating from a single somatic cell. Due to its genomic instability, it continuously acquires mutations and evolves, resulting in intratumor heterogeneity that includes cells with diverse genomic variations. During this evolutionary process of cancer, the tumor microenvironment (TME) acts as a significant selective pressure (or evolutionary pressure) and can serve as a key target for regulating cancer cell metastasis and overcoming drug resistance.

[0007] In this regard, large international cancer genomics consortia such as the TCGA and ICGC have been sequencing the genomes, transcriptomes, and epigenomes of human cancer tissues over the past decade and identifying human cancer genomic mutations. However, genomic studies targeting bulk tissue have had limitations in investigating the interactions between the various subclones present within the cancer tissue and the tumor microenvironment.

[0008] Meanwhile, the inventors of the present invention focused on patient-derived organoids (PDOs). More specifically, a PDO is a cell culture created in a three-dimensional culture state by isolating cells from normal or diseased tissues provided by a patient who has consented to research. This culture is produced in a medium specialized for each organ that can regulate differentiation and stem cell activity. Organoids cultured in this way possess the characteristic of being able to reproduce the histological structural similarity and various physiological and genetic features of the parent tissue. In other words, since the PDO model not only histologically and genetically reflects actual patient cancer cells but also preserves and reproduces the tumor microenvironment, it can be effective for drug screening for personalized treatment.

[0009] Accordingly, the inventors of the present invention recognized that intratumoral heterogeneity according to the type of gastric cancer and the resulting differential treatment methods could be overcome through PDO, and consequently, the inventors of the present invention developed a PDO capable of representing intratumoral heterogeneity for gastric cancer and an analysis method based thereon.

[0010] Furthermore, the inventors of the present invention have discovered that by disassembling gastric cancer samples isolated from an individual into a specific number and region, producing individual organoids from them, and analyzing their genomes, it is possible to determine intratumoral heterogeneity regarding gastric cancer. Furthermore, through signal analysis of organoids in the region where intratumoral heterogeneity occurred, it is possible to determine effective treatment methods and drugs for individuals with intratumoral heterogeneity.

[0011] Ultimately, through the above process, the inventors of the present invention have discovered a new drug capable of treating gastric cancer exhibiting intratumoral heterogeneity.

[0012] Accordingly, the problem that the present invention aims to solve is to provide a pharmaceutical composition for the prevention or treatment of gastric cancer comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, or DHFR.

[0013] In addition, another problem that the present invention aims to solve is to provide a screening method for candidate substances for the prevention or treatment of gastric cancer that exhibit intratumoral heterogeneity.

[0014] In addition, another problem that the present invention aims to solve is to provide a pharmaceutical composition for the prevention or treatment of gastric cancer for co-administration with cisplatin comprising a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1 and MTHFD1 as an active ingredient.

[0015] In addition, another problem that the present invention aims to solve is to provide a biomarker composition for diagnosing intratumoral heterogeneity in gastric cancer patients comprising SHMT1, TYMS, MTHFD1, or DHFR.

[0016] In addition, another problem that the present invention aims to solve is to provide a composition for diagnosing intratumoral heterogeneity in gastric cancer patients comprising, as an active ingredient, a preparation for measuring the level of a protein or gene encoding at least one of SHMT1, TYMS, MTHFD1, and DHFR.

[0017] In addition, another problem that the present invention aims to solve is to provide a method for preventing or treating gastric cancer comprising the step of administering to an individual a pharmaceutical composition comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, and DHFR.

[0018] In addition, another problem that the present invention aims to solve is to provide a method for diagnosing intratumoral heterogeneity in a gastric cancer patient, comprising the step of measuring the level of a protein or a gene encoding at least one of SHMT1, TYMS, MTHFD1, and DHFR from a biological sample from a gastric cancer patient.

[0019] The problems of the present invention are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0020] In order to solve the problem described above, a pharmaceutical composition for the prevention or treatment of gastric cancer is provided, comprising as an active ingredient a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, and DHFR according to one embodiment of the present invention.

[0021] According to a feature of the present invention, the protein activity inhibitor may be at least one selected from the group consisting of a compound, peptide, peptide mimic, substrate analog, aptamer, and antibody that specifically binds to the protein.

[0022] According to another feature of the present invention, the gene expression inhibitor may be at least one selected from the group consisting of antisense nucleotides, RNAi, siRNA, miRNA, shRNA, and ribozymes that bind complementarily to the mRNA of the gene.

[0023] According to another feature of the present invention, the gastric cancer may be of the intestinal type or the diffuse type, and preferably may be of the intestinal type.

[0024] According to another feature of the present invention, the long type may include the genetic features of the lesser type.

[0025] According to another feature of the present invention, the gastric cancer may be characterized by heterogeneity within the tumor.

[0026] According to another feature of the present invention, the compound that specifically binds to the SHMT1 protein may be SHIN1.

[0027] According to another feature of the present invention, the compound that specifically binds to the TYMS protein may be at least one of pemetrexed and raltitrexed.

[0028] According to another feature of the present invention, the compound that specifically binds to the MTHFD1 protein may be at least one of LY 345899, DS18561882 and TH9619.

[0029] According to another feature of the present invention, the compound that specifically binds to the DHFR protein may be at least one of MTX (Methotrexate), Pemetrexed, Raltitrexed, Pralatrexate, and aminopterin.

[0030] According to another feature of the present invention, the composition may further include cisplatin.

[0031] In order to solve the problem described above, a screening method for candidate substances for the prevention or treatment of gastric cancer according to another embodiment of the present invention is provided.

[0032] According to a feature of the present invention, the method comprises the steps of: treating a cell containing at least one gene or protein among SHMT1, TYMS, MTHFD1, and DHFR with a test substance to be analyzed; measuring the expression level of at least one gene among SHMT1, TYMS, MTHFD1, and DHFR in the cell; and, if the test substance reduces the measured gene expression level, selecting the test substance as a candidate substance for prevention or treatment of gastric cancer exhibiting intratumoral heterogeneity.

[0033] According to another feature of the present invention, the gastric cancer may be pathologically intestinal in type and genomically diffuse in type.

[0034] According to another feature of the present invention, the measurement may be performed by at least one of the following methods: Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real-time Polymerase Chain Reaction (real-time PCR), Northern Blot, Enzyme Linked Immunosorbent Assay (ELISA), Radioimmunoassay, Radioimmunodiffusion, Immunohistochemical Analysis, and Fluorescence-Activated Cell Sorting (FACS).

[0035] In order to solve the problem described above, a pharmaceutical composition for the prevention or treatment of gastric cancer for co-administration with cisplatin is provided, comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1 and MTHFD1 according to another embodiment of the present invention.

[0036] To solve the problem described above, a biomarker composition for diagnosing intratumoral heterogeneity in gastric cancer patients comprising SHMT1, TYMS, MTHFD1, or DHFR according to another embodiment of the present invention is provided.

[0037] According to a feature of the present invention, the gastric cancer patient may be a patient diagnosed with intestinal-type gastric cancer pathologically.

[0038] In order to solve the problem described above, a composition for diagnosing intratumoral heterogeneity in gastric cancer patients is provided, comprising as an active ingredient a preparation for measuring the level of a protein or a gene encoding at least one of SHMT1, TYMS, MTHFD1, and DHFR according to another embodiment of the present invention.

[0039] According to a feature of the present invention, the gastric cancer patient may be a patient diagnosed with intestinal-type gastric cancer pathologically.

[0040] According to another feature of the present invention, the agent for measuring the protein level may be any one selected from the group consisting of an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a PNA, or an aptamer that specifically binds to the protein.

[0041] According to another feature of the present invention, the measurement of the protein level may be performed by one or more methods selected from the group consisting of Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), and protein chip.

[0042] According to another feature of the present invention, the agent for measuring the gene level may be an antisense oligonucleotide, a primer pair, or a probe that specifically binds to a gene.

[0043] According to another feature of the present invention, the measurement at the gene level may be performed by one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern blotting, and DNA chips.

[0044] In order to solve the problem described above, a method for preventing or treating gastric cancer according to another embodiment of the present invention is provided.

[0045] According to a feature of the present invention, the preventive or therapeutic method comprises the step of administering to an individual a pharmaceutical composition comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, and DHFR.

[0046] According to another feature of the present invention, the gastric cancer may be pathologically intestinal in type and genomically diffuse in type.

[0047] In order to solve the problem described above, a method for diagnosing intratumoral heterogeneity in a gastric cancer patient according to another embodiment of the present invention is provided.

[0048] According to a feature of the present invention, the diagnostic method comprises the step of measuring the level of a protein or a gene encoding at least one of SHMT1, TYMS, MTHFD1, and DHFR from a biological sample from a gastric cancer patient.

[0049] According to another feature of the present invention, the gastric cancer patient may be a patient who has been pathologically diagnosed with intestinal-type gastric cancer.

[0050] The present invention can serve as a new therapeutic strategy for gastric cancer, which involves resistance to and therapeutic limitations of conventional anticancer drugs due to intratumoral heterogeneity.

[0051] The effects according to the present invention are not limited to those exemplified above, and various other effects are included in this specification.

[0052] FIG. 1 is a flowchart of a screening method for candidate substances for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0053] FIG. 2 is a schematic diagram of the process of constructing a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0054] Figure 3 is the result of immunohistochemistry on intestinal tissue in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0055] Figure 4a is a UMAP visualization result of the sequencing analysis result of an organoid sample in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0056] FIG. 4b is the origin sample and cell distribution results for the sequencing analysis results of an organoid sample in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0057] FIG. 4c is the KEGG pathway analysis result for the sequencing analysis result of an organoid sample in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0058] Figure 5 shows the results of the analysis of CSC-related pathways in diffuse (hGC143) organoids.

[0059] Figures 6a and 6b are the results of CSC marker expression in organoids according to gastric cancer type in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0060] Figure 7 is the result of KEGG pathway analysis for organoids derived from the intestinal type (hGC141) in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0061] Figures 8a and 8b are CD44 high / low classification results for T2 organoids derived from the intestinal type (hGC141) in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0062] FIG. 9 is a schematic diagram of one-carbon metabolism (one carbon pool by folate) according to one embodiment of the present invention.

[0063] FIG. 10a is the result of one-carbon metabolism-related gene expression in T2 organoids derived from intestinal type (hGC141) in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0064] FIG. 10b is the result of evaluating relative cell viability according to anticancer agents (SHIN1, DS18561882, cisplatin, or 5-FU) in T2 organoids derived from an intestinal type (hGC141) according to one embodiment of the present invention.

[0065] FIG. 10c is the result of evaluating cell viability compared to a negative control (DMSO) in T2 organoids derived from an intestinal type (hGC141) according to one embodiment of the present invention, after treatment with cisplatin of SHIN1 or DS18561882.

[0066] Figure 11a shows the results of HSA (Highest Single Agent) and Bliss Independence analysis to confirm the synergistic effect of combined treatment with SHIN1 and cisplatin according to one embodiment of the present invention.

[0067] FIG. 11b is the result of HSA (Highest Single Agent) and Bliss Independence analysis to confirm the synergistic effect of combined treatment of DS18561882 and cisplatin according to one embodiment of the present invention.

[0068] Figures 12a and 12b are the results of a genetic population analysis of clones of genetically diffuse intestinal gastric cancer cells in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0069] When it is mentioned that a component (e.g., a second component) is "directly connected" or "directly coupled," it may be understood that no other component (e.g., a third component) exists between said component and said other component.

[0070] As used in this document, the expression “configured to” may be replaced, depending on the context, with, for example, “suitable for,” “having the capacity to,” “designed to,” “adapted to,” “made to,” or “capable of.” The term “configured to” does not necessarily mean only “specifically designed to” in hardware. Instead, in some situations, the expression “device configured to” may mean that the device is “capable of” together with other devices or components. For example, the phrase “a processor configured (or set) to perform A, B, and C” may mean a dedicated processor for performing said operations (e.g., an embedded processor), or a generic-purpose processor (e.g., a CPU or an application processor) capable of performing said operations by executing one or more software programs stored in a memory device.

[0071] The terms used in this document are used merely to describe specific embodiments and are not intended to limit the scope of other embodiments. Singular expressions may include plural expressions unless the context clearly indicates otherwise. Terms used herein, including technical or scientific terms, may have the same meaning as generally understood by those skilled in the art described in this document. Terms used in this document that are defined in general dictionaries may be interpreted as having the same or similar meaning as they have in the context of the relevant technology, and are not to be interpreted in an ideal or overly formal sense unless explicitly defined in this document. In some cases, even terms defined in this document may not be interpreted to exclude the embodiments of this document.

[0072] The features of each of the various embodiments of the present invention may be combined or combined with one another, either partially or wholly, and as will be fully understood by those skilled in the art, various technical interlocking and operation are possible, and each embodiment may be implemented independently of one another or together in an interlocking relationship.

[0073] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0074] In the following, terms used within this specification are explained for clarity of explanation.

[0075] In this document, expressions such as "have," "can have," "include," or "can include" refer to the existence of the relevant feature (e.g., numerical values, functions, actions, or components, etc.) and do not exclude the existence of additional features.

[0076] In this document, "or" means "and / or" unless otherwise noted. Expressions such as "A or B," "at least one of A or / and B," or "one or more of A or / and B" may include all possible combinations of items listed together. For example, "A or B," "at least one of A and B," or "at least one of A or B" may refer to cases including (1) at least one A, (2) at least one B, or (3) both at least one A and at least one B.

[0077] The term “SHTM1” used in the present invention refers to Serine Hydroxymethyltransferase 1, which is expressed by the SHMT1 gene, catalyzes the reaction that converts serine into glycine to produce 5,10-methylene-tetrahydrofolate, and plays a key role in the folic acid metabolic pathway.

[0078] The term “TYMS” used in the present invention refers to Thymidylate Synthase expressed by the TYMS gene, which catalyzes the reaction of converting dUMP to dTMP and plays an essential role in DNA synthesis and cell proliferation.

[0079] The term “MTHFD1” used in the present invention refers to Methylenetetrahydrofolate Dehydrogenase 1, which is expressed by the MTFD1 gene and is a multifunctional enzyme that interconverts various forms of tetrahydrofolate (THF) and is involved in folate metabolism and methylation reactions.

[0080] The term “DHFR” used in the present invention refers to Dihydrofolate Reductase expressed by the DHFR gene, which catalyzes the reaction of reducing dihydrofolate (DHF) to tetrahydrofolate (THF) and is an enzyme essential for maintaining folate metabolism and DNA synthesis.

[0081] The term "expression" as used in the present invention refers to the production of a protein or nucleic acid in a cell. "Protein" is used interchangeably with "polypeptide" or "peptide" and refers to a polymer of amino acid residues, for example, as generally found in proteins in their natural state. "Polynucleotide" or "nucleic acid" refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) in a single- or double-stranded form. Unless otherwise limited, it also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. "mRNA" is RNA that transmits genetic information (gene-specific base sequences) from a specific gene to a ribosome that determines the amino acid sequence during the process of protein synthesis.

[0082] In the present invention, the term "primer" refers to a short single-strand oligonucleotide that acts as a starting point for DNA synthesis. The primer specifically binds to a polynucleotide that serves as a template under suitable buffer and temperature conditions, and DNA is synthesized by DNA polymerase adding a nucleoside triphosphate having a base complementary to the template DNA to the primer and linking them. The primer generally consists of a sequence of 15 to 30 bases, and the melting temperature (Tm) at which it binds to the template strand varies depending on the base composition and length. Additionally, the primer is a sense and antisense nucleic acid having a sequence of 7 to 50 nucleotides, and may incorporate additional features that do not alter the basic properties of the primer acting as a starting point for DNA synthesis. The sequence of the primer does not need to be completely complementary to a portion of the template's base sequence; it is sufficient to have sufficient complementarity within the range that allows it to hybridize with the template and perform its inherent function.

[0083] In addition, primers for an amplification reaction consist of a set (pair) that binds complementarily to the template (or sense) and the opposite end (antisense) of a specific segment of the target mRNA to be amplified. Primers can be easily designed by a person skilled in the art by referring to the target mRNA or cDNA sequence.

[0084] In the present invention, the term "probe" refers to a fragment of a polynucleotide, such as RNA or DNA, with a length ranging from a few to hundreds of base pairs, capable of specifically binding to the mRNA or cDNA (complementary DNA) of a specific gene, and is labeled so that the presence or absence and expression level of the target mRNA or cDNA to be bound can be confirmed.

[0085] The above primer or probe can be chemically synthesized using a phosphoramidite solid support synthesis method or other widely known methods. Additionally, the primer or probe can be modified in various ways according to methods known in the art, to the extent that hybridization with the target mRNA is not interfered with. Examples of such modifications include methylation, capping, substitution with one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), and the binding of fluorescent or enzymatic labeling materials.

[0086] In the present invention, "the level is increased" means that something that was not previously detected is detected, or that the amount detected is relatively higher than the normal level. To a person skilled in the art, the meaning of the opposite term can be understood as having the opposite meaning in accordance with the above definition.

[0087] As used herein, the term “about” refers to a normal margin of error for each value that is readily known to those skilled in the art. In this specification, the designation of an “about” value or parameter includes an example relating to the value or parameter itself. Furthermore, unless otherwise stated or otherwise evident from the context, the term “about” indicates a range of values ​​corresponding to within 10% in either direction (greater than or less than) a mentioned reference value.

[0088] As used herein, the term “patient or individual” refers interchangeably to any single animal requiring treatment, more preferably a mammal (such as non-human animals, including, e.g., cats, dogs, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates). In various embodiments of this specification, the patient referred to may be a human.

[0089] As used in this specification, the term "composition for treating or preventing gastric cancer" may mean a pharmaceutical composition having a therapeutic effect on gastric cancer.

[0090] In this case, when the pharmaceutical composition is formulated as a liquid solution, it may be diluted by a pharmaceutically acceptable carrier. More specifically, a pharmaceutically acceptable carrier may refer to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the administered compound. For example, acceptable pharmaceutical carriers for a pharmaceutical composition formulated as a liquid solution may be sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Additionally, other conventional additives, such as antioxidants, buffers, and bacteriostatic agents, may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added so that the composition for treating or preventing gastric cancer can be formulated into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0091] A composition for the treatment or prevention of gastric cancer may be administered orally or parenterally, or by application or spraying to the affected area. The composition for the treatment or prevention of gastric cancer may also be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or parenterally via local administration.

[0092] Suitable application, spraying, and dosage of a composition for the treatment or prevention of gastric cancer may vary depending on factors such as the formulation method, method of administration, age, body weight, sex, severity of disease symptoms, food, time of administration, route of administration, excretion rate, and response sensitivity. Additionally, a skilled physician or veterinarian can easily determine and prescribe a dosage of a composition for the treatment or prevention of gastric cancer that is effective for the intended treatment.

[0093] Oral formulations containing a composition for treating or preventing gastric cancer as an active ingredient may be tablets, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups, or elixirs. In this case, to formulate into formulations such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch or sweet potato starch; lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be included, and in the case of capsule formulations, a liquid carrier such as fatty oil may be further included in addition to the aforementioned substances.

[0094] Pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention

[0095] A pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention is a composition specialized for gastric cancer containing intratumoral heterogeneity. In the case of cancer containing intratumoral heterogeneity, the metastatic ability and response to anticancer agents may differ among cancer cells, which may result in limitations in the anticancer effect of conventional anticancer agents. Accordingly, the pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention can serve as a new therapeutic alternative for individuals containing intratumoral heterogeneity who exhibit resistance to or show insufficient efficacy against conventional anticancer agents.

[0096] A pharmaceutical composition for preventing or treating gastric cancer according to one embodiment of the present invention is a pharmaceutical composition for preventing or treating gastric cancer comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, and DHFR, and may be a drug specialized for gastric cancer including intratumoral heterogeneity.

[0097] For example, if an individual's gastric cancer is pathophysiologically diagnosed as intestinal type but gastric cancer cells in some regions contain diffuse genomic characteristics, a pharmaceutical composition for preventing or treating gastric cancer according to one embodiment of the present invention may be used.

[0098] Accordingly, gastric cancer in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention may be of the intestinal type or diffuse type, and preferably, may be gastric cancer that is pathologically intestinal type and includes genomic characteristics of the diffuse type.

[0099] At this time, the protein activity inhibitor is at least one of a compound, peptide, peptide mimic, substrate analog, aptamer, and antibody that specifically binds to a protein, and the gene expression inhibitor may be at least one of an antisense nucleotide, RNAi, siRNA, miRNA, shRNA, and ribozyme that binds complementarily to the mRNA of a gene, but is not limited thereto.

[0100] Furthermore, a compound that specifically binds to the SHMT1 protein may be SHIN1, but is not limited thereto. In this case, SHIN1 is an SHMT inhibitor that can inhibit the production of glycine and CH2-THF and is used in cancers other than gastric cancer.

[0101] The above “SHIN1” has the molecular formula C 24 H 24 It is a SHMT1 selective inhibitor having N4O2, and its IUPAC name is 6-amino-4-[3-(hydroxymethyl)-5-phenylphenyl]-3-methyl-4-propan-2-yl-2H-pyrano[2,3-c]pyrazole-5-carbonitrile, and its molecular weight is 400.5 g / mol.

[0102] Compounds that specifically bind to TYMS proteins may be at least one of Pemetrexed and Raltitrexed, but are not limited thereto.

[0103] The above “Pemtrexed” has the molecular formula C 10 H 21 It is a multiple folate metabolism enzyme inhibitor having N5O6, and its IUPAC name is (2S)-2-[[4-[2-(2-amino-4-oxo-3,7-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioic acid, and it has synonyms such as 137281-23-3 and LY231514, and its molecular weight is 427.4 g / mol. Pemetrexed is an antimetabolite that inhibits enzymes involved in the folate-related pathway and is mainly used in lung cancer. The related enzymes may be, but are not limited to, TYMS (Thymidylate synthase), DHFR (dihydrofolate reductase), and GARFT (glycinamide ribonucleotide formyltransferase).

[0104] The above “Raltitrexed” has the molecular formula C 21 H 22It is a thymidylate synthase (TYMS) inhibitor containing N4O6S, with the IUPAC name (2S)-2-[[5-[methyl-[(2-methyl-4-oxo-3H-quinazolin-6-yl)methyl]amino]thiophene-2-carbonyl]amino]pentanedioic acid, synonyms such as 112887-68-0 and ZD1694, and a molecular weight of 458.5 g / mol. Raltitrexed is a TYMS-associated folic acid inhibitor that inhibits the formation of precursor pyrimidine nucleotides and can inhibit the formation of DNA or RNA (inhibiting the growth and survival of both normal and cancer cells).

[0105] Compounds that specifically bind to the MTHFD1 protein may be at least one of LY 345899, DS18561882 and TH9619, but are not limited thereto.

[0106] The above “LY345899” has the molecular formula C 20 H 21 It is an MTHFD1 inhibitor having N7O7, and its IUPAC name is (2S)-2-[[4-[(6aR)-3-amino-1,9-dioxo-5,6,6a,7-tetrahydro-2H-imidazo[1,5-f]pteridin-8-yl]benzoyl]amino]pentanedioic acid, and it has synonyms such as CHEMBL1233930 and BDBM50535669, and its molecular weight is 471.4 g / mol.

[0107] The above “DS18561882” is molecular formula C 28 H 31It is an MTHFD1 inhibitor having F3N4O6S, and its IUPAC name is N-[4-[8-[(3S)-3,4-dimethylpiperazin-1-yl]-7-methyl-5-oxo-2,4-dihydro-1H-chromeno[3,4-c]pyridine-3-carbonyl]-2-(trifluoromethoxy)phenyl]methanesulfonamide, and it has synonyms such as 2227149-22-4 and CHEMBL4463968, and its molecular weight is 608.6 g / mol. DS18561882 is reported to be an MTHFD1 / 2 inhibitor that can be used in breast and colorectal cancers.

[0108] The above “TH9619” has the molecular formula C 17 H 18 It is an MTHFD1 inhibitor having FN7O7, and its IUPAC name is (2S)-2-[[5-[[2-(2,4-diamino-6-oxo-1H-pyrimidin-5-yl)acetyl]amino]-3-fluoropyridine-2-carbonyl]amino]pentanedioic acid, and it has synonyms such as 2379556-22-4 and SCHEMBL21494659, and its molecular weight is 451.4 g / mol.

[0109] Compounds that specifically bind to DHFR protein may be at least one of MTX (Methotrexate), Pemetrexed, Raltitrexed, Pralatrexate, and aminopterin, but are not limited thereto.

[0110] The above “Methotrexate” has the molecular formula C 20 H 22It is a DHFR inhibitor having N8O5, and its IUPAC name is (2S)-2-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino]benzoyl]amino]pentanedioic acid, and it has synonyms such as Rheumatrex, Amethopterin, and Metatrexan, and its molecular weight is 454.4 g / mol.

[0111] The above “Pralatrexate” has the molecular formula C 23 H 23 It is a DHFR inhibitor having N7O5, and its IUPAC name is (2S)-2-[[4-[1-(2,4-diaminopteridin-6-yl)pent-4-yn-2-yl]benzoyl]amino]pentanedioic acid, and it has synonyms such as Folotyn and pralatrexato, and its molecular weight is 477.5 g / mol. Pralatrexate is an antifolate and DHFR inhibitor that has a high affinity for reduced folate carriers and is used in lymphoma, non-small cell lung cancer, breast cancer, and bladder cancer.

[0112] The above “Aminopterin” has the molecular formula C 19 H 20 It is a DHFR inhibitor having N8O5, and its IUPAC name is (2S)-2-[[4-[(2,4-diaminopteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid, and it has synonyms such as 4-Aminofolic acid and Aminopterine, and its molecular weight is 440.4 g / mol.

[0113] A pharmaceutical composition for preventing or treating gastric cancer according to one embodiment of the present invention has the effect of significantly improving the efficacy of preventing and treating gastric cancer when used in combination with cisplatin.

[0114] Accordingly, a pharmaceutical composition for the prevention or treatment of gastric cancer may be a pharmaceutical composition for the prevention or treatment of gastric cancer for co-administration with cisplatin, but is not limited thereto.

[0115] As described above, the compound used in the pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention is not used for gastric cancer and may be a composition effective for other cancers such as lung cancer or breast cancer. However, the compound used in the pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention may have an effective anticancer effect in gastric cancer including tumor heterogeneity, rather than general gastric cancer.

[0116] A biomarker for diagnosing intratumoral heterogeneity in gastric cancer patients and a diagnostic composition using the same according to one embodiment of the present invention

[0117] The present invention provides a biomarker composition for diagnosing intratumoral heterogeneity in gastric cancer patients comprising SHMT1, TYMS, MTHFD1, or DHFR.

[0118] More specifically, in the case of gastric cancer including intratumoral heterogeneity, for example, gastric cancer including pathologically intestinal type and genomically diffuse type features, it may include features of increased expression for at least one marker among SHMT1, TYMS, MTHFD1 and DHFR.

[0119] The inventors have confirmed that SHMT1, TYMS, MTHFD1, and DHFR are overexpressed in specific regions within the tumor heterogeneity, that is, genomically diffuse-type characteristics, of gastric cancer tissue in patients pathologically diagnosed with intestinal type, and such overexpression has significant diagnostic value as a biological indicator reflecting tumor heterogeneity.

[0120] In particular, SHMT1, TYMS, MTHFD1, and DHFR are enzymes involved in the folate metabolism pathway and are closely related to the proliferation and metabolic activity of cancer cells. According to the present invention, immunohistochemical analysis confirmed that the expression levels of these proteins are significantly increased in heterogeneous sites within gastric cancer tissue, and based on this, tumor heterogeneity can be effectively diagnosed.

[0121] Accordingly, the present invention provides a composition for diagnosing intratumoral heterogeneity in gastric cancer patients comprising, as an active ingredient, a preparation for measuring the level of a protein or a gene encoding at least one of SHMT1, TYMS, MTHFD1, and DHFR.

[0122] At this time, the preparation for measuring the protein level may be a preparation for confirming the presence and degree of expression of the protein, and may be for measuring the amount of protein. As analytical methods for this purpose, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorter (FACS), protein chip, etc. may be used, and may be an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA, or aptamer that specifically binds to the protein.

[0123] The above antibody is a term known in the art and refers to a specific protein molecule directed toward an antigenic site. Each gene can be cloned into an expression vector according to conventional methods to obtain a protein encoded by the marker gene, and can be manufactured from the obtained protein by conventional methods. The form of the above antibody is not particularly limited and may be a monoclonal antibody, a polyclonal antibody, or a chimeric antibody, and optionally may include special antibodies such as humanized antibodies.

[0124] The above-mentioned aptamer refers to a single-stranded DNA (ssDNA) or RNA that has high specificity and affinity for a specific substance. Aptamers have very high affinity for a specific substance, are stable, can be synthesized by a relatively simple method, can be modified in various ways to increase binding strength, and can target cells, proteins, and even small organic substances. Therefore, their specificity and stability are much higher than those of antibodies that have already been developed.

[0125] The agent for measuring the gene level described above is a agent for confirming the presence and expression level of mRNA, and may be used to measure the amount of mRNA. For this purpose, analytical methods such as reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern blotting, DNA chips, etc. may be used, and may be an antisense oligonucleotide, primer pair, or probe that specifically binds to the mRNA.

[0126] The above antisense oligonucleotide may refer to an oligomer having a backbone between nucleotide base sequences and subunits, wherein the antisense oligomer hybridizes with a target sequence within RNA via Watson-Crick base pairing, thereby allowing the formation of a mRNA and RNA:oligomer heterodimer within the target sequence. The oligomer may have exact or approximate sequence complementarity with respect to the target sequence. This antisense oligomer may block or inhibit the translation of mRNA and alter the processing of mRNA to produce splice variants of mRNA.

[0127] The primer may refer to a short nucleic acid sequence having a short free 3-terminal hydroxyl group, capable of forming base pairs with a complementary template, and functioning as a starting point for template strand replication. The primer may initiate DNA synthesis in the presence of a reagent for a polymerization reaction (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.

[0128] The above probe may refer to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to hundreds of bases in length, capable of forming a specific binding with mRNA, and is labeled so that the presence or absence of a specific mRNA can be confirmed. The probe may be constructed in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of a suitable probe and hybridization conditions may be modified based on those known in the art.

[0129] The above antisense oligonucleotides, primers, or probes, etc., can be chemically synthesized using the phosphoramidite solid support method or other widely known methods. These nucleic acid sequences can also be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution with one or more homologues of natural nucleotides, and modification between nucleotides (e.g., modification to uncharged linkages such as methylphosphonates, phosphotriesters, phosphoramidates, carbamates, or charged linkages such as phosphorothioates, phosphorodithioates).

[0130] Accordingly, when a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention includes an inhibitor capable of reducing the aforementioned marker, the effect of preventing or treating gastric cancer including intratumoral heterogeneity may be excellent.

[0131] Screening method for candidate substances for the prevention or treatment of gastric cancer according to one embodiment of the present invention

[0132] Furthermore, the aforementioned marker can be used in a drug screening method for gastric cancer, including intratumoral heterogeneity.

[0133] Hereinafter, with reference to FIG. 1, a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention will be described.

[0134] FIG. 1 is a flowchart of a screening method for candidate substances for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0135] Accordingly, referring to FIG. 1, a screening method for a candidate substance for the prevention or treatment of gastric cancer according to one embodiment of the present invention may include the steps of treating a test substance to be analyzed in a cell containing at least one gene or protein among SHMT1, TYMS, MTHFD1, and DHFR; measuring the expression level of at least one gene among SHMT1, TYMS, MTHFD1, and DHFR in the cell; and, if the test substance reduces the measured gene expression level, selecting the test substance as a candidate substance for the prevention or treatment of gastric cancer containing intratumoral heterogeneity, wherein the gastric cancer may be gastric cancer containing intratumoral heterogeneity, that is, gastric cancer having pathologically intestinal type and genomically diffuse type characteristics.

[0136] Furthermore, the measurement may be performed by at least one of the following methods: Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real-time Polymerase Chain Reaction (real-time PCR), Northern Blot, Enzyme Linked Immunosorbent Assay (ELISA), Radioimmunoassay, Radioimmunodiffusion, Immunohistochemical Analysis, and Fluorescence-Activated Cell Sorting (FACS), but is not limited thereto.

[0137] Accordingly, the screening method for candidate substances for the prevention or treatment of gastric cancer according to one embodiment of the present invention can select candidate substances capable of effectively preventing or treating gastric cancer including tumor heterogeneity.

[0138] Ultimately, since the pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention can provide a new target gene, it can provide information for selecting a new effective drug other than existing anticancer drugs. Ultimately, the pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention can present a new treatment strategy for gastric cancer in which intratumoral heterogeneity occurs.

[0139] Method for preventing or treating gastric cancer according to one embodiment of the present invention

[0140] The present invention provides a method for preventing or treating gastric cancer comprising the step of administering to an individual a pharmaceutical composition comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, and DHFR.

[0141] More specifically, the gastric cancer may be pathologically intestinal in type and genomically diffuse in type, but is not limited thereto.

[0142] The pharmaceutical composition of the present invention may be administered in a therapeutically effective amount or a pharmaceutically effective amount.

[0143] In the present invention, the term "therapeutically effective amount" refers to a pharmaceutically acceptable amount of salt of a composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, the patient's condition, etc. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0144] In the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0145] A method for diagnosing intratumoral heterogeneity in a gastric cancer patient according to one embodiment of the present invention

[0146] The present invention provides a method for diagnosing intratumoral heterogeneity in a gastric cancer patient, comprising the step of measuring the level of a protein or a gene encoding at least one of SHMT1, TYMS, MTHFD1, and DHFR from a biological sample from a gastric cancer patient.

[0147] More specifically, the gastric cancer patient may be a patient diagnosed with intestinal-type gastric cancer pathologically, but is not limited thereto.

[0148] In gastric cancer patients who are pathologically diagnosed as having the intestinal type but genomically exhibit diffuse characteristics, at least one protein among SHMT1, TYMS, MTHFD1, and DHFR, or the gene encoding it, may be overexpressed in tissues containing intratumoral heterogeneity. Therefore, measuring the level of at least one protein among SHMT1, TYMS, MTHFD1, and DHFR, or the gene encoding it, in diagnostic samples can provide information more quickly and effectively on whether the target gastric cancer patient exhibits intratumoral heterogeneity.

[0149] The measurement of the above protein or gene level can be performed without limitation using techniques known in the field, and it can be determined whether the patient has tumor heterogeneity by comparing with normal levels of protein or gene expression.

[0150] In this case, the above normal level refers to the average expression levels of SHMT1, TYMS, MTHFD1, and / or DHFR measured in non-tumorous tissue of the gastric mucosa or gastric tissue of healthy individuals, which may be established based on accredited bioinformatics databases or clinically validated gene expression profiles of normal populations. Additionally, the average expression level of gastric cancer tissue judged to be free of intratumoral heterogeneity within the same individual may also be used as a normal standard.

[0151] The present invention will be explained in more detail below through examples. However, since these examples are merely illustrative of the present invention, the scope of the present invention should not be interpreted as being limited by these examples.

[0152] Confirmation of a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention

[0153] Hereinafter, with reference to FIGS. 2 to 12, the verification process and results for a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention will be described.

[0154] FIG. 2 is a schematic diagram of the process of constructing a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0155] First, raw specimens for gastric cancer were collected by surgical resection from individuals of each type, intestinal and diffuse, and contain gastric cancer lesions. For organoid culture, one or more culture specimens may be collected from the collected raw specimens, and in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention, after the raw specimen is washed with PBS, four culture specimens (t1, t2, t3, t4) at different locations are each 1 mm 3 They were collected in sizes, with each culture specimen having a minimum spacing of 10 mm.

[0156] Next, the respective culture specimens for the intestinal type and diffuse type were cultured into organoids. More specifically, 1 mm 3 Culture specimens of the size were immersed in basal medium containing 0.5 mg / ml collagenase type I and cultured in a roller shaker at 37°C for about 1 hour and 30 minutes. Then, the cultured specimens were passed through a 70 μm mesh, washed twice with PBS, and resuspended using Matrigel. The resuspended specimens were plated at 25 μL per well in a 48-well plate and cultured in a 37°C incubator for 1 to 2 weeks. During this time, the medium was replaced every 2 to 3 days during culture, and the medium used included 50% Wnt-3a medium, 10% R-spondin medium, 1 mM N-Acetylcysteine ​​(Sigma-Aldrich), 1 nM [Leu]-Gastrin I 50 ng / ml mEGF (Invitrogen), 100 ng / ml Noggin (peprotech), 200 ng / ml FGF10 (peprotech), 10 mM Nicotinamide (Invitrogen), 10 µM RHOKi Y-27632 (Wako), and 1X (100 µg / ml) Primocin (Invitrogen). Furthermore, the samples grown into organoids were cultured as Matrigel droplets at a rate of 25 μL per well in 48-well plates and subcultured every 1 to 2 weeks.

[0157] Finally, the cultured organoids were used for single-nucleus RNA sequencing analysis. For the single-nucleus RNA sequencing analysis, the frozen organoids were homogenized to count the nuclei, and then the nuclei were separated using a flow cytometer. Furthermore, a cDNA library containing barcodes for snRNA sequencing was generated from the sorted nuclei using the 10X Genomics Chromium Instrument and a cDNA synthesis kit (10x Genomics: Chromium Next GEM Single Cell 3′Library and Gel Bead Kit v3.1). The quality of the cDNA library was determined using Agilent Bioanalyzer, and genomic analysis of the organoids was performed using paired-end 200bp Flow Cells of the Illumina NovaSeq 5000 / 6000 S1 Rgt Kit v1.5 (200 cycles / read lengths: 28 bp Read1, 10 bp I7 Index, 10 bp I5 Index, and 90 bp Read2).

[0158] Figure 3 shows the results of immunohistochemistry on intestinal tissue in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention. Immunohistochemistry (IHC) was performed using a Ventana XT automated staining instrument (Ventana Medical Systems, Tucson, AZ, USA), and antibodies against C-erb2, MLH1, PMS2, MLH2, MLH6, and HE were used according to the instructions of each manufacturer.

[0159] Referring to FIG. 3, all four sites of the collected specimen and four sites of the cultured organoid were classified as microsatellite stable (MSS) showing positive reactions to MLH1, PMS2, MLH2, and MLH6, and all showed negative results for C-erb2, which indicates that the organoid cultured according to one embodiment of the present invention is a preclinical model that accurately represents patient tissue.

[0160] Figure 4a shows the UMAP visualization results of the sequencing analysis of organoid samples in a pharmaceutical composition for the prevention or treatment of gastric cancer according to an embodiment of the present invention. In this case, the sequencing analysis results (fastq files) were further analyzed using 10X Genomics CellRanger software (v5.0.0), and GRCh38 was used as the reference genome. Furthermore, objects were generated using the R package Seurat, cell clusters were detected based on gene expression, and markers expressed in each cluster were identified. Additionally, cells with fewer than 500 or more than 6,000 features, and cells with mitochondrial genes accounting for more than 5%, were excluded from the analysis, and finding variable genes were calculated using the vst method. The FindMarkers function was used for differential gene analysis (only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25). Cell clustering was performed at a resolution of 0.5, and the results were visualized using UMAPs.

[0161] Referring to Figure 4a, both the intestinal type (hGC141) and the diffuse type (hGC143) are shown to have 9 cell clusters, and the density of each cell differs depending on the type of gastric cancer.

[0162] In this regard, FIG. 4b shows the origin sample and cell distribution results for the sequencing analysis of an organoid sample in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention. Herein, T1, T2, T3, and T4 may refer to organoid-derived cells cultured from respective samples for the t1, t2, t3, and t4 regions, respectively (hereinafter, T1, T2, T3, and T4 are all the same).

[0163] Referring to Figure 4b(a), for the long type (hGC141), the proportions for T1, T2, T3, and T4 cells are 42%, 8.7%, 28%, and 21.3%, respectively.

[0164] For diffuse type (hGC143), the proportions for T1, T2, T3, and T4 cells were found to be 28.2%, 24.8%, 27.9%, and 19%, respectively.

[0165] Referring to Fig. 4b(b), for the long type (hGC141), the distribution ratios for clusters 0 to 8 are 27.6%, 27.4%, 12.7%, 12.8%, 8.7%, 2.3%, 5%, and 0.5%, respectively.

[0166] For the less than type (hGC143), the distribution ratios for clusters 0 to 8 were found to be 28.5%, 19.7%, 15.1%, 8.2%, 9.1%, 7.4%, 7%, 3.8%, and 1.1%, respectively.

[0167] In other words, cultured organoids are shown to have different cell distributions depending on the type of gastric cancer.

[0168] Furthermore, FIG. 4c is a KEGG pathway analysis result for the sequencing analysis result of an organoid sample in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0169] Referring to Figure 4c, the long type (hGC141) is shown to affect propanoate metabolism, glucagon signaling pathway, D-glutamine and D-glutamate metabolism, Rap1 signaling pathway, Pyruvate metabolism, and Chemokine signaling pathway.

[0170] In the case of the diffuse form (hGC143), it is shown to affect the Hippo signaling pathway, p53 signaling pathway, NF-kappa B signaling pathway, Estrogen signaling pathway, Oxidative Phosphorylation, PI3K-Akt signaling pathway, ECM-receptor interaction, cAMP signaling pathway, Glycolysis / Gluconeogenesis, ErbB signaling pathway, and HIF-1 signaling pathway.

[0171] In other words, depending on the type of gastric cancer, the transcripts of cultured organoids appear to be involved in different pathways.

[0172] Ultimately, based on the results of Figures 4a to 4c, it can be confirmed that the genomic composition of cultured organoids differs according to each gastric cancer type, such as the intestinal type (hGC141) and the diffuse type (hGC143).

[0173] Figure 5 shows the results of the analysis of CSC-related pathways in diffuse (hGC143) organoids. In this case, CSCs (cancer stem cells) are stem cells involved in the development, drug resistance, and recurrence of cancer, and CSCs can refer to a specific group of cells within a tumor that simultaneously possess the ability to self-replicate and the ability to differentiate into other types of cells. Furthermore, since CSCs can form new cancerous masses, cancer may recur if CSCs remain, even if the cancerous tumor cells have been completely removed through surgical removal.

[0174] Referring to Figure 5, in the case of the diffuse type (hGC143), it appears that cancer stem cell (CSC) related pathways, including HEDGEHOG signaling, NOTCH signaling, P53, and TNFA signaling via NFKB, are upregulated.

[0175] Figures 6a and 6b show the results of CSC marker expression in organoids according to gastric cancer type in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention. In this case, CD44 was used as a gastric cancer stem cell marker.

[0176] First, referring to Figure 6a(a), the expression level of CD44, a marker for cancer stem cells (CSC), is higher in the diffuse type (hGC143) than in the long type (hGC141).

[0177] Furthermore, referring to Figure 6a(b), the expression level of CD44 is found to be higher in the less than 10 type (hGC143) than in the long type (hGC141), equally across all clusters.

[0178] Referring to Fig. 6b, the results of CSC marker expression in organoids according to sample sites for different types of gastric cancer are shown, where T1, T2, T3, and T4 may represent organoid-derived cells cultured from respective samples for sites t1, t2, t3, and t4, respectively, as described above in Fig. 4b.

[0179] The expression levels of CD44 in T1, T3, and T4 cells are also found to be higher in the diffuse type (hGC143) than in the long type (hGC141).

[0180] On the other hand, CD44 expression in T2 cells appears to be higher in the long type (hGC141) than in the diffuse type (hGC143).

[0181] In this regard, FIG. 7 is the result of KEGG pathway analysis for organoids derived from the intestinal type (hGC141) in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0182] Referring to Figure 7, the genes for T2 organoid cells derived from the long type (hGC141) appear to affect the PI3K-Akt signaling pathway, ErbB signaling pathway, p53 signaling pathway, NF-kappa B signaling pathway, HIF-1 signaling pathway, and TNF signaling pathway.

[0183] Furthermore, these PI3K-Akt signaling pathway, ErbB signaling pathway, p53 signaling pathway, NF-kappa B signaling pathway, HIF-1 signaling pathway and TNF signaling pathway are shown to be included in the KEGG pathway for diffuse (hGC143) organoids, as illustrated in the aforementioned Figure 4c.

[0184] In other words, this implies that T2 organoids derived from the long-form (hGC141) genetically possess diffuse-form (hGC143) characteristics.

[0185] Figures 8a and 8b show the CD44 high / low classification results for T2 organoids derived from the intestinal type (hGC141) in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention. In this case, the CD44 high / low classification was classified according to the presence or absence of CD44 marker expression in the aforementioned 6a(b). As shown in 6a(b), clusters 1, 3, 6, and 7 were found to express CD44 markers, so clusters 1, 3, 6, and 7 were grouped as CD44 high, and clusters 0, 2, 4, 5, and 8 were grouped as CD44 low.

[0186] Referring to Figure 8a, in the CD44 high group, E2F targets, estrogen response (early), estrogen response (late), G2M checkpoint, MTORC1 signaling, MYC targets (v1), MYC targets (v2), NOTCH signaling, oxidative phosphorylation, PI3K-AKT-mTOR signaling, reactive oxygen species pathway, unfolded protein response, WNT-beta catenin signaling, and CD44-related pathways are upregulated.

[0187] Furthermore, referring to Figure 8b, the CD44 high group appears to affect DNA replication, One carbon pool by folate, Alanine, Aspartate and glutamate metabolism, and Pyrimidine metabolism in the KEGG pathway.

[0188] At this time, referring to Fig. 9, one-carbon metabolism (one carbon pool by folate) can regulate nucleotide metabolism, NADPH ratio, and ROS, etc.

[0189] Accordingly, referring again to Fig. 8a, it can be seen that the CD44 high group has upregulated carbon-1 metabolism-related pathways such as E2F targets, G2M checkpoint, and reactive oxygen species pathway.

[0190] FIG. 10a is the result of one-carbon metabolism-related gene expression in T2 organoids derived from the intestinal type (hGC141) in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0191] Referring to Figure 10a, the CD44 high group was found to have higher expression levels of the TYMS, SHMT1, MTHFD1, and DHFR genes than the CD44 low group, and these genes could be new therapeutic targets for gastric cancer.

[0192] For example, as previously mentioned, in the case of gastric cancer that is histopathologically intestinal type and includes some diffuse type characteristics genomically, inhibitors targeting TYMS, SHMT1, MTHFD1, and DHFR may be used as a new therapeutic strategy. More specifically, at least one of Pemetrexed and Raltitrexed may be used as a TYMS inhibitor, SHIN1 may be used as a SHMT1 inhibitor, at least one of LY 345899, DS18561882, and TH9619 may be used as an MTHFD1 inhibitor, and at least one of Methotrexate (MTX), Pemetrexed, Raltitrexed, Pralatrexate, and aminopterin may be used as a DHFR inhibitor.

[0193] FIG. 10b shows the results of evaluating relative cell viability according to anticancer agents (SHIN1, DS18561882, cisplatin, or 5-FU) in T2 organoids derived from an intestinal type (hGC141) according to one embodiment of the present invention.

[0194] Referring to Figure 10b, SHIN1 and DS18561882, which are inhibitors of SHMT1 and MTHFD1, appear to be able to inhibit and kill gastric cancer cells.

[0195] In particular, SHIN1 appears to be able to inhibit and kill gastric cancer cells at a level similar to that of conventional anticancer drugs such as Cisplatin and 5FU.

[0196] FIG. 10c shows the results of evaluating cell viability compared to a negative control (DMSO) in T2 organoids derived from an intestinal type (hGC141) according to one embodiment of the present invention, after combined treatment with cisplatin of SHIN1 or DS18561882.

[0197] Referring to Figure 10c, SHIN1 and DS18561882, inhibitors of SHMT1 and MTHFD1, were found to have an enhanced effect on inhibiting cell viability when combined with the anticancer drug cisplatin compared to treatment alone.

[0198] FIGS. 11a and 11b show the results of HSA (Highest Single Agent) and Bliss Independence analysis to confirm the synergistic effect of combined treatment with SHIN1 or DS18561882 and cisplatin according to one embodiment of the present invention.

[0199] Referring to Figure 11a, SHIN1 had a limited inhibitory effect on cell viability when treated alone, but when SHIN1 and cisplatin were treated together, the HAS score was 10 or higher and the Bliss score was 0.1 or higher, suggesting that SHIN1 and cisplatin have a strong synergistic effect.

[0200] Referring to Figure 11b, DS18561882 had a limited inhibitory effect on cell viability when treated alone, but when DS18561882 and cisplatin were treated together, the HAS score was 5 to 10 and the Bliss score was 0.025 to 1, suggesting that DS18561882 and cisplatin have a synergistic effect.

[0201] Figures 12a and 12b are the results of a genetic population analysis of clones of genetically diffuse intestinal gastric cancer cells in a pharmaceutical composition for the prevention or treatment of gastric cancer according to one embodiment of the present invention.

[0202] Referring to Figure 12a, the results of the Hallmark pathway analysis are shown, and in diffuse type organoids, compared to intestinal type organoids, TNF-alpha signaling via NF-kB, Apoptosis, Estrogen response Early, Interferon alpha response, Estrogen response late, Interferon gamma response, p53 pathway, Glysolysis, G2-M checkpoint, mTORC1 signaling, KRAS signaling UP, Cholesterol homeostasis, Reactive oxygen species pathway, hypoxia, and Oxidative phosphorylation are upregulated.

[0203] Meanwhile, in the case of gastric cancer organoids that are genetically diffuse type and histopathologically intestinal type (141 intestinal type), estrogen response late, oxidative phosphorylation, and estrogen response early are found to be upregulated compared to general intestinal type organoids, and these upregulated pathways appear to be included in the previously mentioned diffuse type (143 diffuse type) organoid pathway results.

[0204] Furthermore, referring to Fig. 12b, the results of the KEGG pathway analysis are shown, and in the case of diffuse type (143 diffuse type) organoids, compared to intestinal type (141 intestinal type) organoids, apoptosis, TNF signaling pathway, NOD-like receptor signaling pathway, Hippo signaling pathway, p53 signaling pathway, NK-kappa B signaling pathway, IL-17 signaling pathway, Estrogen signaling pathway, Oxidative phosphorylation, Cell cycle, PI3K-Akt signaling pathway, cAMP signaling pathway, Glycolysis / Gluconeogenesis, HIF-1 signaling pathway, and steroid biosynthesis are upregulated.

[0205] Meanwhile, in the case of gastric cancer organoids that are genetically diffuse type and histopathologically intestinal type (141 intestinal type), steroid biosynthesis, phenylalanine metabolism, nitrogen metabolism, and oxidative phosphorylation are found to be upregulated compared to general intestinal type organoids, and among these, steroid biosynthesis and oxidative phosphorylation are found to be included in the results of the diffuse type (143 diffuse type) organoid pathways mentioned above.

[0206] Ultimately, it can be confirmed through the aforementioned Figures 12a and 12b that gastric cancer organoids of the intestinal type (141 intestinal type) histopathologically differ genetically from the general intestinal type and have a genetic composition similar to the diffuse type, and based on this, a treatment strategy differentiated from existing intestinal type gastric cancer can be proposed.

[0207] Although embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments and may be modified in various ways within the scope of the technical spirit of the present invention. Accordingly, the embodiments disclosed in the present invention are intended to explain, not limit, the technical spirit of the present invention, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of protection of the present invention shall be interpreted by the claims below, and all technical spirits within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

[0208]

[0209] [National R&D projects that supported this invention]

[0210] [Project ID] 1711188688

[0211] [Assignment No.] 2021R1A2C2011296

[0212] [Ministry Name] Ministry of Science and ICT

[0213] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0214] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0215] [Project Title] Study on the Origin of Mutations in Diffuse Gastric Cancer through Mutation Signature Analysis Using Organoids

[0216] [Name of Project Performing Organization] Yonsei University

[0217] [Research Period] 2023.03.01 ~ 2024.02.29

[0218]

[0219] [Project ID] 2460002777

[0220] [Assignment No.] KH049403

[0221] [Ministry Name] Ministry of Health and Welfare

[0222] [Name of Project Management (Specialized) Agency] Korea Health Industry Development Institute

[0223] [Research Project Name] Development of Research-Oriented Hospitals (R&D)

[0224] [Project Title] Development of Innovative Treatment Technologies for Obesity and Fatty Liver Disease Based on Intestine-Liver-Adipose Tissue Interactions

[0225] [Name of Project Performing Organization] Yonsei University Industry-Academic Cooperation Foundation

[0226] [Research Period] July 26, 2018 – December 31, 2026

Claims

1. A pharmaceutical composition for the prevention or treatment of gastric cancer comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, and DHFR, wherein The above-mentioned protein activity inhibitor is, At least one of a compound, peptide, peptide mimic, substrate analog, aptamer, and antibody that specifically binds to the above protein, and The above-mentioned gene expression inhibitor is, A pharmaceutical composition for the prevention or treatment of gastric cancer, comprising at least one of an antisense nucleotide that binds complementarily to the mRNA of the above gene, RNAi, siRNA, miRNA, shRNA, and ribozyme.

2. In Paragraph 1, The above gastric cancer is, A pharmaceutical composition for the prevention or treatment of gastric cancer, which is of the intestinal type or diffuse type.

3. In Paragraph 2, The above gastric cancer is, A pharmaceutical composition for the prevention or treatment of gastric cancer.

4. In Paragraph 3, The above-mentioned long type is, A pharmaceutical composition for the prevention or treatment of gastric cancer comprising diffuse genomic features.

5. In Paragraph 1, The above gastric cancer is, A pharmaceutical composition for the prevention or treatment of gastric cancer, characterized by intratumoral heterogeneity.

6. In Paragraph 1, The compound that specifically binds to the SHMT1 protein is, A pharmaceutical composition for the prevention or treatment of gastric cancer, which is SHIN1.

7. In Paragraph 1, The compound that specifically binds to the above TYMS protein is, A pharmaceutical composition for the prevention or treatment of gastric cancer, comprising at least one of pemetrexed and raltitrexed.

8. In Paragraph 1, The compound that specifically binds to the above MTHFD1 protein is, A pharmaceutical composition for the prevention or treatment of gastric cancer, at least one of LY 345899, DS18561882 and TH9619.

9. In Paragraph 1, A compound that specifically binds to the above DHFR protein is, A pharmaceutical composition for the prevention or treatment of gastric cancer, comprising at least one of MTX (Methotrexate), Pemetrexed, Raltitrexed, Pralatrexate, and aminopterin.

10. In Paragraph 1, The above composition is a pharmaceutical composition for the prevention or treatment of gastric cancer, further comprising cisplatin.

11. A step of treating a test substance to be analyzed into a cell containing at least one gene or protein among SHMT1, TYMS, MTHFD1, and DHFR; A step of measuring the gene expression level of at least one of SHMT1, TYMS, MTHFD1, and DHFR for the above cells, and If the above test substance reduces the measured gene expression level, A screening method for a candidate substance for the prevention or treatment of gastric cancer, comprising the step of selecting the above test substance as a candidate substance for the prevention or treatment of gastric cancer exhibiting intratumoral heterogeneity.

12. In Paragraph 11, The above gastric cancer is, Pathologically it is of the intestinal type, A screening method for candidate substances for the prevention or treatment of gastric cancer that include genomic diffuse type features.

13. In Paragraph 11, The above measurement is, A screening method for candidate substances for the prevention or treatment of gastric cancer, performed by at least one of Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real-time Polymerase Chain Reaction (real-time PCR), Northern Blot, Enzyme Linked Immunosorbent Assay (ELISA), Radioimmunoassay, Radioimmunodiffusion, Immunohistochemical Analysis, and Fluorescence-Activated Cell Sorting (FACS).

14. A pharmaceutical composition for the prevention or treatment of gastric cancer for co-administration with cisplatin, comprising as an active ingredient a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1 and MTHFD1.

15. A biomarker composition for diagnosing intratumoral heterogeneity in gastric cancer patients, comprising SHMT1, TYMS, MTHFD1, or DHFR.

16. In Paragraph 15, A biomarker composition for diagnosing intratumoral heterogeneity in a gastric cancer patient, wherein the gastric cancer patient is a patient pathologically diagnosed with intestinal-type gastric cancer.

17. A composition for diagnosing intratumoral heterogeneity in gastric cancer patients, comprising as an active ingredient a preparation for measuring the level of a protein or a gene encoding at least one of SHMT1, TYMS, MTHFD1, and DHFR.

18. In Paragraph 17, A composition for diagnosing intratumoral heterogeneity in a gastric cancer patient, wherein the gastric cancer patient is a patient who has been pathologically diagnosed with intestinal-type gastric cancer.

19. In Paragraph 17, A composition for diagnosing intratumoral heterogeneity in gastric cancer patients, wherein the preparation for measuring the protein level is any one selected from the group consisting of an oligopeptide that specifically binds to a protein, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a PNA, or an aptamer.

20. In Paragraph 19, A composition for diagnosing intratumoral heterogeneity in gastric cancer patients, wherein the measurement of the protein level is performed using one or more methods selected from the group consisting of Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), and protein chip.

21. In Paragraph 17, A composition for diagnosing intratumoral heterogeneity in gastric cancer patients, wherein the agent for measuring the gene level is an antisense oligonucleotide, primer pair, or probe that specifically binds to a gene.

22. In Paragraph 21, A composition for diagnosing intratumoral heterogeneity in gastric cancer patients, wherein the above-mentioned gene level measurement is performed by one or more methods selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction, RNase-protected assay, Northern blotting, and DNA chips.

23. A method for preventing or treating gastric cancer, comprising the step of administering to an individual a pharmaceutical composition comprising, as an active ingredient, a protein activity inhibitor or gene expression inhibitor for at least one of SHMT1, TYMS, MTHFD1, and DHFR.

24. In Paragraph 23, The above gastric cancer is, Pathologically it is of the intestinal type, A method for the prevention or treatment of gastric cancer that includes genomic diffuse type characteristics.

25. A method for diagnosing intratumoral heterogeneity in a gastric cancer patient, comprising the step of measuring the level of a protein or a gene encoding at least one of SHMT1, TYMS, MTHFD1, and DHFR from a biological sample from a gastric cancer patient.

26. In Paragraph 25, A method for diagnosing intratumoral heterogeneity in a gastric cancer patient, wherein the gastric cancer patient is a patient who has been pathologically diagnosed with intestinal-type gastric cancer.

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