Pharmaceutical composition for treating, suppressing, or preventing progression of t lymphocyte-positive pancreatic carcinoma

Abstract

The present invention provides: a pharmaceutical composition for treating, suppressing, or preventing progression of T lymphocyte-positive pancreatic carcinoma, the pharmaceutical composition containing siRNA that suppresses expression of the CHST15 gene; and a method for suppressing T lymphocyte-positive pancreatic carcinoma.

Description

Pharmaceutical composition for treating, suppressing, or preventing the progression of T lymphocyte-positive pancreatic cancer.

[0001] The present invention relates to a pharmaceutical composition for treating, suppressing, or preventing T lymphocyte-positive pancreatic cancer, comprising CHST15 siRNA as an active ingredient.

[0002] Pancreatic cancer, in particular, presents a highly immunosuppressive environment and has a low number of tumor-infiltrating lymphocytes (TILs), making treatment with immunotherapy such as immune checkpoint inhibitors difficult. It is believed that increasing and activating TILs, especially tumor-infiltrating CD8-positive T lymphocytes, is key to treatment. Previously, immunohistochemical analysis of surgically resected specimens of resectable pancreatic cancer has reported that the presence of intratumoral CD8-positive cells correlates with a favorable postoperative prognosis. However, the presence of intratumoral CD8-positive cells at the time of initial diagnosis remained unclear. Furthermore, there has been little analysis of TILs in advanced, inoperable pancreatic cancer, and no systematic reports have been conducted. Considering that the cancer has progressed without surgery, it is presumed that immunosuppression is more advanced than in patients who are eligible for surgery, and therefore, the number of TILs is likely to be lower. In fact, reports from clinical trials targeting second-line treatment for unresectable advanced pancreatic cancer have shown that baseline histological analysis reveals that TILs are at a level of less than 1 / 10 to 1 / 100 of those in surgically resected specimens (References 1-4).

[0003] Recently, a systematic report on tumor infiltrating lymphocytes (TIL) and prognosis of life, as well as prognosis of chemotherapy, in unresectable advanced pancreatic cancer has been made (Reference 5). This is a study analyzing the relationship between baseline intratumoral CD8-positive cells before treatment and prognosis in 170 cases diagnosed with unresectable advanced pancreatic cancer by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), which is the largest-scale study of its kind to date. The possibility of the baseline intratumoral CD8-positive area (%) as a prognostic factor was analyzed in all cases, and the possibility as a predictive factor for the effect of chemotherapy was analyzed in the population receiving chemotherapy (GEM-related regimens and fluorouracil-related regimens). Using the median (0.1%) of the baseline intratumoral CD8-positive area (%) as a cut-off, the group with a higher value was designated as CD8-H and the group with a lower value as CD8-L. As a result, in unresectable advanced pancreatic cancer, it was found that it is difficult to assume baseline CD8 as a prognostic factor or as a predictive factor for the effect of chemotherapy. The result that the presence of CD8 does not affect the effect of chemotherapy has been considered sufficient as a practical clinical sense from the perspective of the mechanism of action of chemotherapy or from the perspective of predictive factors for the effect of chemotherapy speculated in previous reports, and it has also been reported as actual systematic clinical research data.

[0004] CHST15 (Carbohydratesulfotransferase 15), a glycosylsulfotransferase, is a type II transmembrane Golgi protein that transfers a sulfate group to the 6-position of the GalNAc (4SO ４ residue of chondroitin sulfate A (CS-A) to synthesize highly sulfated chondroitin sulfate E (CS-E) (References 6, 7). It is scarcely expressed in normal human tissues, but its expression is known to be enhanced in inflammation / fibrosis and cancer. It has been reported in cardiac fibrosis disease, pulmonary fibrosis disease, and gastrointestinal fibrosis disease that suppression of mRNA expression by CHST15 siRNA inhibits the synthesis of CS-E and suppresses fibrosis (References 8-13). In cancer, it is known to have an antitumor effect by suppressing cancer cell proliferation on the human pancreatic cancer cell line Panc-1, and an effect of suppressing cancer cell invasion and cancer stroma on the human pancreatic cancer cell line BxPC-3 (References 14, 15).

[0005] Fujisawa T, Tsuchiya T, Kato M, et al. STNM01, the RNA oligonucleotide targeting carbohydrate sulfotransferase 15, as second-line therapy for chemotherapy-refractory patients with unresectable pancreatic cancer: An open label, phase I / IIa trial. EClinicalMedicine. 55: 101731, 2022.Orhan A, Vogelsang RP, Andersen MB, et al. The prognostic value of tumour-infiltrating lymphoc as an additional co-author by his contribution to PFS generation es in pancreatic cancer: a systematic review and meta-analysis. Eur J Cancer. 132:71-84, 2020.Kiryu S, Ito Z, Suka M, et al. Prognostic value of immune factors in the tumor microenvironment of patients with pancreatic ductal adenocarcinoma. BMC Cancer. 2:1197, 2021.Muller M, Haghnejad V, Schaefer M, et al. The Immune Landscape of Human Pancreatic Ductal Carcinoma: Key Players, Clinical Implications, and Challenges. Cancers (Basel). 14: 995, 2022.Tanisaka Y, Ryozawa S, Mizuide M, et al.The correlation between tumoral CD8 expression and clinical course in patients with unresectable pancreatic cancer using tissue samples acquired by endoscopic ultrasound-guided tissue acquisition. J Hepatobiliary Pancreat Sci. 2024.Ohtake S, Kondo S, Morisaki T, et al. Expression of sulfotransferase involved in the biosynthesis of chondroitin sulfate E in the bone marrow derived mast cells. Biochemical Biophysica Acta 1780: 687-95, 2008.Habuchi O, Moroi R, Ohtake S, et al. Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage. Anal Biochem 310: 129-36, 2002.Kai Y, Tomoda K, Yoneyama H et al. Silencing of carbohydrate sulfotransferase 15 hinders murine pulmonary fibrosis development. Mol Ther Nucleic Acid 6: 163-172, 2017.Sato H, Sagara S, Nakajima S, et al. Prevention of esophageal stricture after endoscopic submucosal dissection, using siRNA-based silencing of carbohydrate sulfotransferase 15 in pig.Endoscopy 49: 491-497, 2017.Suzuki K, Yokoyama J, Kawauchi Y, et al. Phase 1 clinical study of siRNA targeting carbohydrate sulfotransferase 15 in Crohn’s disease patients with active mucosal lesions. J Crohns Colitis 11: 221-228, 2017.Suzuki K, Arumugam S, Yokoyama J, et al. Pivotal role of carbohydrate sulfotransferase 15 in fibrosis and mucosal healing in mouse colitis. PLoS One 11: e0158977, 2016.Watanabe K, Arumugam S, Sreedhar R, et al. Small interfering RNA therapy against carbohydrate sulfotransferase 15 inhibits cardiac remodeling in rats with dilated cardiomyopathy. Cell Signal 27: 1517-1524, 2015.Yamada S and Sugahara K. Potential therapeutic Application of chondroitin sulfate / dermatan sulfate. Current Drug Discovery Technologies 5: 289-301, 2008.Takakura K, Shibazaki Y, Yoneyama H, et al. Inhibition of Cell Proliferation and Growth of Pancreatic Cancer by Silencing of Carbohydrate Sulfotransferase 15 In Vitro and in a Xenograft Model. PLoS One10:e0142981, 2015.Ye J, Suizu F, Yamakawa K, et al. Intratumoral administration of CHST15 siRNA remodels tumor microenvironment and augments tumor-infiltrating T cells in pancreatic cancer. Mol Ther Oncol. 32: 200812, 2024.

[0006] The present invention aims to provide a pharmaceutical composition for treating, suppressing, or preventing T lymphocyte-positive pancreatic cancer.

[0007] The inventors have created an siRNA (hereinafter referred to as CHST15 siRNA) that suppresses the expression of the CHST15 gene, given that the CHST15 gene is strongly expressed in pancreatic cancer cells and is involved in cancer cell invasion and fibrosis of the cancer stroma. They have conducted non-clinical and clinical trials targeting pancreatic cancer patients and have shown that CHST15 siRNA can suppress the cancer stroma. On the other hand, since T lymphocytes do not express the CHST15 gene, it was not considered at all possible that CHST15 siRNA would act on T lymphocytes. However, the inventors have discovered a phenomenon that could not be predicted at all from conventional techniques, i.e., the results of standard chemotherapy: the survival period was extremely good in the group showing a pre-treatment (baseline) intratumoral CD8-positive area above the median. This suggests that in pancreatic cancer patients with severe immunosuppression, the therapeutic response to CHST15 siRNA may be higher in the group with a large number of remaining CD8-positive cells.

[0008] More specifically, the present invention provides the following [1] to

[11] : [1] A pharmaceutical composition for treating, suppressing or preventing the progression of T lymphocyte-positive pancreatic cancer, comprising an siRNA that suppresses the expression of the CHST15 gene. [2] The pharmaceutical composition according to [1], wherein the T lymphocytes are cytotoxic T lymphocytes. [3] The pharmaceutical composition according to [2], wherein cytotoxic T lymphocyte positivity is determined by the detection of CD8. [4] The pharmaceutical composition according to [2], wherein the area ratio of cytotoxic T lymphocytes on the surface of cancer cells is 0.15% or more. [5] The pharmaceutical composition according to any one of [1] to [4], wherein the siRNA that suppresses the expression of the CHST15 gene comprises RNA having the sequence shown in SEQ ID NO: 1 and RNA having the sequence shown in SEQ ID NO: 2. [6] The pharmaceutical composition according to any one of [1] to [5], wherein the pancreatic cancer is a chemotherapy-refractory cancer. [7] The pharmaceutical composition according to any one of [1] to [6], which is administered in combination with other chemotherapy agents. [8] A method for treating, suppressing, or preventing T lymphocyte-positive pancreatic cancer by administering siRNA that suppresses the expression of the CHST15 gene. [9] The method according to [8], wherein the T lymphocytes are cytotoxic T lymphocytes.

[10] The method according to [9], wherein cytotoxic T lymphocyte positivity is determined by the detection of CD8.

[11] The method according to [9] or

[10] , wherein the area ratio of cytotoxic T lymphocytes on the surface of cancer cells is 0.15% or more.

[0009] Pancreatic cancer is characterized by a particularly strong immunosuppressive environment and, compared to other solid tumors, exhibits absolutely minimal lymphocyte infiltration. Especially in unresectable advanced pancreatic cancer, the absolute number of lymphocytes infiltrating the tumor is inherently small, and therefore, in clinical practice, it has not been considered that the magnitude of this infiltration has any clinically significant impact on treatment effectiveness or prognosis. Clinical studies have also reported no statistically significant correlation between the degree of lymphocyte infiltration and the effectiveness of chemotherapy (Reference 5). In this context, it has become clear for the first time that the effectiveness of CHST15 siRNA can be predicted based on the degree of lymphocyte infiltration into the tumor. According to this invention, it becomes possible to effectively treat T-lymphocyte-positive pancreatic cancer patients who have not responded at all to conventional anticancer drug therapy in immunosuppressed pancreatic cancer with CHST15 siRNA. Furthermore, it is conceivable that combining it with chemotherapy and molecular targeted drugs may reduce their side effects while enhancing their therapeutic effects. Moreover, it can be applied to multidisciplinary treatment and recurrence prevention of pancreatic cancer, such as enhancing the effects of surgery and radiotherapy.

[0010] Figure 1 shows the results of baseline tumor infiltration CD3 and CD8 positive area (%) and survival (OS) after CHST15 siRNA treatment in an ancillary study of a clinical trial involving patients with unresectable advanced pancreatic cancer. (A) Patients were classified into High (H) and Low (L) groups based on the median baseline tumor infiltration CD3 (%), and OS was compared between the two groups. (B) Patients were classified into High (H) and Low (L) groups based on the median baseline tumor infiltration CD8 (%), and OS was compared between the two groups. Figure 2 shows the regimens of a randomized trial that evaluated the efficacy of adding CHST15 siRMA to the standard treatment group in each group, which was classified into High (H) and Low (L) groups based on baseline tumor infiltration CD8 (%).

[0011] The present invention will now be described in detail. The inventors have discovered that suppressing the expression of the CHST15 (Carbohydrate sulfotransferase 15) gene has an inhibitory effect on T lymphocyte-positive pancreatic cancer. More specifically, the inventors have discovered that suppressing the expression of the CHST15 gene by RNAi (RNA interference) has an inhibitory effect on T lymphocyte-positive pancreatic cancer.

[0012] The CHST15 gene of the present invention is not particularly limited, but is usually of animal origin, more preferably of mammalian origin, and most preferably of human origin. The CHST15 of the present invention is also known as GalNAc4S-6ST (N-acetylgalactosamine 4-sulfate 6-0 sulfotransferase).

[0013] The sequence of CHST15 (GalNAc4S-6ST) of the present invention can be obtained, for example, based on accession number NM_015892. As an example, the nucleotide sequence of the CHST15 gene of the present invention is described in Sequence ID No. 3. Not only proteins consisting of amino acid sequences derived from the nucleotide sequence described in Sequence ID No. 3, but also proteins that have a high degree of identity with it (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) and have the function of the above protein are included in the CHST15 protein of the present invention. The above protein is, for example, a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted, substituted, or inserted in the amino acid sequence of the CHST15 protein, and the number of amino acids that usually change is within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids.

[0014] The genes described above in this invention include, for example, endogenous genes in other organisms corresponding to DNA consisting of the base sequence described in Sequence ID No. 3 (such as homologs of the above human gene). Furthermore, endogenous DNA in other organisms corresponding to DNA consisting of the base sequence described in Sequence ID No. 3 generally have a high degree of identity (homology) with the DNA described in Sequence ID No. 3. High identity means homology of preferably 70% or more, more preferably 80% or more, and more preferably 90% or more (for example, 95% or more, and even more preferably 96%, 97%, 98%, or 99% or more). This homology can be determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7). Furthermore, when isolated from a living organism, this DNA is thought to hybridize with the DNA described in Sequence ID No. 3 under stringent conditions. Examples of "stringent conditions" include "2×SSC, 0.1%SDS, 50°C", "2×SSC, 0.1%SDS, 42°C", "1×SSC, 0.1%SDS, 37°C", and even more stringent conditions such as "2×SSC, 0.1%SDS, 65°C", "0.5×SSC, 0.1%SDS, 42°C", and "0.2×SSC, 0.1%SDS, 65°C".

[0015] In this specification, the "siRNA that suppresses the expression of the CHST15 gene" may also be expressed as "CHST15 siRNA," and is preferably an siRNA having a structure in which bases overhang at the 3' end of one strand and the 3' end of the other strand, and more preferably an siRNA having a structure in which the RNAs described in SEQ ID NOs: 1 and 2 are hybridized.

[0016] In the present invention, the siRNA does not necessarily have to consist entirely of ribonucleotides (RNA). That is, in the present invention, one or more ribonucleotides constituting the siRNA may be corresponding deoxyribonucleotides, as long as the molecule itself has the function of suppressing the expression of the CHST15 gene. "Corresponding" here means that although the structure of the sugar portion is different, they are the same type of base (adenine, guanine, cytosine, thymine (uracil)). For example, a deoxyribonucleotide corresponding to a ribonucleotide containing adenine is a deoxyribonucleotide containing adenine. Furthermore, the term "multiple" is not particularly limited, but preferably refers to a small number of about two to five.

[0017] The siRNA of the present invention can be appropriately prepared by those skilled in the art using commercially available nucleic acid synthesizers. Furthermore, general contract synthesis services can be used for the synthesis of desired RNAs.

[0018] In this invention, T lymphocyte-positive pancreatic cancer refers to pancreatic cancer in which the CD8-positive area in a biopsy pathological specimen is 0.01% or more, or 0.1%, preferably 0.15%, or 0.2% or more of the total pancreatic tumor area.

[0019] The present invention provides a pharmaceutical composition for treating, suppressing, or preventing the progression of T lymphocyte-positive pancreatic cancer, comprising CHST15 siRNA as an active ingredient. Alternatively, the present invention provides a method for suppressing T lymphocyte-positive pancreatic cancer comprising the step of administering siRNA that suppresses the expression of the CHST15 gene; siRNA that suppresses the expression of the CHST15 gene for use in a method for suppressing T lymphocyte-positive pancreatic cancer; use of siRNA that suppresses the expression of the CHST15 gene in the manufacture of a reagent; and a method for producing a T lymphocyte-positive pancreatic cancer inhibitor comprising the step of using siRNA that suppresses the expression of CHST15 (formulating and / or mixing with a pharmaceutically or physiologically acceptable carrier).

[0020] The cancers targeted for treatment or prevention in the present invention are not particularly limited as long as the siRNA that suppresses the expression of the CHST15 gene in the present invention exerts a therapeutic effect on pancreatic cancer, but include invasive ductal pancreatic carcinoma, acinar cell carcinoma, mucinous pancreatic cancer, adenosquamous carcinoma, medullary carcinoma, anaplastic carcinoma, intraductal papillary mucinous neoplasm (IPMN), mucinous cystic neoplasm (MCN), solid pseudopapillary neoplasm (SPN), serous cystic neoplasm (SCN), and pancreatic neuroendocrine neoplasm (P-NET).

[0021] In the present invention, "suppressing T lymphocyte-positive pancreatic cancer" means preventing the progression and metastasis of cancer, and / or enhancing the effectiveness of cancer treatment. It should be noted that "treatment" in the present invention is not necessarily limited to cases with complete therapeutic effect, but may also include cases with partial effect.

[0022] The pharmaceutical composition for suppressing T lymphocyte-positive pancreatic cancer of the present invention can be mixed with a pharmaceutically or physiologically acceptable carrier, excipient, or diluent and administered orally or parenterally. Oral formulations may be in the form of granules, powders, tablets, capsules, solvents, emulsions, or suspensions. Parenteral formulations may be in the form of injections, intravenous infusions, topical agents, inhalants (nebulizers), or suppositories. Injectable formulations may include subcutaneous injections, intramuscular injections, intraperitoneal injections, intracranial injections, or intranasal injections. Topical agents may include nasal injections or ointments. Formulation techniques for producing the above-mentioned formulations containing the main component of the pharmaceutical composition of the present invention are publicly known.

[0023] For example, tablets for oral administration can be manufactured by adding excipients, disintegrants, binders, and lubricants to the pharmaceutical composition for restoring tissue integrity of the present invention, mixing the mixture, and then compressing and molding it. Common excipients include lactose, starch, or mannitol. Common disintegrants include calcium carbonate and calcium carboxymethylcellulose. Common binders include gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone. Known lubricants include talc and magnesium stearate.

[0024] The tablets containing the pharmaceutical composition for suppressing T lymphocyte-positive pancreatic cancer of the present invention can be coated with known coatings for masking or to create an enteric-coated formulation. Ethyl cellulose, polyoxyethylene glycol, and the like can be used as coating agents.

[0025] The injectable preparation can be obtained by dissolving the main component of the present invention, a pharmaceutical composition for suppressing T lymphocyte-positive pancreatic cancer, with a suitable dispersant, dissolving it in a dispersion medium, or dispersing it. Depending on the choice of dispersion medium, it can be either an aqueous solvent or an oily solvent. For aqueous solvents, distilled water, physiological saline, or Ringer's solution can be used as the dispersion medium. For oily solvents, various vegetable oils or propylene glycol can be used as the dispersion medium. In this case, preservatives such as parabens can be added as needed. In addition, known isotonic agents such as sodium chloride or glucose can be added to the injectable preparation. Furthermore, analgesics such as benzalkonium chloride or procaine hydrochloride can be added.

[0026] Furthermore, the pharmaceutical composition for suppressing T lymphocyte-positive pancreatic cancer of the present invention can be made into a topical preparation by being a solid, liquid, or semi-solid composition with or without excipients or carriers. For solid or liquid compositions, topical preparations can be made by using compositions similar to those described above. Semi-solid compositions can be prepared by adding a thickener as needed to a suitable solvent. The solvent can be water, ethyl alcohol, or polyethylene glycol. The thickeners commonly used are bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone. Preservatives such as benzalkonium chloride can be added to this composition. Additionally, by combining it with an oily base material such as cocoa butter or an aqueous gel base material such as a cellulose derivative as a carrier, it can also be made into a suppository.

[0027] When the pharmaceutical composition for suppressing T lymphocyte-positive pancreatic cancer of the present invention is used as a gene therapy agent, methods include direct administration of the cancer invasion inhibitor or pharmaceutical composition of the present invention by injection, as well as administration of a vector incorporating nucleic acid. Examples of such vectors include adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, vaccinia virus vectors, retrovirus vectors, lentivirus vectors, etc., and these viral vectors can be used to administer the drug efficiently.

[0028] Furthermore, it is also possible to introduce the pharmaceutical composition for suppressing T lymphocyte-positive pancreatic cancer of the present invention into phospholipid vesicles such as liposomes and administer the vesicles. The vesicles containing siRNA are introduced into designated cells by lipofection. The resulting cells are then administered systemically, for example, intravenously or intra-arterially.

[0029] The present invention also provides a method for suppressing cancer invasion in a subject, comprising the step of administering the pharmaceutical composition for suppressing T lymphocyte-positive pancreatic cancer of the present invention to an individual (e.g., a patient) or its tumor tissue. The subject of the present invention is not particularly limited as long as it is an organism capable of developing tumors, but is preferably a human. Administration to the subject can be carried out by methods known to those skilled in the art, such as oral administration, intratumor administration, intradermal administration, subcutaneous administration, or intravenous injection. Systemic administration or direct local administration into tumor tissue is possible. Furthermore, a commercially available gene transfer kit can be used to introduce the siRNA of the present invention into target cells, tissues, or organs.

[0030] The pharmaceutical composition for suppressing T-lymphocyte-positive pancreatic cancer of the present invention is administered to mammals, including humans, in the necessary amount (effective dose) within a range of doses considered safe. The dosage of the pharmaceutical composition for suppressing cancer invasion of the present invention can ultimately be appropriately determined by a person skilled in the art (physician or veterinarian), taking into consideration the type of dosage form, method of administration, age and weight of the subject, symptoms of the subject, etc. For example, although it will vary depending on age, sex, symptoms, route of administration, number of administrations, and dosage form, the dosage of CHST15 siRNA of the present invention administered topically is about 1 pM to 1 mM once a day, and can be administered as a single dose, at intervals of one week to one month (e.g., every week, every two weeks, or every month), or daily.

[0031] Furthermore, all prior art documents cited herein are incorporated herein by reference.

[0032] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples. The "CHST15 siRNA" used in these examples is an siRNA with a structure hybridized with the RNAs described in SEQ ID NOs: 1 and 2. The base sequence of the GalNac 4S-6ST siRNA drug used in these examples is shown below. The sequence is not necessarily limited to this example. [human GalNac4-6STsiRNA] (Gene Bank accession number NM_015892) (Manufactured by Hokkaido System Science Co., Ltd.) 5'-ggagcagagcaagaugaauacaauc-ag -3' (SEQ ID NO: 1) 3'-ua-ccucgucucguucuacuuauguuag -5' (SEQ ID NO: 2)

[0033] <Example 1> Using human pancreatic cancer specimens stained according to the method described in Reference 1 (Fujisawa T, Tsuchiya T, Kato M, et al. STNM01, the RNA oligonucleotide targeting carbohydrate sulfotransferase 15, as second-line therapy for chemotherapy-refractory patients with unresectable pancreatic cancer: An open label, phase I / IIa trial. EClinicalMedicine. 55: 101731, 2022), the area percentage was calculated by observation or measurement according to the following procedure. (1) Calculation method for CD3 and CD8 "positive area %" 1) Five fields of view were randomly photographed at 400x magnification per individual. 2) Based on the captured images, the area of ​​each field of view for CD8 and CD3 was measured using Image J software (National Institutes of Health). 1. The section image was opened from File. 2. Colors were separated using Image > Color > Split channels. 3. The image with the Blue filter was selected. 4. The threshold was determined using Image > Adjust > Threshold. 5. Analyze was selected, and Area and Area fraction were checked under Set measurement. 6. The positive area was calculated using Analyze > Measure. 7. The positive area of ​​5 fields was calculated for each individual, and the average of these was used as the individual value. 8. For the area of ​​the part without an intercept, Freehand selection was selected, and the periphery was traced. 9. The area of ​​the part without an intercept was calculated using Analyze > Measure. 3) The value of the area of ​​the part without an intercept was subtracted from the captured area. 4) The positive area percentage was divided by the actual intercept area, and the final data was displayed as a percentage.

[0034] <Example 2> A subgroup analysis was performed using OS data observed in the clinical trial conducted (Reference 1). In this example, the median (0.2%) was set as the threshold (cutoff value). In the subgroup analysis, the median OS (mOS) of the group with baseline intratumoral CD3-positive area greater than or equal to the median (CD3-H group) was 6.3 months, and the mOS of the group with baseline area less than the median (CD3-L group) was 7.9 months, with no statistically significant difference between the two groups (Figure 1A). In contrast, the mOS of the group with baseline intratumoral CD8-positive area greater than or equal to the median (CD8-H group) was 14.3 months, and the mOS of the group with baseline area less than the median (CD8-L group) was 6.2 months, with a statistically significant difference observed between the two groups (Figure 1B). Therefore, it was suggested that in patients with unresectable advanced pancreatic cancer in a severely immunosuppressed state, the group with a large number of remaining CD8-positive cells may have a higher treatment response.

[0035] <Example 3> Patients with unresectable pancreatic cancer are classified into two cohorts based on the percentage of CD8-positive area within the tumor (hereinafter, CD8 ≥ threshold is referred to as CD8-high, and CD8 < threshold is referred to as CD8-low). In this clinical trial, the threshold is set to 0.15%. Statistical simulations showed that the results were equally good whether the threshold was set to 0.2% or 0.15%, so the threshold was set to 0.15% in order to obtain a larger number of target patients. In each cohort, each subject is randomized to either the nal-IRI / FL therapy group or the STNM01 + nal-IRI / FL combination therapy group using a minimization method with age, stage, and ECOG PS as minimization factors (allocation ratio = 1:1), and after receiving intervention, they are followed up until the observation point of the required number of events or 24 months after randomization for the final subject. The primary endpoints are overall survival in the CD8-high group and overall survival in the general population, independent of CD8-positive area percentage, with a Family Wise Error (FWE) of 5% or less controlled by the Holm method. This design allows for evaluation of whether CHST15 siRNA can treat and suppress T lymphocyte-positive pancreatic cancer.

[0036] <Example 4> The following is an overview of the clinical trial.

[0037] Discussion: CHST15 siRNA was suggested to treat and suppress T lymphocyte-positive pancreatic cancer.

[0038] The CHST15 siRNA of the present invention showed effects in treating, suppressing, or preventing the progression of T lymphocyte-positive pancreatic cancer, suggesting that it may lead to new cancer treatment effects.

Claims

1. A pharmaceutical composition for treating, suppressing, or preventing T lymphocyte-positive pancreatic cancer, comprising siRNA that suppresses the expression of the CHST15 gene.

2. The pharmaceutical composition according to claim 1, wherein the T lymphocytes are cytotoxic T lymphocytes.

3. The pharmaceutical composition according to claim 2, wherein cytotoxic T lymphocyte positivity is determined by the detection of CD8.

4. The pharmaceutical composition according to claim 2, wherein the area ratio of cytotoxic T lymphocytes on the surface of cancer cells is 0.15% or more.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the siRNA that suppresses the expression of the CHST15 gene comprises RNA having the sequence shown in SEQ ID NO: 1 and RNA having the sequence shown in SEQ ID NO:

2.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pancreatic cancer is a cancer that is refractory to chemotherapy.

7. A pharmaceutical composition according to any one of claims 1 to 6, which is administered in combination with other chemotherapy agents.

8. A method for treating, suppressing, or preventing T-lymphocyte-positive pancreatic cancer by administering siRNA that suppresses the expression of the CHST15 gene.

9. The method according to claim 8, wherein the T lymphocytes are cytotoxic T lymphocytes.

10. The method according to claim 9, wherein cytotoxic T lymphocyte positivity is determined by the detection of CD8.

11. The method according to claim 9 or 10, wherein the area ratio of cytotoxic T lymphocytes on the surface of cancer cells is 0.15% or more.

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