Method for producing tumor-reactive t cell

Abstract

[Problem] To provide a technique that makes it possible to easily and efficiently select tumor-reactive T cells using an expression molecular marker as an index, while minimizing time, cost and labor. [Solution] The problem is solved by a method for producing tumor-reactive T cells, the method being characterized by comprising: (1) a candidate isolation step for isolating CD8-positive T cells from tumor-infiltrating lymphocytes (TIL) or whole blood obtained from a cancer patient, using, among antibodies found on the cell surfaces, PD-1 and / or 4-1BB as markers; (2) a tumor reactivity evaluation step for transducing a T cell receptor (TCR) gene of the T cells isolated in the candidate isolation step into T cells and identifying and evaluating a TCR having tumor reactivity; and (3) a treatment T cell acquisition step for proliferating T cells that react with a tumor in vitro to obtain tumor treatment T cells to be administered to the cancer patient.

Description

Method for producing tumor-reactive T cells

[0001] The present invention relates to a method for producing tumor-reactive T cells, and the like.

[0002] Recent advances in anti-checkpoint antibody therapy, using antibodies against molecules such as PD-1 and PD-L1, have revealed that cancer patients possess T cells that recognize tumors, particularly mutated antigens resulting from genomic mutations in individual tumors. Reactivation of suppressed T cells can lead to tumor regression and prolonged prognosis. Furthermore, TIL therapy, which involves the infusion of expanded tumor-infiltrating lymphocytes (TILs), has proven effective, particularly in the treatment of malignant melanoma. It has been reported that T cells specific for mutated antigens contained in TILs contribute to this efficacy. Furthermore, it has recently become clear that the administration of T cells newly tuned for tumor reactivity via chimeric antigen receptors (CARs) or T cell receptors (TCRs) (CAR-T cells, TCR-T cells) can also lead to tumor regression. Therefore, there is growing hope that tumor-reactive T cells that recognize tumor antigens, including mutated antigens, or therapies utilizing their TCRs, may be effective treatments for solid tumors. Given this background, attempts have been made to identify and obtain tumor-reactive T cells in various cancers, but these studies have mainly focused on malignant melanoma. At present, it has been extremely difficult in practice to reliably obtain tumor-reactive T cells from peripheral blood, as well as from TILs, which are thought to contain large numbers of tumor-reactive T cells, in other carcinomas, including colorectal cancer (Non-Patent Documents 1 to 3).

[0003] Publication WO2014-017533A1

[0004] PD-1 identifies the patient-specific CD8+ tumor-reactive repertoireinfiltrating human tumors. J Clin Invest. 2014 May;124(5):2246-59.Bystander CD8+ T cells are abundant and phenotypically distinct inhuman tumour infiltrates. Nature. 2018 May;557(7706):575-579.Isolation of T-Cell Receptors Specifically Reactive with MutatedTumor-Associated Antigens from Tumor-Infiltrating Lymphocytes Based on CD137Expression. Clin Cancer Res. 2017 May 15; 23(10): 2491-2505.Fujii et al., Identification of an immunogenic neo-epitope encodedby mouse sarcoma using CXCR3 ligand mRNAs as sensors, Oncoimmunology. 2017 Mar20;6(5):e1306617.Hanson HL.et al., Eradication of Established Tumors by DUC18T CellAdoptive Immunotherapy, Immunity 2000, 13:265-76.

[0005] It has been reported that the expression of PD-1, LAG3, and TIM3 in TILs can serve as indicators of tumor-reactive T cells. However, this study focused on malignant melanoma, and no suitable indicators have been identified for other cancers. Other methods have also been reported, including (1) co-culturing antigen-presenting cells (e.g., peptides or tumor lysates) with TILs to selectively culture and expand tumor-specific T cells, and (2) performing single-cell RNA sequencing and TCR sequencing on TILs to select tumor-reactive T cells using characteristic expression groups at the mRNA level as indicators. However, these methods require significant time, effort, and cost, making them impractical for application to individual cancer patients. The present invention was developed in light of the above-mentioned circumstances. Its purpose is to provide a technology that enables efficient selection of tumor-reactive T cells using expressed molecular markers as indicators, while minimizing time, cost, and effort.

[0006] The present invention provides a method for producing tumor-reactive T cells, which comprises: (1) a candidate isolation step of isolating CD-8+ T cells from tumor-infiltrating lymphocytes (TILs) or whole blood collected from a cancer patient using PD-1 and / or 4-1BB as cell surface antigen markers; (2) a tumor reactivity evaluation step of transducing T cell receptor (TCR) genes of the T cells isolated in the candidate isolation step into target cells to identify and evaluate tumor-reactive TCRs; and (3) a therapeutic T cell acquisition step of expanding T cells with tumor-reactive TCRs in vitro to obtain tumor-therapeutic T cells for administration to the cancer patient. In the present invention, the term "target cells" refers to cells that can exhibit tumor reactivity by expressing a specific TCR gene, and includes, for example, T cells and peripheral blood mononuclear cells (PBMCs). In the above invention, the solid tumor is preferably at least one selected from the group consisting of brain tumor, tongue cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, lung cancer, thyroid cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, rhabdomyosarcoma, and leiomyoma. Furthermore, the candidate isolation step is preferably characterized by further using a tetraspanin as a marker. In this case, the tetraspanin is preferably at least one selected from the group consisting of CD9, TSPAN2, CD151, CD53, CD37, CD82, CD81, and CD63.

[0007] <Outline of the present invention> 1. Method for identifying TCRs that react to autologous tumors Figure 1 shows an overview of a method for searching for cell surface antigens on tumor-reactive T cells. Tumor tissue is obtained from a cancer patient with a solid tumor (e.g., a colon cancer patient) (1). Generally, tumor tissue contains tumor cells and TILs in addition to normal cells. Patient tumor cells are maintained in vitro by creating cancer tissue-originating spheroids (CTOS) from the tumor cells contained in this tumor tissue (5). Meanwhile, CD8-positive TILs (CD8 + TILs) were identified (2), and CD8 TILs were identified using the expression of PD-1 and 4-1BB as indicators. + The T cell population was subdivided, and each cell population (PD-1 positive and 4-1BB negative (PD-1 + 4-1BB － ), PD-1 positive and 4-1BB positive (PD-1 + 4-1BB + ), PD-1 and 4-1BB negative (PD-1 － 4-1BB － Individual T cells belonging to the CTOS subtype are isolated (candidate isolation step), and their T cell receptors (TCRs) are isolated by single-cell PCR. Repertoire analysis is then performed using the gene sequences of these TCRs (3). Focusing on TCRs with large repertoire sizes, individual TCRs are transfected into peripheral blood mononuclear cells (PBMCs) from healthy donors using retroviral vectors (4). These are then co-cultured with autologous tumor-derived CTOS for 24 hours, and cytokines (IFN-γ) in the culture supernatant are measured to identify TCRs that react with the autologous tumor (6: tumor reactivity evaluation step). The upper right corner of the figure shows an image obtained by staining with an anti-CEA antibody for CEA (carcinoembryonic antigen), which is highly expressed in colorectal cancer tissue, to confirm the "tumor potential" of CTOS.

[0008] 2. Method for verifying TCR recognition against mutant antigens Figure 2 shows the verification method used to analyze the reactivity of tumor-reactive TCRs that respond to CTOS to mutant antigens (antigens derived from genetic mutations in individual tumor genomes). Next-generation sequencing (3) analyzes nucleic acids (DNA / RNA) obtained from individual patient tumor tissues (1) and normal tissue nucleic acids (DNA) obtained from the patient's peripheral blood (2) to identify genetic mutations associated with tumor-specific amino acid mutations (4). Further in silico analysis is performed to select mutant antigens that are likely to be presented to the patient's HLA and are expected to be highly immunogenic (5). Plasmids (Tandem Mini Genes; TMGs) containing 8–10 tandem sequences encoding these mutant antigens (27-mer amino acid sequences (13 amino acids on either side of the mutated amino acid)) are constructed (6), and mRNA is then produced by in vitro transcription. Meanwhile, CD8 TCRs are isolated from the patient's tumor tissue. + Identify and isolate CD8 TILs + The clonality of TILs is analyzed (7). TCR genes are transduced (8), and tumor reactivity is evaluated using CTOS (9: candidate isolation step). The previously prepared mRNA is expressed and transduced into LCLs (immortalized B cell line) prepared from the patient's B cells using electroporation. The cells are then co-cultured with T cells transduced with tumor-reactive TCRs, and their reactivity is analyzed using the ELISPOT assay (tumor reactivity evaluation step). If the TCR recognizes a specific mutant antigen, it will react with LCLs transduced with TMG mRNA. Since TMG contains 8–10 different mutant antigens, newly preparing and analyzing individual TMGs allows the identification of which mutant antigens are recognized (10).

[0009] 3. Immune cell therapy using TIL or PBMC from cancer patients Figure 3 shows an overview of immune cell therapy using TIL or PBMC derived from cancer patients. Tumors or PBMCs are collected from cancer patients, and PD-1 + 4-1BB + CD9 + Triple Positive CD8+ T cells are isolated as TIL candidates (1: candidate isolation step). From these, TCR genes are analyzed to obtain tumor-reactive TCRs (2). This TCR is then transduced into T cells (3: tumor reactivity evaluation step). Separately, PD-1 is isolated from the patient's PBMCs. + 4-1BB + CD9 + This allows the generation of tumor-reactive TCRs (4). The cells obtained in (3) and / or (4) are then expanded in vitro (5: therapeutic T cell generation process) and administered to patients (6). By administering these T cells, an immune response can be induced to convert a non-immunogenic (low-immunogenic) tumor (cold tumor) into a highly immunogenic tumor (hot tumor) (7), leading to a cure (8).

[0010] The present invention provides T cells for tumor therapy in cancer patients. These T cells can be effectively used to treat the cancer patient. Possible treatment methods include, for example: 1. genetically modified T cell infusion therapy using the TCR of tumor-reactive T cells; 2. a treatment method in which a population of tumor-reactive T cells is expanded and infused without modification; 3. a treatment method in which tetraspanin molecules are co-expressed simultaneously with genetic modification of the TCR / CAR to enhance efficacy; and 4. the application of extracellular vesicles (EVs) containing exosomes from tumor-reactive T cells to cancer treatment.

[0011] This figure shows the results of analyzing tumor-reactive TCRs by ELISPOT analysis after transfection of LCLs derived from CCP1 patients with mRNA prepared from four types of TMG. This figure shows the results of analyzing tumor-reactive TCRs by ELISPOT analysis after transfection of LCLs derived from CCP1 patients with mRNA prepared from four types of TMG. This figure shows the results of analyzing tumor-reactive TCRs by ELISPOT analysis after transfection of LCLs derived from CCP3 ... This figure shows the results of analyzing tumor-reactive TCRs by ELISPOT analysis after transfection of LCLs derived from CCP3 patients with mRNA prepared from four types of TMG. This figure shows the results of analyzing tumor-reactive TCRs by ELISPOT analysis after transfection of LCLs derived from CCP3 patients with mRNA prepared from four types of TMG. This figure shows the results of analyzing tumor-reactive TCRs by ELISPOT analysis after transfection of LCLs derived from CCP3 patients with mRNA prepared from four types of TMG. This figure shows the results of analyzing tumor-reactive TCRs by ELISPOT analysis after transfection of LCLs derived from CCP3 patients with mRNA prepared from four types of TMG. This figure shows the results of analyzing tumor-reactive TCRs by ELISPOT analysis after transfection of LCLs derived from CCP3 Figures 1A and 1B show the results of analyzing tumor-reactive TCRs for CCP5. (A) A pie chart showing the division of TILs into two types of cell populations based on the presence or absence of PD-1 expression, and a diagram showing the results of repertoire analysis of individual TCR genes obtained from each cell population. (B) A graph showing the results of ELISPOT analysis of mRNAs prepared from two types of TMGs after transfection into LCLs derived from CCP5 patients. (C) A graph showing the results of examining the reactivity of TMs obtained by individually dividing the genes in TMG1. Figures 1A and 1B show the results of analyzing tumor-reactive TCRs for CCP15. (A) A pie chart showing the division of TILs into three types of cell populations based on the presence or absence of PD-1 and 4-1BB expression. (B) A graph showing the results of ELISPOT analysis of mRNAs prepared from four types of TMGs after transfection into LCLs derived from CCP15 patients. (C) A graph showing the results of examining the reactivity of TMs obtained by individually dividing the genes in TMG1. CD8 TCRs obtained from 12 CCP cases. + Figure 1 shows the results of examining the expression frequency of PD-1 and 4-1BB in tumor-reactive TCRs among TILs. (A) PD-1 + 4-1BB +(B) Comparison of the proportion of DP (Double Positive) populations in CCP1, (C) Pie chart showing the results of examining the presence or absence of PD-1 and 4-1BB expression in CCP1, and (D) Pie chart showing the three types of cell populations in CCP1 based on the presence or absence of PD-1 and 4-1BB expression in TIL. + PD-1 in TILs + 4-1BB + (A) PD-1 + 4-1BB + (A) A pie chart showing the results of examining the DP population of PD-1 and a graph showing the results of examining their reactivity to CTOS; (B) A graph showing similar RNA expression profiles for two types of TCR; (C) A figure showing the results of RNA expression analysis of DP5-6 and DP53-1 and other cells. Figure 1 shows the results of analyzing tumor-reactive TCRs for CCP9. (A) PD-1 + (B) Pie chart showing the results of TCR analysis using the PD-1 + CD9 + (A) CD8 used in the IFN-γ ICS method. +(A) A diagram showing the collection schedule of CD8+ T cells, (B) A graph showing the results of the IFN-γ ICS method, and (C) A diagram showing the results of an investigation into the effectiveness of mutant Snd1 peptides and adjuvants (CHP: hydrophobic polysaccharide) in CMS7 tumor-bearing mice (the upper panel is the experimental protocol, and the lower panel is a graph examining the relationship between tumor length and tumor administration). Figure 1 shows the results of an investigation into whether mutant Snd1-specific CD8+ T cells express CD9 molecules. (A) A diagram showing the collection schedule of CD8+ T cells, (B) A graph showing the results of the IFN-γ ICS method, and (C) A diagram showing the results of an investigation into whether mutant Snd1 peptide-specific CD8+ T cells express CD9 molecules by the ELISPOT method. + This is a graph showing the results of examining the frequency of CD8 cells. + CD9 + CD81 + Figure 1 shows the results of TCR repertoire analysis of T cells. (A) A diagram showing the splenocyte collection schedule. (B) Splenic CD8 + CD9 + CD81 + (C) Results of TCR analysis of T cells. (D) CD8 specific for mutant Snd1. + Figure 1 shows the results of analyzing CD81 expression in T cells. + CD9 + CD81 + Figure 1 shows the results of TCR repertoire analysis of T cell populations and analysis of TCR reactivity to CMS7. (A) Graph showing the results of measurement of mouse IFN-γ (mIFN-γ), (B) Graph showing the results of measurement of mouse TNFα (mTNFα), and (C) Pie chart showing the results of TCR specifically recognizing CMS7 found in three cells. + CD9 + CD81 + Figure 1 shows the results of TCR repertoire analysis of T cell populations. (A) A diagram showing the collection schedule of mouse spleens. (B) CD9+ of CD8+ cells in the spleens on Day 21. + CD81 + This figure shows the results of cell repertoire analysis. + CD9+ CD81 + Figures showing the results of investigating the characteristics of T cell populations. (A) A diagram showing the mouse spleen collection schedule, (B) and (C) graphs showing the results of measuring mouse IFN-γ (mIFN-γ). Figures showing the results of an experiment to verify that CD9 expression enhances antitumor effects. (A) A diagram showing the test schedule, (B) A graph showing the relationship between tumor administration and tumor size.

[0012] Next, embodiments of the present invention will be described with reference to the figures and diagrams. However, the technical scope of the present invention is not limited to these embodiments, and various forms can be implemented without changing the gist of the invention. <Materials and Test Methods> 1. Ethics regarding human samples Written informed consent was obtained from patients and healthy volunteers in accordance with the guidelines of the Declaration of Helsinki. The test protocol was approved by the Ethics Committee of the Mie University School of Medicine. 2. Ethics regarding mouse experiments All animal experiments were performed using protocols approved by the Animal Care and Use Committee of the Mie University Life Science Center. 3. Mice Female BALB / c mice were purchased from Shizuoka Animal Research Institute. H-2K d mERK2 136-144 The NOD / Shi-scid / IL-2Rγ transgenic DUC18 mice, known as NOG mice, were established as previously described. null Mice were purchased from the Central Institute of Laboratory Animals. All mice were housed in a specific pathogen-free environment and used at 8-10 weeks of age.

[0013] 4. Antibodies. Fluorescein isothiocyanate (FITC)-conjugated anti-human CD3 monoclonal antibody (mAb, OKT3), phycoerythrin (PE)-Cy7-conjugated anti-human CD8 mAb (RPA-T8), PerCP-Cy5.5-conjugated anti-human CD9 (HI9a), allophycocyanin (APC)-conjugated anti-human PD-1 mAb (EH12.2H7), PE-conjugated anti-human 4-1BB (CD137) mAb (4B4-1), and eFluor780-FixableViability Dye were purchased from BioLegend. PerCP-Cy5.5-conjugated anti-mouse CD3 mAb (145-2C11), APC-Cy7-conjugated anti-mouse CD8 mAb (53-6.7), FITC-conjugated anti-mouse CD9 mAb (MZ3), PE-conjugated anti-mouse CD81 mAb (Eat-2), and PE-Cy7-conjugated anti-mouse PD-1 mAb were purchased from BioLegend. FITC-conjugated anti-CEA mAb (CB30) was purchased from Abcam. For mouse treatment, anti-mouse PD-1 (RMP1-14), anti-mouse CTLA-4 (9D9), and anti-mouse GITR (DTA-1) mAbs were produced by hybridomas and purified on a protein G column. 5. Cell Lines: CMS5 and CMS7 are BALB / c-derived 3-methylcholanthrene-induced sarcoma cell lines. CT26 is a colon epithelial tumor cell line derived from intrarectal injection of N-nitroso-N-methylurethane into BALB / c mice. P1.HTR is a subline of the DBA / 2-derived P815 mastocytoma cell line. Human B-lymphoblastoid cell lines (LCLs) were generated in our laboratory from patient peripheral blood using EBV-containing supernatant. All mouse tumor lines and LCLs were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 50 μM 2-mercaptoethanol (2-ME), and 0.2 mg / mL glutamine.

[0014] 6. Patients and Samples: Patients with colorectal cancer (CRC) who visited the Department of Gastrointestinal and Pediatric Surgery at Mie University Hospital and underwent surgery between 2016 and 2018 were enrolled in this study. Fresh tumor tissue samples were collected from surgically resected CRC. Paired peripheral blood mononuclear cells (PBMCs) from individual patients were isolated from fresh heparinized venous blood samples by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). 7. Preparation of Cancer Tissue-Originated Spheroid (CTOS) Lines and TILs: CTOS were prepared as previously described with appropriate modifications. Briefly, the procedure is as follows. Resected tumors were rapidly mechanically minced and cultured in DMEM / Ham's F12 medium (Wako Pure Chemical Industries, Ltd.) containing 26 units / mL of Liberase DH (Roche) for 90 minutes at 37°C with continuous agitation. After adding 10 μg / mL of DNase I (Roche), the culture was incubated for an additional 15 minutes. The digested culture medium was sequentially filtered through 500, 250, 100, and 40 μm mesh filters (BD Falcon). Fractions between 100-250 μm (Fr. 100-250) and 40-100 μm (Fr. 40-100) were collected. The Fr. 40-100 sample was cultured in STEM PRO hESC SFM (Invitrogen) at 37°C in a 5% CO2, 20% O2 atmosphere for 24-48 hours to form CTOS. The Fr. 100-250 sample was mechanically disrupted by moving the plunger up and down several times using a 27-gauge needle, and then cultured in the same manner as the Fr. 40-100 sample. Cells that passed through a 40-μm mesh filter were collected and frozen at -80°C in Cellbanker-1 medium (Takara Bio). These were used as TILs.

[0015] 8. Purification of DNA and RNA from Human Samples DNA and RNA were isolated from tumor tissue samples measuring 5 mm in diameter. RNA and genomic DNA were purified using the RNeasy and Puregene Core kit (Qiagen) according to the manufacturer's protocol. DNA was isolated using 5 x 10 ６ PBMCs were used. Genomic DNA was purified from PBMCs using the QIAmp DNA Kit (Qiagen) according to the manufacturer's protocol. 9. Next-generation sequencing and data analysis. Genomic DNA extracted from the patient's normal and tumor tissues (200 ng) was cleaved to 150-200 bp fragments. Adapters were ligated using the SureSelect XT HS Kit (Agilent Technologies). Exon regions were hybridized with biotinylated oligo RNA baits consisting of SureSelect Human All Exon V6 with an MHC region added. Samples were collected using avidin beads, PCR amplified to obtain libraries, and 101-bp paired-end Whole Exome Sequencing (WES) was performed using NextSeq550 (Illumina). Total RNA extracted from tumor tissue (300-500 ng) was used for TruSeq analysis. TM　 After adapters were ligated to the mRNA using the Stranded mRNA Library Kit (Ilumina), PCR amplification was performed to obtain a library, after which 76-bp paired-end RNA sequencing (RNA-Seq) was performed using NextSeq550.

[0016] 10. Identification of Somatic Mutations in Tumor Tissues. Using the human reference genome hg38, we performed a comparative analysis of WES data from the patient's normal and tumor tissues to detect tumor-specific somatic mutations. We also used the WES data from the same reference genome and normal tissues to detect germline mutations. Each analysis was performed according to GATK best practices. After filtering the detected mutations to select those with high probability, we used the GENCODE human reference gene model to extract mutations located in the protein translation sequence of the gene. Somatic and germline mutations located in the protein translation sequence of the same gene and at close genomic loci were physically phased to determine haplotypes surrounding the alleles carrying the somatic mutation. Furthermore, based on this haplotype information, we extracted the overall pattern of 8- to 11-residue peptide sequences that could potentially represent the mutant antigen epitopes. To quantify the transcriptional expression level of mRNA translating the mutant antigen, we performed transcriptional expression analysis using RNA-Seq data from the patient's tumor tissue and quantified it at the isoform level using TPM (Transcripts Per Million). This analysis was performed according to the GTEx / TOPMed RNA-Seq pipeline. When multiple transcript isoforms overlapped with the same somatic mutation, the TPM values ​​were summed to obtain TPM_SUM. Furthermore, to estimate the allele-specific transcript expression levels, including somatic mutations, the mutant allele frequency in the RNA-Seq data for somatic mutations detected by WES was calculated, and the TPM or TPM_SUM value was multiplied by this to obtain TPMvar or TPM_SUMvar.

[0017] To rank peptide sequences of 8-11 residues that could serve as mutant antigen epitopes for their potential presentation to the human major histocompatibility complex (MHC), we calculated scores using a presentation prediction model developed by BrightPath Bio. The model was constructed using a generalized linear model (logistic regression model) trained on immunopeptidome data obtained from public databases and random peptides. Parameter estimation was performed using maximum likelihood (ML) estimation, with predicted values ​​from the MHC binding prediction tools NetMHCpan-4.0 and MHCflurry-1.4 and the proteasome cleavage prediction tool NetChop-3.1 as explanatory variables. Using this estimated parameterized linear predictor, the presentation potential of patient-derived mutant antigen epitopes was scored for each patient's HLA allele. Specifically, the presentation score (SCORE) was calculated by soft-plus-transforming the calculated linear predictor value (z) for each peptide, as shown in Equation (1). Furthermore, to correct for transcriptional expression levels, SCOREadj was calculated using Equation (2).

[0018]

[0019] The HLA allele with the highest SCORE value among all HLA alleles possessed by the patient was determined to be the predicted HLA allele for that mutant antigen epitope, and the mutant antigen epitopes were ranked in descending order of SCOREadj of that HLA allele.

[0020] 11. Preparation of DNA Encoding Tandem Minigenes (TMGs) and In Vitro Transcription. The DNA sequence used for the TMG method was designed as follows: A 27-mer peptide sequence centered on the mutant residue was designed and reverse-translated into DNA. For insertion / deletion somatic mutations, a 27-mer peptide sequence centered on the mutated antigen epitope (8-11 mer) was used and reverse-translated into DNA. TMGs were cloned into the MCS of pcDNA3.1(+) using BamHI and XhoI. Plasmid DNA was linearized by digestion with XhoI enzyme, and the DNA was precipitated with sodium acetate and ethanol. Next, 1 μg of DNA was used to generate in vitro transcribed RNA using the mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies) according to the manufacturer's protocol. The resulting capped and tailed RNA was resuspended in 1 μg / μL water and stored at -80°C before use in transfecting LCLs.

[0021] 12. Electroporation: LCLs were harvested, washed twice with phosphate-buffered saline (PBS), and resuspended in RPMI 1640 medium. Next, 10 μg of mRNA was added to a maximum of 1 × 10 6 The cells were mixed with 100 μl of cell suspension containing 1 × 10 cells, transferred to a 2 mm gap cuvette (Genetronics), and electroporated using a BTXECM830 square wave electroporator (Genetronics). After electroporation, the cells were immediately transferred to 2.0 ml of medium and cultured overnight in a 24-well plate at 37°C in a CO2 incubator until use. 13. Intracellular staining (ICS) Intracellular cytokine staining was performed as follows. Splenocytes (1 × 10 cells) were cultured overnight at 37°C in a 24-well plate. 6) were incubated with 50 μM synthetic peptide or DMSO for 15 minutes at room temperature, followed by incubation with GolgiPlug (BD Bioscience) for 4 hours. Cells were stained with CD8α-, CD9-, or CD81-specific mAbs for 15 minutes at 4°C. After permeabilization and fixation using the Cytofix / Cytoperm kit (BD Biosciences) according to the manufacturer's protocol, cells were stained with allophycocyanin-conjugated anti-IFNγ mAb.

[0022] 14. Enzyme-linked immunosorbent assay (ELISA). Human or mouse cytokine concentrations in culture supernatants were determined using an ELISA kit (Thermo Fisher Scientific) according to the manufacturer's protocol. 15. Enzyme-linked immunospot (ELISPOT) assay. Human IFNγ ELISPOT assay was performed as previously described with minor modifications. Briefly, ELISPOT plates (MAHA S4510, Millipore) were coated with anti-human IFNγ mAb (1-D1K, Mabtech). A total of 2 × 10 4 2 x 10 TCR-transduced T cells and 2 x 10 4 TMG mRNA-transduced LCLs were added to each well of the plate. After 22 hours of incubation at 37°C, the plate was washed, and a biotinylated capture antibody (7-B6-1, Mabtech) was added and incubated overnight at 4°C. After washing the wells, the cells were reacted with alkaline phosphatase-conjugated streptavidin and then stained with an alkaline phosphatase-conjugated substrate kit (Bio-Rad). Spots were counted using an ELISPOT plate reader (ImmunoSpot, CTL-Europe GmbH).

[0023] 16. Peptides: MuLV gp70-derived AH-1 (SPSYVYHQF (SEQ ID NO: 1)), mutant ERK2-derived peptide (mERK2-9m: QYIHSANVL (SEQ ID NO: 2)), mutant Snd1-derived peptide (mSnd1: YAPCRGEF (SEQ ID NO: 3)), and non-mutated Snd1-derived peptide (wSnd1: YAPRRGEF (SEQ ID NO: 4)) were obtained from Invitrogen with purity exceeding 80%. All peptides were dissolved in DMSO at a concentration of 10 mM, aliquoted, and stored at -80°C before use. 17. RNA sequencing and TCR sequencing of isolated cells: CCP1 TILs were washed multiple times and resuspended in 1x PBS containing 0.04% BSA. Approximately 5x10 cells with 100% viability were used to generate GEMs (gel beads in emulsion) using the Chromium Controller (10x Genomics). 3 100 cells were used. By this method, RNA molecules were individually tagged in each cell. Molecularly tagged RNA fragments, including those derived from the TCR V(D)J genes, were converted to cDNA, and libraries for next-generation sequencing were established using the Chromium Single Cell V(D)J Reagent Kits v1.1 (10x Genomics). The resulting libraries were quantified using Qubit dsDNA Assay (ThermoFisher Scientific) and TapeStation D1000 (Agilent). The gene expression library and V(D)J library were pooled at a 9:1 ratio and sequenced on an Illumina HiSeq platform with the following configuration: read 1: 26 bp, read 2: 91 bp, and i7 index: 8 bp, resulting in a total of approximately 400M paired-end reads per sample. Image analysis and base calling were performed using the HiSeq instrument software. Raw FASTQ reads were imported into the Cell Ranger V(D)J pipeline (10x Genomics) for mapping, gene expression counts, and clonotype annotation. The isolated cell analysis was performed according to the manufacturer's protocol.

[0024] 18. Preparation of PBMCs: PBMCs were isolated from fresh heparinized venous blood samples by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). 19. Preparation of Mouse Splenocytes: Mouse spleens were pulverized using a microscope slide, treated with ammonium chloride potassium (ACK), and filtered through Cell Trics® 30 μm (Sysmex Partec). Isolated cell suspensions from pooled spleens were incubated with the appropriate peptides at a concentration of 5 × 10 6 The cells were cultured in 96-well V-bottom plates at a density of 100 cells / ml. + Splenic T cells were obtained by positive enrichment using the MACS system (Miltenyi Biotec). Flow cytometry revealed that the T cell fraction contained over 95% CD8 + The presence of T cells was confirmed. 20. Flow cytometry analysis and isolated cell sorting. TILs were thawed and stained with FITC-anti-human CD3 mAb, PE-Cy7-anti-human CD8 mAb, PE-anti-human CD137 (4-1BB) mAb, and APC-anti-human PD-1 mAb. Dead cells were excluded by staining with eFluor 780-Fixable Viability Dye. After staining, cells were analyzed using a FACSAria (Beckton Dickinson). CD8 + PD-1 + CD137 + , CD8 + PD-1 + CD137 - and CD8 + PD-1-CD137 - T cells were isolated in 96-well PCR plates. Mouse splenocytes were stained with PerCP-Cy5.5-conjugated anti-mouse CD3 mAb, APC-Cy7-conjugated anti-mouse CD8 mAb, FITC-conjugated anti-mouse CD9 mAb, and PE-conjugated anti-mouse CD81 mAb. Dead cells were excluded by staining with eFluor780-FixableViabilityDye.

[0025] 21. Amplification of TCR cDNA from Isolated T Cells. T cell receptor (TCR) cloning was performed according to the method described in WO2014-017533A1 (Patent Document 1). Briefly, the procedure is as follows: RNA extracted from each isolated T cell was converted to cDNA, and the Vα and Vβ regions were amplified by multistep nested PCR. The DNA sequences of the PCR products were then determined by direct sequencing. The TCR repertoire was analyzed using the IMGT / V-Quest tool (http: / / www.imgt.org / ). For transduction of human PBMCs or mouse splenocytes, the TCRα and TCRβ chains were linked by a P2A sequence and cloned into the pMX-IRES-EGFP vector. 22. Retrovirus Production. Plat-A or Plat-E cells (Cell Biolabs) were used to generate retroviruses for transducing TCR genes into human PBMCs or mouse splenocytes. 23. Retroviral transduction of human PBMCs. PBMCs obtained from healthy volunteers were stimulated using anti-CD3 antibody-coated plates and RetroNectin (Takara Bio Inc.) in the presence of 600 IU / mL recombinant IL-2. Proliferating lymphocytes were transduced with retroviruses encoding candidate TCRα / β genes and further expanded in vitro. Ten days after transduction, cells were harvested and used for experiments.

[0026] 24. Retroviral transduction of mouse splenocytes. Total splenocytes (1.5 × 10) obtained from BALB / c mice were transduced with 100 μg of ... 7 5 × 10 cells / 5 mL) were stimulated with immobilized anti-CD3 (1 μg / mL; 145-2C11) and soluble anti-CD28 (1 μg / mL; 37.51) in one well of a 6-well plate. One day after stimulation (day 1), 5 × 10 cells were 5Each well of a 24-well plate was coated with RetroNectin (Takara Bio), 1 mL of medium was added, and the cells were transduced with candidate TCR-expressing viral vectors using the RetroNectin-conjugated viral infection method. On day 3, the cells were transferred to a 50 mL flask containing 10 mL of medium and expanded and cultured. On day 5, the cells were harvested and used for experiments. 60 IU / mL of recombinant human IL-2 (Novartis) was added during culture. In some experiments, murine CD9-transduced T cells were prepared using splenic T cells obtained from DUC18 mice. 25. Single-cell RNA sequencing and TCR sequencing analysis using TILs. TILs frozen in liquid nitrogen were thawed in 10% FBS RPMI1640 medium and washed several times with 2% FBS 1x PBS. CD8 in the TILs was then isolated using human CD8 magnet beads (Miltenyi Biotec). + T cells were isolated and subjected to single-cell RNA sequencing and TCR sequencing. Statistical analysis was performed using an unpaired Student's t-test, with a p < 0.05 being considered statistically significant.

[0027] <Test Results> Table 1 summarizes patient information, whether CTOS was established, and whether the TCR gene that recognizes CTOS was acquired.

[0028]

[0029] As shown in the table, CTOS was established in 10 of the 16 colorectal cancer patients (CCP1-CCP16) (marked with a "○" or "△" in the CTOS column). Furthermore, TIL-derived TCRs were obtained in 6 of these 10 cases (marked with a "+" in the TCR CTOS-Reactivity column). Figure 4 shows the results of analyzing tumor-reactive TCRs for CCP1 of the above 6 cases (CCP1, 3, 5, 7, 9, 15). In CCP1 cases, the presence or absence of PD-1 and 4-1BB expression determined the CD8 + TILs were divided into three types of cell populations (Fig. 4(A)). －,4-1BB － : 60 pieces), bottom right (PD-1 ＋ ,4-1BB － : 72 pieces), upper right (PD-1 ＋ , 4-1BB ＋ : 72). The PD-1 in the upper left － ,4-1BB ＋ (No detection was made for PD-1.) Individual TCR genes were obtained from each cell population and repertoire analysis was performed. The pie chart in Figure 4(A) shows the results of TCR repertoire analysis for each cell population, and the corresponding two types of TCRs were found to be PD-1. ＋ 4-1BB ＋ The results show that the TCRs were most frequently enriched in the DP fraction (Figure 4(B)). We also examined the reactivity of these TCRs to CTOS. As shown in Figure 4(B), we isolated two tumor-reactive TCRs (DP5-6 and DP53-1; DP stands for double positive). Reactivity to CTOS was assessed by IFN-γ production in cocultures of CTOS with TCR-transfected T cells. To examine the reactivity of these two TCRs to mutant antigens, we created four TMGs (TM1–TM4). mRNA prepared from each TMG was transfected into CCP1 patient-derived LCLs and analyzed by ELISPOT. As shown in Figure 4(C), both TCRs were reactive to TMG1. Further analysis revealed that both TCRs specifically recognized the mutant CREBBP in TMG1. The amino acid sequences in the figure are "NGTASQSTSPSQPCKKIFKPEELRQAL" (SEQ ID NO: 5) for mutant CRBBP and "NGTASQSTSPSQPRKKIFKPEELRQAL" (SEQ ID NO: 6) for wild-type CRBBP.

[0030] Figure 5 shows the results of analyzing tumor-reactive TCRs for CCP3. In CCP3 cases, as in Figure 4, the expression of PD-1 and 4-1BB was associated with CD8 +TILs were divided into three cell populations (Fig. 5(A)), and individual TCR genes were isolated from each cell population for repertoire analysis. Furthermore, the reactivity of these TCRs to CTOS was examined, and a TCR (DP72-6) showing tumor reactivity was isolated (Fig. 5(B)). The pie chart in Fig. 5(A) shows the results of the TCR repertoire analysis for each cell population. T cells expressing the corresponding TCR express PD-1. + 4-1BB + In the DP fraction of ＋ 4-1BB － The results show that the TCR was detected in only one cell (single cell) in the fraction. To examine the reactivity of this TCR to the mutant antigen, four TMGs (TM1-TM4) were constructed. mRNA prepared from each TMG was transfected into LCLs derived from a CCP3 patient, and ELISPOT analysis revealed reactivity to TMG1 (Figure 5(C)). Further analysis revealed that the TCR specifically recognized the mutant DDX60 in TMG1. The amino acid sequences shown in the figure were "PRVMDMLKLYFLFYLQFLVKEGYLDQE" (SEQ ID NO: 7) for mutant DDX60 and "PRVMDMLKLYFLFSLQFLVKEGYLDQE" (SEQ ID NO: 8) for wild-type DDX60P.

[0031] Figure 6 shows the results of analyzing tumor-reactive TCRs for CCP5. In CCP5 cases, PD-1 expression led to CD8 + TILs were divided into two cell populations (Figure 6(A)), and individual TCR genes were isolated from each cell population for repertoire analysis. Furthermore, the reactivity of these TCRs to CTOS was examined, and a TCR (SP27-2) showing tumor reactivity was isolated. The pie chart in Figure 6(A) shows the results of the TCR repertoire analysis for each cell population, and T cells expressing the corresponding TCR were found to express PD-1. + In the fraction, PD-1 was detected in 3 cells. －This shows that the reactivity of the TCR to the mutant antigen was not observed in the fractions. Two types of TMG (TM1 and TM2) were prepared to examine the reactivity of the TCR to the mutant antigen. mRNA prepared from each TMG was transfected into LCL derived from a CCP5 patient, and analyzed by ELISPOT. Reactivity to TM1 was observed (Figure 6(B)). Further analysis revealed that the TCR specifically recognized the mutant FAM129B in TM1 (Figure 6(C). The 4 in TM1 corresponds to "FAM129B"). The amino acid sequences shown in the figure were "RAQIHMREQMDNAMYTFETLLHQELGK" (SEQ ID NO: 9) for mutant FAM129B and "RAQIHMREQMDNAVYTFETLLHQELGK" (SEQ ID NO: 10) for wild-type FAM129B.

[0032] Figure 7 shows the results of analyzing tumor-reactive TCRs for CCP15. In CCP15 cases, the expression of PD-1 and 4-1BB led to the expression of CD8 + TILs were divided into three cell populations (Fig. 7(A)), and individual TCR genes were extracted from each cell population and subjected to repertoire analysis. The pie chart in Fig. 7(A) shows the results of TCR repertoire analysis for each cell population, indicating that T cells expressing the corresponding TCR express PD-1. + 4-1BB + In the fraction, PD-1 was detected in 3 cells. + 4-1BB － This indicates that the mutant ABCF1 was detected in only one cell (single cell) in the fraction. To examine the reactivity of the TCR to the mutant antigen, four TMGs (TM1-TM4) were constructed. mRNA prepared from each TMG was transfected into LCLs derived from a CCP15 patient, and ELISPOT analysis confirmed that T cells transfected with the DP28-5 TCR exhibited reactivity to TMG1 (Fig. 7(B)). Further analysis revealed that the mutant ABCF1 in TMG1 specifically recognized the mutant ABCF1 (Fig. 7(C)). The amino acid sequences shown in the figure are "TKQAEKQTKEALTQKQQKCRRKNQDEE" (SEQ ID NO: 11) for the mutant ABCF1 and "TKQAEKQTKEALTRKQQKCRRKNQDEE" (SEQ ID NO: 12) for the wild-type ABCF1.

[0033] Figure 8 shows the characteristics of all tumor-reactive TCRs derived from four cases (CCP1, 3, 5, and 15). All tumor-reactive TCRs expressed PD-1 + 4-1BB + The most frequently identified TCRs in the DP population were those with CCP5. All of the mutant antigen-specific TCRs expressed PD-1 in TILs. + 4-1BB + Figure 8(B) shows the analysis results for CCP1. PD-1, including CCP3 and 15, was identified in cases with a high frequency of the group. + 4-1BB + The fraction contained PD-1 + 4-1BB － Compared with the PD-1 fraction, tumor-reactive T cells were more frequently enriched. － Figure 9 shows the prognosis of the 16 analyzed colon cancer cases compared with the "CD8 + PD-1 in TILs + 4-1BB + The results of a log-rank test using "cell population frequency of 3% or more" as the standard are shown. Although no significant difference was observed, in four cases (CCP1, 3, 5, 15: corresponding to graph "A" in the figure), no deaths were observed until 2000 days later, while in the remaining 12 cases (corresponding to graph "B" in the figure), seven cases survived after 2000 days. + PD-1 in TILs + 4-1BB + It was determined that cases with a cell population frequency of 3% or more tended to have a good prognosis. Figure 10 shows the results of sc (single cell) RNA-seq analysis and TCR-seq analysis using TILs from CCP1 cases. The results showed that CD8 + Next, we investigated the RNA expression patterns of CD8 T cells expressing these two types of tumor-reactive TCRs (Fig. 10(B)). + T cells and other CD8 +Comparison of RNA expression between T cells and those expressing these two types of tumor-reactive TCRs showed that CD9 RNA expression was significantly higher in T cells expressing these two types of tumor-reactive TCRs (Figure 10(C)).

[0034] Figure 11 shows the results of an analysis of CCP9 cases, in which CD9 was added to PD-1 as an index and TILs were narrowed down. + Individual CD8 belonging to the TIL cell compartment + We isolated TCRs from T cells and confirmed that two of the TCRs showed reactivity to CTOS derived from CCP9 patients (Fig. 11(A)). Next, we used cryopreserved TILs from CCP9 patients to identify PD-1 + CD9 + CD8 + Individual TCR genes were isolated from the TIL cell fraction and subjected to repertoire analysis. As a result, two types of tumor-reactive TCRs were identified. + CD9 + The CD9 molecule was identified more frequently in the fraction (Fig. 11(B)). These results suggest that CD9 molecules are useful for narrowing down tumor-reactive T cells, and that adding CD9 as an indicator to PD-1 may enable further narrowing down of tumor-reactive T cells.

[0035] The CD9 molecule is a type of molecule known as "tetraspanin." Tetraspanins are a family of transmembrane proteins with a structure that spans the cell membrane four times, and are also called the Transmembrane 4 superfamily (TS4SF). In addition to CD9, other known tetraspanins include TSPAN2, CD151, CD53, CD37, CD82, CD81, and CD63. There have been no reports to date that tetraspanin molecules can be used as indicators for the selection of tumor-reactive T cells. Therefore, we used a mouse model to examine whether the expression of the CD9 molecule can be used as an indicator of tumor reactivity. As a mouse model, we used a CD8 T cell expressing mutant Snd1 (staphylococcal nuclease domain-containing protein 1) specific for the mutant Snd1 (staphylococcal nuclease domain-containing protein 1) that we reported. +We utilized a tumor-bearing mouse treatment model in which T cells are induced (Non-Patent Document 4). In this model, tumors were borne by mouse fibroblastic sarcoma-derived cell line CMS7, and treatment with anti-checkpoint antibodies (anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-GITR antibody) induced CD8 T cells specific to mutant Snd1 encoded by the CMS7 tumor genome in spleen cells 21 days after tumor bearing. + T cells can be seen.

[0036] The test method is shown in Figure 12(A). Mice were subcutaneously administered CMS7 on day 0, and intravenously administered anti-checkpoint antibodies on days 7, 9, and 11. Spleens were harvested on day 21 and 1 × 10 6 A mutant Snd1-derived peptide (SYAPCRGEF (SEQ ID NO: 13)) or a wild-type Snd1-derived peptide (SYAPRRGEF (SEQ ID NO: 14)) was added to spleen cells, and the IFN-γ ICS method (intracellular staining method) was used to detect CD8 cells that reacted to the peptide and produced IFN-γ intracellularly. + T cells can be measured. This response was specific to the mutant Snd1 peptide and was not observed with the wild-type Snd1 peptide or the solvent control DMSO. Furthermore, tumor-bearing mice untreated without anti-checkpoint antibodies showed a very low response to the mutant Snd1 peptide (Figure 12(B)). The proportion of mutant Snd1 (mSnd1) in the antibody-treated group that showed IFN-γ production was extremely high, at 1.6%. Figure 12(C) shows that the mutant Snd1 peptide (31-mer) and adjuvant (polyIC:LC) were effective in treating CMS7 tumor-bearing mice.

[0037] Figure 13 shows the results of investigating whether TILs express CD9 molecules. First, mutant Snd1-specific CD8 TILs were observed in a CMS7 tumor-bearing mouse model treated with an immune checkpoint inhibitor (ICI). + We examined whether T cells expressed CD9 molecules. 6BALB / c mice were inoculated subcutaneously into the backs of the mice with 1×10 cells, and were treated with ICI on days 7, 9, and 11. A control group was treated without ICI. Spleens were removed from the mice on day 21 (Figure 13(A)). 6 The mutant Snd1-derived peptide or control peptide was added to spleen cells, and mutant Snd1-specific CD8 + As a result, as shown in Figure 13(B), in the ICI-treated group, the production of IFN-γ specific to the mutant Snd1-derived peptide was significantly increased by CD8 + The majority of these cells belonged to the CD9 molecule-expressing cell population (the two right data points in the upper row). On the other hand, in the control peptide-added group, IFN-γ-producing CD8 + T cells were not observed (two data points on the right side of the middle row), and in the ICI-untreated group, mutant Snd1-derived peptide-specific CD8 + The frequency of T cells was extremely low (two data points on the right side of the bottom row).

[0038] Next, to examine the possibility that mutant Snd1-derived peptides may increase the expression of CD9 molecules, spleen cells were prepared from three mice in the ICI treatment group and CD8 was isolated using a mouse CD8 isolation kit (Miltenyi Biotec). － Spleen cells and CD8 + T cells were obtained. A portion of the obtained CD8 cells was stained with PE-conjugated mouse CD9 antibody, and then CD8 was detected using anti-PE beads (Miltenyi Biotec). + CD9 + T cell populations and CD8 + CD9 － T cell populations were obtained. These three types of 1 × 10 5 Whole CD8 cell population (CD8 + CD9 － , CD8 + CD9 + ) and 1 × 10 mutant Snd1 peptides were added. 6 CD8 －Co-culture with splenocytes and mutant Snd1 peptide-specific CD8 + The frequency of mutant Snd1-specific CD8 cells was analyzed using the ELISPOT assay. + T cells stimulated with peptides did not increase the expression of CD9 molecules, but rather increased the expression of mutant Snd1-specific CD8 in the spleens of tumor-bearing mice. + The majority of T cells are CD9 + This was revealed to be the case (Figure 13(C)).

[0039] CD9 is a type of molecule belonging to the tetraspanin family, and many other molecules also belong to the tetraspanin family. Among them, it is known that female mice in which CD9 or CD81 is knocked out have problems with cell fusion during fertilization and become infertile, and functional similarities between tetraspanin molecules have been reported. Therefore, we investigated the CD81 molecule by analyzing the mutant antigen Snd1-specific CD8 + The expression of CD81 molecules in T cells was analyzed. The results are shown in Figure 14. + The majority of T cells expressed CD81 molecules, as well as CD9 (Fig. 14(C)). + CD9 + CD81 + Tumor-reactive CD8 T cell population + To test this idea, we investigated the presence of mutant Snd1-specific CD8 T cells. + CD8 T cells in the spleens of CMS7 tumor-bearing mice without ICI treatment showed extremely low frequencies. + CD9 + CD81 + T cells (CD8 + TCR repertoire analysis was performed on the TCRs of the 36 analyzed cells (1.9% of all T cells). The results are shown on the right side of Figure 14 (B). Among the TCRs of the 36 analyzed cells, two types of TCRs were found to have the same TCR gene sequence, in three and two cells, respectively.

[0040] CD8 in the spleen of CMS7 tumor-bearing mice without ICI treatment + CD9 + CD81 +Based on the results of TCR repertoire analysis of the T cell population, two types of TCRs were found to express multiple TCRs, and two types of TCRs were obtained as single TCRs. These TCR-introduced mouse T cells (1 × 10 5 cells) and CMS7 tumor cells (5 × 10 4 The cells were co-cultured with CMS7 for 24 hours, and mouse IFN-γ and TNF-α levels in the supernatant were measured. A mutant Snd1-specific TCR (mSnd1 22-1 TCR) that specifically reacts with CMS7 was used as a positive control. As shown in Figure 15, the 11-3 TCR, which was identical in all three cell lines, specifically recognized CMS7 and released these cytokines. This reaction was specific to CMS7; cytokine release was not observed in CMS5, a mouse fibroblastic sarcoma cell line, which is similar to CMS7.

[0041] Next, CD8 in the spleen of CT26 tumor-bearing mice was + CD9 + CD81 + TCR repertoire analysis of T cell populations was performed. To examine whether similar results could be obtained with mouse tumor cell lines other than CMS7, we used the CT26 cell line, a tumor cell line derived from mouse colon cancer. As shown in Figure 16(A), on day 0, 1 × 10 6 BALB / c mice were inoculated subcutaneously with CT26 cells into the back, and on day 21, CD8 + CD9 + CD81 + We performed TCR repertoire analysis of the T cell population. As shown in Figure 16(B), analysis of the TCRs derived from the 27 cells revealed that one type of TCR was expressed in two cells, and all the others were expressed in a single form.

[0042] Next, we selected one TCR that was found in two cells and two TCRs that existed as a single TCR, and examined their reactivity to CT26 tumor cells. First, we transduced each TCR into BALB / c mouse T cells using a retroviral vector. These TCR-transduced mouse T cells (1 × 10 5 cells) and CT26 tumor cells (5 × 10 4 CT26 tumor-reactive TCRs (1 × 10 TCRs) were co-cultured with P1.HTR cells (H-2d) for 24 hours, and mouse IFN-γ in the supernatant was measured. The results revealed that a single TCR (23-1 TCR) specifically recognized CT26 (Fig. 17(B)). It has been reported that CT26 tumor-bearing mice elicit a CD8+ T cell immune response against the AH-1 peptide derived from the endogenous murine leukemia virus envelope gp70. Therefore, we investigated the possibility that the AH-1 peptide is the antigen recognized by the CT26 tumor-reactive TCR identified in this study. Using P1.HTR cells, which share the same mouse MHC (major histocompatibility complex) H-2d, TCR-transduced mouse T cells (1 × 10 TCRs) were transfected. 5 cells) and P1.HTR cells (5 × 10 4 The mice were co-cultured with CT26 tumor-bearing mice for 24 hours, and the levels of mouse IFN-γ in the supernatant were measured (Fig. 17(C)). The results demonstrated that the antigen peptide recognized by the identified TCR was indeed AH-1. AH-1-specific CD8+ T cells in the spleen of CT26 tumor-bearing mice were + The frequency of T cells is estimated to be less than 1%, and these results are thought to indicate that the tetraspanin molecules CD9 and CD81 are extremely useful for narrowing down tumor-reactive CD8-positive T cells.

[0043] The function of tetraspanins, including CD9 molecules, has not yet been fully elucidated. This study revealed that they play an important role in the interaction between T cells and tumor cells, suggesting that CD9 expression may enhance the efficacy of immune cell therapy, which involves the infusion of T cells transfected with antigen-specific TCR / CAR. Therefore, to verify that CD9 expression enhances the antitumor effect in antigen-specific T cell infusion therapy, we obtained the following data. We used a transgenic mouse (DUC18, non-patent literature 5) carrying a TCR that specifically recognizes a complex between H-2Kd and a peptide derived from the mutant ERK2 antigen (mERK2-9m) encoded by the mouse fibrosarcoma-derived cell line CMS5. As shown in Figure 18(A), on day 0, CMS5 (1 × 10 6 BALB / c mice were inoculated subcutaneously with 2 × 10 cells into the back of the mice, and cell transfusion (ACT) was performed on day 3. Three treatment groups (n = 4 / group) were established: (1) no treatment (control), (2) T cells (2 × 10 cells) stimulated and expanded for 5 days using anti-CD3 and anti-CD28 antibodies in DUC18 mouse spleen cells, and (3) T cells (2 × 10 cells) stimulated and expanded for 5 days using anti-CD3 and anti-CD28 antibodies in DUC18 mouse spleen cells. 6 (3) T cells (2 × 10) stimulated and proliferated for 4 days after transfection with DUC18 mouse spleen cells using anti-CD3 and anti-CD28 antibodies and retrovirally transfected with mouse CD9 gene the next day. 6 The results showed that the therapeutic effect was enhanced by the infusion of T cells that strongly express CD9 molecules, as shown in Figure 18(B). Thus, this embodiment provides T cells for tumor treatment in cancer patients. These T cells can be effectively used in the treatment of cancer patients.

Claims

1. A method for producing tumor-reactive T cells, comprising: a candidate isolation step of isolating CD-8 positive T cells from tumor-infiltrating lymphocytes (TIL) collected from a cancer patient or whole blood, using PD-1 and / or 4-1BB as markers for antigens on the cell surface; a tumor reactivity evaluation step of transducing the T cell receptor (TCR) gene of the T cells isolated in the candidate isolation step into target cells to identify and evaluate a TCR having tumor reactivity; and a therapeutic T cell acquisition step of proliferating in vitro T cells having a TCR reactive to a tumor to obtain therapeutic T cells for administration to the cancer patient.

2. The method for producing tumor-reactive T cells according to claim 1, wherein the solid tumor is at least one selected from the group consisting of brain tumor, tongue cancer, esophageal cancer, gastric cancer, small intestine cancer, colorectal cancer, liver cancer, kidney cancer, bladder cancer, lung cancer, thyroid cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, rhabdomyosarcoma, and leiomyosarcoma.

3. The method for producing tumor-reactive T cells according to claim 1 or 2, wherein in the candidate isolation step, tetraspanin is further used as a marker.

4. The method for producing tumor-reactive T cells according to claim 3, wherein the tetraspanin is at least one selected from the group consisting of CD9, TSPAN2, CD151, CD53, CD37, CD82, CD81, and CD63.

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