WO2023074713A1 - T cell quality evaluation method, and reagent to be used therein - Google Patents

T cell quality evaluation method, and reagent to be used therein Download PDF

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WO2023074713A1
WO2023074713A1 PCT/JP2022/039808 JP2022039808W WO2023074713A1 WO 2023074713 A1 WO2023074713 A1 WO 2023074713A1 JP 2022039808 W JP2022039808 W JP 2022039808W WO 2023074713 A1 WO2023074713 A1 WO 2023074713A1
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cells
amino acid
acid sequence
polypeptide
gene
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PCT/JP2022/039808
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French (fr)
Japanese (ja)
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新 金子
洋平 河合
佳奈 山口
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国立大学法人京都大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention provides a method for evaluating the quality of T cells, a reagent for evaluating the quality of T cells used therein, a pharmaceutical composition containing high-quality T cells obtained by the method for evaluating T cells, and a cancer using the pharmaceutical composition. It relates to the prevention or treatment method of
  • T cells antigen-specific cytotoxic T-lymphocytes (CTL) related to the disease
  • CTL cytotoxic T-lymphocytes
  • T cells unmodified T cells
  • Expectations are high for cancer immunotherapy, which treats cancer by returning cells to the original patient.
  • Patent Document 1 discloses a method of inducing CD8 ⁇ + ⁇ + cytotoxic T cells by culturing CD4 + CD8 + T cells in a medium containing interleukin 7 and a TCR (T cell receptor) activator. is described.
  • T cell quality evaluation items include, for example, analysis of differentiation markers expressed at each stage of T cells, analysis of cytokines produced after TCR stimulation, proliferation ability, cytotoxic activity, animal experiments and cancer cell-suppressing activity.
  • the quality of T cells is comprehensively evaluated by these items, but such evaluations are often complicated and require a long period of time. Furthermore, since the cost of evaluation is high, there is a demand for a method that can evaluate the quality of T cells more simply and in a short period of time.
  • Non-Patent Document 1 As a method for evaluating the quality of such T cells, for example, it has been found that there is a relationship between the expression level of PD-1, lag3, etc. and the exhaustion of T cells (Non-Patent Document 1). , using these proteins as T cell exhaustion markers, a method of evaluating T cell exhaustion based on the expression level thereof.
  • the present invention has been made in view of the above-mentioned problems of the prior art, and provides a T cell quality evaluation method capable of evaluating the quality of relatively young T cells using a new index, and a T cell quality evaluation reagent used therein. intended to provide
  • the present inventors have made intensive studies to solve the above problems. Analysis and transcriptome analysis were performed. Next, from these analysis results, we identified signal transduction pathways involving genes and proteins with large differences in expression levels between high-proliferation T cells and low-proliferation T cells, and identified proteins involved in such signal transduction pathways (CD39 and CD73) were selected as candidate markers for T cell quality assessment. Next, when the selected proteins were examined to see if they could be used as quality evaluation markers for T cells, the expression level of a specific protein (CD39), which is a type of T cell-derived secretome, and the proliferative ability of T cells, We found that there is a correlation between exhaustion. Therefore, the present inventors have found that the quality of relatively young T cells can also be evaluated by using such a protein as a new marker and using this expression level as an index, thus completing the present invention.
  • CD39 specific protein
  • a method for assessing T cell quality comprising: (i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
  • the detection step detects the expression product using a probe molecule that specifically binds to the expression product of at least one gene selected from the group consisting of the genes (i) to (iii)
  • the method according to [1] which is a step.
  • the expression product is a polypeptide encoded by any one of the genes (i) to (iii), and the probe molecule specifically binds to the polypeptide
  • the method of [2] which is an antibody and/or an aptamer.
  • the expression product is a polypeptide encoded by any one of the genes (i) to (iii), and the probe molecule specifically binds to the polypeptide
  • the reagent of [5] which is an antibody and/or an aptamer.
  • [11] A method of treating or preventing cancer, comprising administering the pharmaceutical composition of [8] to a subject who has or is at risk of having cancer.
  • a method for treating or preventing cancer comprising administering T cells to a subject having cancer or at risk of having cancer, wherein A detection step of detecting the expression of at least one gene selected from the group consisting of the genes of ⁇ (III); an evaluation step of evaluating the quality of T cells using the expression of the gene as an index; A method, including (i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
  • a method for producing T cells comprising a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (I) to (III) in the T cells; an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
  • a method of producing a T cell comprising: (i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
  • the present invention it is possible to provide a T cell quality evaluation method capable of evaluating T cell quality using a new index, and a T cell quality evaluation reagent used for the method.
  • (a) is a graph in which the Y-axis is the expression level of the gene encoding CD39
  • the expression level of CD45RA in T cells after 1 stimulation treatment and 10 stimulation treatments (Y-axis), and CD45RO, CD39, or PD-1 in the T cells is a graph showing the relationship between the expression level (X-axis) of (a) is a graph when the X axis is the expression level of CD45RO, (b) is a graph when the X axis is the expression level of CD39, and (c) is a graph when the X axis is the expression level of PD-1 It is a graph when it is a quantity.
  • the method for evaluating the quality of T cells of the present invention comprises a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells; an evaluation step of evaluating the quality of T cells using the expression of the gene as an index; (hereinafter sometimes simply referred to as "the method of the present invention").
  • the T cell in the present invention means a cell expressing an antigen receptor called T cell receptor (TCR) on the surface, alpha beta ( ⁇ ) T cells and gamma delta ( ⁇ ) containing T cells.
  • TCR T cell receptor
  • T cells in the present invention include, for example, helper T cells, cytotoxic T cells, regulatory T cells, natural killer T cells, naive T cells, memory T cells (e.g., stem cell memory T cells (TSCM), central memory T cells). cells (TCM), effector memory T cells, etc.), antigen-stimulated memory T cells, or terminal effector T cells, combinations thereof, or subpopulations thereof.
  • helper T cells cytotoxic T cells
  • regulatory T cells e.g., regulatory T cells, natural killer T cells, naive T cells
  • memory T cells e.g., stem cell memory T cells (TSCM), central memory T cells).
  • TCM stem cell memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • TCM central memory T cells
  • Helper T cells are CD4-positive cells, and are further classified into Th1 cells, Th2 cells, Th17 cells, etc. according to the cytokines they express (e.g., J Allergy Clin. Immunol., 135(3): 626-635, 2012).
  • Th1 cells include, for example, cells expressing IFN- ⁇ , IL-2, TNF- ⁇ , and the like.
  • Th2 cells include, for example, cells expressing IL-4, IL-5, IL-6, IL-10, IL-13, and the like.
  • Th17 cells include, for example, cells expressing IL-17 or IL-6.
  • Cytotoxic T cells are CD8-positive cells, and like helper T cells, are further classified into Tc1 cells, Tc2 cells, etc. according to the cytokines they express.
  • Tc1 cells include cells expressing IFN- ⁇ , IL-2, TNF- ⁇ , and the like.
  • Tc2 cells include cells that express IL-4, IL-5, IL-6, IL-10, IL-13, and the like.
  • Regulatory T cells preferably include, for example, CD4(+) CD25(+) FoxP3(+) cells.
  • Naive T cells include, for example, CD4 (+) CD45RA (+) CD62L (+) CCR7 (+) cells, CD8 (+) CD45RA (+) CD62L (+) CCR7 (+) cells, CD4 (+) CCR7 ( +)CD45RA(+)CD95(-)CD45RO(-) cells or CD8(+)CCR7(+)CD45RA(+)CD95(-)CD45RO(-) cells are preferred.
  • Stem cell memory T cells include, for example, CD4 (+) CD45RA (+) CD62L (+) CCR7 (+) CD95 (+) cells, CD8 (+) CD45RA (+) CD62L (+) CCR7 (+) CD95 (+) ) cells, CD4(+)CCR7(+)CD45RA(+)CD95(+)CD45RO(+) cells or CD8(+)CCR7(+)CD45RA(+)CD95(+)CD45RO(+) cells are preferred. be done.
  • Central memory T cells include, for example, CD4 (+) CD45RA (-) CD62L (+) CCR7 (+) CD95 (+) cells, CD8 (+) CD45RA (-) CD62L (+) CCR7 (+) CD95 (+) ) cells, CD4(+)CCR7(+)CD45RA(-)CD45RO(+) cells, CD8(+)CCR7(+)CD45RA(-)CD45RO(+) cells, and the like are preferable.
  • effector memory T cells include preferably CD4(+) CCR7(-) CD45RA(-) CD45RO(+) cells or CD8(+) CCR7(-) CD45RA(-) CD45RO(+) cells.
  • Terminal effector T cells preferably include, for example, CD4(+)CD45RA(+)CD62L(-) cells or CD8(+)CD45RA(+)CD62L(-) cells.
  • T cells are not particularly limited, and may be animal-derived or human-derived. or from a person or animal suffering from a malignant tumor, an infectious disease, or an autoimmune disease).
  • T cells differentiated from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); T cells differentiated from somatic stem cells such as hematopoietic stem cells; It may be a T cell.
  • T cells are isolated from peripheral blood mononuclear cells (PBMC: Peripheral Blood Monoclear Cells) isolated from peripheral blood using conventionally known methods or commercially available kits. It can be isolated.
  • PBMC peripheral blood mononuclear Cells
  • T cells include T cells that have not been artificially genetically modified (hereinafter sometimes referred to as “unmodified T cells”), as well as artificial genes in the unmodified T cells. Also included are “genetically modified T cells” that have been modified.
  • artificial genetic modification includes modification of T cell functions by gene transfer to T cells or gene editing of T cells. The artificial genetic modification may be modification of the gene itself possessed by T cells, deletion of the gene itself possessed by T cells, or modification resulting from the introduction of a foreign gene. A combination of two or more types may also be used.
  • the method of artificial genetic modification includes conventionally known methods and methods based thereon, and is not particularly limited.
  • Gene introduction into T cells includes, for example, the gene itself (DNA, mRNA, miRNA, antagomir, ODN (oligodeoxyribonucleotide), etc.) that modifies the function of T cells, or a vector into which the gene is inserted (lentiviral vector , ⁇ - or ⁇ -retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, equine encephalopathic viral vectors, non-viral vectors such as the transposon vector piggyBac®).
  • lentiviral vector , ⁇ - or ⁇ -retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, equine encephalopathic viral vectors, non-viral vectors such as the transposon vector piggyBac®.
  • Gene editing of T cells includes, for example, gene editing of T cells using site-specific nucleases (meganuclease, zinc finger nuclease, TALEN, PPR, CRISPR-Cas, etc.) (genome Edit).
  • site-specific nucleases meganuclease, zinc finger nuclease, TALEN, PPR, CRISPR-Cas, etc.
  • the artificial genetic modification method may be one of these alone or a combination of two or more.
  • Examples of the gene that modifies the function of T cells include proteins secreted from T cells; fusion proteins containing proteins expressed on the surface of T cells and at least one intracellular signaling domain; proteins expressed on the surface of T cells; Fusion proteins comprising at least one co-stimulatory domain and at least one intracellular signaling domain include, more specifically, cell surface enzymes, cell adhesion factors, receptors (e.g., chimeric antigen receptors), and these and subunits that make up the
  • Detection process (target gene)
  • expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells is detected.
  • a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence
  • the polypeptide encoded by any one of the genes (i) to (iii) is preferably contained in the T cell-derived secretome.
  • secretome refers to a protein secreted from a cell or exposed outside the cell through the cell membrane, and includes a secretory protein endogenous to the cell.
  • the gene whose expression is detected (hereinafter sometimes referred to as "target gene") is human-derived and typically contains "(i) the amino acid sequence set forth in SEQ ID NO: 1 A gene encoding a polypeptide".
  • the amino acid sequence set forth in SEQ ID NO: 1 is CD39, a secretome derived from T cells (UniProt No.: P49961).
  • CD39 also known as SPG64, is also known to have ENTPD1 (EctoNucleoside TriphosPhate Diphosphohydrolase 1) activity, ATPase activity, ADPase activity, and NTPDase-1 activity.
  • the target gene is a homologous gene (e.g., a counterpart gene in an organism other than human) of "(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1".
  • a homologous gene e.g., a counterpart gene in an organism other than human
  • the nucleotide sequence of a gene can be mutated in nature (that is, non-artificially) due to mutation thereof, etc., such natural mutants can also be used as the target gene in the present invention.
  • a person skilled in the art can modify the nucleotide sequence and modify the function, although it is different from the amino acid sequence that codes for it. Since proteins can also be prepared, such variants may also be used as the target gene.
  • amino acid sequence in which amino acid residues are substituted, deleted, inserted, and/or added means an amino acid sequence in which amino acid residues are substituted, deleted, inserted, or added, or It shows that the amino acid sequence is a combination of two or more of Further, “plurality” means, for example, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 is preferably an integer of, but not limited to.
  • One or more amino acid residues is preferably 1 to 10 amino acid residues, more preferably 1 to 5, still more preferably 1 to 3, particularly preferably 2 or less. is.
  • aspects of the target gene according to the present invention also include "(iv) a nucleotide sequence that hybridizes under stringent conditions with the complementary strand of the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1".
  • Hybridization reactions are typically performed under stringent conditions to isolate homologous genes.
  • stringent conditions means that the washing operation of the membrane after hybridization is performed at a high temperature in a low-salt solution. trisodium citrate, 150 mM sodium chloride) and 0.5% SDS solution at 60°C for 20 minutes.
  • hybridization can be performed, for example, according to the method described in the instruction manual attached to the known ECL direct DNA/RNA labeling/detection system (manufactured by GE Healthcare Bioscience).
  • ECL direct DNA/RNA labeling/detection system manufactured by GE Healthcare Bioscience.
  • the above conditions are only examples, and the necessary stringency can be achieved by appropriately combining DNA concentration, DNA length, hybridization reaction time, and the like.
  • a protein encoded by a homologous gene obtained by such a method usually has a high degree of identity with the amino acid sequence set forth in SEQ ID NO:1. Therefore, the target gene aspect of the present invention also includes "(iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO: 1". Amino acid sequence identity can be determined, for example, using the BLASTP program (Altschul et al., J. Mol. Biol., 215, 1990, p. 403-410).
  • identity with the amino acid sequence set forth in SEQ ID NO: 1 is typically preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (eg, 96% or more, 97% or more, 98% or more, 99% or more).
  • detecting gene expression includes both detection of the presence or absence of gene expression and detection of the degree of expression.
  • the expression of the target gene is detected.
  • the gene expression level can be grasped as an absolute amount or as a relative amount. When grasping the relative amount, for example, it can be judged by comparing with the gene expression level of a prepared standard sample.
  • a "standard sample” refers to a sample for which it has been specified in advance whether or not the target gene is expressed, and if so, the amount of expression. For example, T cells that have been previously identified as having good or bad quality can be used as the standard sample.
  • “expression of a gene” means both transcription and translation of a gene. Therefore, “detection of gene expression” in the present invention includes detection at the transcription level (mRNA level) and detection at the translation level (protein level) (that is, detection of the polypeptide encoded by the gene). both are included.
  • splicing occurs in which introns in the pre-mRNA are removed and the exons before and after are recombined (splicing). , which produces a variety of mature mRNAs. In turn, various proteins are translated thereby. Various mRNAs and proteins resulting from such splicing differences are called "splicing variants". Therefore, detection of gene expression in the present invention includes detection of the splicing variant as long as it is specifically recognized by the following probe molecule.
  • the detection step according to the method of the present invention includes binding specifically to the expression product of at least one gene selected from the group consisting of the genes (i) to (iii) (that is, the target gene).
  • the expression product is detected using a probe molecule that
  • the expression product of the target gene is mRNA transcribed from the target gene (hereinafter sometimes referred to as "target polynucleotide").
  • the target polynucleotide also includes cDNA using the mRNA as a template.
  • Detection methods include, for example, Northern blotting, dot blotting, and RNase protection assay using oligonucleotide probes designed to hybridize to appropriate positions in the base sequence of the target polynucleotide as the probe molecule. , DNA microarray analysis, in situ hybridization, and the like to detect the target polynucleotide.
  • the oligonucleotide probe one labeled with a labeling substance is used, a signal corresponding to the labeling substance is detected, and the detected signal amount is the amount of the target polynucleotide (i.e., expression level of the target gene).
  • labeling substances at this time include fluorescent substances such as FITC (fluorescein isothiocyanate), FAM (fluorescein amidite), DEAC (7-(diethylamino)coumarin), R6G (rhodamine 6G), TexRed, Cy5, and BODIPY FL; enzymes such as ⁇ -D-glucosidase, luciferase, HRP (horseradish peroxidase); radioactive isotopes such as 3 H, 14 C, 32 P, 35 S, 123 I; affinity substances such as biotin and streptavidin; Luminescent substances such as luminol, luciferin, and lucigenin are included.
  • fluorescent substances such as FITC (fluorescein isothiocyanate), FAM (fluorescein amidite), DEAC (7-(diethylamino)coumarin), R6G (rhodamine 6G), TexRed, Cy5, and BODIPY FL
  • enzymes
  • the "signal” includes coloration (coloration), reflected light, luminescence, quenching, fluorescence, radiation from a radioactive isotope, and the like, and in addition to those that can be confirmed with the naked eye, depending on the type of signal It also includes those that can be confirmed by measuring methods and equipment.
  • oligonucleotide primers designed to sandwich appropriate positions in the target polynucleotide are used, PCR method, RT-PCR method, TRC (Transcription Reverse Transcription Concerned) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, TMA (Transcription-Mediated Amplification) method, or the like may also be used to amplify and detect the target polynucleotide.
  • a signal (fluorescence) obtained by intercalating the obtained amplification product with a fluorescent dye e.g., ethidium bromide, SYBR Green (trade name), SYTO63 (trade name), etc.
  • a fluorescent dye e.g., ethidium bromide, SYBR Green (trade name), SYTO63 (trade name), etc.
  • the detected signal amount fluorescence intensity
  • detection may be performed in combination with the oligonucleotide probe (double dye probe method, etc.).
  • the amount of the target polynucleotide may be determined by directly subjecting a sample containing the target polynucleotide to a sequencer for analysis.
  • oligonucleotide probes and oligonucleotide primers can be designed by a person skilled in the art based on the sequence of the target polynucleotide by a known method or a method based thereon.
  • the length of the oligonucleotide probe and oligonucleotide primer are each independently preferably at least 15 bases, usually 15 to 100 bases, preferably 17 to 30 bases, more preferably 20-25 bases.
  • the oligonucleotide probes and oligonucleotide primers can be synthesized by, for example, a commercially available oligonucleotide synthesizer.
  • nucleotides deoxyribonucleotides and/or ribonucleotides
  • PNA polyamide nucleic acid
  • LNA locked nucleic acid
  • ENA registered trademark, 2'- O,4'-C-Ethylene-bridged nucleic acids
  • the expression product of the target gene is a polypeptide encoded by the target gene (hereinafter sometimes referred to as "target polypeptide"). More specifically, it is a polypeptide encoded by any one of the genes (i) to (iii), that is, the group consisting of the following polypeptides (i') to (iii') A polypeptide selected from (i') a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (ii') in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or added (iii') A polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
  • these target polypeptides to be detected are preferably the above secretomes (T cell-derived secretomes).
  • Detection methods for detection at the translation level include, for example, antibodies that specifically bind to the target polypeptide (hereinafter sometimes referred to as "anti-polypeptide antibodies”) and/or aptamers (hereinafter , In some cases, simply referred to as "aptamer”), immune cell staining method, imaging cytometry, flow cytometry, ELISA (Enzyme-Linked Immuno Sorbent Assay) method, radioimmunoassay, immunoprecipitation method, immunoblotting (Western blotting method etc.), methods of detection using antibodies such as antibody arrays and in vivo imaging (immunological techniques); methods of detection using aptamers instead of the antibodies.
  • the immunological method may be performed automatically using an enzyme immunoassay device such as AIA-900 or AIA-CL2400 (both manufactured by Tosoh Corporation) after preparing necessary reagents.
  • an “antibody” may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody.
  • “Antibody” also includes all classes and subclasses of immunoglobulins.
  • a “functional fragment” of an antibody refers to a portion (partial fragment) of an antibody that specifically recognizes the polypeptide encoded by the target gene in the present invention. Specifically, Fab, Fab′, F(ab′) 2 , variable region fragment (Fv), disulfide-bonded Fv, single-chain Fv (scFv), sc(Fv) 2 , diabody, multispecific antibodies, nanobodies, and polymers thereof.
  • the anti-polypeptide antibody according to the present invention is a polyclonal antibody
  • an immunized animal is immunized with an antigen (target polypeptide, its partial peptide, or cells expressing these, etc.), and the antiserum is obtained by conventional means ( Salting out, centrifugation, dialysis, column chromatography, etc.) can be appropriately purified and obtained.
  • Monoclonal antibodies can also be produced by the hybridoma method or recombinant DNA method.
  • Hybridoma methods include, for example, Kohler and Milstein's method (Kohler & Milstein, Nature, 256:495 (1975)).
  • Recombinant DNA methods include, for example, DNA encoding the anti-polypeptide antibody.
  • B cells or the like It is cloned from B cells or the like, incorporated into an appropriate vector, introduced into host cells (mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.), and the anti-polypeptide antibody is produced as a recombinant antibody.
  • host cells mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.
  • the anti-polypeptide antibody is produced as a recombinant antibody.
  • the anti-polypeptide antibody and the aptamer are each labeled with a labeling substance, the signal corresponding to the labeling substance is detected, and the amount of the detected signal is calculated as the amount of the target polypeptide. (that is, the expression level of the target gene).
  • the labeling substance at this time is not particularly limited as long as it can bind to the anti-polypeptide antibody or aptamer and can be detected by a chemical or optical method.
  • fluorescent substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC)
  • enzymes such as peroxidase, ⁇ -D-galactosidase, microperoxidase, horseradish peroxidase (HRP) and alkaline phosphatase, and radioactive substances.
  • the anti-polypeptide antibody and/or aptamer When the anti-polypeptide antibody and/or aptamer is used, a method of using a secondary antibody bound with the labeling substance, or a method of using a polymer binding the secondary antibody and the labeling substance. Indirect detection methods such as can also be used.
  • the "secondary antibody” is an antibody that exhibits specific binding to the anti-polypeptide antibody and/or aptamer.
  • an anti-rabbit IgG antibody can be used as the secondary antibody.
  • Labeled secondary antibodies that can be used for antibodies derived from various species such as rabbits, goats, and mice are commercially available. Antibodies can be selected and used.
  • protein G for example, derived from Streptococcus spp.
  • protein A for example, derived from Staphylococcus aureus
  • protein L derived from Peptostreptococcus magnus
  • Detection with labeled amino acids include, for example, a method of culturing T cells in a medium supplemented with a labeled amino acid and detecting a target polypeptide labeled with the labeled amino acid.
  • a labeling step of culturing T cells in a T cell labeling medium containing a labeled amino acid in which the amino acid is labeled instead of at least one essential amino acid for T cell proliferation and containing a common ⁇ chain family cytokine; a detection step a of detecting the T cell-derived polypeptide labeled with the labeled amino acid; is preferred.
  • T cell labeling medium refers to a medium containing at least labeling amino acids for culturing and labeling T cells.
  • essential amino acids for T cell proliferation refers to amino acids essential for T cell proliferation, more specifically, valine, isoleucine, leucine, methionine, lysine, phenylalanine, tryptophan, threonine, histidine.
  • the T cell labeling medium contains a labeled amino acid in which the amino acid is labeled instead of at least one of the essential amino acids, that is, does not contain at least one of the essential amino acids and the essential amino acid that is not contained Contains labeled amino acids, where the amino acids are labeled amino acids.
  • "Containing no essential amino acids” means that the T cell labeling medium does not substantially contain the essential amino acids. More specifically, the content of the essential amino acids in the T cell labeling medium is is 1 ⁇ 10 ⁇ 3 mmol/L or less, preferably less than 1 ⁇ 10 ⁇ 4 mmol/L.
  • the essential amino acids excluded from the T cell labeling medium may be any one of the above or a combination of two or more of them. Among them, methionine is preferred from the viewpoint of efficient incorporation into the protein synthesis pathway.
  • the "labeled amino acid” refers to an essential amino acid partially modified while maintaining the structure of the essential amino acid, and detectable based on the modification.
  • the above-mentioned “detectable” includes those that can be directly confirmed by visual observation such as coloring (color development), quenching, reflected light, luminescence, and fluorescence, as well as those that can be confirmed by a specific measuring method or measuring device.
  • labeled amino acids examples include isotope-labeled amino acids in which some of the atoms of the amino acid are substituted with pulse stable isotopes (e.g., 2 H, 13 C, 15 N); Examples include structural analogs of amino acids modified by modifying groups such as groups, and corresponding to the essential amino acids, any one of them or a combination of two or more of them good.
  • the labeling amino acid contained in the T cell labeling medium instead of methionine may be, for example, 2 H-labeled methionine, 13 C-labeled methionine, and 15 N-labeled methionine, which are isotopically-labeled methionines substituted with the stable isotope; L-azidohomoalanine (AHA), which are structural analogues of methionine; -homopropargylglycine (HPG: L-Homopropargylglycine), L-homoallylglycine (HAG: L-Homoallylglycine), any one of which may be used or a combination of two or more thereof may be used.
  • HPG L-Homopropargylglycine
  • HOG L-homoallylglycine
  • the content of the labeled amino acids includes the T cell uptake efficiency, the culture efficiency, the T cell It can be determined as appropriate in consideration of the toxicity etc., and is not particularly limited. Preferably, it is 0.001 to 0.02 mmol/L. More specifically, for example, when the labeled amino acid is AHA, it is preferably 0.0005 to 0.03 mmol/L, more preferably 0.001 to 0.03 mmol/L. More preferably 0.002 to 0.03 mmol/L, even more preferably 0.005 to 0.02 mmol/L.
  • the cells tend to weaken or die, whereas when the content is less than the lower limit, the polypeptide labeled with the labeled amino acid is secreted or exposed outside the cell. As a result, the number of cells to be detected tends to decrease, and the detection accuracy of the polypeptide tends to decrease.
  • the T cell labeling medium further contains common ⁇ -chain family cytokines.
  • a "common ⁇ chain family cytokine” refers to a cytokine that acts via a receptor containing a common ⁇ chain ( ⁇ subunit). More specifically, interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 15 (IL-15) , and interleukin 21 (IL-21), any one of which may be used in combination.
  • interleukin 7, interleukin 15, interleukin 21, and combinations of two or more of these are more preferable from the viewpoint of better maintenance of cell survival and growth.
  • the content of the common ⁇ -chain family cytokine (when there are two or more common ⁇ -chain family cytokines, the total content thereof; the same shall apply hereinafter) is determined according to the properties of T cells, etc. Although it is not limited to this, it is preferably 1 to 1000 ng/mL, more preferably 5 to 100 ng/mL, and 5 to 20 ng/mL. It is even more preferable to have When the content of the common ⁇ -chain family cytokine exceeds the upper limit, the effect of containing the common ⁇ -chain family cytokine tends to decrease or cell proliferation tends to be inhibited. The number of cells that secrete or extracellularly expose a polypeptide labeled with a labeled amino acid tends to decrease, resulting in a decrease in the detection accuracy of the polypeptide.
  • the T cell labeling medium preferably does not contain serum or, if it does contain serum, is dialyzed serum from which the essential amino acids have been removed.
  • the T cell labeling medium may also contain cytoprotective additives such as serum albumin and ITS (insulin-transferrin-sodium selenite); scaffolding agents such as ECM (extracellular matrix) components.
  • T cell labeling media or their basal media are not particularly limited as long as they do not contain the desired essential amino acids, and are DMEM (Dulbecco's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium), ⁇ MEM ( ⁇ -modified basal medium such as Eagle minimum essential medium); AIM V TM medium (manufactured by Thermo Fisher Scientific) which is a serum-free medium exclusively for T cells; PRIME-XV T cell Expansion XSFM (manufactured by Fujifilm Wako Pure Chemical) or a commercially available medium for T cells can be used as appropriate.
  • DMEM Disbecco's Modified Eagle Medium
  • RPMI Roswell Park Memorial Institute Medium
  • ⁇ MEM ⁇ -modified basal medium such as Eagle minimum essential medium
  • AIM V TM medium manufactured by Thermo Fisher Scientific
  • PRIME-XV T cell Expansion XSFM manufactured by Fujifilm Wako Pure Chemical
  • a commercially available medium for T cells
  • T cells are cultured in the T cell labeling medium.
  • the labeled amino acid is taken up by T cells, and a polypeptide labeled with the labeled amino acid (T cell-derived polypeptide) is secreted from the T cells or exposed outside the cells.
  • the culture method in the labeling step is not particularly limited, and known T cell proliferation culture conditions can be appropriately adopted, and the characteristics and concentration of T cells can be taken into consideration and adjusted as appropriate.
  • the culture temperature is 35 to 37.5° C., preferably 36 to 37° C.
  • the culture time is 2 to 48 hours, preferably 8 to 24 hours.
  • the culture apparatus for the labeling step is not particularly limited, and examples thereof include plates, dishes, columns, flasks, culture bags, and the like. Ejection means (sensors, valves, pumps, tanks, etc.) may also be provided.
  • the T-cell-derived polypeptide that has been labeled with the labeling amino acid and has been secreted or exposed outside the cell is detected.
  • the detection step a may be performed simultaneously (in real time) with the above labeling step.
  • a detection method in the detection step a a detection method suitable for the type of the labeled amino acid can be appropriately employed.
  • Such a detection method is not particularly limited, and a known method or a method based thereon can be appropriately employed.
  • the labeled amino acid is an isotope-labeled amino acid or a structural analogue of an amino acid
  • the medium after the labeling step or during the labeling step that is, during the culture
  • gas isotope ratio mass spectrometry By performing mass spectrometry such as infrared spectroscopy, liquid chromatography mass spectrometry (LC-MS/MS), a signal corresponding to the labeled amino acid is detected, and the amount of the detected signal is the amount of the target polypeptide (i.e. , expression level of the target gene).
  • the labeled amino acid when it is a structural analogue of an amino acid, it can be detected by staining the modified group specifically.
  • the modifying group is an azide group (such as AHA)
  • the azide group can be stained with a fluorescent reagent such as Click-iT TM Alexa Fluor 488 sDIBO Alkyne (manufactured by Thermo Fisher Scientific).
  • a fluorescent reagent such as Click-iT TM Alexa Fluor 488 sDIBO Alkyne (manufactured by Thermo Fisher Scientific).
  • a signal color development, fluorescence, etc.
  • the amount of the detected signal is the amount of the target polypeptide (i.e., the amount of the target gene). expression level).
  • detection methods for detection at the translation level include, for example, a method of recognizing and detecting the sugar chain of the target polypeptide. Such methods include, for example, methods using commercially available reagents such as Click-iT TM (manufactured by Thermo Fisher Scientific). The above detection method for detection at the translation level may be used in combination with other methods as appropriate.
  • detection of the expression of the gene is preferably detection at the translation level (detection of the target polypeptide) from the viewpoint of convenience. Methods that detect polypeptides are preferred.
  • the quality of T cells is evaluated using the presence or absence or the expression level of the target gene detected in the detection step as an index.
  • the quality of T cells to be evaluated in the present invention includes proliferative ability and cancer cytotoxic activity.
  • the method of the present invention is preferable as a method for evaluating the proliferative ability of T cells. More specifically, the term "proliferative ability" indicates the ability to activate T cells and increase cells having cytotoxic activity in vitro or in vivo by TCR stimulation or antigen stimulation.
  • T cells The proliferative ability of T cells can be confirmed, for example, by stimulating T cells with TCR-stimulating magnetic beads by the method shown in the Examples below, and checking the ratio of the number of cells before stimulation to the number of cells 14 days after stimulation. However, it is not limited to this.
  • the lower the expression level of the target gene detected in the detection step the higher the growth ability, that is, the better the quality. It can be evaluated as having low growth ability, ie, poor quality.
  • the evaluation criteria for the quality of the T cells may be appropriately set according to the purpose, and are not particularly limited.
  • the detection step if even a little expression of the target gene is not detected according to the expression level of the target gene (transcription level or translation level of the target gene), that is, the target polynucleotide or the target polypeptide If even a little is not detected, the proliferative ability of the T cells may be evaluated as high, and if the expression level of the target gene above a certain threshold is not detected, the proliferative ability may be evaluated as high. Further, the degree of T cell proliferation ability may be evaluated according to the expression level of the target gene.
  • the expression level of the target gene transcription level or translation level of the target gene
  • the expression level of the target gene detected in a standard sample such as T cells that have been specified in advance to have low proliferative ability it is below a certain value, or below the average expression level of the target gene.
  • a signal amount of 4 SD or less, 3 SD or less, or 2 SD or less may be evaluated as having a high proliferative capacity, ie, good quality.
  • the T cell quality evaluation reagent of the present invention is a reagent (composition) for use in the T cell quality evaluation method of the present invention, and is selected from the group consisting of the following genes (i) to (iii): is a composition (hereinafter sometimes simply referred to as "reagent of the present invention") containing a probe molecule that specifically binds to the expression product of at least one gene.
  • the genes (i) to (iii) and expression products of the genes are as described in the method of the present invention, including preferred embodiments thereof.
  • probe molecules related to the reagent of the present invention include oligonucleotide probes, oligonucleotide primers, anti-polypeptide antibodies, and aptamers mentioned in the method of the present invention described above. in the method of the present invention.
  • the probe molecule may be one of these or a combination of two or more of them. Among them, the anti-polypeptide antibody and/or the aptamer are preferable, and the anti-polypeptide antibody is more preferable.
  • the reagent of the present invention may be liquid or solid such as powder. Additional ingredients may be included.
  • the T cell evaluation kit of the present invention is a kit for use in the T cell quality evaluation method of the present invention, and includes at least one gene selected from the group consisting of the genes (i) to (iii) above. (hereinafter, sometimes simply referred to as "the kit of the present invention").
  • the probe molecule according to the kit of the present invention is preferably the reagent of the present invention.
  • the kit of the present invention also contains a buffer, a solution for dilution or suspension such as physiological saline; a reagent for extracting and/or purifying DNA or protein; a blocking agent; a chelating agent; The labeling substance; the fluorescent dye; reagents necessary for the detection step; reagents such as pH adjusters; standard samples; instructions for use;
  • the present invention provides high-quality T cells obtained by the above T-cell evaluation method and pharmaceutical compositions containing the high-quality T cells as active ingredients.
  • the pharmaceutical composition containing T cells as an active ingredient of the present invention can be used to treat cancer patients.
  • the pharmaceutical composition of the present invention can be produced by a method commonly used in the field of formulation technology, such as the method described in the Japanese Pharmacopoeia.
  • the pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives. Examples of such additives include cell culture media, physiological saline, and suitable buffers (eg, phosphate buffers).
  • the pharmaceutical composition of the present invention can be produced by suspending T cells in physiological saline, an appropriate buffer (eg, phosphate buffer), or the like. It is preferable to contain, for example, 1 ⁇ 10 7 cells or more in a single dose so as to achieve the desired therapeutic effect. It is more preferably 1 ⁇ 10 8 or more, still more preferably 1 ⁇ 10 9 or more.
  • the content of cells can be appropriately adjusted in consideration of the sex, age, weight, condition of the affected area, condition of cells, etc. of the subject.
  • the pharmaceutical composition of the present invention may contain dimethylsulfoxide (DMSO), serum albumin, and the like for the purpose of protecting cells.
  • DMSO dimethylsulfoxide
  • Antibiotics and the like may be added for the purpose of preventing bacterial contamination, and vitamins, cytokines, and the like may be included for the purpose of promoting activation and differentiation of cells.
  • other pharmaceutically acceptable components e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc. It may be included in the pharmaceutical composition of the present invention.
  • a pharmaceutical composition containing the T cells of the present invention as an active ingredient can be cryopreserved.
  • the temperature is not particularly limited as long as it is suitable for cell preservation. Examples include -20°C, -80°C and -120 to -196°C.
  • cells can be stored in suitable containers such as vials. Manipulations to minimize the risk of cell damage during freezing and thawing of T cells are well known to those of skill in the art.
  • T cells are collected from the culture medium, washed with buffer or culture medium, counted, concentrated by centrifugation, etc., and placed in a freezing medium (e.g., containing 10% DMSO). culture solution), and cryopreserved.
  • T cells can be made into a single lot by combining cells cultured in multiple culture vessels.
  • the pharmaceutical composition containing T cells of the present invention contains, for example, 5 ⁇ 10 4 to 9 ⁇ 10 10 T cells per container such as a vial. It can be changed according to the route or the like.
  • Examples of administration routes of the pharmaceutical composition containing the T cells of the present invention include infusion, intratumoral injection, arterial injection, portal vein injection, intraperitoneal administration, and the like.
  • the administration route is not limited to this as long as the T cells, which are the active ingredients in the pharmaceutical composition of the present invention, are delivered to the affected area.
  • Dosage schedules can be single doses or multiple doses. For the period of multiple administrations, for example, a method of repeating administration once every 2 to 4 weeks, a method of repeating administration once every six months to a year, or the like can be adopted.
  • the sex, age, body weight, disease state, etc. of the target patient can be taken into consideration.
  • cancer patients from whom T cells are collected or The non-cancer patient preferably has the same HLA type as the cancer patient to whom the pharmaceutical composition of the present invention is administered for cancer treatment. Moreover, it is more preferable that the cancer patient to whom the pharmaceutical composition of the present invention is administered and the cancer patient from whom T cells are collected are the same person. That is, cancer treatment by autologous transplantation is more preferable than allogeneic transplantation.
  • the pharmaceutical composition of the present invention is used for prevention or treatment of cancer.
  • Cancers include ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, and glioblastoma. including, but not limited to, blastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, colorectal cancer, and B-cell non-Hodgkin's lymphoma.
  • the timing of administration of the pharmaceutical composition and cancer vaccine of the present invention is not limited,
  • the pharmaceutical composition of the present invention and cancer vaccine may be administered to the subject at the same time or at different times.
  • the pharmaceutical composition of the present invention and cancer vaccine may be formulated separately, or may be a combination drug in which both are mixed.
  • the dosage of the pharmaceutical composition and cancer vaccine of the present invention may conform to the dosage used clinically, and can be appropriately selected depending on the disease, administration subject, administration route, combination with drugs, and the like.
  • the present invention provides a method for treating or preventing cancer, comprising administering T cells to a subject who has cancer or is at risk of having cancer, wherein the T cells have the following (I A detection step of detecting expression of at least one gene selected from the group consisting of genes from ) to (III); an evaluation step of evaluating the quality of T cells using the expression of the gene as an index; (I) A gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (II) In the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted or deleted , a gene encoding a polypeptide comprising an inserted and/or added amino acid sequence (III) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1. )I will provide a.
  • a subject in the present invention is a vertebrate.
  • the vertebrate is a mammal.
  • Such mammals include, but are not limited to, farm animals (eg, cows), sport animals, pet/companion animals (eg, cats, dogs, and horses), primates, mice, and rats.
  • the mammal is human.
  • the present invention provides a method for producing T cells, comprising a detection step of detecting the expression of at least one gene selected from the group consisting of the following genes (I) to (III) in the T cells; an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
  • a method for producing a T cell ((I) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (II) in the amino acid sequence set forth in SEQ ID NO: 1, wherein one or more amino acid residues are Gene (III) encoding a polypeptide comprising a substituted, deleted, inserted and/or added amino acid sequence
  • a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 encoding genes.).
  • High-proliferation T cells (1 type): T cell receptors (TCR) of naive T cells (CD45RA + , CCR7 + ) collected from human peripheral blood mononuclear cells were treated with Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (manufactured by Thermo Fisher Scientific) (hereinafter referred to as "TCR stimulation magnetic beads") was used for stimulation, medium A (15% (w/v) fetal bovine serum (FBS, BioWest), 5 ng/ The cells were cultured for 14 days under conditions of 37° C.
  • TCR stimulation magnetic beads T cell receptors of naive T cells collected from human peripheral blood mononuclear cells were treated with Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (manufactured by Thermo Fisher Scientific) (hereinafter referred to as "TCR stimulation magnetic beads”) was used for stimulation, medium A (15% (w/v) fetal
  • T cells with a proliferation rate of 81 times that before stimulation T cells with a proliferation rate of 81 times that before stimulation;
  • T cells whose proliferation rate is 20 times or less before stimulation T cells whose proliferation rate is 20 times or less before stimulation.
  • TCR of the high-proliferation T cells and low-proliferation T cells of (1) above is stimulated using the TCR-stimulating magnetic beads, and medium A is exchanged as appropriate under conditions of 5% CO 2 and 37°C. was cultured for 6 days.
  • AHA secreted into the culture supernatant was extracted using Click-iT TM Protein Enrichment Kit, for click chemistry capture of azide-modified protein (manufactured by Thermo Fisher Scientific).
  • the labeled protein (secretome) was extracted by chaptering the azide groups of AHA onto alkyne agarose resin using click chemistry.
  • Transcriptome analysis of high-proliferation T cells and low-proliferation T cells (1) The T cells stimulated and cultured in (2) of 1 above were collected, and total RNA was extracted using RNeasy Micro Kit (manufactured by QIAGEN). Extracted. cDNA was synthesized and amplified from 10 ng of total RNA using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (manufactured by Clontech).
  • Pathway analysis (1) Secretome analysis results obtained in 1 (5) above (quantitative values of identified proteins) and transcriptome analysis results obtained in 2 above (3) (expression level for each mapped gene ) were integrated, and the high-proliferation T cells and the low-proliferation T-cell group were compared, and the following conditions: secretome analysis results of unique peptide 2 or more, and transcriptome analysis results of expression ratio 0.67 Signals to which proteins encoded by genes whose expression levels differ significantly between high-proliferation T cells and low-proliferation T cells by KeyMolnet analysis using the correlation search method, narrowed down to less than or 1.5 or more Transduction pathways (signaling pathways with high scores calculated by the following formula; in the following formula, molecules belonging to pathways indicate proteins belonging to the signaling pathways). The score is a value indicating the overall degree of involvement calculated by the following formula based on a statistical method (hypergeometric distribution), and the higher the value, the more significant the signal transduction pathway.
  • Table 1 below shows the 1st to 10th signal transduction pathways and their scores (Scores) in descending order of scores obtained by pathway analysis. From the pathways shown in Table 1, the adenosine signaling pathway at position 5 and the P2Y signaling pathway at position 9 were selected and analyzed in Trends in Molecular Medicine, Vol. 19, 2013, p. 355-367, CD39 and CD73, which are proteins involved in these signaling pathways, were selected as candidate markers for quality evaluation of T cells.
  • T cells 22 types with different proliferative potentials, which is one of the evaluation items of T cell quality.
  • Primary T cells (9 types): T cells that have been stimulated with different culture conditions or stimulation times to na ⁇ ve CTL-derived T cells, and that have shown different proliferative abilities;
  • iPS cell-derived T cells 13 types: T cells derived from different iPS cells and having different proliferative potentials.
  • the TCR of the primary T cells or iPS cell-derived T cells of (1) above is stimulated using the TCR-stimulating magnetic beads, and the medium is replaced with medium A as appropriate under conditions of 5% CO 2 and 37°C. for 14 days.
  • the cultured T cells were collected, and transcriptome analysis was performed in the same manner as in 2 of ⁇ Selection of Marker Candidates for Evaluation of T Cells> above to determine the expression level of the gene encoding CD39 or CD73.
  • FIG. 1 shows a graph in which the proliferative capacity (fold expansion) of each T cell is plotted on the X axis and the expression level of the gene encoding CD39 or CD73 in the T cells (CD39 expression or CD73 expression) is plotted on the Y axis.
  • the proliferative ability of each T cell is the ratio of the number of cells before stimulation to the number of cells 14 days after stimulation (number of cells after 14 days/stimulation cell number).
  • CD39 can be used as a marker for T cell proliferative ability evaluation (for example, an exhaustion marker).
  • CD39 is represented by the amino acid sequence shown in SEQ ID NO:1.
  • T cell evaluation marker candidate 2> The expression times of CD39 selected in ⁇ Selection of T cell evaluation marker candidates> and PD-1, which is a known exhaustion marker, were compared.
  • iPS cell-derived T cells exhibiting naive traits were stimulated with the mitogen PHA (Phytohemagglutinin) and a peripheral blood mononuclear cell feeder, and medium A was exchanged as appropriate. , 37° C., 5% CO 2 for 14 days, and stimulation treatment was repeated 10 times.
  • the expression levels of CD39, CD45RA, CD45RO, and PD-1 on the cell surface after one stimulation treatment and after ten stimulation treatments were measured by flow cytometry.
  • the present invention it is possible to provide a T cell quality evaluation method capable of evaluating T cell quality using a new index, and a T cell quality evaluation reagent used for the method.

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Abstract

A T cell quality evaluation method that comprises a detection step for detecting the expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells, and an evaluation step for evaluating the qualities of the T cells with the use of the expression of the gene as an indicator. (i) A gene encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1. (ii) A gene encoding a polypeptide containing an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and/or added in the amino acid sequence represented by SEQ ID NO: 1. (iii) A gene encoding a polypeptide containing an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1.

Description

Ｔ細胞の品質評価方法、および当該方法に用いる試薬Method for evaluating the quality of T cells and reagents used in the method
　本発明は、Ｔ細胞の品質評価方法、それに用いるＴ細胞の品質評価用試薬、Ｔ細胞の評価方法により得られた高品質Ｔ細胞を含有する医薬組成物及び該医薬組成物を用いる、がんの予防または治療方法等に関する。 The present invention provides a method for evaluating the quality of T cells, a reagent for evaluating the quality of T cells used therein, a pharmaceutical composition containing high-quality T cells obtained by the method for evaluating T cells, and a cancer using the pharmaceutical composition. It relates to the prevention or treatment method of
　悪性腫瘍等のがんや慢性難治性感染症などの疾患に対する新しい治療方法として、当該疾患に関連する抗原特異的な細胞傷害性Ｔ細胞（ＣＴＬ：Ｃｙｔｏｔｏｘｉｃ　Ｔ　Ｌｙｍｐｈｏｃｙｔｅ）を用いた細胞免疫療法が知られている。近年では、がん治療に有効な手段として、患者からＴ細胞（未改変Ｔ細胞）を採取し、がん攻撃能力が高まるように遺伝子改変して遺伝子改変Ｔ細胞とした後、当該遺伝子改変Ｔ細胞を元の患者に戻してがんを治療するがん免疫療法が期待を集めている。 Cellular immunotherapy using antigen-specific cytotoxic T-lymphocytes (CTL) related to the disease is known as a new treatment method for diseases such as cancer such as malignant tumors and chronic intractable infectious diseases. It is In recent years, as an effective means for cancer therapy, T cells (unmodified T cells) are collected from patients, genetically modified to enhance cancer-attacking ability, and then genetically modified T cells are obtained. Expectations are high for cancer immunotherapy, which treats cancer by returning cells to the original patient.
　また、患者から採取したＴ細胞では、細胞疲弊が起こりやすかったり、増殖培養（又は拡大培養）が困難であるといった課題を有していることから、用いることができる細胞数に制限がないｉＰＳ細胞に由来するＴ細胞を利用した、がん免疫療法の開発も進められている。例えば、特許文献１には、ＣＤ４＋ＣＤ８＋Ｔ細胞を、インターロイキン７及びＴＣＲ（Ｔ細胞受容体）活性化剤を含む培地で培養し、ＣＤ８α＋β＋細胞傷害性Ｔ細胞を誘導する方法が記載されている。 In addition, T cells collected from patients have problems such as cell exhaustion and difficulty in proliferation culture (or expansion culture), so there is no limit to the number of iPS cells that can be used. Cancer immunotherapy is also being developed using T cells derived from For example, Patent Document 1 discloses a method of inducing CD8α + β + cytotoxic T cells by culturing CD4 + CD8 + T cells in a medium containing interleukin 7 and a TCR (T cell receptor) activator. is described.
　ｉＰＳ細胞に由来するＴ細胞では、ｉＰＳ細胞株や培養条件等によって品質が異なるため、得られたＴ細胞においては、その品質を評価して前記がん免疫療法に適したものを選択する必要がある。また、得られた遺伝子改変Ｔ細胞についても、その品質を評価して目的の遺伝子改変Ｔ細胞として適したものを選択する必要がある。このようなＴ細胞の品質評価項目としては、具体的に、例えば、Ｔ細胞の各段階で発現する分化マーカーの分析、ＴＣＲ刺激後に産生されるサイトカインの分析、増殖能、細胞傷害活性、動物実験でのがん細胞抑制活性等が挙げられる。Ｔ細胞の品質は、これらの項目によって総合的に評価されるが、かかる評価は、煩雑であったり長期間を要する場合が多い。さらに、評価にかかるコストも高額なことから、より簡便かつ短期間でＴ細胞の品質を評価できる方法が求められている。 Since the quality of iPS cell-derived T cells varies depending on the iPS cell line, culture conditions, etc., it is necessary to evaluate the quality of the obtained T cells and select those suitable for the cancer immunotherapy. be. In addition, it is also necessary to evaluate the quality of the obtained genetically modified T cells and select those suitable as the desired genetically modified T cells. Specific examples of such T cell quality evaluation items include, for example, analysis of differentiation markers expressed at each stage of T cells, analysis of cytokines produced after TCR stimulation, proliferation ability, cytotoxic activity, animal experiments and cancer cell-suppressing activity. The quality of T cells is comprehensively evaluated by these items, but such evaluations are often complicated and require a long period of time. Furthermore, since the cost of evaluation is high, there is a demand for a method that can evaluate the quality of T cells more simply and in a short period of time.
　このようなＴ細胞の品質評価方法としては、例えば、ＰＤ－１やｌａｇ３等の発現量とＴ細胞の疲弊化との間に関係があることが見出されているため（非特許文献１）、これらのタンパク質をＴ細胞の疲弊化マーカーとして、その発現量によってＴ細胞の疲弊化を評価する方法が挙げられる。 As a method for evaluating the quality of such T cells, for example, it has been found that there is a relationship between the expression level of PD-1, lag3, etc. and the exhaustion of T cells (Non-Patent Document 1). , using these proteins as T cell exhaustion markers, a method of evaluating T cell exhaustion based on the expression level thereof.
国際公開第２０１８／１３５６４６号WO2018/135646
　ｉｎ　ｖｉｔｒｏにおいて、上記の疲弊化マーカーは、長期間培養されてかなりＴ細胞の疲弊化が進んでから発現する。そのため、ｉＰＳ細胞から分化誘導した直後のＴ細胞等、比較的若いＴ細胞の品質を評価するためには不向きであることを本発明者らは見出した。従来、このように比較的若いＴ細胞の品質も評価することができるマーカーは特定されていない。 In vitro, the above-mentioned exhaustion markers are expressed after long-term culture and the exhaustion of T cells has progressed considerably. Therefore, the present inventors found that it is unsuitable for evaluating the quality of relatively young T cells such as T cells immediately after being induced to differentiate from iPS cells. So far, no markers have been identified that can also assess the quality of relatively young T cells in this way.
　本発明は、上記従来技術の有する課題に鑑みてなされたものであり、新たな指標によって比較的若いＴ細胞の品質も評価できるＴ細胞の品質評価方法、及びそれに用いるＴ細胞の品質評価用試薬を提供することを目的とする。 The present invention has been made in view of the above-mentioned problems of the prior art, and provides a T cell quality evaluation method capable of evaluating the quality of relatively young T cells using a new index, and a T cell quality evaluation reagent used therein. intended to provide
　本発明者らは上記課題を解決すべく鋭意検討を重ね、先ず、増殖能が高いＴ細胞（高増殖Ｔ細胞）及び増殖能が比較的低いＴ細胞（低増殖Ｔ細胞）についてセクレトーム（ｓｅｃｒｅｔｏｍｅ）解析及びトランスクリプトーム（ｔｒａｎｓｃｒｉｐｔｏｍｅ）解析を行なった。次いで、これらの解析結果から、高増殖Ｔ細胞と低増殖Ｔ細胞のとの間で発現量の差が大きい遺伝子及びタンパク質が関与するシグナル伝達経路を特定し、かかるシグナル伝達経路に関わるタンパク質（ＣＤ３９及びＣＤ７３）をＴ細胞の品質評価用のマーカー候補として選定した。次いで、選定したタンパク質について、Ｔ細胞の品質評価用マーカーとして利用可能か否かについて検証したところ、Ｔ細胞由来セクレトームの１種である特定のタンパク質（ＣＤ３９）の発現量とＴ細胞の増殖能や疲弊化との間に相関関係があることを見出した。そのため、かかるタンパク質を新たなマーカーとし、この発現量を指標とすることによって、比較的若いＴ細胞の品質も評価できることを見出し、本発明を完成させるに至った。 The present inventors have made intensive studies to solve the above problems. Analysis and transcriptome analysis were performed. Next, from these analysis results, we identified signal transduction pathways involving genes and proteins with large differences in expression levels between high-proliferation T cells and low-proliferation T cells, and identified proteins involved in such signal transduction pathways (CD39 and CD73) were selected as candidate markers for T cell quality assessment. Next, when the selected proteins were examined to see if they could be used as quality evaluation markers for T cells, the expression level of a specific protein (CD39), which is a type of T cell-derived secretome, and the proliferative ability of T cells, We found that there is a correlation between exhaustion. Therefore, the present inventors have found that the quality of relatively young T cells can also be evaluated by using such a protein as a new marker and using this expression level as an index, thus completing the present invention.
　すなわち、本発明の態様は以下のとおりである。 That is, the aspects of the present invention are as follows.
　［１］　Ｔ細胞における、下記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
を含む、Ｔ細胞の品質評価方法。
（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。 [1] a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells;
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
A method for assessing T cell quality, comprising:
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
　［２］　前記検出工程が、前記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現産物に特異的に結合するプローブ分子を用いて前記発現産物を検出する工程である、［１］に記載の方法。 [2] The detection step detects the expression product using a probe molecule that specifically binds to the expression product of at least one gene selected from the group consisting of the genes (i) to (iii) The method according to [1], which is a step.
　［３］　前記発現産物が、前記（ｉ）～（ｉｉｉ）の遺伝子のうちのいずれか１つにコードされるポリペプチドであり、かつ、前記プローブ分子が、前記ポリペプチドに特異的に結合する抗体及び／又はアプタマーである、［２］に記載の方法。 [3] The expression product is a polypeptide encoded by any one of the genes (i) to (iii), and the probe molecule specifically binds to the polypeptide The method of [2], which is an antibody and/or an aptamer.
　［４］　前記評価工程が、前記遺伝子の発現を指標としてＴ細胞の増殖能を評価する工程である、［１］～［３］のいずれか一項に記載の方法。 [4] The method according to any one of [1] to [3], wherein the evaluation step is a step of evaluating T cell proliferation ability using the expression of the gene as an index.
　［５］　［２］～［４］のいずれか一項に記載の方法に用いるための試薬であり、下記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現産物に特異的に結合するプローブ分子を含有する、Ｔ細胞の品質評価用試薬。
（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。 [5] A reagent for use in the method according to any one of [2] to [4], containing at least one gene selected from the group consisting of the following genes (i) to (iii) A T cell quality assessment reagent containing a probe molecule that specifically binds to an expression product.
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
　［６］　前記発現産物が、前記（ｉ）～（ｉｉｉ）の遺伝子のうちのいずれか１つにコードされるポリペプチドであり、かつ、前記プローブ分子が、前記ポリペプチドに特異的に結合する抗体及び／又はアプタマーである、［５］に記載の試薬。 [6] The expression product is a polypeptide encoded by any one of the genes (i) to (iii), and the probe molecule specifically binds to the polypeptide The reagent of [5], which is an antibody and/or an aptamer.
［７］［１］～［４］のいずれかに記載のＴ細胞の評価方法により得られた高品質Ｔ細胞。 [7] High-quality T cells obtained by the method for evaluating T cells according to any one of [1] to [4].
［８］［７］に記載の高品質Ｔ細胞を含有する医薬組成物。 [8] A pharmaceutical composition containing the high-quality T cells of [7].
［９］　がんの予防又は治療に使用するための［８］に記載の医薬組成物。 [9] The pharmaceutical composition according to [8] for use in preventing or treating cancer.
［１０］［８］に記載の医薬組成物を用いる、がんの予防または治療方法。 [10] A method for preventing or treating cancer using the pharmaceutical composition of [8].
［１１］がんを有するかまたはがんを有する危険性のある被験者に、［８］に記載の医薬組成物を投与することを含む、がんを治療または予防する方法。 [11] A method of treating or preventing cancer, comprising administering the pharmaceutical composition of [8] to a subject who has or is at risk of having cancer.
［１２］がんを有するかまたはがんを有する危険性のある被験者に、Ｔ細胞を投与することを含む、がんを治療または予防する方法であって、該Ｔ細胞における、下記（Ｉ）～（ＩＩＩ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
を含む、方法。
（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。 [12] A method for treating or preventing cancer, comprising administering T cells to a subject having cancer or at risk of having cancer, wherein A detection step of detecting the expression of at least one gene selected from the group consisting of the genes of ~ (III);
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
A method, including
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
［１３］Ｔ細胞の製造方法であって、該Ｔ細胞における、下記（Ｉ）～（ＩＩＩ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
を含む、Ｔ細胞の製造方法。
（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。
  [13] A method for producing T cells, comprising a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (I) to (III) in the T cells;
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
A method of producing a T cell, comprising:
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
　本発明によれば、新たな指標によってＴ細胞の品質を評価できるＴ細胞の品質評価方法、及びそれに用いるＴ細胞の品質評価用試薬を提供することが可能となる。 According to the present invention, it is possible to provide a T cell quality evaluation method capable of evaluating T cell quality using a new index, and a T cell quality evaluation reagent used for the method.
「Ｔ細胞評価用マーカー候補の検証１」における、各Ｔ細胞の増殖能（Ｘ軸）と、当該Ｔ細胞におけるＣＤ３９又はＣＤ７３をコードする遺伝子の発現量（Ｙ軸）との関係を示すグラフである。（ａ）はＹ軸がＣＤ３９をコードする遺伝子の発現量であるときのグラフであり、（ｂ）はＹ軸がＣＤ７３をコードする遺伝子の発現量であるときのグラフである。A graph showing the relationship between the proliferative ability of each T cell (X axis) and the expression level of the gene encoding CD39 or CD73 in the T cell (Y axis) in "Verification of T cell evaluation marker candidates 1" be. (a) is a graph in which the Y-axis is the expression level of the gene encoding CD39, and (b) is a graph in which the Y-axis is the expression level of the gene encoding CD73. 「Ｔ細胞評価用マーカー候補の検証２」における、刺激処理１回後及び刺激処理１０回後のＴ細胞におけるＣＤ４５ＲＡの発現量（Ｙ軸）と、当該Ｔ細胞におけるＣＤ４５ＲＯ、ＣＤ３９、又はＰＤ－１の発現量（Ｘ軸）との関係を示すグラフである。（ａ）はＸ軸がＣＤ４５ＲＯの発現量であるときのグラフであり、（ｂ）はＸ軸がＣＤ３９の発現量であるときのグラフであり、（ｃ）はＸ軸がＰＤ－１の発現量であるときのグラフである。In "Verification of Marker Candidates for T Cell Evaluation 2", the expression level of CD45RA in T cells after 1 stimulation treatment and 10 stimulation treatments (Y-axis), and CD45RO, CD39, or PD-1 in the T cells is a graph showing the relationship between the expression level (X-axis) of (a) is a graph when the X axis is the expression level of CD45RO, (b) is a graph when the X axis is the expression level of CD39, and (c) is a graph when the X axis is the expression level of PD-1 It is a graph when it is a quantity.
　以下、本発明をその好適な実施形態に即して詳細に説明する。 The present invention will be described in detail below in accordance with its preferred embodiments.
　＜Ｔ細胞の品質評価方法＞
　本発明のＴ細胞の品質評価方法は、
　Ｔ細胞における、下記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
を含む方法（以下、場合により、単に「本発明の方法」という）である。
（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。 <Method for evaluating the quality of T cells>
The method for evaluating the quality of T cells of the present invention comprises
a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells;
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
(hereinafter sometimes simply referred to as "the method of the present invention").
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
　［Ｔ細胞］ [T cells]
  本発明におけるＴ細胞とは、表面にＴ細胞受容体（Ｔ  ｃｅｌｌ  ｒｅｃｅｐｔｏｒ、ＴＣＲ）と称される抗原受容体を発現している細胞を意味し、アルファベータ(αβ)T細胞およびガンマデルタ(γδ)T細胞を含む。 The T cell in the present invention means a cell expressing an antigen receptor called T cell receptor (TCR) on the surface, alpha beta (αβ) T cells and gamma delta (γδ ) containing T cells.
  本発明におけるＴ細胞は、例えば、ヘルパーＴ細胞、細胞傷害性Ｔ細胞、制御性Ｔ細胞、ナチュラルキラーＴ細胞、ナイーブＴ細胞、メモリーＴ細胞（例えば、ステムセルメモリーＴ細胞（ＴＳＣＭ）、セントラルメモリーＴ細胞（ＴＣＭ））、エフェクターメモリーＴ細胞等）、抗原刺激されたメモリーＴ細胞、又はターミナルエフェクターＴ細胞、それらの組み合わせ、またはそれらの亜集団を含むが、これらに限定されることはない。 T cells in the present invention include, for example, helper T cells, cytotoxic T cells, regulatory T cells, natural killer T cells, naive T cells, memory T cells (e.g., stem cell memory T cells (TSCM), central memory T cells). cells (TCM), effector memory T cells, etc.), antigen-stimulated memory T cells, or terminal effector T cells, combinations thereof, or subpopulations thereof.
  ヘルパーＴ細胞はＣＤ４陽性細胞であり、更に発現するサイトカインによりＴｈ１細胞、Ｔｈ２細胞及びＴｈ１７細胞等に分類される（例えば、J Allergy Clin. Immunol., 135(3): 626-635,2012）。 Helper T cells are CD4-positive cells, and are further classified into Th1 cells, Th2 cells, Th17 cells, etc. according to the cytokines they express (e.g., J Allergy Clin. Immunol., 135(3): 626-635, 2012).
  Ｔｈ１細胞としては、例えば、ＩＦＮ－γ、ＩＬ－２又はＴＮＦ－α等を発現する細胞等が挙げられる。Ｔｈ２細胞としては、例えば、ＩＬ－４、ＩＬ－５、ＩＬ－６、ＩＬ－１０又はＩＬ－１３等を発現する細胞等が挙げられる。Ｔｈ１７細胞としては、例えば、ＩＬ－１７又はＩＬ－６等を発現する細胞等が挙げられる。 Th1 cells include, for example, cells expressing IFN-γ, IL-2, TNF-α, and the like. Th2 cells include, for example, cells expressing IL-4, IL-5, IL-6, IL-10, IL-13, and the like. Th17 cells include, for example, cells expressing IL-17 or IL-6.
  細胞傷害性Ｔ細胞は、ＣＤ８陽性細胞であり、ヘルパーＴ細胞同様に、更に発現するサイトカインによりＴｃ１細胞及びＴｃ２細胞等に分類される。   Cytotoxic T cells are CD8-positive cells, and like helper T cells, are further classified into Tc1 cells, Tc2 cells, etc. according to the cytokines they express.
  Ｔｃ１細胞としては、例えば、ＩＦＮ－γ、ＩＬ－２又はＴＮＦ－α等を発現する細胞等が挙げられる。Ｔｃ２細胞としては、例えば、ＩＬ－４、ＩＬ－５、ＩＬ－６、ＩＬ－１０、ＩＬ－１３等を発現する細胞等が挙げられる。 Examples of Tc1 cells include cells expressing IFN-γ, IL-2, TNF-α, and the like. Examples of Tc2 cells include cells that express IL-4, IL-5, IL-6, IL-10, IL-13, and the like.
  制御性Ｔ細胞としては、例えば、ＣＤ４（＋）ＣＤ２５（＋）ＦｏｘＰ３（＋）細胞が好ましく挙げられる。 Regulatory T cells preferably include, for example, CD4(+) CD25(+) FoxP3(+) cells.
  ナイーブＴ細胞としては、例えば、ＣＤ４（＋）ＣＤ４５ＲＡ（＋）ＣＤ６２Ｌ（＋）ＣＣＲ７（＋）細胞、ＣＤ８（＋）ＣＤ４５ＲＡ（＋）ＣＤ６２Ｌ（＋）ＣＣＲ７（＋）細胞、ＣＤ４（＋）ＣＣＲ７（＋）ＣＤ４５ＲＡ（＋）ＣＤ９５（－）ＣＤ４５ＲＯ（－）細胞又はＣＤ８（＋）ＣＣＲ７（＋）ＣＤ４５ＲＡ（＋）ＣＤ９５（－）ＣＤ４５ＲＯ（－）細胞等が好ましく挙げられる。 Naive T cells include, for example, CD4 (+) CD45RA (+) CD62L (+) CCR7 (+) cells, CD8 (+) CD45RA (+) CD62L (+) CCR7 (+) cells, CD4 (+) CCR7 ( +)CD45RA(+)CD95(-)CD45RO(-) cells or CD8(+)CCR7(+)CD45RA(+)CD95(-)CD45RO(-) cells are preferred.
ステムセルメモリーＴ細胞としては、例えば、ＣＤ４（＋）ＣＤ４５ＲＡ（＋）ＣＤ６２Ｌ（＋）ＣＣＲ７（＋）ＣＤ９５（＋）細胞、ＣＤ８（＋）ＣＤ４５ＲＡ（＋）ＣＤ６２Ｌ（＋）ＣＣＲ７（＋）ＣＤ９５（＋）細胞、ＣＤ４（＋）ＣＣＲ７（＋）ＣＤ４５ＲＡ（＋）ＣＤ９５（＋）ＣＤ４５ＲＯ（＋）細胞又はＣＤ８（＋）ＣＣＲ７（＋）ＣＤ４５ＲＡ（＋）ＣＤ９５（＋）ＣＤ４５ＲＯ（＋）細胞等が好ましく挙げられる。 Stem cell memory T cells include, for example, CD4 (+) CD45RA (+) CD62L (+) CCR7 (+) CD95 (+) cells, CD8 (+) CD45RA (+) CD62L (+) CCR7 (+) CD95 (+) ) cells, CD4(+)CCR7(+)CD45RA(+)CD95(+)CD45RO(+) cells or CD8(+)CCR7(+)CD45RA(+)CD95(+)CD45RO(+) cells are preferred. be done.
  セントラルメモリーＴ細胞としては、例えば、ＣＤ４（＋）ＣＤ４５ＲＡ（－）ＣＤ６２Ｌ（＋）ＣＣＲ７（＋）ＣＤ９５（＋）細胞、ＣＤ８（＋）ＣＤ４５ＲＡ（－）ＣＤ６２Ｌ（＋）ＣＣＲ７（＋）ＣＤ９５（＋）細胞、ＣＤ４（＋）ＣＣＲ７（＋）ＣＤ４５ＲＡ（－）ＣＤ４５ＲＯ（＋）細胞又はＣＤ８（＋）ＣＣＲ７（＋）ＣＤ４５ＲＡ（－）ＣＤ４５ＲＯ（＋）細胞等が好ましく挙げられる。 Central memory T cells include, for example, CD4 (+) CD45RA (-) CD62L (+) CCR7 (+) CD95 (+) cells, CD8 (+) CD45RA (-) CD62L (+) CCR7 (+) CD95 (+) ) cells, CD4(+)CCR7(+)CD45RA(-)CD45RO(+) cells, CD8(+)CCR7(+)CD45RA(-)CD45RO(+) cells, and the like are preferable.
  エフェクターメモリーＴ細胞としては、例えば、ＣＤ４（＋）ＣＣＲ７（－）ＣＤ４５ＲＡ（－）ＣＤ４５ＲＯ（＋）細胞又はＣＤ８（＋）ＣＣＲ７（－）ＣＤ４５ＲＡ（－）ＣＤ４５ＲＯ（＋）細胞等が好ましく挙げられる。 Examples of effector memory T cells include preferably CD4(+) CCR7(-) CD45RA(-) CD45RO(+) cells or CD8(+) CCR7(-) CD45RA(-) CD45RO(+) cells.
  ターミナルエフェクターＴ細胞としては、例えば、ＣＤ４（＋）ＣＤ４５ＲＡ（＋）ＣＤ６２Ｌ（－）細胞又はＣＤ８（＋）ＣＤ４５ＲＡ（＋）ＣＤ６２Ｌ（－）細胞等が好ましく挙げられる。 Terminal effector T cells preferably include, for example, CD4(+)CD45RA(+)CD62L(-) cells or CD8(+)CD45RA(+)CD62L(-) cells.
　本発明において、Ｔ細胞の由来としては特に制限はなく、動物由来であっても、人由来であってもよく、健常者由来のＴ細胞であっても、患者（例えば、免疫機能が低下している人又は動物や、悪性腫瘍、感染症又は自己免疫疾患を患っている人又は動物）由来のＴ細胞であってもよい。さらには、人工多能性幹細胞（ｉＰＳ細胞）、胚性幹細胞（ＥＳ細胞）等の多能性幹細胞から分化誘導したＴ細胞；造血幹細胞等の体性幹細胞から分化誘導したＴ細胞；株化したＴ細胞であってもよい。 In the present invention, the origin of T cells is not particularly limited, and may be animal-derived or human-derived. or from a person or animal suffering from a malignant tumor, an infectious disease, or an autoimmune disease). Furthermore, T cells differentiated from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); T cells differentiated from somatic stem cells such as hematopoietic stem cells; It may be a T cell.
　人から採取されたＴ細胞の場合には、例えば、末梢血から分離された末梢血単核細胞（ＰＢＭＣ：Ｐｅｒｉｐｈｅｒａｌ　Ｂｌｏｏｄ　Ｍｏｎｏｎｕｃｌｅａｒ　Ｃｅｌｌｓ）より、従来公知の方法や市販のキット等を用いてＴ細胞を単離することができる。 In the case of T cells collected from humans, for example, T cells are isolated from peripheral blood mononuclear cells (PBMC: Peripheral Blood Monoclear Cells) isolated from peripheral blood using conventionally known methods or commercially available kits. It can be isolated.
　また、本発明において、「Ｔ細胞」には、人為的な遺伝子改変がなされていないＴ細胞（以下、場合により「未改変Ｔ細胞」という）の他、前記未改変Ｔ細胞に人為的な遺伝子改変がなされた「遺伝子改変Ｔ細胞」も含む。なお、本発明において、「人為的な遺伝子改変」には、Ｔ細胞への遺伝子導入又はＴ細胞の遺伝子編集によりＴ細胞の機能を改変することを含む。前記人為的な遺伝子改変としては、Ｔ細胞が有する遺伝子そのものの改変であっても、Ｔ細胞が有する遺伝子そのものの欠損であっても、外来の遺伝子が導入された改変であっても、これらの２種以上を組み合わせたものであってもよい。 In addition, in the present invention, "T cells" include T cells that have not been artificially genetically modified (hereinafter sometimes referred to as "unmodified T cells"), as well as artificial genes in the unmodified T cells. Also included are "genetically modified T cells" that have been modified. In the present invention, "artificial genetic modification" includes modification of T cell functions by gene transfer to T cells or gene editing of T cells. The artificial genetic modification may be modification of the gene itself possessed by T cells, deletion of the gene itself possessed by T cells, or modification resulting from the introduction of a foreign gene. A combination of two or more types may also be used.
　前記人為的な遺伝子改変の方法としては、従来公知の方法やそれに準じた方法が挙げられ、特に限定されない。Ｔ細胞への遺伝子導入としては、例えば、Ｔ細胞の機能を改変させる遺伝子そのもの（ＤＮＡ、ｍＲＮＡ、ｍｉＲＮＡ、アンタゴｍｉｒ、ＯＤＮ（オリゴデオキシリボヌクレオチド）等）、又は当該遺伝子を挿入したベクター（レンチウイルスベクター、γ－又はα－レトロウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、ヘルペスウイルスベクター、ウマエンセファロパシーウイルスベクター等のウイルスベクター、トランスポゾンベクターであるpｉｇｇｙＢａｃ（登録商標）の非ウイルスベクター）のＴ細胞への導入が挙げられ、Ｔ細胞の遺伝子編集としては、例えば、部位特異的ヌクレアーゼ（メガヌクレアーゼ、ジンクフィンガーヌクレアーゼ、ＴＡＬＥＮ、ＰＰＲ、ＣＲＩＳＰＲ－Ｃａｓ等）を用いたＴ細胞の遺伝子の編集（ゲノム編集）が挙げられる。本発明において、前記人為的な遺伝子改変の方法としては、これらの１種単独であっても２種以上を組み合わせたものであってもよい。 The method of artificial genetic modification includes conventionally known methods and methods based thereon, and is not particularly limited. Gene introduction into T cells includes, for example, the gene itself (DNA, mRNA, miRNA, antagomir, ODN (oligodeoxyribonucleotide), etc.) that modifies the function of T cells, or a vector into which the gene is inserted (lentiviral vector , γ- or α-retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, equine encephalopathic viral vectors, non-viral vectors such as the transposon vector piggyBac®). Gene editing of T cells includes, for example, gene editing of T cells using site-specific nucleases (meganuclease, zinc finger nuclease, TALEN, PPR, CRISPR-Cas, etc.) (genome Edit). In the present invention, the artificial genetic modification method may be one of these alone or a combination of two or more.
　前記Ｔ細胞の機能を改変させる遺伝子としては、例えば、Ｔ細胞より分泌されるタンパク質；Ｔ細胞表面に発現するタンパク質及び少なくとも１つの細胞内シグナリングドメインを含む融合タンパク質；Ｔ細胞表面に発現するタンパク質、少なくとも１つの共刺激ドメイン、及び少なくとも１つの細胞内シグナリングドメインを含む融合タンパク質が挙げられ、より具体的には、細胞表面酵素、細胞接着因子、受容体（例えば、キメラ抗原受容体）、及びこれらを構成するサブユニットが挙げられる。 Examples of the gene that modifies the function of T cells include proteins secreted from T cells; fusion proteins containing proteins expressed on the surface of T cells and at least one intracellular signaling domain; proteins expressed on the surface of T cells; Fusion proteins comprising at least one co-stimulatory domain and at least one intracellular signaling domain include, more specifically, cell surface enzymes, cell adhesion factors, receptors (e.g., chimeric antigen receptors), and these and subunits that make up the
　［検出工程］
　（標的遺伝子）
　本発明の方法に係る検出工程においては、Ｔ細胞における、下記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する。
（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。 [Detection process]
(target gene)
In the detection step according to the method of the present invention, expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells is detected.
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
　前記（ｉ）～（ｉｉｉ）の遺伝子のうちのいずれか１つにコードされるポリペプチドは、Ｔ細胞由来のセクレトームに含まれることが好ましい。本発明において、「セクレトーム」とは、細胞から分泌される又は細胞膜の中から細胞外に露出されるタンパク質を示し、細胞に内在する分泌タンパク質も含む。 The polypeptide encoded by any one of the genes (i) to (iii) is preferably contained in the T cell-derived secretome. In the present invention, the term "secretome" refers to a protein secreted from a cell or exposed outside the cell through the cell membrane, and includes a secretory protein endogenous to the cell.
　本発明において、発現が検出される遺伝子（以下、場合により「標的遺伝子」という）は、ヒト由来のものであれば、典型的には、「（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子」である。配列番号１に記載のアミノ酸配列は、Ｔ細胞由来のセクレトームである、ＣＤ３９である（ＵｎｉＰｒｏｔ　Ｎｏ．：Ｐ４９９６１）。ＣＤ３９は、ＳＰＧ６４としても知られており、また、ＥＮＴＰＤ１（ＥｃｔｏＮｕｃｌｅｏｓｉｄｅ　ＴｒｉｐｈｏｓＰｈａｔｅ　Ｄｉｐｈｏｓｐｈｏｈｙｄｒｏｌａｓｅ　１）活性、ＡＴＰａｓｅ活性、ＡＤＰａｓｅ活性、及びＮＴＰＤａｓｅ－１活性を有することも知られている。 In the present invention, the gene whose expression is detected (hereinafter sometimes referred to as "target gene") is human-derived and typically contains "(i) the amino acid sequence set forth in SEQ ID NO: 1 A gene encoding a polypeptide". The amino acid sequence set forth in SEQ ID NO: 1 is CD39, a secretome derived from T cells (UniProt No.: P49961). CD39, also known as SPG64, is also known to have ENTPD1 (EctoNucleoside TriphosPhate Diphosphohydrolase 1) activity, ATPase activity, ADPase activity, and NTPDase-1 activity.
　本発明においては、上記の「（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子」の相同遺伝子（例えば、ヒト以外の生物におけるカウンターパート遺伝子）を前記標的遺伝子とすることもできる。また、遺伝子のヌクレオチド配列は、その変異などにより、自然界において（すなわち、非人工的に）変異し得ることから、本発明においては、このような天然の変異体も、前記標的遺伝子とすることができる。さらに、現在の技術水準においては、当業者であれば、例えば、上記（ｉ）の遺伝子情報が得られた場合、そのヌクレオチド配列を改変し、そのコードするアミノ酸配列とは異なるが、機能改変したタンパク質を調製することもできるため、このような改変体も、前記標的遺伝子としてよい。 In the present invention, the target gene is a homologous gene (e.g., a counterpart gene in an organism other than human) of "(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1". can also In addition, since the nucleotide sequence of a gene can be mutated in nature (that is, non-artificially) due to mutation thereof, etc., such natural mutants can also be used as the target gene in the present invention. can. Furthermore, in the current state of the art, for example, when the genetic information of (i) above is obtained, a person skilled in the art can modify the nucleotide sequence and modify the function, although it is different from the amino acid sequence that codes for it. Since proteins can also be prepared, such variants may also be used as the target gene.
　したがって、本発明に係る標的遺伝子の態様には、「（ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が、置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子」も含まれる。ここで、「アミノ酸残基が、置換、欠失、挿入、及び／又は付加されたアミノ酸配列」とは、アミノ酸残基が、置換、欠失、挿入、若しくは付加されたアミノ酸配列、又は、これらの２種以上の組み合わせがなされたアミノ酸配列であることを示す。また、「複数個」とは、例えば、２０、１９、１８、１７、１６、１５、１４、１３、１２、１１、１０、９、８、７、６、５、４、３、又は２個の整数であることが好ましいが、これに制限されない。１若しくは複数個のアミノ酸残基とは、好ましくはアミノ酸残基が１個以上１０個以下、より好ましくは１個以上５個以下、さらに好ましくは１個以上３個以下、特に好ましくは２個以下である。 Therefore, in the aspect of the target gene according to the present invention, "(ii) one or more amino acid residues are substituted, deleted, inserted, and/or added in the amino acid sequence set forth in SEQ ID NO: 1 A gene that encodes a polypeptide comprising an amino acid sequence is also included. Here, the term "amino acid sequence in which amino acid residues are substituted, deleted, inserted, and/or added" means an amino acid sequence in which amino acid residues are substituted, deleted, inserted, or added, or It shows that the amino acid sequence is a combination of two or more of Further, "plurality" means, for example, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 is preferably an integer of, but not limited to. One or more amino acid residues is preferably 1 to 10 amino acid residues, more preferably 1 to 5, still more preferably 1 to 3, particularly preferably 2 or less. is.
　さらに、現在の技術水準においては、当業者であれば、例えば、上記（ｉ）の遺伝子情報が得られた場合、そのヌクレオチド配列に基づいて、ハイブリダイゼーション技術（Ｓｏｕｔｈｅｒｎ，Ｅ．Ｍ．，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，９８：５０３－５１７，１９７５）や、ポリメラーゼ連鎖反応（ＰＣＲ）技術（Ｓａｉｋｉ，Ｒ．Ｋ．，ｅｔ　ａｌ．Ｓｃｉｅｎｃｅ，２３０：１３５０－１３５４，１９８５、Ｓａｉｋｉ，Ｒ．Ｋ．ｅｔ　ａｌ．Ｓｃｉｅｎｃｅ，２３９：４８７－４９１，１９８８）等により、他の生物や細胞から相同遺伝子を取得することが可能である。 Furthermore, in the current state of the art, for example, when the genetic information of (i) above is obtained, a person skilled in the art can perform hybridization techniques (Southern, EM, J. Mol. Mol. Biol., 98: 503-517, 1975) and polymerase chain reaction (PCR) technology (Saiki, RK, et al. Science, 230: 1350-1354, 1985, Saiki, RK et al. al.
　したがって、本発明に係る標的遺伝子の態様には、「（ｉｖ）配列番号１に記載のアミノ酸配列をコードするヌクレオチド配列の相補鎖とストリンジェントな条件下でハイブリダイズするヌクレオチド配列」も含まれる。相同遺伝子を単離するためには、通常、ストリンジェントな条件下でハイブリダイゼーション反応を行う。「ストリンジェントな条件」とは、ハイブリダイゼーション後のメンブレンの洗浄操作を、高温下、低塩濃度溶液中で行うことを示し、かかる条件としては、例えば、２×ＳＳＣ濃度（１×ＳＳＣ：１５ｍＭクエン酸３ナトリウム、１５０ｍＭ塩化ナトリウム）、０．５％　ＳＤＳ溶液中で６０℃、２０分間の洗浄条件が挙げられる。また、ハイブリダイゼーションは、例えば、公知であるＥＣＬダイレクトＤＮＡ／ＲＮＡラベリング・検出システム（ＧＥヘルスケア　バイオサイエンス株式会社製）に添付の使用説明書に記載の方法にしたがって行うことができる。ハイブリダイゼーションの条件が厳しくなるほど、高い相同性を有するＤＮＡの単離を期待し得る。ただし、上記の条件は例示であり、ＤＮＡの濃度、ＤＮＡの長さ、ハイブリダイゼーションの反応時間等を適宜組み合わせることにより、必要な厳密性（ストリンジェンシー）を実現することが可能である。 Therefore, aspects of the target gene according to the present invention also include "(iv) a nucleotide sequence that hybridizes under stringent conditions with the complementary strand of the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1". Hybridization reactions are typically performed under stringent conditions to isolate homologous genes. The term "stringent conditions" means that the washing operation of the membrane after hybridization is performed at a high temperature in a low-salt solution. trisodium citrate, 150 mM sodium chloride) and 0.5% SDS solution at 60°C for 20 minutes. Also, hybridization can be performed, for example, according to the method described in the instruction manual attached to the known ECL direct DNA/RNA labeling/detection system (manufactured by GE Healthcare Bioscience). The more stringent the hybridization conditions, the more highly homologous DNA can be expected to be isolated. However, the above conditions are only examples, and the necessary stringency can be achieved by appropriately combining DNA concentration, DNA length, hybridization reaction time, and the like.
　さらに、例えばこのような方法にて取得された相同遺伝子がコードするタンパク質は、通常、配列番号１に記載のアミノ酸配列と高い同一性を有する。したがって、本発明に係る標的遺伝子の態様には、「（ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子」も含まれる。アミノ酸配列の同一性は、例えば、ＢＬＡＳＴＰのプログラム（Ａｌｔｓｃｈｕｌら，Ｊ．Ｍｏｌ．Ｂｉｏｌ．，２１５，１９９０年、ｐ．４０３－４１０）を用いて決定することができる。また、配列番号１に記載のアミノ酸配列との同一性は、典型的には、７０％以上であることが好ましく、より好ましくは、７５％以上、８０％以上、８５％以上、９０％以上、９５％以上（例えば、９６％以上、９７％以上、９８％以上、９９％以上）である。 Furthermore, for example, a protein encoded by a homologous gene obtained by such a method usually has a high degree of identity with the amino acid sequence set forth in SEQ ID NO:1. Therefore, the target gene aspect of the present invention also includes "(iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO: 1". Amino acid sequence identity can be determined, for example, using the BLASTP program (Altschul et al., J. Mol. Biol., 215, 1990, p. 403-410). In addition, the identity with the amino acid sequence set forth in SEQ ID NO: 1 is typically preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (eg, 96% or more, 97% or more, 98% or more, 99% or more).
　（遺伝子の発現の検出）
　本発明において、「遺伝子の発現を検出する」とは、遺伝子の発現の有無の検出及び発現の程度の検出の双方を含む意であり、本発明においては、前記標的遺伝子の発現を検出する。遺伝子の発現量は、絶対量として又は相対量として把握することができる。相対量を把握する場合には、例えば、用意した標準試料の遺伝子の発現量と比較して判断することができる。「標準試料」とは、標的遺伝子を発現しているか否か、又は発現している場合にはその量が事前に特定されている試料を示す。例えば、品質が良い又は悪いことが事前に特定されているＴ細胞を前記標準試料とすることができる。 (Detection of gene expression)
In the present invention, "detecting gene expression" includes both detection of the presence or absence of gene expression and detection of the degree of expression. In the present invention, the expression of the target gene is detected. The gene expression level can be grasped as an absolute amount or as a relative amount. When grasping the relative amount, for example, it can be judged by comparing with the gene expression level of a prepared standard sample. A "standard sample" refers to a sample for which it has been specified in advance whether or not the target gene is expressed, and if so, the amount of expression. For example, T cells that have been previously identified as having good or bad quality can be used as the standard sample.
　本発明において、「遺伝子の発現」とは、遺伝子の転写及び翻訳の双方を含む意である。したがって、本発明における「遺伝子の発現の検出」には、転写レベル（ｍＲＮＡレベル）での検出、及び翻訳レベル（タンパク質レベル）での検出（すなわち、前記遺伝子にコードされるポリペプチドの検出）の双方が含まれる。 In the present invention, "expression of a gene" means both transcription and translation of a gene. Therefore, "detection of gene expression" in the present invention includes detection at the transcription level (mRNA level) and detection at the translation level (protein level) (that is, detection of the polypeptide encoded by the gene). both are included.
　また、真核細胞では、遺伝子の転写過程で、ｍＲＮＡ前駆体中のイントロンを除去し、前後のエキソンを再結合する反応（スプライシング）が生じるが、エキソンの再結合に多様性が生じる場合があり、これにより様々な成熟ｍＲＮＡが生産される。ひいては、それにより様々なタンパク質が翻訳される。このようなスプライシング（ｓｐｌｉｃｉｎｇ）の違いにより生じる多様なｍＲＮＡやタンパク質を「スプライシングバリアント（ｓｐｌｉｃｉｎｇ　ｖａｒｉａｎｔ）」という。したがって、本発明における遺伝子の発現の検出には、下記のプローブ分子に特異的に認識される限り、当該スプライシングバリアントの検出も含まれる。 In eukaryotic cells, in the process of gene transcription, a reaction (splicing) occurs in which introns in the pre-mRNA are removed and the exons before and after are recombined (splicing). , which produces a variety of mature mRNAs. In turn, various proteins are translated thereby. Various mRNAs and proteins resulting from such splicing differences are called "splicing variants". Therefore, detection of gene expression in the present invention includes detection of the splicing variant as long as it is specifically recognized by the following probe molecule.
　本発明における遺伝子の発現の検出には、適宜公知の手法又はそれに準じた手法を用いることができる。中でも、本発明の方法に係る検出工程としては、前記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子（すなわち、前記標的遺伝子）の発現産物に特異的に結合するプローブ分子を用いて前記発現産物を検出することが好ましい。 For the detection of gene expression in the present invention, a known technique or a technique based thereon can be used as appropriate. Among them, the detection step according to the method of the present invention includes binding specifically to the expression product of at least one gene selected from the group consisting of the genes (i) to (iii) (that is, the target gene). Preferably, the expression product is detected using a probe molecule that
　〔転写レベルでの検出（標的ポリヌクレオチドの検出）〕
　転写レベルでの検出において、前記標的遺伝子の発現産物は、当該標的遺伝子から転写されたｍＲＮＡ（以下、場合により「標的ポリヌクレオチド」という）である。前記標的ポリヌクレオチドには、前記ｍＲＮＡを鋳型とするｃＤＮＡも含む。この場合の検出方法としては、例えば、前記プローブ分子として、前記標的ポリヌクレオチドの塩基配列中の適切な位置にハイブリダイズするように設計したオリゴヌクレオチドプローブを用い、ノーザンブロッティング、ドットブロット、ＲＮａｓｅプロテクションアッセイ、ＤＮＡマイクロアレイ解析法、ｉｎ　ｓｉｔｕハイブリダイゼーション法等によって、前記標的ポリヌクレオチドを検出する方法が挙げられる。 [Detection at transcription level (detection of target polynucleotide)]
In detection at the transcriptional level, the expression product of the target gene is mRNA transcribed from the target gene (hereinafter sometimes referred to as "target polynucleotide"). The target polynucleotide also includes cDNA using the mRNA as a template. Detection methods in this case include, for example, Northern blotting, dot blotting, and RNase protection assay using oligonucleotide probes designed to hybridize to appropriate positions in the base sequence of the target polynucleotide as the probe molecule. , DNA microarray analysis, in situ hybridization, and the like to detect the target polynucleotide.
　この場合には、例えば、前記オリゴヌクレオチドプローブとして、標識物質によって標識されたものを用い、当該標識物質に応じたシグナルを検出して、検出されたシグナル量を前記標的ポリヌクレオチドの量（すなわち、標的遺伝子の発現量）とすることができる。このときの標識物質としては、例えば、ＦＩＴＣ（フルオレセインイソチオシアネート）、ＦＡＭ（フルオレセインアミダイト）、ＤＥＡＣ（７－（ジエチルアミノ）クマリン）、Ｒ６Ｇ（ローダミン６Ｇ）、ＴｅｘＲｅｄ、Ｃｙ５、ＢＯＤＩＰＹ　ＦＬ等の蛍光物質；β－Ｄ－グルコシダ―ゼ、ルシフェラーゼ、ＨＲＰ（ホースラディッシュペルオキシダーゼ）等の酵素；３Ｈ、１４Ｃ、３２Ｐ、３５Ｓ、１２３Ｉ等の放射性同位体；ビオチン、ストレプトアビジン等の親和性物質；ルミノール、ルシフェリン、ルシゲニン等の発光物質が挙げられる。 In this case, for example, as the oligonucleotide probe, one labeled with a labeling substance is used, a signal corresponding to the labeling substance is detected, and the detected signal amount is the amount of the target polynucleotide (i.e., expression level of the target gene). Examples of labeling substances at this time include fluorescent substances such as FITC (fluorescein isothiocyanate), FAM (fluorescein amidite), DEAC (7-(diethylamino)coumarin), R6G (rhodamine 6G), TexRed, Cy5, and BODIPY FL; enzymes such as β-D-glucosidase, luciferase, HRP (horseradish peroxidase); radioactive isotopes such as 3 H, 14 C, 32 P, 35 S, 123 I; affinity substances such as biotin and streptavidin; Luminescent substances such as luminol, luciferin, and lucigenin are included.
　なお、本発明において、「シグナル」には、呈色（発色）、反射光、発光、消光、蛍光、放射性同位体による放射線等が含まれ、肉眼で確認できるものの他、シグナルの種類に応じた測定方法・装置によって確認できるものも含まれる。 In the present invention, the "signal" includes coloration (coloration), reflected light, luminescence, quenching, fluorescence, radiation from a radioactive isotope, and the like, and in addition to those that can be confirmed with the naked eye, depending on the type of signal It also includes those that can be confirmed by measuring methods and equipment.
　また、例えば、前記プローブ分子として、前記標的ポリヌクレオチド中の適切な位置を挟み込むように設計したオリゴヌクレオチドプライマーを用い、ＰＣＲ法、ＲＴ－ＰＣＲ法、ＴＲＣ（Ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｒｅｖｅｒｓｅ　ｔｒａｎｓｃｒｉｐｔｉｏｎ　Ｃｏｎｃｅｒｔｅｄ）法、ＮＡＳＢＡ（Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｓｅｑｕｅｎｃｅ－Ｂａｓｅｄ　Ａｍｐｌｉｆｉｃａｔｉｏｎ）法、ＴＭＡ（Ｔｒａｎｓｃｒｉｐｔｉｏｎ－Ｍｅｄｉａｔｅｄ　Ａｍｐｌｉｆｉｃａｔｉｏｎ）法等によって、前記標的ポリヌクレオチドを増幅して検出する方法も挙げられる。 Further, for example, as the probe molecule, oligonucleotide primers designed to sandwich appropriate positions in the target polynucleotide are used, PCR method, RT-PCR method, TRC (Transcription Reverse Transcription Concerned) method, NASBA (Nucleic Acid Sequence-Based Amplification) method, TMA (Transcription-Mediated Amplification) method, or the like may also be used to amplify and detect the target polynucleotide.
　この場合には、例えば、得られた増幅産物に蛍光色素（例えば、エチジウムブロマイド、ＳＹＢＲ　Ｇｒｅｅｎ（商品名）、ＳＹＴＯ６３（商品名）等）をインターカレートして得られたシグナル（蛍光）を検出して、検出されたシグナル量（蛍光強度）を、前記標的ポリヌクレオチドの量（すなわち、標的遺伝子の発現量）とすることができる。また、前記オリゴヌクレオチドプローブと組み合わせて検出してもよい（ダブルダイプローブ法等）。さらに、前記標的ポリヌクレオチドを含む試料を直接シーケンサーに供して解析することにより、前記標的ポリヌクレオチドの量としてもよい。 In this case, for example, a signal (fluorescence) obtained by intercalating the obtained amplification product with a fluorescent dye (e.g., ethidium bromide, SYBR Green (trade name), SYTO63 (trade name), etc.) is detected. Then, the detected signal amount (fluorescence intensity) can be used as the amount of the target polynucleotide (that is, the expression level of the target gene). Moreover, detection may be performed in combination with the oligonucleotide probe (double dye probe method, etc.). Furthermore, the amount of the target polynucleotide may be determined by directly subjecting a sample containing the target polynucleotide to a sequencer for analysis.
　このようなオリゴヌクレオチドプローブ及びオリゴヌクレオチドプライマーは、当業者であれば、前記標的ポリヌクレオチドの配列に基づいて、公知の方法又はそれに準じた方法で設計することができる。また、前記オリゴヌクレオチドプローブ及びオリゴヌクレオチドプライマーの長さは、それぞれ独立に、少なくとも１５塩基であることが好ましく、通常は、１５～１００塩基であり、好ましくは１７～３０塩基であり、より好ましくは２０～２５塩基である。前記オリゴヌクレオチドプローブ及びオリゴヌクレオチドプライマーは、例えば、市販のオリゴヌクレオチド合成機により合成することができる。また、天然のヌクレオチド（デオキシリボヌクレオチド及び／又はリボヌクレオチド）のみから構成されていなくともよく、例えば、ＰＮＡ（ｐｏｌｙａｍｉｄｅ　ｎｕｃｌｅｉｃ　ａｃｉｄ）、ＬＮＡ（登録商標、ｌｏｃｋｅｄ　ｎｕｃｌｅｉｃ　ａｃｉｄ）、ＥＮＡ（登録商標、２’－Ｏ，４’－Ｃ－Ｅｔｈｙｌｅｎｅ－ｂｒｉｄｇｅｄ　ｎｕｃｌｅｉｃ　ａｃｉｄｓ）等の非天然型のヌクレオチドにてその一部又は全部が構成されていてもよい。 Such oligonucleotide probes and oligonucleotide primers can be designed by a person skilled in the art based on the sequence of the target polynucleotide by a known method or a method based thereon. In addition, the length of the oligonucleotide probe and oligonucleotide primer are each independently preferably at least 15 bases, usually 15 to 100 bases, preferably 17 to 30 bases, more preferably 20-25 bases. The oligonucleotide probes and oligonucleotide primers can be synthesized by, for example, a commercially available oligonucleotide synthesizer. In addition, it may not consist only of natural nucleotides (deoxyribonucleotides and/or ribonucleotides), for example, PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), ENA (registered trademark, 2'- O,4'-C-Ethylene-bridged nucleic acids) may be partially or wholly composed of non-natural nucleotides.
　〔翻訳レベルでの検出（標的ポリペプチドの検出）〕
　翻訳レベルでの検出において、前記標的遺伝子の発現産物は、当該標的遺伝子にコードされるポリペプチド（以下、場合により「標的ポリペプチド」という）である。より具体的には、前記（ｉ）～（ｉｉｉ）の遺伝子のうちのいずれか１つにコードされるポリペプチドであり、すなわち、下記（ｉ’）～（ｉｉｉ’）のポリペプチドからなる群から選択されるポリペプチドである。
（ｉ’）配列番号１に記載のアミノ酸配列を含むポリペプチド
（ｉｉ’）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチド
（ｉｉｉ’）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチド。 [Detection at translation level (detection of target polypeptide)]
In translational level detection, the expression product of the target gene is a polypeptide encoded by the target gene (hereinafter sometimes referred to as "target polypeptide"). More specifically, it is a polypeptide encoded by any one of the genes (i) to (iii), that is, the group consisting of the following polypeptides (i') to (iii') A polypeptide selected from
(i') a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (ii') in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or added (iii') A polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
　本発明において、検出対象となるこれらの標的ポリペプチドとしては、上記のセクレトーム（Ｔ細胞由来セクレトーム）であることが好ましい。 In the present invention, these target polypeptides to be detected are preferably the above secretomes (T cell-derived secretomes).
　〈抗ポリペプチド抗体及び／又はアプタマーによる検出〉
　翻訳レベルでの検出の場合の検出方法としては、例えば、前記プローブ分子として、前記標的ポリペプチドに特異的に結合する抗体（以下、場合により「抗ポリペプチド抗体」という）及び／又はアプタマー（以下、場合により、単に「アプタマー」という）を用い、免疫細胞染色法、イメージングサイトメトリー、フローサイトメトリー、ＥＬＩＳＡ（Ｅｎｚｙｍｅ－Ｌｉｎｋｅｄ　Ｉｍｍｕｎｏ　Ｓｏｒｂｅｎｔ　Ａｓｓａｙ）法、ラジオイムノアッセイ、免疫沈降法、イムノブロッティング（ウェスタンブロット法等）、抗体アレイ、ｉｎ　ｖｉｖｏ　イメージング等の抗体を用いて検出する方法（免疫学的手法）；前記抗体に代えてアプタマーを用いて検出する方法が挙げられる。また、前記免疫学的手法は、必要な試薬を調製した上で、ＡＩＡ－９００やＡＩＡ－ＣＬ２４００（いずれも東ソー（株）製）等のエンザイムイムノアッセイ装置を用いて自動的に行ってもよい。 <Detection by anti-polypeptide antibody and/or aptamer>
Detection methods for detection at the translation level include, for example, antibodies that specifically bind to the target polypeptide (hereinafter sometimes referred to as "anti-polypeptide antibodies") and/or aptamers (hereinafter , In some cases, simply referred to as "aptamer"), immune cell staining method, imaging cytometry, flow cytometry, ELISA (Enzyme-Linked Immuno Sorbent Assay) method, radioimmunoassay, immunoprecipitation method, immunoblotting (Western blotting method etc.), methods of detection using antibodies such as antibody arrays and in vivo imaging (immunological techniques); methods of detection using aptamers instead of the antibodies. In addition, the immunological method may be performed automatically using an enzyme immunoassay device such as AIA-900 or AIA-CL2400 (both manufactured by Tosoh Corporation) after preparing necessary reagents.
　本発明において、「抗体」は、ポリクローナル抗体であっても、モノクローナル抗体であってもよく、また、抗体の機能的断片であってもよい。また、「抗体」には、免疫グロブリンの全てのクラス及びサブクラスが含まれる。抗体の「機能的断片」とは、抗体の一部分（部分断片）であって、本発明においては、前記標的遺伝子にコードされるポリペプチドを特異的に認識するものを示す。具体的には、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、可変領域断片（Ｆｖ）、ジスルフィド結合Ｆｖ、一本鎖Ｆｖ（ｓｃＦｖ）、ｓｃ（Ｆｖ）２、ダイアボディー（Ｄｉａｂｏｄｙ）、多特異性抗体、ナノボディー、及びこれらの重合体等が挙げられる。 In the present invention, an "antibody" may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody. "Antibody" also includes all classes and subclasses of immunoglobulins. A "functional fragment" of an antibody refers to a portion (partial fragment) of an antibody that specifically recognizes the polypeptide encoded by the target gene in the present invention. Specifically, Fab, Fab′, F(ab′) 2 , variable region fragment (Fv), disulfide-bonded Fv, single-chain Fv (scFv), sc(Fv) 2 , diabody, multispecific antibodies, nanobodies, and polymers thereof.
　本発明に係る抗ポリペプチド抗体は、ポリクローナル抗体であれば、抗原（標的ポリペプチド、その部分ペプチド、又はこれらを発現する細胞等）で免疫動物を免疫し、その抗血清から、従来の手段（塩析、遠心分離、透析、カラムクロマトグラフィー等）によって、適宜精製して取得することができる。また、モノクローナル抗体は、ハイブリドーマ法や組換えＤＮＡ法によって作製することができる。ハイブリドーマ法としては、例えば、コーラー及びミルスタインの方法（Ｋｏｈｌｅｒ＆Ｍｉｌｓｔｅｉｎ，Ｎａｔｕｒｅ，２５６：４９５（１９７５））が挙げられ、組換えＤＮＡ法としては、例えば、上記抗ポリペプチド抗体をコードするＤＮＡをハイブリドーマやＢ細胞等からクローニングし、適当なベクターに組み込んで、これを宿主細胞（哺乳類細胞株、大腸菌、酵母細胞、昆虫細胞、植物細胞等）に導入し、前記抗ポリペプチド抗体を組換え抗体として産生させる手法が挙げられる（例えば、Ｐ．Ｊ．Ｄｅｌｖｅｓ，Ａｎｔｉｂｏｄｙ　Ｐｒｏｄｕｃｔｉｏｎ：Ｅｓｓｅｎｔｉａｌ　Ｔｅｃｈｎｉｑｕｅｓ，１９９７　ＷＩＬＥＹ；Ｐ．Ｓｈｅｐｈｅｒｄ　ａｎｄ　Ｃ．Ｄｅａｎ　Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ，２０００　ＯＸＦＯＲＤ　ＵＮＩＶＥＲＳＩＴＹ　ＰＲＥＳＳ；Ｖａｎｄａｍｍｅ　Ａ．Ｍ．ｅｔ　ａｌ．，Ｅｕｒ．Ｊ．Ｂｉｏｃｈｅｍ．１９２：７６７－７７５（１９９０））。 If the anti-polypeptide antibody according to the present invention is a polyclonal antibody, an immunized animal is immunized with an antigen (target polypeptide, its partial peptide, or cells expressing these, etc.), and the antiserum is obtained by conventional means ( Salting out, centrifugation, dialysis, column chromatography, etc.) can be appropriately purified and obtained. Monoclonal antibodies can also be produced by the hybridoma method or recombinant DNA method. Hybridoma methods include, for example, Kohler and Milstein's method (Kohler & Milstein, Nature, 256:495 (1975)). Recombinant DNA methods include, for example, DNA encoding the anti-polypeptide antibody. It is cloned from B cells or the like, incorporated into an appropriate vector, introduced into host cells (mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.), and the anti-polypeptide antibody is produced as a recombinant antibody. (For example, P. J. Delves, Antibody Production: Essential Techniques, 1997 WILEY; P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; Vandamme AM et al., Eur.J. Biochem. 192:767-775 (1990)).
　この場合には、前記抗ポリペプチド抗体及びアプタマーとして、それぞれ、標識物質によって標識されたものを用い、当該標識物質に応じたシグナルを検出して、検出されたシグナル量を前記標的ポリペプチドの量（すなわち、標的遺伝子の発現量）とすることができる。このときの標識物質としては、前記抗ポリペプチド抗体又はアプタマーに結合することができ、化学的又は光学的方法で検出できるものであれば特に制限されることはなく、例えば、フィコエリスリン（ＰＥ）、フルオレセインイソチオシアネート（ＦＩＴＣ）、ローダミンイソチオシアネート（ＲＩＴＣ）等の蛍光物質や、ペルオキシダーゼ、β－Ｄ－ガラクトシダーゼ、マイクロペルオキシダーゼ、ホースラディッシュペルオキシダーゼ（ＨＲＰ）、アルカリホスファターゼ等の酵素、放射性物質が挙げられる。 In this case, the anti-polypeptide antibody and the aptamer are each labeled with a labeling substance, the signal corresponding to the labeling substance is detected, and the amount of the detected signal is calculated as the amount of the target polypeptide. (that is, the expression level of the target gene). The labeling substance at this time is not particularly limited as long as it can bind to the anti-polypeptide antibody or aptamer and can be detected by a chemical or optical method. ), fluorescent substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC), enzymes such as peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP) and alkaline phosphatase, and radioactive substances. .
　また、前記抗ポリペプチド抗体及び／又はアプタマーを用いる場合には、前記標識物質を結合させた二次抗体を利用する方法や、二次抗体と前記標識物質とを結合させたポリマーを利用する方法などの間接的検出方法を利用することもできる。ここで、「二次抗体」とは、前記抗ポリペプチド抗体及び／又はアプタマーに特異的な結合性を示す抗体である。例えば、上記抗ポリペプチド抗体をウサギ抗体として調製した場合には、二次抗体として抗ウサギＩｇＧ抗体を使用することができる。ウサギ、ヤギ、マウスなどの様々な生物種に由来する抗体に対して、使用可能な標識二次抗体が市販されており、用いる抗ポリペプチド抗体の由来する生物種に応じて、適切な二次抗体を選択し、使用することができる。また、二次抗体に代えて、標識物質を結合させたプロテインＧ（例えば、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ属菌由来）、プロテインＡ（例えば、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ　ａｕｒｅｕｓ由来）、プロテインＬ（Ｐｅｐｔｏｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｍａｇｎｕｓ由来）等を用いることも可能である。 When the anti-polypeptide antibody and/or aptamer is used, a method of using a secondary antibody bound with the labeling substance, or a method of using a polymer binding the secondary antibody and the labeling substance. Indirect detection methods such as can also be used. Here, the "secondary antibody" is an antibody that exhibits specific binding to the anti-polypeptide antibody and/or aptamer. For example, when the anti-polypeptide antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. Labeled secondary antibodies that can be used for antibodies derived from various species such as rabbits, goats, and mice are commercially available. Antibodies can be selected and used. In place of the secondary antibody, protein G (for example, derived from Streptococcus spp.), protein A (for example, derived from Staphylococcus aureus), protein L (derived from Peptostreptococcus magnus), etc. to which a labeling substance is bound can also be used. be.
　〈標識アミノ酸による検出〉
　翻訳レベルでの検出の場合の検出方法としては、他にも例えば、標識アミノ酸を添加した培地でＴ細胞を培養し、前記標識アミノ酸で標識された標的ポリペプチドを検出する方法が挙げられる。このような方法としては、
　少なくとも１種のＴ細胞増殖用必須アミノ酸に代えてそのアミノ酸が標識された標識アミノ酸を含有し、かつ、共通γ鎖ファミリーサイトカインを含有するＴ細胞標識用培地でＴ細胞を培養する標識工程と、
　前記標識アミノ酸で標識されたＴ細胞由来ポリペプチドを検出する検出工程ａと、
を含む方法が好ましい。 <Detection with labeled amino acids>
Other detection methods for detection at the translation level include, for example, a method of culturing T cells in a medium supplemented with a labeled amino acid and detecting a target polypeptide labeled with the labeled amino acid. As such a method,
a labeling step of culturing T cells in a T cell labeling medium containing a labeled amino acid in which the amino acid is labeled instead of at least one essential amino acid for T cell proliferation and containing a common γ chain family cytokine;
a detection step a of detecting the T cell-derived polypeptide labeled with the labeled amino acid;
is preferred.
　ここで、「Ｔ細胞標識用培地」とは、Ｔ細胞を培養して標識するための標識アミノ酸を少なくとも含有する培地のことを示す。また、「Ｔ細胞増殖用必須アミノ酸（以下、場合により単に「必須アミノ酸」という）」とは、Ｔ細胞が増殖するために必須のアミノ酸を示し、より具体的には、バリン、イソロイシン、ロイシン、メチオニン、リジン、フェニルアラニン、トリプトファン、スレオニン、ヒスチジンである。 Here, "T cell labeling medium" refers to a medium containing at least labeling amino acids for culturing and labeling T cells. In addition, “essential amino acids for T cell proliferation (hereinafter, sometimes simply referred to as “essential amino acids”)” refers to amino acids essential for T cell proliferation, more specifically, valine, isoleucine, leucine, methionine, lysine, phenylalanine, tryptophan, threonine, histidine.
　前記Ｔ細胞標識用培地は、前記必須アミノ酸の少なくとも１種に代えてそのアミノ酸が標識された標識アミノ酸を含有する、すなわち、前記必須アミノ酸の少なくとも１種を含有せず、かつ、当該含有されない必須アミノ酸が標識されたアミノ酸である標識アミノ酸を含有する。「必須アミノ酸を含有しない」とは、前記Ｔ細胞標識用培地中に前記必須アミノ酸を実質的に含有しないことをいい、より具体的には、当該必須アミノ酸の含有量がＴ細胞標識用培地中に１×１０－３ｍｍｏｌ／Ｌ以下であることをいい、１×１０－４ｍｍｏｌ／Ｌ未満であることが好ましい。前記Ｔ細胞標識用培地から除かれる（すなわち、前記Ｔ細胞標識用培地に実質的に含有されない）必須アミノ酸としては、上記のいずれか１種であっても、２種以上の組み合わせであってもよいが、中でも、タンパク質合成経路への効率的な取り込みの観点から、メチオニンが好ましい。 The T cell labeling medium contains a labeled amino acid in which the amino acid is labeled instead of at least one of the essential amino acids, that is, does not contain at least one of the essential amino acids and the essential amino acid that is not contained Contains labeled amino acids, where the amino acids are labeled amino acids. "Containing no essential amino acids" means that the T cell labeling medium does not substantially contain the essential amino acids. More specifically, the content of the essential amino acids in the T cell labeling medium is is 1×10 −3 mmol/L or less, preferably less than 1×10 −4 mmol/L. The essential amino acids excluded from the T cell labeling medium (that is, substantially not contained in the T cell labeling medium) may be any one of the above or a combination of two or more of them. Among them, methionine is preferred from the viewpoint of efficient incorporation into the protein synthesis pathway.
　前記「標識アミノ酸」とは、前記必須アミノ酸の構造を維持したままでその一部が修飾され、その修飾に基づいて検出可能であるものを示す。前記「検出可能」には、呈色（発色）、消光、反射光、発光、蛍光等の目視で直接的に確認できるものの他、特定の測定方法や測定装置によって確認できることを含む。 The "labeled amino acid" refers to an essential amino acid partially modified while maintaining the structure of the essential amino acid, and detectable based on the modification. The above-mentioned "detectable" includes those that can be directly confirmed by visual observation such as coloring (color development), quenching, reflected light, luminescence, and fluorescence, as well as those that can be confirmed by a specific measuring method or measuring device.
　このような標識アミノ酸としては、例えば、アミノ酸の原子の一部をパルス安定同位体（例えば、２Ｈ、１３Ｃ、１５Ｎ）に置換した同位体標識アミノ酸；アミノ酸の一部がアジド基、アルキン基等の修飾基によって修飾されたアミノ酸の構造的類似体（ａｎａｌｏｇ）が挙げられ、前記必須アミノ酸に対応して、これらのいずれか１種であっても、２種以上の組み合わせであってもよい。 Examples of such labeled amino acids include isotope-labeled amino acids in which some of the atoms of the amino acid are substituted with pulse stable isotopes (e.g., 2 H, 13 C, 15 N); Examples include structural analogs of amino acids modified by modifying groups such as groups, and corresponding to the essential amino acids, any one of them or a combination of two or more of them good.
　例えば、前記Ｔ細胞標識用培地が、前記必須アミノ酸のメチオニンを含有しない場合、メチオニンに代えて前記Ｔ細胞標識用培地に含有される標識アミノ酸としては、例えば、メチオニンを構成する原子の一部を前記安定同位体に置換した同位体標識メチオニンである２Ｈ標識メチオニン、１３Ｃ標識メチオニン、１５Ｎ標識メチオニン；メチオニンの構造的類似体であるＬ－アジドホモアラニン（ＡＨＡ：Ｌ－Ａｚｉｄｏｈｏｍｏａｌａｎｉｎｅ）、Ｌ－ホモプロパルギルグリシン（ＨＰＧ：Ｌ－Ｈｏｍｏｐｒｏｐａｒｇｙｌｇｌｙｃｉｎｅ）、Ｌ－ホモアリルグリシン（ＨＡＧ：Ｌ－Ｈｏｍｏａｌｌｙｌｇｌｙｃｉｎｅ）が挙げられ、これらのいずれか１種であっても、２種以上の組み合わせであってもよい。 For example, when the T cell labeling medium does not contain the essential amino acid methionine, the labeling amino acid contained in the T cell labeling medium instead of methionine may be, for example, 2 H-labeled methionine, 13 C-labeled methionine, and 15 N-labeled methionine, which are isotopically-labeled methionines substituted with the stable isotope; L-azidohomoalanine (AHA), which are structural analogues of methionine; -homopropargylglycine (HPG: L-Homopropargylglycine), L-homoallylglycine (HAG: L-Homoallylglycine), any one of which may be used or a combination of two or more thereof may be used.
　前記Ｔ細胞標識用培地において、前記標識アミノ酸の含有量（標識アミノ酸が２種以上である場合にはそれらの合計含有量、以下同じ）としては、Ｔ細胞の取り込み効率や培養効率、同Ｔ細胞への毒性等を考慮して適宜決定することができ、特に制限されないが、例えば、０．０００１～０．１ｍｍｏｌ／Ｌの範囲が挙げられ、０．００１～０．０５ｍｍｏｌ／Ｌであることが好ましく、０．００１～０．０２ｍｍｏｌ／Ｌであることがより好ましい。より具体的に、例えば、前記標識アミノ酸がＡＨＡである場合には、０．０００５～０．０３ｍｍｏｌ／Ｌであることが好ましく、０．００１～０．０３ｍｍｏｌ／Ｌであることがより好ましく、０．００２～０．０３ｍｍｏｌ／Ｌであることがさらに好ましく、０．００５～０．０２ｍｍｏｌ／Ｌであることがさらにより好ましい。前記標識アミノ酸の含有量が前記上限を超えると、細胞が弱ったり死滅し易くなる傾向にあり、他方、前記下限未満であると、前記標識アミノ酸で標識されたポリペプチドを分泌又は細胞外に露出する細胞数が少なくなり、ポリペプチドの検出精度が低下する傾向にある。 In the T cell labeling medium, the content of the labeled amino acids (the total content of the labeled amino acids when there are two or more labeled amino acids; the same shall apply hereinafter) includes the T cell uptake efficiency, the culture efficiency, the T cell It can be determined as appropriate in consideration of the toxicity etc., and is not particularly limited. Preferably, it is 0.001 to 0.02 mmol/L. More specifically, for example, when the labeled amino acid is AHA, it is preferably 0.0005 to 0.03 mmol/L, more preferably 0.001 to 0.03 mmol/L. More preferably 0.002 to 0.03 mmol/L, even more preferably 0.005 to 0.02 mmol/L. When the content of the labeled amino acid exceeds the upper limit, the cells tend to weaken or die, whereas when the content is less than the lower limit, the polypeptide labeled with the labeled amino acid is secreted or exposed outside the cell. As a result, the number of cells to be detected tends to decrease, and the detection accuracy of the polypeptide tends to decrease.
　前記Ｔ細胞標識用培地は、共通γ鎖ファミリーサイトカインをさらに含有する。これを含有することにより、前記標識アミノ酸で標識されたポリペプチドを発現する細胞を高効率で得ることが可能となる。「共通γ鎖ファミリーサイトカイン」とは、共通のγ鎖（ｃｏｍｍｏｎ　γ　ｃｈａｉｎ：γサブユニット）を含む受容体を介して作用するサイトカインを示す。より具体的には、インターロイキン２（ＩＬ－２）、インターロイキン４（ＩＬ－４）、インターロイキン７（ＩＬ－７）、インターロイキン９（ＩＬ－９）、インターロイキン１５（ＩＬ－１５）、及びインターロイキン２１（ＩＬ－２１）が挙げられ、これらのいずれか１種であっても、２種以上の組み合わせであってもよい。中でも、細胞の生存及び増殖をより維持する観点からは、インターロイキン７、インターロイキン１５、インターロイキン２１、及びこれらの２種以上の組み合わせがより好ましい。 The T cell labeling medium further contains common γ-chain family cytokines. By containing this, it becomes possible to obtain cells expressing the polypeptide labeled with the labeled amino acid with high efficiency. A "common γ chain family cytokine" refers to a cytokine that acts via a receptor containing a common γ chain (γ subunit). More specifically, interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 15 (IL-15) , and interleukin 21 (IL-21), any one of which may be used in combination. Among them, interleukin 7, interleukin 15, interleukin 21, and combinations of two or more of these are more preferable from the viewpoint of better maintenance of cell survival and growth.
　前記Ｔ細胞標識用培地において、前記共通γ鎖ファミリーサイトカインの含有量（共通γ鎖ファミリーサイトカインが２種以上である場合にはそれらの合計含有量、以下同じ）としては、Ｔ細胞の性状等を考慮して適宜決定することができるため、これに限定されるものではないが、１～１０００ｎｇ／ｍＬであることが好ましく、５～１００ｎｇ／ｍＬであることがより好ましく、５～２０ｎｇ／ｍＬであることがさらに好ましい。前記共通γ鎖ファミリーサイトカインの含有量が前記上限を超えると、共通γ鎖ファミリーサイトカインを含有させることの効果が低下したり細胞増殖を阻害する傾向にあり、他方、前記下限未満であると、前記標識アミノ酸で標識されたポリペプチドを分泌又は細胞外に露出する細胞数が少なくなり、ポリペプチドの検出精度が低下する傾向にある。 In the T cell labeling medium, the content of the common γ-chain family cytokine (when there are two or more common γ-chain family cytokines, the total content thereof; the same shall apply hereinafter) is determined according to the properties of T cells, etc. Although it is not limited to this, it is preferably 1 to 1000 ng/mL, more preferably 5 to 100 ng/mL, and 5 to 20 ng/mL. It is even more preferable to have When the content of the common γ-chain family cytokine exceeds the upper limit, the effect of containing the common γ-chain family cytokine tends to decrease or cell proliferation tends to be inhibited. The number of cells that secrete or extracellularly expose a polypeptide labeled with a labeled amino acid tends to decrease, resulting in a decrease in the detection accuracy of the polypeptide.
　前記Ｔ細胞標識用培地としては、前記必須アミノ酸を除くため、血清を含有しないか、含有する場合には前記必須アミノ酸が除かれた透析血清であることが好ましい。また、前記Ｔ細胞標識用培地としては、他に、血清アルブミンやＩＴＳ（インスリン－トランスフェリン－亜セレン酸ナトリウム）などの細胞保護添加剤；ＥＣＭ（細胞外基質）成分等の足場剤等を含有していてもよい。これらのＴ細胞標識用培地又はその基礎培地としては、目的の必須アミノ酸を含有しない限り特に制限されず、ＤＭＥＭ（ダルベッコ改変イーグル培地）、ＲＰＭＩ（ロズウェルパーク記念研究所培地）、αＭＥＭ（α改変型イーグル最小必須培地）等の基礎培地；Ｔ細胞専用の無血清培地であるＡＩＭ　ＶＴＭ培地（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）；ＰＲＩＭＥ－ＸＶ　Ｔ　ｃｅｌｌ　Ｅｘｐａｎｓｉｏｎ　ＸＳＦＭ（富士フイルム和光純薬社製）等の公知や市販のＴ細胞用の培地を適宜採用することができる。 In order to remove the essential amino acids, the T cell labeling medium preferably does not contain serum or, if it does contain serum, is dialyzed serum from which the essential amino acids have been removed. In addition, the T cell labeling medium may also contain cytoprotective additives such as serum albumin and ITS (insulin-transferrin-sodium selenite); scaffolding agents such as ECM (extracellular matrix) components. may be These T cell labeling media or their basal media are not particularly limited as long as they do not contain the desired essential amino acids, and are DMEM (Dulbecco's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium), αMEM (α-modified basal medium such as Eagle minimum essential medium); AIM V TM medium (manufactured by Thermo Fisher Scientific) which is a serum-free medium exclusively for T cells; PRIME-XV T cell Expansion XSFM (manufactured by Fujifilm Wako Pure Chemical) or a commercially available medium for T cells can be used as appropriate.
　前記標識工程では、前記Ｔ細胞標識用培地でＴ細胞を培養する。これにより、前記標識アミノ酸をＴ細胞に取り込ませ、Ｔ細胞から前記標識アミノ酸で標識されたポリペプチド（Ｔ細胞由来ポリペプチド）を分泌又は細胞外に露出させる。前記標識工程における培養方法としては、特に制限されず、公知のＴ細胞の増殖培養条件を適宜採用することができ、また、Ｔ細胞の性状や濃度等を考慮して適宜調整することができるが、例えば、培養温度３５～３７．５℃、好ましくは３６～３７℃において、培養時間２～４８時間、好ましくは８～２４時間の条件が挙げられる。前記標識工程の培養装置としては、特に制限されず、例えば、プレート、ディッシュ、カラム、フラスコ、培養バッグ等が挙げられ、前記Ｔ細胞標識用培地等を供給するための供給手段や排出するための排出手段（センサ、バルブ、ポンプ、タンク等）をさらに備えていてもよい。 In the labeling step, T cells are cultured in the T cell labeling medium. As a result, the labeled amino acid is taken up by T cells, and a polypeptide labeled with the labeled amino acid (T cell-derived polypeptide) is secreted from the T cells or exposed outside the cells. The culture method in the labeling step is not particularly limited, and known T cell proliferation culture conditions can be appropriately adopted, and the characteristics and concentration of T cells can be taken into consideration and adjusted as appropriate. For example, the culture temperature is 35 to 37.5° C., preferably 36 to 37° C., and the culture time is 2 to 48 hours, preferably 8 to 24 hours. The culture apparatus for the labeling step is not particularly limited, and examples thereof include plates, dishes, columns, flasks, culture bags, and the like. Ejection means (sensors, valves, pumps, tanks, etc.) may also be provided.
　前記検出工程ａでは、前記標識アミノ酸で標識され、分泌又は細胞外に露出されたＴ細胞由来ポリペプチドを検出する。前記検出工程ａとしては、上記の標識工程と同時（リアルタイム）で行ってもよい。前記検出工程ａにおける検出方法としては、前記標識アミノ酸の種類に応じた検出方法を適宜採用することができる。このような検出方法としては、特に制限されず、適宜公知の方法又はそれに準じた方法を適宜採用することができる。前記標識アミノ酸が同位体標識アミノ酸又はアミノ酸の構造的類似体である場合には、例えば、前記標識工程後又は標識工程中（すなわち培養途中）の培地について、ガス同位体比質量分析、同位体比赤外分光、液体クロマトグラフ質量分析（ＬＣ－ＭＳ／ＭＳ）等の質量分析を行うことによって、前記標識アミノ酸に応じたシグナルを検出し、検出されたシグナル量を前記標的ポリペプチドの量（すなわち、標的遺伝子の発現量）とすることができる。 In the detection step a, the T-cell-derived polypeptide that has been labeled with the labeling amino acid and has been secreted or exposed outside the cell is detected. The detection step a may be performed simultaneously (in real time) with the above labeling step. As a detection method in the detection step a, a detection method suitable for the type of the labeled amino acid can be appropriately employed. Such a detection method is not particularly limited, and a known method or a method based thereon can be appropriately employed. When the labeled amino acid is an isotope-labeled amino acid or a structural analogue of an amino acid, for example, the medium after the labeling step or during the labeling step (that is, during the culture) is subjected to gas isotope ratio mass spectrometry, By performing mass spectrometry such as infrared spectroscopy, liquid chromatography mass spectrometry (LC-MS/MS), a signal corresponding to the labeled amino acid is detected, and the amount of the detected signal is the amount of the target polypeptide (i.e. , expression level of the target gene).
　また、前記標識アミノ酸がアミノ酸の構造的類似体である場合には、前記修飾基に特異的な染色をすることによって検出することができる。例えば、前記修飾基がアジド基である場合（ＡＨＡ等）には、当該アジド基をＣｌｉｃｋ－ｉＴＴＭ　Ａｌｅｘａ　Ｆｌｕｏｒ　４８８　ｓＤＩＢＯ　Ａｌｋｙｎｅ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）等の蛍光試薬によって染色することができるため、蛍光顕微鏡、フローサイトメトリー、イメージングサイトメトリー等を用いて、前記標識アミノ酸に応じたシグナル（発色、蛍光等）を検出し、検出されたシグナル量を前記標的ポリペプチドの量（すなわち、標的遺伝子の発現量）とすることができる。 Moreover, when the labeled amino acid is a structural analogue of an amino acid, it can be detected by staining the modified group specifically. For example, when the modifying group is an azide group (such as AHA), the azide group can be stained with a fluorescent reagent such as Click-iT ™ Alexa Fluor 488 sDIBO Alkyne (manufactured by Thermo Fisher Scientific). Using fluorescence microscopy, flow cytometry, imaging cytometry, etc., a signal (color development, fluorescence, etc.) corresponding to the labeled amino acid is detected, and the amount of the detected signal is the amount of the target polypeptide (i.e., the amount of the target gene). expression level).
　また、翻訳レベルでの検出の場合の検出方法としては、他にも例えば、標的ポリペプチドの糖鎖を認識して検出する方法も挙げられる。このような方法としては、例えば、Ｃｌｉｃｋ－ｉＴＴＭ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）等の市販の試薬を用いる方法が挙げられる。上記の翻訳レベルでの検出の場合の検出方法は、適宜他の方法と組み合わせて用いてもよい。 Other detection methods for detection at the translation level include, for example, a method of recognizing and detecting the sugar chain of the target polypeptide. Such methods include, for example, methods using commercially available reagents such as Click-iT ™ (manufactured by Thermo Fisher Scientific). The above detection method for detection at the translation level may be used in combination with other methods as appropriate.
　本発明において、前記遺伝子の発現の検出としては、簡便性の観点から、翻訳レベルでの検出（前記標的ポリペプチドの検出）であることが好ましく、特に、前記抗ポリペプチド抗体を用いて前記標的ポリペプチドを検出する方法が好ましい。 In the present invention, detection of the expression of the gene is preferably detection at the translation level (detection of the target polypeptide) from the viewpoint of convenience. Methods that detect polypeptides are preferred.
　［評価工程］
　本発明の方法に係る評価工程においては、前記検出工程で検出された標的遺伝子の発現の有無又はその発現量を指標として、Ｔ細胞の品質を評価する。本発明において評価されるＴ細胞の品質としては、増殖能、がん細胞傷害活性が挙げられ、中でも、本発明の方法は、Ｔ細胞の増殖能を評価する方法として好ましい。「増殖能」とは、より具体的には、Ｉｎ　ｖｉｔｒｏ又はＩｎ　ｖｉｖｏにおいて、ＴＣＲ刺激又は抗原刺激によってＴ細胞が活性化し、細胞傷害活性を有する細胞が増加する能力を示す。Ｔ細胞の増殖能は、例えば、下記の実施例に示す方法でＴ細胞をＴＣＲ刺激磁性ビーズで刺激し、刺激前の細胞数と刺激から１４日後の細胞数との比で確認することができるが、これに制限されるわけではない。 [Evaluation process]
In the evaluation step according to the method of the present invention, the quality of T cells is evaluated using the presence or absence or the expression level of the target gene detected in the detection step as an index. The quality of T cells to be evaluated in the present invention includes proliferative ability and cancer cytotoxic activity. Among them, the method of the present invention is preferable as a method for evaluating the proliferative ability of T cells. More specifically, the term "proliferative ability" indicates the ability to activate T cells and increase cells having cytotoxic activity in vitro or in vivo by TCR stimulation or antigen stimulation. The proliferative ability of T cells can be confirmed, for example, by stimulating T cells with TCR-stimulating magnetic beads by the method shown in the Examples below, and checking the ratio of the number of cells before stimulation to the number of cells 14 days after stimulation. However, it is not limited to this.
　本発明においては、前記検出工程で検出された標的遺伝子の発現量が少ないほど、増殖能が高い、すなわち品質が良いと評価でき、前記検出工程で検出された標的遺伝子の発現量が多いほど、増殖能が低い、すなわち品質が悪いと評価できる。前記Ｔ細胞の品質の評価基準としては、目的に応じて適宜設定すればよく、特に限定されるものではない。例えば、前記検出工程で、前記標的遺伝子の発現レベル（標的遺伝子の転写レベルや翻訳レベル）に応じた標的遺伝子の発現が少しでも検出されなければ、すなわち、前記標的ポリヌクレオチドや前記標的ポリペプチドが少しでも検出されなければ、Ｔ細胞の増殖能が高いと評価してもよく、一定の閾値以上の標的遺伝子の発現量が検出されなければ増殖能が高いと評価をしてもよい。また前記標的遺伝子の発現量に応じて、Ｔ細胞の増殖能の度合いを評価してもよい。例えば、増殖能が低いことが事前に特定されているＴ細胞等の標準試料で検出される前記標的遺伝子の発現量に対して、一定の値以下、又は当該標的遺伝子の発現量の平均値の４ＳＤ以下、３ＳＤ以下、若しくは２ＳＤ以下のシグナル量であれば、増殖能が高い、すなわち品質が良いと評価してもよい。 In the present invention, the lower the expression level of the target gene detected in the detection step, the higher the growth ability, that is, the better the quality. It can be evaluated as having low growth ability, ie, poor quality. The evaluation criteria for the quality of the T cells may be appropriately set according to the purpose, and are not particularly limited. For example, in the detection step, if even a little expression of the target gene is not detected according to the expression level of the target gene (transcription level or translation level of the target gene), that is, the target polynucleotide or the target polypeptide If even a little is not detected, the proliferative ability of the T cells may be evaluated as high, and if the expression level of the target gene above a certain threshold is not detected, the proliferative ability may be evaluated as high. Further, the degree of T cell proliferation ability may be evaluated according to the expression level of the target gene. For example, with respect to the expression level of the target gene detected in a standard sample such as T cells that have been specified in advance to have low proliferative ability, it is below a certain value, or below the average expression level of the target gene. A signal amount of 4 SD or less, 3 SD or less, or 2 SD or less may be evaluated as having a high proliferative capacity, ie, good quality.
　＜Ｔ細胞の品質評価用試薬、Ｔ細胞評価用キット＞
　本発明のＴ細胞の品質評価用試薬は、上記本発明のＴ細胞の品質評価方法に用いるための試薬（組成物）であり、下記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現産物に特異的に結合するプローブ分子を含有する組成物（以下、場合により、単に「本発明の試薬」という）である。
（ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。 <T cell quality evaluation reagent, T cell evaluation kit>
The T cell quality evaluation reagent of the present invention is a reagent (composition) for use in the T cell quality evaluation method of the present invention, and is selected from the group consisting of the following genes (i) to (iii): is a composition (hereinafter sometimes simply referred to as "reagent of the present invention") containing a probe molecule that specifically binds to the expression product of at least one gene.
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
　本発明の試薬において、上記（ｉ）～（ｉｉｉ）の遺伝子、及び遺伝子の発現産物としては、その好ましい態様も含めて、上記の本発明の方法において述べたとおりである。本発明の試薬に係るプローブ分子としては、上記の本発明の方法において挙げた、オリゴヌクレオチドプローブ、オリゴヌクレオチドプライマー、抗ポリペプチド抗体、及びアプタマーが挙げられ、それぞれ、その好ましい態様も含めて、上記の本発明の方法において述べたとおりである。前記プローブ分子としては、これらのいずれか１種であっても２種以上の組み合わせであってもよい。中でも、前記抗ポリペプチド抗体及び／又は前記アプタマーが好ましく、前記抗ポリペプチド抗体がより好ましい。 In the reagent of the present invention, the genes (i) to (iii) and expression products of the genes are as described in the method of the present invention, including preferred embodiments thereof. Examples of probe molecules related to the reagent of the present invention include oligonucleotide probes, oligonucleotide primers, anti-polypeptide antibodies, and aptamers mentioned in the method of the present invention described above. in the method of the present invention. The probe molecule may be one of these or a combination of two or more of them. Among them, the anti-polypeptide antibody and/or the aptamer are preferable, and the anti-polypeptide antibody is more preferable.
　本発明の試薬は、液体であっても粉末状等の固体であってもよく、前記プローブ分子に加えて、緩衝液、生理食塩水、安定剤、保存剤、防腐剤、培地等の他の成分がさらに含有されていてもよい。 The reagent of the present invention may be liquid or solid such as powder. Additional ingredients may be included.
　本発明のＴ細胞評価用キットは、上記本発明のＴ細胞の品質評価方法に用いるためのキットであり、上記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現産物に特異的に結合するプローブ分子を備えるキット（以下、場合により、単に「本発明のキット」という）である。 The T cell evaluation kit of the present invention is a kit for use in the T cell quality evaluation method of the present invention, and includes at least one gene selected from the group consisting of the genes (i) to (iii) above. (hereinafter, sometimes simply referred to as "the kit of the present invention").
　本発明のキットに係るプローブ分子としては、上記本発明の試薬であることが好ましい。また、本発明のキットは、前記プローブ分子に加えて、緩衝液、生理食塩水等の希釈又は懸濁用液；ＤＮＡ又はタンパク質を抽出及び／又は精製するための試薬；ブロッキング剤；キレート剤；前記標識物質；前記蛍光色素；前記検出工程に必要な試薬；ｐＨ調整剤等の試薬；標準試料；使用説明書；安全データシート（Ｓａｆｅｔｙ　Ｄａｔａ　Ｓｈｅｅｔ）等をさらに備えていてもよい。 The probe molecule according to the kit of the present invention is preferably the reagent of the present invention. In addition to the probe molecule, the kit of the present invention also contains a buffer, a solution for dilution or suspension such as physiological saline; a reagent for extracting and/or purifying DNA or protein; a blocking agent; a chelating agent; The labeling substance; the fluorescent dye; reagents necessary for the detection step; reagents such as pH adjusters; standard samples; instructions for use;
　本発明は、前記のＴ細胞の評価方法により得られた高品質Ｔ細胞および該高品質Ｔ細胞を有効成分として含有する医薬組成物を提供する。 The present invention provides high-quality T cells obtained by the above T-cell evaluation method and pharmaceutical compositions containing the high-quality T cells as active ingredients.
　本発明の、T細胞を有効成分として含む医薬組成物は、がん治療対象を処置するために使用することができる。本発明の医薬組成物は、製剤技術分野において慣用の方法、例えば、日本薬局方に記載の方法等により製造することができる。本発明の医薬組成物は、薬学的に許容される添加剤を含んでいてもよい。該添加剤としては、例えば、細胞培養液、生理食塩水や適当な緩衝液（例えば、リン酸系緩衝液）等が挙げられる。 The pharmaceutical composition containing T cells as an active ingredient of the present invention can be used to treat cancer patients. The pharmaceutical composition of the present invention can be produced by a method commonly used in the field of formulation technology, such as the method described in the Japanese Pharmacopoeia. The pharmaceutical composition of the present invention may contain pharmaceutically acceptable additives. Examples of such additives include cell culture media, physiological saline, and suitable buffers (eg, phosphate buffers).
　本発明の医薬組成物は、T細胞を生理食塩水や適当な緩衝液（例えばリン酸系緩衝液）等に懸濁することによって製造することができる。所望の治療効果が発揮されるように、一回投与分の量として、例えば1×10７個以上の細胞を含有させることが好ましい。より好ましくは１×108個以上、さらに好ましくは１×109個以上である。細胞の含有量は、適用対象の性別、年齢、体重、患部の状態、細胞の状態等を考慮して適宜調整することができる。本発明の医薬組成物には、T細胞の他、細胞を保護する目的でジメチルスルフォキシド（DMSO）および血清アルブミン等を含有させてもよい。また、細菌の混入を阻止する目的で抗生物質等ならびに細胞の活性化および分化を促す目的でビタミン類やサイトカイン等を含有させてもよい。さらに、製剤上許容される他の成分（例えば、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤、生理食塩水等）を本発明の医薬組成物に含有させてもよい。 The pharmaceutical composition of the present invention can be produced by suspending T cells in physiological saline, an appropriate buffer (eg, phosphate buffer), or the like. It is preferable to contain, for example, 1×10 7 cells or more in a single dose so as to achieve the desired therapeutic effect. It is more preferably 1×10 8 or more, still more preferably 1×10 9 or more. The content of cells can be appropriately adjusted in consideration of the sex, age, weight, condition of the affected area, condition of cells, etc. of the subject. In addition to T cells, the pharmaceutical composition of the present invention may contain dimethylsulfoxide (DMSO), serum albumin, and the like for the purpose of protecting cells. Antibiotics and the like may be added for the purpose of preventing bacterial contamination, and vitamins, cytokines, and the like may be included for the purpose of promoting activation and differentiation of cells. Furthermore, other pharmaceutically acceptable components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) It may be included in the pharmaceutical composition of the present invention.
　本発明のT細胞を有効成分として含む医薬組成物は、低温凍結保存することができる。低温凍結保存する場合、温度は、細胞の保存に適した温度であれば特に限定されない。例えば、－20℃、－80℃および－120～－196℃が挙げられる。低温凍結保存する場合、細胞は、バイアル等の適切な容器中で保存することができる。T細胞の凍結時および解凍時の細胞損傷リスクを最小限にするための操作は、当業者には周知である。 A pharmaceutical composition containing the T cells of the present invention as an active ingredient can be cryopreserved. In the case of low-temperature cryopreservation, the temperature is not particularly limited as long as it is suitable for cell preservation. Examples include -20°C, -80°C and -120 to -196°C. For cryopreservation, cells can be stored in suitable containers such as vials. Manipulations to minimize the risk of cell damage during freezing and thawing of T cells are well known to those of skill in the art.
　T細胞の冷凍保存においては、T細胞を培養液から回収し、緩衝液または培養液により洗浄し、細胞数を計数し、遠心分離等により濃縮して、凍結媒質（例えば、10% DMSOを含む培養液）中に懸濁した後、低温凍結保存する。T細胞は、複数の培養容器で培養した細胞を合わせ、単一ロットにすることができる。本発明のT細胞を含む医薬組成物は、バイアル等の容器当たり、例えば、5×104個～9×1010個のT細胞を含むが、対象とするがんの種類、投与対象、投与経路等に応じて変更することができる。 For cryopreservation of T cells, T cells are collected from the culture medium, washed with buffer or culture medium, counted, concentrated by centrifugation, etc., and placed in a freezing medium (e.g., containing 10% DMSO). culture solution), and cryopreserved. T cells can be made into a single lot by combining cells cultured in multiple culture vessels. The pharmaceutical composition containing T cells of the present invention contains, for example, 5×10 4 to 9×10 10 T cells per container such as a vial. It can be changed according to the route or the like.
　本発明のT細胞を含む医薬組成物の投与経路としては、例えば、輸注、腫瘍内注射、動注、門脈注、腹腔内投与等が挙げられる。但し、本発明の医薬組成物中の有効成分であるT細胞が患部に送達される限り、投与経路はこれに限られるものではない。投与スケジュールとしては、単回投与または複数回投与とすることができる。複数回投与の期間は例えば、２～４週間に１回投与を繰り返す方法または半年から１年に１回投与を繰り返す方法等を採用することができる。投与スケジュールの作成においては、対象患者の性別、年齢、体重、病態等を考慮することができる。 Examples of administration routes of the pharmaceutical composition containing the T cells of the present invention include infusion, intratumoral injection, arterial injection, portal vein injection, intraperitoneal administration, and the like. However, the administration route is not limited to this as long as the T cells, which are the active ingredients in the pharmaceutical composition of the present invention, are delivered to the affected area. Dosage schedules can be single doses or multiple doses. For the period of multiple administrations, for example, a method of repeating administration once every 2 to 4 weeks, a method of repeating administration once every six months to a year, or the like can be adopted. In preparing the administration schedule, the sex, age, body weight, disease state, etc. of the target patient can be taken into consideration.
　本発明のT細胞を含む医薬組成物を、がんの予防または治療に使用する場合において、他家移植である場合、拒絶反応を起こしにくいという観点から、T細胞を採取されるがん患者または非がん患者は、がん治療のために、本発明の医薬組成物を投与されるがん患者と、HLAの型が一致していることが好ましい。また、本発明の医薬組成物が投与されるがん患者と、T細胞を採取されるがん患者とは同一人であることがより好ましい。すなわち、他家移植より自家的な移植によるがん治療がより好ましい。 In the case of using the pharmaceutical composition containing the T cells of the present invention for the prevention or treatment of cancer, in the case of allotransplantation, from the viewpoint that rejection is unlikely to occur, cancer patients from whom T cells are collected or The non-cancer patient preferably has the same HLA type as the cancer patient to whom the pharmaceutical composition of the present invention is administered for cancer treatment. Moreover, it is more preferable that the cancer patient to whom the pharmaceutical composition of the present invention is administered and the cancer patient from whom T cells are collected are the same person. That is, cancer treatment by autologous transplantation is more preferable than allogeneic transplantation.
　本発明の医薬組成物は、がんの予防または治療のために用いられる。がんとしては、卵巣がん、肝芽腫、肝細胞がん、胃がん、食道がん、膵臓がん、腎細胞がん、乳がん、悪性黒色腫、非小細胞肺がん、子宮頸がん、膠芽腫、前立腺がん、神経芽腫瘍、慢性リンパ性白血病、甲状腺乳頭がん、大腸がん、およびB細胞非ホジキンリンパ腫が挙げられるが、これらに限定されない。 The pharmaceutical composition of the present invention is used for prevention or treatment of cancer. Cancers include ovarian cancer, hepatoblastoma, hepatocellular carcinoma, gastric cancer, esophageal cancer, pancreatic cancer, renal cell carcinoma, breast cancer, malignant melanoma, non-small cell lung cancer, cervical cancer, and glioblastoma. including, but not limited to, blastoma, prostate cancer, neuroblastoma, chronic lymphocytic leukemia, papillary thyroid cancer, colorectal cancer, and B-cell non-Hodgkin's lymphoma.
　がんの予防または治療のために、本発明のT細胞を含む医薬組成物とがんワクチンとを組み合わせて使用する場合、本発明の医薬組成物およびがんワクチンの投与時期は限定されず、本発明の医薬組成物およびがんワクチンの投与を、投与対象に対し、同時に投与してもよく、また時間差をおいて投与してもよい。本発明の医薬組成物とがんワクチンとは別々に製剤化されていてもよく、また両者が混合された合剤であってもよい。本発明の医薬組成物およびがんワクチンの投与量は、臨床上用いられている投与量に準ずればよく、疾患、投与対象、投与経路、薬物との組み合わせ等により適宜選択することができる。 When the pharmaceutical composition containing T cells of the present invention and a cancer vaccine are used in combination for the prevention or treatment of cancer, the timing of administration of the pharmaceutical composition and cancer vaccine of the present invention is not limited, The pharmaceutical composition of the present invention and cancer vaccine may be administered to the subject at the same time or at different times. The pharmaceutical composition of the present invention and cancer vaccine may be formulated separately, or may be a combination drug in which both are mixed. The dosage of the pharmaceutical composition and cancer vaccine of the present invention may conform to the dosage used clinically, and can be appropriately selected depending on the disease, administration subject, administration route, combination with drugs, and the like.
　本発明は、がんを有するかまたはがんを有する危険性のある被験者に、Ｔ細胞を投与することを含む、がんを治療または予防する方法であって、該Ｔ細胞における、下記（Ｉ）～（ＩＩＩ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
を含む、方法
（（Ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ＩＩ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ＩＩＩ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。）を提供する。 The present invention provides a method for treating or preventing cancer, comprising administering T cells to a subject who has cancer or is at risk of having cancer, wherein the T cells have the following (I A detection step of detecting expression of at least one gene selected from the group consisting of genes from ) to (III);
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
(I) A gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (II) In the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted or deleted , a gene encoding a polypeptide comprising an inserted and/or added amino acid sequence (III) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1. )I will provide a.
　本発明における被験者は、脊椎動物である。特定の実施形態では、該脊椎動物は、哺乳動物である。該哺乳動物には、家畜(例えば、ウシ)、スポーツ動物、ペット/コンパニオン動物(例えば、ネコ、イヌ、及びウマ)、霊長類、マウス、及びラットが含まれるが、これらに限定されない。特定の実施形態では、哺乳動物は、ヒトである。 A subject in the present invention is a vertebrate. In certain embodiments, the vertebrate is a mammal. Such mammals include, but are not limited to, farm animals (eg, cows), sport animals, pet/companion animals (eg, cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal is human.
　本発明は、Ｔ細胞の製造方法であって、該Ｔ細胞における、下記（Ｉ）～（ＩＩＩ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
を含む、Ｔ細胞の製造方法
（（Ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
（ＩＩ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
（ＩＩＩ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。）を提供する。
  The present invention provides a method for producing T cells, comprising a detection step of detecting the expression of at least one gene selected from the group consisting of the following genes (I) to (III) in the T cells;
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
A method for producing a T cell ((I) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (II) in the amino acid sequence set forth in SEQ ID NO: 1, wherein one or more amino acid residues are Gene (III) encoding a polypeptide comprising a substituted, deleted, inserted and/or added amino acid sequence A polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 encoding genes.).
　以下、実施例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.
　＜Ｔ細胞評価用マーカー候補の選定＞
　１．高増殖Ｔ細胞及び低増殖Ｔ細胞のセクレトーム解析
　（１）高増殖Ｔ細胞及び低増殖Ｔ細胞として、それぞれ以下に示す細胞を用いた。
高増殖Ｔ細胞（１種類）：人の末梢血単核球から採取したナイーブＴ細胞（ＣＤ４５ＲＡ＋、ＣＣＲ７＋）のＴ細胞受容体（ＴＣＲ）を、Ｄｙｎａｂｅａｄｓ　Ｈｕｍａｎ　Ｔ－Ａｃｔｉｖａｔｏｒ　ＣＤ３／ＣＤ２８　ｆｏｒ　Ｔ　Ｃｅｌｌ　Ｅｘｐａｎｓｉｏｎ　ａｎｄ　Ａｃｔｉｖａｔｉｏｎ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）（以下、「ＴＣＲ刺激磁性ビーズ」という）を用いて刺激し、培地Ａ（１５％（ｗ／ｖ）ウシ胎児血清（ＦＢＳ、ＢｉｏＷｅｓｔ社製）、５ｎｇ／ｍＬインターロイキン７、及び５ｎｇ／ｍＬインターロイキン１５を添加したαＭＥＭ（α改変型イーグル最小必須）培地）で適宜培地交換しながら、３７℃、５％ＣＯ２の条件下で１４日間培養した結果、増殖率が刺激前の８１倍となったＴ細胞；
低増殖Ｔ細胞（５種類）：ｉＰＳ細胞由来Ｔ細胞のＴＣＲを前記ＴＣＲ刺激磁性ビーズを用いて刺激し、培地Ａで適宜培地交換しながら、３７℃、５％ＣＯ２の条件下で１４日間培養した結果、増殖率が刺激前の２０倍以下となったＴ細胞。 <Selection of marker candidates for T cell evaluation>
1. Secretome analysis of high-proliferation T cells and low-proliferation T cells (1) As high-proliferation T cells and low-proliferation T cells, the following cells were used, respectively.
High-proliferation T cells (1 type): T cell receptors (TCR) of naive T cells (CD45RA + , CCR7 + ) collected from human peripheral blood mononuclear cells were treated with Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (manufactured by Thermo Fisher Scientific) (hereinafter referred to as "TCR stimulation magnetic beads") was used for stimulation, medium A (15% (w/v) fetal bovine serum (FBS, BioWest), 5 ng/ The cells were cultured for 14 days under conditions of 37° C. and 5% CO 2 while appropriately replacing the medium with αMEM (α-modified Eagle minimum essential) medium supplemented with mL interleukin 7 and 5 ng/mL interleukin 15. T cells with a proliferation rate of 81 times that before stimulation;
Low-proliferation T cells (5 types): TCR of iPS cell-derived T cells was stimulated using the TCR-stimulating magnetic beads, and medium A was changed as appropriate for 14 days at 37°C and 5% CO2 . As a result of culturing, T cells whose proliferation rate is 20 times or less before stimulation.
　（２）上記（１）の高増殖Ｔ細胞及び低増殖Ｔ細胞のＴＣＲを前記ＴＣＲ刺激磁性ビーズを用いて刺激し、培地Ａで適宜培地交換しながら、５％ＣＯ２、３７℃の条件下で６日間培養した。 (2) The TCR of the high-proliferation T cells and low-proliferation T cells of (1) above is stimulated using the TCR-stimulating magnetic beads, and medium A is exchanged as appropriate under conditions of 5% CO 2 and 37°C. was cultured for 6 days.
　（３）上記（２）で培養した各Ｔ細胞をそれぞれ１×１０６ｃｅｌｌｓ回収し、１５％（ｗ／ｖ）透析ＦＢＳ、４ｍｍｏｌ／Ｌ　Ｌ－グルタミン、ＩＴＳ（インシュリン－トランスフェリン－亜セレン酸ナトリウム）サプリメント（×１）、５０μｇ／ｍＬアスコルビン酸、５ｎｇ／ｍＬ　ＩＬ－７、及び５ｎｇ／ｍＬ　ＩＬ－１５を添加したαＭＥＭ（－Ｍｅｔ）培地（α改変型イーグル最小必須培地、メチオニン不含有）で懸濁して、３７℃、５％ＣＯ２の条件下で１時間培養後、０．００６２５ｍＭ　Ｌ－アジドホモアラニン（ＡＨＡ）、１５％（ｗ／ｖ）透析ＦＢＳ、４ｍＭ　Ｌ－Ｇｌｕｔａｍｉｎｅ、ＩＴＳ（×１）、５０μｇ／ｍＬアスコルビン酸、５ｎｇ／ｍＬ　ＩＬ－７、及び５ｎｇ／ｍＬ　ＩＬ－１５を添加したαＭＥＭ（－Ｍｅｔ）培地で、３７℃、５％ＣＯ２の条件下で２４時間培養した。培養後、培養上清０．４ｍＬを回収し、Ｃｌｉｃｋ－ｉＴＴＭ　Ｐｒｏｔｅｉｎ　Ｅｎｒｉｃｈｍｅｎｔ　Ｋｉｔ，ｆｏｒ　ｃｌｉｃｋ　ｃｈｅｍｉｓｔｒｙ　ｃａｐｔｕｒｅ　ｏｆ　ａｚｉｄｅ－ｍｏｄｉｆｉｅｄ　ｐｒｏｔｅｉｎ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を用いて、培養上清に分泌された、ＡＨＡで標識されたタンパク質（セクレトーム）をＡＨＡのアジド基をクリックケミストリーを用いてアルキンアガロース樹脂にチャプターすることによって抽出した。 (3) Collect 1×10 6 cells of each T cell cultured in (2) above, add 15% (w/v) dialyzed FBS, 4 mmol/L L-glutamine, ITS (insulin-transferrin-sodium selenite ) in αMEM (-Met) medium (α-modified Eagle minimal essential medium, no methionine) supplemented with supplements (×1), 50 μg/mL ascorbic acid, 5 ng/mL IL-7, and 5 ng/mL IL-15 After suspending and culturing for 1 hour at 37° C., 5% CO 2 , 0.00625 mM L-azidohomoalanine (AHA), 15% (w/v) dialyzed FBS, 4 mM L-glutamine, ITS (× 1), cultured for 24 hours at 37° C., 5% CO 2 in αMEM (-Met) medium supplemented with 50 μg/mL ascorbic acid, 5 ng/mL IL-7, and 5 ng/mL IL-15. After culturing, 0.4 mL of the culture supernatant was collected, and AHA secreted into the culture supernatant was extracted using Click-iT ™ Protein Enrichment Kit, for click chemistry capture of azide-modified protein (manufactured by Thermo Fisher Scientific). The labeled protein (secretome) was extracted by chaptering the azide groups of AHA onto alkyne agarose resin using click chemistry.
　（４）上記（３）で抽出したタンパク質をＬＣ－ＭＳ／ＭＳ（Ｕｌｔｉｍａｔｅ　３０００　ＲＳＬＣｎａｎｏ及びＱ　Ｅｘａｃｔｉｖｅ；いずれもＴｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）で解析した。解析データから、Ｐｒｏｔｅｏｍｅ　Ｄｉｓｃｏｖｅｒｅｒソフトウェア（ｖｅｒ．２．２、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）を用いて、抽出したタンパク質のアミノ酸配列を抽出した。 (4) The proteins extracted in (3) above were analyzed by LC-MS/MS (Ultimate 3000 RSLCnano and Q Exactive; both manufactured by Thermo Fisher Scientific). From the analyzed data, the amino acid sequences of the extracted proteins were extracted using Proteome Discoverer software (ver.2.2, manufactured by Thermo Fisher Scientific).
　（５）Ｍａｓｃｏｔソフトウェア（ｖｅｒ．２．５、Ｍａｔｒｉｘ　Ｓｃｉｅｎｃｅ　社製）を用い、下記（Ａ）～（Ｃ）に記載のアミノ酸配列を組み合わせて構築したデータベースと照合することで、抽出したアミノ酸配列からタンパク質を同定した。なお、有意なタンパク質の同定は、同定のスコア閾値を、Ｆａｌｓｅ　ｄｉｓｃｏｖｅｒｙ　ｒａｔｅ（ＦＤＲ）が１％になるように調整して行なった。同定したタンパク質は、Ｐｒｏｔｅｏｍｅ　Ｄｉｓｃｏｖｅｒｅｒ　２．２のＬａｂｅｌ　Ｆｒｅｅ　Ｑｕａｎｔｉｆｉｃａｔｉｏｎ（ＬＦＱ）を用いて定量した。
（Ａ）ＳｗｉｓｓＰｒｏｔ（ｈｔｔｐ：／／ｗｗｗ．ｕｎｉｐｒｏｔ．ｏｒｇ／）２０１８＿１１版に登録されているヒト（Ｈｏｍｏ　ｓａｐｉｅｎｓ）由来のタンパク質エントリー（計２０４１３件）；
（Ｂ）ＳｗｉｓｓＰｒｏｔ（ｈｔｔｐ：／／ｗｗｗ．ｕｎｉｐｒｏｔ．ｏｒｇ／）２０１８＿１１版に登録されているウシ（Ｂｏｓ　ｔａｕｒｕｓ）由来のタンパク質エントリー（計５９９２件）；
（Ｃ）Ｔｈｅ　Ｇｌｏｂａｌ　Ｐｒｏｔｅｏｍｅ　Ｍａｃｈｉｎｅ　Ｏｒｇａｎｉｚａｔｉｏｎ（ｈｔｔｐ：／／ｗｗｗ．ｔｈｅｇｐｍ．ｏｒｇ／ｃｒａｐ／ｉｎｄｅｘ．ｈｔｍｌ）から取得した、ヒト由来及びウシ由来以外のタンパク質汚染物のアミノ酸配列（計４６件）。 (5) Using Mascot software (ver.2.5, manufactured by Matrix Science), by matching with a database constructed by combining the amino acid sequences described in (A) to (C) below, from the extracted amino acid sequence protein was identified. The identification of significant proteins was performed by adjusting the identification score threshold so that the false discovery rate (FDR) was 1%. Identified proteins were quantified using Label Free Quantification (LFQ) in Proteome Discoverer 2.2.
(A) Homo sapiens-derived protein entries registered in SwissProt (http://www.uniprot.org/) version 2018_11 (20413 in total);
(B) protein entries from bovine (Bos taurus) registered in SwissProt (http://www.uniprot.org/) version 2018_11 (5992 in total);
(C) Amino acid sequences of human and non-bovine protein contaminants obtained from The Global Proteome Machine Organization (http://www.thegpm.org/crap/index.html) (46 in total).
　２．高増殖Ｔ細胞及び低増殖Ｔ細胞のトランスクリプトーム解析
　（１）上記１の（２）で刺激及び培養したＴ細胞をそれぞれ回収し、ＲＮｅａｓｙ　Ｍｉｃｒｏ　Ｋｉｔ（ＱＩＡＧＥＮ社製）を用いて、Ｔｏｔａｌ　ＲＮＡを抽出した。１０ｎｇのＴｏｔａｌ　ＲＮＡから、ＳＭＡＲＴ－Ｓｅｑ　ｖ４　Ｕｌｔｒａ　Ｌｏｗ　Ｉｎｐｕｔ　ＲＮＡ　Ｋｉｔ　ｆｏｒ　Ｓｅｑｕｅｎｃｉｎｇ（Ｃｌｏｎｔｅｃｈ社製）を用いて、ｃＤＮＡを合成・増幅した。 2. Transcriptome analysis of high-proliferation T cells and low-proliferation T cells (1) The T cells stimulated and cultured in (2) of 1 above were collected, and total RNA was extracted using RNeasy Micro Kit (manufactured by QIAGEN). Extracted. cDNA was synthesized and amplified from 10 ng of total RNA using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (manufactured by Clontech).
　（２）上記（１）で得られたｃＤＮＡ　１ｎｇ分を使用して、Ｎｅｘｔｅｒａ　ＸＴ　ＤＮＡ　Ｌｉｂｒａｒｙ　Ｐｒｅｐａｒａｔｉｏｎ　Ｋｉｔ（Ｉｌｌｕｍｉｎａ社製）及びＮｅｘｔｅｒａ　ＸＴ　ｖ２　Ｉｎｄｅｘ　Ｋｉｔ　Ｓｅｔ　Ａ（Ｉｌｌｕｍｉｎａ社製）を用いてライブラリー調製を行ない、Ｎｅｘｔ－ｓｅｑ５００（ｉｌｌｕｍｉｎａ社製）を用いて、リード長：７５ｂｐ、ｓｉｎｇｌｅ　ｅｎｄ　ｒｅａｄの条件でシークエンス解析を行うことにより、１試料あたり５００万リード以上の遺伝子を解読した。 (2) Using 1 ng of cDNA obtained in (1) above, prepare a library using Nextera XT DNA Library Preparation Kit (manufactured by Illumina) and Nextera XT v2 Index Kit Set A (manufactured by Illumina). Using Next-seq500 (manufactured by Illumina Inc.), sequence analysis was performed under the conditions of read length: 75 bp, single end read, and more than 5 million reads of genes per sample were decoded.
　（３）上記（２）で解読した遺伝子（シークエンスデータ）をＴｏｐＨａｔ２（ＪＯＨＮＳ　ＨＯＰＫＩＮＳ大学）及びＢｏｗｔｉｅ２（ＪＯＨＮＳ　ＨＯＰＫＩＮＳ大学）を用いて、ヒトゲノム配列にマップした。なお、ヒトゲノム配列情報及びヒト遺伝子情報には、ＮＣＢＩ（Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｌｏｇｉｃａｌ　Ｉｎｆｏｒｍａｔｉｏｎ）より公開されているＢＵＩＬＤ　ＧＲＣｈ３８を使用した。マップした遺伝子について、Ｃｕｆｆｌｉｎｋｓ（Ｗａｓｈｉｎｇｔｏｎ大学）によって、解読した各遺伝子のリード数から、ＦＰＫＭ（Ｆｒａｇｍｅｎｔｓ　Ｐｅｒ　Ｋｉｌｏｂａｓｅ　ｏｆ　ｅｘｏｎ　ｐｅｒ　Ｍｉｌｌｉｏｎ　ｒｅａｄｓ　ｍａｐｐｅｄ）を単位とする遺伝子ごとの発現量を求めた。 (3) The genes (sequence data) decoded in (2) above were mapped to the human genome sequence using TopHat2 (JOHNS HOPKINS University) and Bowtie2 (JOHNS HOPKINS University). For human genome sequence information and human gene information, BUILD GRCh38 published by NCBI (National Center for Biological Information) was used. For the mapped genes, the expression level for each gene was determined in units of FPKM (fragments per kilobase of exon per million reads mapped) from the number of reads of each gene decoded by Cufflinks (University of Washington).
　３．パスウェイ解析
　（１）上記１の（５）で得られたセクレトーム解析結果（同定したタンパク質の定量値）及び上記２の（３）で得られたトランスクリプトーム解析結果（マップした遺伝子ごとの発現量）を統合し、高増殖Ｔ細胞と低増殖Ｔ細胞群とを比較して、次の条件：セクレトーム解析結果についてはｕｎｉｑｕｅ　ｐｅｐｔｉｄｅ　２以上、かつ、トランスクリプトーム解析結果については発現量比０．６７以下又は１．５以上で絞りこみ、相互関係検索法を用いたＫｅｙＭｏｌｎｅｔ解析により、高増殖Ｔ細胞と低増殖Ｔ細胞のとの間で発現量の差が大きかった遺伝子がコードするタンパク質が属するシグナル伝達経路（下記式で算出されるスコアが大きいシグナル伝達経路；下記式中、パスウェイに属している分子とは、シグナル伝達経路に属しているタンパク質を示す）を特定した。前記スコアは、統計学的手法(超幾何分布)に基づいて下記式で算出される総合的な関与度を示す値であり、値が大きいほどそのシグナル伝達経路が有意であることを示す。 3. Pathway analysis (1) Secretome analysis results obtained in 1 (5) above (quantitative values of identified proteins) and transcriptome analysis results obtained in 2 above (3) (expression level for each mapped gene ) were integrated, and the high-proliferation T cells and the low-proliferation T-cell group were compared, and the following conditions: secretome analysis results of unique peptide 2 or more, and transcriptome analysis results of expression ratio 0.67 Signals to which proteins encoded by genes whose expression levels differ significantly between high-proliferation T cells and low-proliferation T cells by KeyMolnet analysis using the correlation search method, narrowed down to less than or 1.5 or more Transduction pathways (signaling pathways with high scores calculated by the following formula; in the following formula, molecules belonging to pathways indicate proteins belonging to the signaling pathways). The score is a value indicating the overall degree of involvement calculated by the following formula based on a statistical method (hypergeometric distribution), and the higher the value, the more significant the signal transduction pathway.
Figure JPOXMLDOC01-appb-M000001
Figure JPOXMLDOC01-appb-M000001
　パスウェイ解析により得られたスコアが大きい順に、１位～１０位のシグナル伝達経路及びそのスコア（Ｓｃｏｒｅ）を下記の表１に示す。表１に示す経路の中から、５位のアデノシンシグナル伝達経路及び９位のＰ２Ｙシグナル伝達経路を選択し、Ｔｒｅｎｄｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｍｅｄｉｃｉｎｅ，Ｖｏｌ．１９，２０１３年，ｐ．３５５－３６７より、これらのシグナル伝達経路に関わるタンパク質であるＣＤ３９及びＣＤ７３をＴ細胞の品質評価用マーカー候補として選定した。 Table 1 below shows the 1st to 10th signal transduction pathways and their scores (Scores) in descending order of scores obtained by pathway analysis. From the pathways shown in Table 1, the adenosine signaling pathway at position 5 and the P2Y signaling pathway at position 9 were selected and analyzed in Trends in Molecular Medicine, Vol. 19, 2013, p. 355-367, CD39 and CD73, which are proteins involved in these signaling pathways, were selected as candidate markers for quality evaluation of T cells.
Figure JPOXMLDOC01-appb-T000002
  
 
Figure JPOXMLDOC01-appb-T000002
  
 
　＜Ｔ細胞評価用マーカー候補の検証１＞
　上記＜Ｔ細胞評価用マーカー候補の選定＞で選定したＣＤ３９及びＣＤ７３がＴ細胞の品質評価用マーカーとして利用可能かについて検証した。 <Verification of marker candidate for T cell evaluation 1>
It was verified whether CD39 and CD73 selected in <Selection of T cell evaluation marker candidates> can be used as T cell quality evaluation markers.
　（１）Ｔ細胞の品質の評価項目の１つである増殖能の異なるＴ細胞（２２種類）として、以下に示す細胞を用いた。
初代Ｔ細胞（９種類）：ｎａｉｖｅ　ＣＴＬ由来Ｔ細胞に異なる培養条件又は刺激回数で刺激を与え、それぞれ異なる増殖能を示すようになったＴ細胞；
ｉＰＳ細胞由来Ｔ細胞（１３種類）：異なるｉＰＳ細胞に由来し、それぞれ異なる増殖能を有するＴ細胞。 (1) The following cells were used as T cells (22 types) with different proliferative potentials, which is one of the evaluation items of T cell quality.
Primary T cells (9 types): T cells that have been stimulated with different culture conditions or stimulation times to naïve CTL-derived T cells, and that have shown different proliferative abilities;
iPS cell-derived T cells (13 types): T cells derived from different iPS cells and having different proliferative potentials.
　（２）上記（１）の初代Ｔ細胞又はｉＰＳ細胞由来Ｔ細胞のＴＣＲを前記ＴＣＲ刺激磁性ビーズを用いて刺激し、培地Ａで適宜培地交換しながら、５％ＣＯ２、３７℃の条件下で１４日間培養した。培養したＴ細胞をそれぞれ回収し、上記＜Ｔ細胞評価用マーカー候補の選定＞の２と同様にしてトランスクリプトーム解析を行い、ＣＤ３９又はＣＤ７３をコードする遺伝子の発現量を求めた。 (2) The TCR of the primary T cells or iPS cell-derived T cells of (1) above is stimulated using the TCR-stimulating magnetic beads, and the medium is replaced with medium A as appropriate under conditions of 5% CO 2 and 37°C. for 14 days. The cultured T cells were collected, and transcriptome analysis was performed in the same manner as in 2 of <Selection of Marker Candidates for Evaluation of T Cells> above to determine the expression level of the gene encoding CD39 or CD73.
　各Ｔ細胞の増殖能（Ｆｏｌｄ　ｅｘｐａｎｓｉｏｎ）をＸ軸に、当該Ｔ細胞におけるＣＤ３９又はＣＤ７３をコードする遺伝子の発現量（ＣＤ３９　ｅｘｐｒｅｓｓｉｏｎ又はＣＤ７３　ｅｘｐｒｅｓｓｉｏｎ）をＹ軸に、それぞれプロットしたグラフを図１に示す。各Ｔ細胞の増殖能は、上記（２）でＴ細胞をＴＣＲ刺激磁性ビーズで刺激したときの、刺激前の細胞数と刺激から１４日後の細胞数との比（１４日後の細胞数／刺激前の細胞数）で求めた値である。ＣＤ３９をコードする遺伝子の発現量は、増殖能の違いに基づいて変化した。具体的には、増殖能が低いＴ細胞の方がＣＤ３９をコードする遺伝子の発現量は高かった（図１（ａ）、Ｒ２＝０．５０８５）。このことから、ＣＤ３９は、Ｔ細胞の増殖能評価用マーカー（例えば、疲弊化マーカー）として利用できることが確認された。なお、ＣＤ３９は、配列番号１に記載のアミノ酸配列で示される。他方、ＣＤ７３をコードする遺伝子の発現量は、増殖能の違いによる変化がほとんどなく（図１（ｂ）、Ｒ２＝０．００６８）、Ｔ細胞の増殖能評価用マーカーとしては適さないことが確認された（ＣＤ７３：比較例）。 FIG. 1 shows a graph in which the proliferative capacity (fold expansion) of each T cell is plotted on the X axis and the expression level of the gene encoding CD39 or CD73 in the T cells (CD39 expression or CD73 expression) is plotted on the Y axis. . The proliferative ability of each T cell is the ratio of the number of cells before stimulation to the number of cells 14 days after stimulation (number of cells after 14 days/stimulation cell number). Expression levels of the gene encoding CD39 varied based on differences in proliferative capacity. Specifically, the expression level of the gene encoding CD39 was higher in T cells with lower proliferation ability (Fig. 1(a), R2 = 0.5085). From this, it was confirmed that CD39 can be used as a marker for T cell proliferative ability evaluation (for example, an exhaustion marker). CD39 is represented by the amino acid sequence shown in SEQ ID NO:1. On the other hand, the expression level of the gene encoding CD73 hardly changes due to differences in proliferation ability (Fig. 1(b), R2 = 0.0068), and is not suitable as a marker for evaluating T cell proliferation ability. It was confirmed (CD73: Comparative Example).
　＜Ｔ細胞評価用マーカー候補の検証２＞
　上記＜Ｔ細胞評価用マーカー候補の選定＞で選定したＣＤ３９と公知の疲弊化マーカーであるＰＤ－１との発現時期を比較した。 <Verification of T cell evaluation marker candidate 2>
The expression times of CD39 selected in <Selection of T cell evaluation marker candidates> and PD-1, which is a known exhaustion marker, were compared.
　（１）ナイーブ形質（ＣＤ４５ＲＡ＋、ＣＣＲ７＋）を示すｉＰＳ細胞由来Ｔ細胞に対して、マイトジェンであるＰＨＡ（Ｐｈｙｔｏｈｅｍａｇｇｌｕｔｉｎｉｎ）及び末梢血単核球フィーダーにより刺激を与え、培地Ａで適宜培地交換しながら、３７℃、５％ＣＯ２の条件下で１４日間培養する刺激処理を１０回繰り返した。刺激処理１回後及び刺激処理１０回後のＣＤ３９、ＣＤ４５ＲＡ、ＣＤ４５ＲＯ、及びＰＤ－１の細胞表面での発現量をフローサイトメトリーで測定した。 (1) iPS cell-derived T cells exhibiting naive traits (CD45RA + , CCR7 + ) were stimulated with the mitogen PHA (Phytohemagglutinin) and a peripheral blood mononuclear cell feeder, and medium A was exchanged as appropriate. , 37° C., 5% CO 2 for 14 days, and stimulation treatment was repeated 10 times. The expression levels of CD39, CD45RA, CD45RO, and PD-1 on the cell surface after one stimulation treatment and after ten stimulation treatments were measured by flow cytometry.
　刺激処理１回後及び刺激処理１０回後の各Ｔ細胞におけるＣＤ４５ＲＡの発現量をＹ軸に、当該Ｔ細胞におけるＣＤ４５ＲＯ、ＣＤ３９、又はＰＤ－１の発現量をＸ軸に、それぞれプロットした結果を図２に示す。前記刺激処理１回後に比べて、前記刺激処理１０回後においては、ナイーブ細胞のマーカーであるＣＤ４５ＲＡの発現量が低下し、かつ、ＣＤ４５ＲＯの発現量が上昇して、Ｔ細胞の分化が進んだことが確認された（図２（ａ））。ＣＤ３９の発現量は、ＴＣＲの刺激の繰り返しにより上昇し、ＣＤ４５ＲＡの発現量と負の相関関係を示した（図２（ｂ））。他方、ＰＤ－１の発現量は、刺激処理１０回後でも上昇は軽微であった。このことから、ＣＤ３９は、従来公知の疲弊化マーカーであるＰＤ－１よりも初期に発現する疲弊化マーカーとして利用できることが示唆された。 The results of plotting the expression levels of CD45RA in each T cell after 1 stimulation treatment and 10 stimulation treatments on the Y axis and the expression levels of CD45RO, CD39, or PD-1 in the T cells on the X axis, respectively. Shown in FIG. After 10 stimulation treatments compared to after 1 stimulation treatment, the expression level of CD45RA, a marker for naive cells, was decreased and the expression level of CD45RO was increased, and T cell differentiation progressed. It was confirmed (Fig. 2(a)). The CD39 expression level was elevated by repeated TCR stimulation, and showed a negative correlation with the CD45RA expression level (Fig. 2(b)). On the other hand, the expression level of PD-1 showed a slight increase even after 10 stimulation treatments. This suggests that CD39 can be used as an exhaustion marker that is expressed earlier than PD-1, which is a conventionally known exhaustion marker.
　本発明によれば、新たな指標によってＴ細胞の品質を評価できるＴ細胞の品質評価方法、及びそれに用いるＴ細胞の品質評価用試薬を提供することが可能となる。 According to the present invention, it is possible to provide a T cell quality evaluation method capable of evaluating T cell quality using a new index, and a T cell quality evaluation reagent used for the method.

Claims (13)

  1. 　Ｔ細胞における、下記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
    　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
    を含む、Ｔ細胞の品質評価方法。
    （ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
    （ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
    （ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。     a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells;
    an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
    A method for assessing T cell quality, comprising:
    (i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
  2. 　前記検出工程が、前記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現産物に特異的に結合するプローブ分子を用いて前記発現産物を検出する工程である、請求項１に記載の方法。 The detecting step is a step of detecting the expression product using a probe molecule that specifically binds to the expression product of at least one gene selected from the group consisting of the genes (i) to (iii). A method according to claim 1.
  3. 　前記発現産物が、前記（ｉ）～（ｉｉｉ）の遺伝子のうちのいずれか１つにコードされるポリペプチドであり、かつ、前記プローブ分子が、前記ポリペプチドに特異的に結合する抗体及び／又はアプタマーである、請求項２に記載の方法。 The expression product is a polypeptide encoded by any one of the genes (i) to (iii), and the probe molecule is an antibody that specifically binds to the polypeptide and/or or an aptamer.
  4. 　前記評価工程が、前記遺伝子の発現を指標としてＴ細胞の増殖能を評価する工程である、請求項１～３のいずれか一項に記載の方法。 　The method according to any one of claims 1 to 3, wherein the evaluation step is a step of evaluating the proliferative ability of T cells using the expression of the gene as an index.
  5. 　請求項２～４のいずれか一項に記載のＴ細胞の品質評価方法に用いるための試薬であり、下記（ｉ）～（ｉｉｉ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現産物に特異的に結合するプローブ分子を含有する、Ｔ細胞の品質評価用試薬。
    （ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
    （ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
    （ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。     A reagent for use in the T cell quality evaluation method according to any one of claims 2 to 4, wherein at least one gene selected from the group consisting of the following genes (i) to (iii) A T cell quality assessment reagent containing a probe molecule that specifically binds to an expression product.
    (i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
  6. 　前記発現産物が、前記（ｉ）～（ｉｉｉ）の遺伝子のうちのいずれか１つにコードされるポリペプチドであり、かつ、前記プローブ分子が、前記ポリペプチドに特異的に結合する抗体及び／又はアプタマーである、請求項５に記載の試薬。 The expression product is a polypeptide encoded by any one of the genes (i) to (iii), and the probe molecule is an antibody that specifically binds to the polypeptide and/or or an aptamer, the reagent of claim 5.
  7. 　請求項１～４のいずれか一項に記載のＴ細胞の評価方法により得られた高品質Ｔ細胞。 High-quality T cells obtained by the T cell evaluation method according to any one of claims 1 to 4.
  8. 　請求項７に記載の高品質Ｔ細胞を含有する医薬組成物。 A pharmaceutical composition containing the high-quality T cells according to claim 7.
  9. 　がんの予防又は治療に使用するための請求項８に記載の医薬組成物。 The pharmaceutical composition according to claim 8 for use in preventing or treating cancer.
  10. 　請求項８に記載の医薬組成物を用いる、がんの予防または治療方法。 A method for preventing or treating cancer using the pharmaceutical composition according to claim 8.
  11. 　がんを有するかまたはがんを有する危険性のある被験者に、請求項８に記載の医薬組成物を投与することを含む、がんを治療または予防する方法。 A method of treating or preventing cancer, comprising administering the pharmaceutical composition of claim 8 to a subject who has cancer or is at risk of having cancer.
  12. 　がんを有するかまたはがんを有する危険性のある被験者に、Ｔ細胞を投与することを含む、がんを治療または予防する方法であって、該Ｔ細胞における、下記（Ｉ）～（ＩＩＩ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
    　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
    を含む、方法。
    （ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
    （ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
    （ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。     A method of treating or preventing cancer, comprising administering T cells to a subject who has cancer or is at risk of having cancer, wherein the following (I) to (III) in the T cells ) detecting the expression of at least one gene selected from the group consisting of genes;
    an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
    A method, including
    (i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
  13. 　Ｔ細胞の製造方法であって、該Ｔ細胞における、下記（Ｉ）～（ＩＩＩ）の遺伝子からなる群から選択される少なくとも１種の遺伝子の発現を検出する検出工程と、
    　前記遺伝子の発現を指標としてＴ細胞の品質を評価する評価工程と、
    を含む、Ｔ細胞の製造方法。
    （ｉ）配列番号１に記載のアミノ酸配列を含むポリペプチドをコードする遺伝子
    （ｉｉ）配列番号１に記載のアミノ酸配列において、１若しくは複数個のアミノ酸残基が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を含むポリペプチドをコードする遺伝子
    （ｉｉｉ）配列番号１に記載のアミノ酸配列と７０％以上の同一性を有するアミノ酸配列を含むポリペプチドをコードする遺伝子。     A method for producing T cells, comprising a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (I) to (III) in the T cells;
    an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
    A method of producing a T cell, comprising:
    (i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170233808A1 (en) * 2014-10-17 2017-08-17 Dana-Farber Cancer Institute, Inc. Compositions and methods for identification, assessment, prevention, and treatment of t-cell exhaustion using cd39 biomarkers and modulators
JP2019519246A (en) * 2016-06-28 2019-07-11 ジーニアス・バイオテクノロジー・インコーポレイテッド T cell composition for immunotherapy
WO2020251046A1 (en) * 2019-06-14 2020-12-17 サイアス株式会社 Medicinal composition
JP2021505615A (en) * 2017-12-08 2021-02-18 ジュノー セラピューティクス インコーポレイテッド Phenotypic markers for cell therapy and related methods

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170233808A1 (en) * 2014-10-17 2017-08-17 Dana-Farber Cancer Institute, Inc. Compositions and methods for identification, assessment, prevention, and treatment of t-cell exhaustion using cd39 biomarkers and modulators
JP2019519246A (en) * 2016-06-28 2019-07-11 ジーニアス・バイオテクノロジー・インコーポレイテッド T cell composition for immunotherapy
JP2021505615A (en) * 2017-12-08 2021-02-18 ジュノー セラピューティクス インコーポレイテッド Phenotypic markers for cell therapy and related methods
WO2020251046A1 (en) * 2019-06-14 2020-12-17 サイアス株式会社 Medicinal composition

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FERNANDO P. CANALE, MARIA C. RAMELLO, NICOLAS NUNEZ, CINTIA L. ARAUJO FURLAN, SABRINA N. BOSSIO, MELISA GOROSITO SERRAN, JIMENA TO: "CD39 Expression Defines Cell Exhaustion in Tumor-Infiltrating CD8 + T Cells", CANCER RESEARCH, vol. 78, no. 1, 1 January 2018 (2018-01-01), US, pages 115 - 128, XP055662774, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-16-2684 *

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