WO2023074713A1 - Procédé d'évaluation de qualité de cellules t, et réactif mettant en œuvre ce procédé - Google Patents

Procédé d'évaluation de qualité de cellules t, et réactif mettant en œuvre ce procédé 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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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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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10Cells modified by introduction of foreign genetic material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881Nucleic 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; 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

L'invention concerne un procédé d'évaluation de qualité de cellules T qui inclut : une étape de détection au cours de laquelle l'expression d'au moins une sorte de gène choisie dans un groupe constitué de gènes (i) à (iii), est détectée dans des cellules T ; et une étape d'évaluation au cours de laquelle la qualité des cellules T est évaluée sur la base de l'expression dudit gène. (i) Gène codant un polypeptide contenant une séquence d'acides aminés représentée par SEQ ID NO : 1. (ii) Gène codant un polypeptide contenant une séquence d'acides aminés dans laquelle un ou plusieurs résidus d'acides aminés sont substitués, délétés, insérés et/ou additionnés dans une séquence d'acides aminés représentée par SEQ ID NO : 1. (iii) Gène codant un polypeptide contenant une séquence d'acides aminés à au moins 70% identique à une séquence d'acides aminés représentée par SEQ ID NO : 1.
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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 (ja) * 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 免疫療法向けのt細胞組成物
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JP2021505615A (ja) * 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 細胞療法および関連方法のための表現型マーカー

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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 (ja) * 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 免疫療法向けのt細胞組成物
JP2021505615A (ja) * 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 細胞療法および関連方法のための表現型マーカー
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