WO2023074711A1 - Procédé d'évaluation de cellules t, et composition destinée à l'évaluation de cellules t - Google Patents

Procédé d'évaluation de cellules t, et composition destinée à l'évaluation de cellules t Download PDF

Info

Publication number
WO2023074711A1
WO2023074711A1 PCT/JP2022/039806 JP2022039806W WO2023074711A1 WO 2023074711 A1 WO2023074711 A1 WO 2023074711A1 JP 2022039806 W JP2022039806 W JP 2022039806W WO 2023074711 A1 WO2023074711 A1 WO 2023074711A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
cells
acid sequence
gene
polypeptide
Prior art date
Application number
PCT/JP2022/039806
Other languages
English (en)
Japanese (ja)
Inventor
新 金子
洋平 河合
佳奈 山口
勇一 熊木
達 二見
Original Assignee
国立大学法人京都大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人京都大学 filed Critical 国立大学法人京都大学
Publication of WO2023074711A1 publication Critical patent/WO2023074711A1/fr

Links

Images

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • C12Q1/6813—Hybridisation assays
    • 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 T cells, a composition for evaluating T cells, a pharmaceutical composition containing high-quality T cells obtained by the method for evaluating T cells, and cancer prevention or treatment using the pharmaceutical composition. It relates to treatment methods and the like.
  • 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 describes a method of inducing CD8 ⁇ + ⁇ + CTL by culturing CD4 + CD8 + T cells in a medium containing interleukin 7 and a TCR (T cell receptor) activator.
  • 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 evaluation method capable of evaluating the quality of relatively young T cells using a new index, and a T cell evaluation reagent used therein. for the purpose.
  • secretome secretome
  • high-quality T cells high-quality T cells
  • low-quality T cells T cells with relatively low proliferative potential
  • PCA principal component analysis
  • genes with a high principal component loading value of the first principal component are expressed in cells with a high PC1 score, that is, high-quality T cells, and genes with a low principal component loading value of the first principal component are expressed in It was found to be expressed in cells with low PC1 score, ie low quality T cells.
  • the present inventors used genes with a large absolute value of the principal component loading with respect to the first principal component as markers for quality evaluation of T cells, and used their expression as an index to determine the quality of T cells. We found that quality can be evaluated. Furthermore, since these genes are genes that are expressed relatively early after TCR stimulation of T cells, it was found that the quality of relatively young T cells can also be evaluated by using their expression as an index, and the present invention has been made. I came to complete it.
  • a method for evaluating T cells comprising: (I) a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-77 (II) one or more of the amino acid sequences set forth in any one of SEQ ID NOs: 1-77 70% or more of the amino acid sequence described in any one of gene (III) SEQ ID NOS: 1 to 77 encoding a polypeptide comprising an amino acid sequence in which amino acid residues are substituted, deleted, inserted, and / or added A gene encoding a polypeptide comprising an amino acid sequence having the identity of
  • 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.
  • At least one gene selected from the group consisting of the genes (I) to (III) is at least one gene selected from the group consisting of the following genes (I') to (III') The method according to any one of [1] to [4], which is a gene.
  • At least one gene selected from the group consisting of the genes (I) to (III) is at least one gene selected from the group consisting of the following genes (I') to (III') The method according to any one of [1] to [5], which is a gene.
  • 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 [6] which is an antibody and/or an aptamer.
  • At least one gene selected from the group consisting of the genes (I) to (III) is at least one gene selected from the group consisting of the following genes (I') to (III')
  • At least one gene selected from the group consisting of the genes (I) to (III) is at least one gene selected from the group consisting of the following genes (I') to (III')
  • [15] A method of treating or preventing cancer, comprising administering the pharmaceutical composition of [12] 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 any one of SEQ ID NOs: 1-77 (II) one or more of the amino acid sequences set forth in any one of SEQ ID NOs: 1-77 70% or more of the amino acid sequence described in any one of gene (III) SEQ ID NOS: 1 to 77 encoding a polypeptide comprising an amino acid sequence in which amino acid residues are substituted, deleted, inserted, and / or added A gene encoding a polypeptide comprising an amino acid sequence having the identity of
  • 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 any one of SEQ ID NOs: 1-77 (II) one or more of the amino acid sequences set forth in any one of SEQ ID NOs: 1-77 70% or more of the amino acid sequence described in any one of gene (III) SEQ ID NOS: 1 to 77 encoding a polypeptide comprising an amino acid sequence in which amino acid residues are substituted, deleted, inserted, and / or added A gene encoding a polypeptide comprising an amino acid sequence having the identity of
  • the present invention it is possible to provide a T cell evaluation method that can evaluate the quality of T cells using a new index, and a T cell evaluation reagent used for the method.
  • FIG. 3 is a scatter plot obtained by performing principal component analysis (PCA) on data for all 37,912 genes in 23 T cell lines.
  • FIG. 3 is a diagram showing the relationship between the rank of proliferation rate (proliferation rank) and the rank of PC1 score (PC1 rank) of 23 types of T cell lines.
  • the method for evaluating T cells of the present invention comprises 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 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 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.
  • pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells)
  • ES 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 encephalitis viral vectors, non-viral vectors such as transposon vector piggyBac (registered trademark) to T cells
  • Gene editing of T cells includes, for example, gene editing of T cells using site-specific nucleases (meganuclease, zinc finger nuclease, TALEN, PPR (Pentatricpeptide repeat), CRISPR-Cas, etc.) (genome editing).
  • 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)
  • the expression of at least one gene selected from the group consisting of genes (I) to (III) below is detected in T cells.
  • a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-77 (II) one or more of the amino acid sequences set forth in any one of SEQ ID NOs: 1-77 70% or more of the amino acid sequence described in any one of gene (III) SEQ ID NOS: 1 to 77 encoding a polypeptide comprising an amino acid sequence in which amino acid residues are substituted, deleted, inserted, and / or added
  • the gene (I), the gene (II), and the gene (III) are independently of each other. Two or more types may be used.
  • the polypeptide encoded by any one of the genes (I) to (III) is preferably contained in the T cell-derived secretome.
  • secretome refers to proteins that are secreted from cells or that are exposed outside the cells through cell membranes, and include embodiments in which all or part of these proteins are endogenous to cells.
  • the gene whose expression is to be detected (hereinafter sometimes referred to as "target gene") is, if it is human-derived, typically "(I) any one of SEQ ID NOS: 1 to 77 A gene encoding a polypeptide comprising the amino acid sequence described in .
  • Tables 1 to 5 below show the sequence numbers and GenBank Accession Nos. of each amino acid sequence for the amino acid sequences set forth in SEQ ID NOS: 1 to 77. (GenBank No.); SEQ ID NO of a typical nucleotide sequence of a gene encoding a polypeptide consisting of each amino acid sequence, its open reading frame (ORF position), and GenBank Accession No.
  • Tables 1 to 5 show the origins from which each gene was obtained in the examples below, namely the following (a) to (d): (a) Genes mapped by transcriptome analysis in day0 samples (transcriptome day0) (b) genes mapped by transcriptome analysis in day6 samples (transcriptome day6); (c) genes mapped by transcriptome analysis in day14 samples (transcriptome day14); (d) genes encoding proteins identified by secretome analysis (secretome) are shown together with the "Origin data" column. Furthermore, the principal component loading of the first principal component of the expression level of each gene obtained in (a) to (d) above by the principal component analysis (PCA) of the following example is also included. show.
  • PCA principal component analysis
  • the present inventors have found that in high-quality T cells and low-quality T cells, there is a high correlation between the PC1 score obtained by principal component analysis (PCA) for gene expression levels and the quality of T cells.
  • PCA principal component analysis
  • Genes with high values of principal component loading of one principal component are expressed in cells with high PC1 score, i.e. high-quality T cells, and genes with low values of principal component loading of the first principal component have low PC1 scores. cells, namely low quality T cells.
  • the described genes encoding polypeptides consisting of amino acid sequences represented by SEQ ID NOs: 26 to 77 have a high contribution rate to the first principal component (specifically, the main The absolute value of the component load is 0.9 or more) and encodes a protein other than a transmembrane protein.
  • homologous genes e.g., counterpart genes in organisms other than humans
  • (I) a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 77
  • the target gene can also be the target gene.
  • 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.
  • 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.
  • (IV) hybridizes under stringent conditions with the complementary strand of the nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 77 Also included are “nucleotide sequences”. 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. 2 ⁇ SSC) in 0.5% (w/v) SDS solution at 60° C. for 20 minutes.
  • hybridization can be carried out, for example, according to the method described in the instructions 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.
  • these conditions are only examples, and the required stringency can be achieved by appropriately combining DNA concentration, DNA length, hybridization reaction time, and the like.
  • proteins encoded by homologous genes obtained by such methods usually have a high degree of identity with the amino acid sequence set forth in any one of SEQ ID NOS: 1-77. Therefore, in the embodiment of the target gene according to the present invention, "(III) encodes a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 77 Also includes “genes”. 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 any one of SEQ ID NOs: 1 to 77 is typically preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more. % or more, 90% or more, 95% or more (eg, 96% or more, 97% or more, 98% or more, 99% or more).
  • the target gene is preferably at least one gene selected from the group consisting of the following genes (I') to (III') among the genes listed in Tables 1 to 5. Since the proteins expressed from these genes are exposed outside the membrane as membrane proteins of T cells, after detection, the membrane proteins are used to produce high-quality (or low-quality) T cells. This is because capture, separation, or purification is also possible.
  • (I') a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 7, 15, 27 and 44 (more preferably SEQ ID NOs: 5, 15 and 27) (II') ) Substitution or deletion of one or more amino acid residues in the amino acid sequence set forth in any one of SEQ ID NOs: 5, 7, 15, 27, and 44 (more preferably SEQ ID NOs: 5, 15, and 27) Gene (III') SEQ ID NOS: 5, 7, 15, 27, and 44 (more preferably SEQ ID NOS: 5, 15, and 27) encoding a polypeptide comprising deleted, inserted, and/or added amino acid sequences
  • a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence described in any one of the above, wherein in the group consisting of the genes (I') to (III'), (I The gene of '), the gene of (II'), and the gene of (III') may be each independently of one type or two
  • the target gene according to the present invention from the viewpoint of being able to evaluate the quality of younger T cells (less days after TCR stimulation), among the genes shown in Tables 1 to 5 above, "derived data" Genes whose column is "transcriptome day 0" are preferred.
  • 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). In some cases, this produces different 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 at least one selected from the group consisting of the genes (I) to (III) (preferably the genes (I') to (III'))
  • the expression product of a gene is detected using a probe molecule that specifically binds to said expression product.
  • 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 FITC (fluorescein isothiocyanate), FAM (fluorescein amidite), DEAC (7-(diethylamino)coumarin), R6G (rhodamine 6G), Texas Red, Cy5, and BODIPY FL (trade name).
  • luminescence substances such as luminol, luciferin, and lucigenin.
  • 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, selected from the group consisting of the following polypeptides (i) to (iii) is a polypeptide that is (i) a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOS: 1-77; (ii) one or more amino acid residues in the amino acid sequence set forth in any one of SEQ ID NOS: 1-77; is a substituted, deleted, inserted, and / or added amino acid sequence (iii) an amino acid sequence having 70% or more identity with the amino acid sequence set forth in any one of SEQ ID NOS: 1 to 77
  • polypeptides (i) to (iii) are each independently one species alone, may be two or more.
  • polypeptides (i) to (iii) include polypeptides encoded by any one of the genes (I') to (III'), that is, the following (i' ) to (iii') are preferably selected from the group consisting of polypeptides.
  • polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 7, 15, 27 and 44 (more preferably SEQ ID NOs: 5, 15 and 27);
  • SEQ ID NO: 5 , 7, 15, 27, and 44 (more preferably SEQ ID NOS: 5, 15, and 27), wherein one or more amino acid residues are substituted, deleted, inserted, and/or an amino acid according to any one of SEQ ID NOs: 5, 7, 15, 27 and 44 (more preferably SEQ ID NOs: 5, 15 and 27), comprising a polypeptide (iii') comprising an added amino acid sequence
  • a polypeptide comprising an amino acid sequence having 70% or more identity with a sequence.
  • polypeptides (i') to (iii') each independently It may be one type alone or two or more types.
  • these target polypeptides to be detected are preferably the above secretomes (T cell-derived secretomes).
  • detection methods for detection at the translation level include detection using anti-polypeptide antibodies and/or aptamers, and detection using labeled amino acids.
  • 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 uses an enzyme immunoassay device such as AIA-900 (manufactured by Tosoh Corporation) or a chemiluminescent enzyme immunoassay device such as AIA-CL2400 (manufactured by Tosoh Corporation) after preparing necessary reagents. can be done automatically.
  • AIA-900 manufactured by Tosoh Corporation
  • AIA-CL2400 manufactured by Tosoh Corporation
  • 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, short-chain antibodies, 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.
  • the hybridoma method includes, for example, Kohler and Milstein's method (Kohler & Milstein, Nature, 256:495 (1975)), and the recombinant DNA method includes, for example, the 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)).
  • 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.
  • 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, one of the atoms constituting methionine.
  • 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 is labeled with the labeling amino acid and is secreted or exposed outside the cell is detected.
  • the detection step A may be performed simultaneously (in real time) with the 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 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 can be confirmed, for example, by stimulating the TCR of T cells with TCR-stimulating magnetic beads by the method shown in the Examples below and comparing the cell count before stimulation to the cell count 14 days after stimulation. can, but is not limited to.
  • genes with a positive principal component loading value have a higher proliferation ability as the expression level of the gene increases. That is, it can be evaluated that the quality is good.
  • a gene with a negative principal component loading value can be evaluated as having a lower proliferation ability, ie, a lower quality, as the expression level of the gene increases.
  • 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 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 small amount is detected, the proliferative potential of T cells may be evaluated as high (or low), and if the expression level of the target gene above a certain threshold is detected, the proliferative potential of T cells is high (or low). 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
  • a certain value or more, or the expression level of the target gene relative 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 high (or low) proliferative ability If the signal amount is 4 SD or more, 3 SD or more, or 2 SD or more of the average value of , the proliferative ability is high (or low), that is, the quality may be evaluated as good (or bad).
  • the T cell evaluation reagent of the present invention is a reagent (composition) for use in the T cell evaluation method of the present invention, and is at least one gene selected from the group consisting of the following genes (I) to (III).
  • a reagent containing a probe molecule that specifically binds to an expression product of a gene of a species hereinafter sometimes simply referred to as "the reagent of the present invention”).
  • the genes (I) to (III) and the 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 kit for evaluating T cells of the present invention is a kit for use in the method of evaluating T cells of the present invention, and contains at least one gene selected from the group consisting of genes (I) to (III). It is a kit comprising a probe molecule that specifically binds to an expression product (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; A method ((I) a gene encoding a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOS: 1-77, comprising , gene (III) encoding a polypeptide comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and/or added amino acid according to any one of SEQ ID NOs: 1 to 77 A gene encoding a polypeptide comprising an amino acid sequence having greater than 70% identity with the sequence.).
  • 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 T cells ((I) a gene encoding a polypeptide comprising an amino acid sequence according to any one of SEQ ID NOS: 1-77 (II) according to any one of SEQ ID NOS: 1-77
  • Any one of gene (III) SEQ ID NOS: 1 to 77 encoding a polypeptide comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and/or added in the amino acid sequence of A gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence described in .).
  • T cells Twenty-four types of T cell lines shown in Table 11 below were thawed and placed in medium A (15% (w/v) fetal bovine serum (FBS, BioWest) in a 96-well plate. , 5 ng/mL interleukin 7 (IL-7), and 5 ng/mL interleukin 15 (IL-15) added to ⁇ MEM ( ⁇ modified Eagle minimal essential medium)) at 1 ⁇ 10 6 cells/well, respectively. sown.
  • medium A 15% (w/v) fetal bovine serum (FBS, BioWest) in a 96-well plate.
  • IL-7 interleukin 7
  • IL-15 interleukin 15
  • T cell receptor (TCR) stimulation Magnetic beads (Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (manufactured by Thermo Fisher Scientific) are used to stimulate the TCR of the seeded T cells. , and cultured for 14 days under conditions of 5% CO 2 and 37° C. while replacing the medium with medium A as appropriate.
  • T cells of the same strain as the T cells classified into high-quality T cells (12 types) and low-quality T cells (12 types) in 1 (3) above TCR unstimulated
  • TCR unstimulated For each, stimulate the TCR using TCR-stimulating magnetic beads in the same manner as in 1 (1) to (2) above, and replace the medium with medium A as appropriate under the conditions of 5% CO 2 and 37 ° C. cultured.
  • the genes (sequence data) decoded in (4) and (3) were mapped to the human genome sequence using TopHat2 (JOHNS HOPKINS University) and Bowtie2 (JOHNS HOPKINS University).
  • TopHat2 JOHNS HOPKINS University
  • Bowtie2 JOHNS HOPKINS University
  • BUILD GRCh38 published by NCBI (National Center for Biological Information) was used.
  • 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).
  • T cells TCR unstimulated
  • T cells classified into high-quality T cells (12 types) and low-quality T cells (12 types) in 1 (3) above Each TCR was stimulated using TCR-stimulating magnetic beads in the same manner as in 1 (1) to (2) above, and cultured under the conditions of 5% CO 2 and 37° C. while exchanging the medium with medium A as appropriate. .
  • ⁇ MEM (-Met) medium supplemented with (insulin-transferrin-sodium selenite) supplement (x1) 50 ⁇ g/mL ascorbic acid, 5 ng/mL IL-7, and 5 ng/mL IL-15, 37 °C and 5% CO2 for 24 hours.
  • PCA Principal component analysis From (4) of the transcriptome analysis in 2 above and (5) of the secretome analysis in 3 above, the following (a) to (d): (a) 12,829 genes mapped by transcriptome analysis in day0 samples (transcriptome day0) and their expression levels, (b) 12,270 genes mapped by transcriptome analysis in day6 samples (transcriptome day6) and their expression levels; (c) 12,013 genes mapped by transcriptome analysis in day14 samples (transcriptome day14) and their expression levels, (d) Data on 800 genes (secretome) encoding proteins identified by secretome analysis and their expression levels were obtained. However, the numbers of genes in (a) to (d) do not include low-expressed or non-expressed genes.
  • PCA principal component analysis
  • the scatter diagram obtained by the PCA is shown in Figure 1.
  • the first principal component (PC1) score was high in high-quality T cells (cells No. 1-12), and the PC1 score was low in low-quality T cells (cells No. 13-21, 23, 24).
  • Table 12 below and FIG. 2 show the relationship between the descending order (proliferation order) of the 23 types of proliferation rate, the PC1 score (PC1), and the descending order (PC1 order).
  • the correlation coefficient (R) between the T cell proliferation rank and the PC1 rank is 0.9615, indicating a high correlation between the cell quality (in this case, proliferative capacity) and the PC1 score. was accepted.
  • the variable with the high value of the principal component loading of the first principal component in this case, the variable is the expression level of each gene
  • a variable that contributes positively to the first principal component and has a low (negative value) value of the principal component loading of the first principal component contributes negatively to the first principal component.
  • genes with a high principal component loading value of the first principal component are expressed in cells with a high PC1 score, that is, high-quality T cells, and genes with a low principal component loading value of the first principal component are expressed in It was found to be expressed in cells with low PC1 score, ie low quality T cells.
  • the principal component loading of the first principal component was determined for each gene expression level obtained in (a) to (d) above.
  • the contribution rate to the first principal component is high (specifically, the absolute value of the principal component loading for the first principal component of the gene expression level is 0.9 or more) and genes encoding transmembrane proteins are the genes shown in Tables 1 and 2 above.
  • the contribution rate to the first principal component is high (specifically, the absolute value of the principal component loading amount with respect to the first principal component of the gene expression level is 0.9 or more), and other than transmembrane proteins
  • Genes encoding proteins are those shown in Tables 3-5 above. In Tables 1 to 5, for each gene, the categories (a) to (d) above are shown in the column of "origin data", respectively, and the principal component loadings for the first principal component obtained above are also included. indicated.
  • ITGB7, BMPR2, and LGALS8 can be said to be markers capable of evaluating the quality of T cells in the state after cell stimulation with TCR.
  • ALCAM, BMPR2, and TP53I3 have absolute values of correlation coefficients of 0.6 or more, and these three proteins can be said to be particularly preferred embodiments of markers capable of evaluating the quality of T cells.
  • the present invention it is possible to provide a T cell evaluation method that can evaluate the quality of T cells using a new index, and a T cell evaluation reagent used for the method.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Sustainable Development (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé d'évaluation 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 l'une des séquences SEQ ID NO : 1 à SEQ ID NO : 77. (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 l'une des séquences SEQ ID NO : 1 à SEQ ID NO : 77. (III) Gène codant un polypeptide contenant une séquence d'acides aminés à au moins 70% identique à une séquence d'acides aminés représentée par l'une des séquences SEQ ID NO : 1 à SEQ ID NO : 77.
PCT/JP2022/039806 2021-10-27 2022-10-26 Procédé d'évaluation de cellules t, et composition destinée à l'évaluation de cellules t WO2023074711A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021175126A JP2023064788A (ja) 2021-10-27 2021-10-27 Ｔ細胞の評価方法及びｔ細胞評価用の組成物
JP2021-175126 2021-10-27

Publications (1)

Publication Number Publication Date
WO2023074711A1 true WO2023074711A1 (fr) 2023-05-04

Family

ID=86158030

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/039806 WO2023074711A1 (fr) 2021-10-27 2022-10-26 Procédé d'évaluation de cellules t, et composition destinée à l'évaluation de cellules t

Country Status (2)

Country Link
JP (1) JP2023064788A (fr)
WO (1) WO2023074711A1 (fr)

Citations (3)

* 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
JP2019154320A (ja) * 2018-03-13 2019-09-19 ロート製薬株式会社 細胞の品質を評価する方法、及び細胞の品質判定キット
JP2021501318A (ja) * 2017-10-27 2021-01-14 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア 疲弊したｔ細胞に関連する疾患を治療するための方法および組成物

Patent Citations (3)

* 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
JP2021501318A (ja) * 2017-10-27 2021-01-14 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア 疲弊したｔ細胞に関連する疾患を治療するための方法および組成物
JP2019154320A (ja) * 2018-03-13 2019-09-19 ロート製薬株式会社 細胞の品質を評価する方法、及び細胞の品質判定キット

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GEORGE ELIAS; BENSON OGUNJIMI; VIGGO VAN TENDELOO: "Tracking Dye‐Independent Approach to Identify and Isolate In Vitro Expanded T Cells", CYTOMETRY A, WILEY-LISS, HOBOKEN, USA, vol. 95, no. 10, 29 July 2019 (2019-07-29), Hoboken, USA, pages 1096 - 1107, XP072332516, ISSN: 1552-4922, DOI: 10.1002/cyto.a.23867 *

Also Published As

Publication number Publication date
JP2023064788A (ja) 2023-05-12

Similar Documents

Publication Publication Date Title
JP6259012B2 (ja) 脱毛症の治療方法
Maazi et al. Activated plasmacytoid dendritic cells regulate type 2 innate lymphoid cell–mediated airway hyperreactivity
US9125869B2 (en) Method for expanding monocytes
JP6416131B2 (ja) 濃縮された腫瘍反応性ｔ細胞集団を腫瘍から作製する方法
Reutner et al. CD27 expression discriminates porcine T helper cells with functionally distinct properties
US20190353643A1 (en) Method For Sorting Highly Effective Stem Cells For Treating Immune Disorder
US20230330145A1 (en) Natural killer cells
CN114929752A (zh) 嵌合细胞因子受体
Talker et al. Monocyte biology conserved across species: Functional insights from cattle
Teteloshvili et al. Involvement of MicroRNAs in the aging-related decline of CD28 expression by human T cells
Richert et al. Expansion of antigen-specific T cells from cerebrospinal fluid of patients with multiple sclerosis
Flinn et al. Thymopoiesis, alterations in dendritic cells and Tregs, and reduced T cell activation in successful extracorporeal photopheresis treatment of GVHD
Davies et al. Robust production of merkel cell polyomavirus oncogene specific T cells from healthy donors for adoptive transfer
Crispim et al. Interferon-γ+ 874 polymorphism in the first intron of the human interferon-γ gene and kidney allograft outcome
WO2023074711A1 (fr) Procédé d'évaluation de cellules t, et composition destinée à l'évaluation de cellules t
WO2023074712A1 (fr) Procédé d'évaluation de cellules t, et composition destinée à l'évaluation de cellules t
WO2023074713A1 (fr) Procédé d'évaluation de qualité de cellules t, et réactif mettant en œuvre ce procédé
US9678062B2 (en) Methods and compositions for expanding cells and improving engraftment
Mumprecht et al. Chronic myelogenous leukemia maintains specific CD8+ T cells through IL‐7 signaling
CN113227357A (zh) 人造血干细胞向具有增强的抗体依赖性细胞毒性活性的成熟自然杀伤细胞的加速的分化
Yu et al. Mechanism study of ubiquitination in T cell development and autoimmune disease
Dawson et al. Functional effects of chimeric antigen receptor co-receptor signaling domains in human Tregs
Hanson et al. CD62L expression marks a functionally distinct subset of memory B cells
Kustermann et al. Adoptively transferred in vitro-generated myeloid-derived suppressor cells improve T-cell function and antigen-specific immunity after traumatic lung injury
Nakano et al. Impact of vaccine therapy using nuclear histone H1 on allograft survival in experimental organ transplantation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22887037

Country of ref document: EP

Kind code of ref document: A1