JP2023064788A - Evaluation method of t-cell, and composition for evaluating t-cell - Google Patents

Abstract

To provide an evaluation method of a T-cell which is capable of evaluating the quality of a T-cell by a new index.SOLUTION: An evaluation method of a T-cell includes: a detection process of detecting an expression of at least one kind of gene selected from the group consisting of genes of the following (I) to (III), in a T-cell; and an evaluation process of evaluating the quality of a T-cell by using the expression of the gene as an index. (I) a gene encoding polypeptide including an amino acid sequence described in any one of specific 77 kinds of sequences. (II) a gene encoding polypeptide including an amino acid sequence in which one or a plurality of amino acid residues are substituted, deleted, inserted, added and/or added, in the amino acid sequence described in any one of specific 77 kinds of sequences. (III) a gene encoding polypeptide including an amino acid sequence having 70% or more of identity with the amino acid sequence described in any one of specific 77 kinds of sequences.SELECTED DRAWING: None

Description

The present invention relates to a method for evaluating T cells and a composition for evaluating T cells.

Cellular immunotherapy using antigen-specific cytotoxic T lymphocytes (CTL) related to the disease is known as a new treatment method for diseases such as cancer such as malignant tumors and chronic intractable infectious diseases. It is In recent years, as an effective means for cancer therapy, T cells (unmodified T cells) are collected from patients, genetically modified to enhance cancer-attacking ability, and then genetically modified T cells are obtained. Expectations are high for cancer immunotherapy, which treats cancer by returning cells to the original patient.

In addition, T cells collected from patients have problems such as cell exhaustion and difficulty in proliferation culture (or expansion culture), so there is no limit to the number of iPS cells that can be used. Cancer immunotherapy is also being developed using T cells derived from For example, Patent Document 1 describes a method of inducing CD8α ⁺ β ⁺ CTL by culturing CD4 ⁺ CD8 ⁺ T cells in a medium containing interleukin 7 and a TCR (T cell receptor) activator. there is

Since the quality of iPS cell-derived T cells varies depending on the iPS cell line, culture conditions, etc., it is necessary to evaluate the quality of the obtained T cells and select those suitable for the cancer immunotherapy. be. In addition, it is also necessary to evaluate the quality of the obtained genetically modified T cells and select those suitable as the desired genetically modified T cells. Specific examples of such T cell quality evaluation items include, for example, analysis of differentiation markers expressed at each stage of T cells, analysis of cytokines produced after TCR stimulation, proliferation ability, cytotoxic activity, animal experiments and cancer cell-suppressing activity. The quality of T cells is comprehensively evaluated by these items, but such evaluations are often complicated and require a long period of time. Furthermore, since the cost of evaluation is high, there is a demand for a method that can evaluate the quality of T cells more simply and in a short period of time.

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.

WO2018/135646

Daniel L. Barber et al., Nature, 439, 2006, p. 682-687

In vitro, the above exhaustion markers are expressed after the exhaustion of T cells has progressed significantly due to long-term culture. Therefore, the present inventors found that it is unsuitable for evaluating the quality of relatively young T cells such as T cells immediately after being induced to differentiate from iPS cells. So far, no markers have been identified that can also assess the quality of such relatively young T cells.

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.

The present inventors have made intensive studies to solve the above problems, and as a result, secretome (secretome) for T cells with high proliferative potential (high-quality T cells) and T cells with relatively low proliferative potential (low-quality T cells) Analysis and transcriptome analysis were performed. The data obtained from these analyzes were subjected to principal component analysis (PCA) of the high-quality T cells and the low-quality T cells. It was found that there is a specific relationship between the expression levels of specific genes at each elapsed day. More specifically, a high correlation was observed between the order of the proliferation fold of T cells and the order of the first principal component (PC1) score. Lowered PC1 score. Therefore, genes with a high principal component loading value of the first principal component are expressed in cells with a high PC1 score, i.e., high-quality T cells, and genes with a low principal component loading of the first principal component are expressed in cells with a high PC1 score. was found to be expressed in cells with low cytotoxicity, i.e., low quality T cells.

Based on these findings, 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.

That is, aspects of the present invention are as follows.

[1] 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;
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

[2] The detecting step detects the expression product using a probe molecule that specifically binds to the expression product of at least one gene selected from the group consisting of the genes (I) to (III). The method according to [1], which is a step.

[3] The expression product is a polypeptide encoded by any one of the genes (I) to (III), and the probe molecule specifically binds to the polypeptide The method of [2], which is an antibody and/or an aptamer.

[4] The method according to any one of [1] to [3], wherein the evaluation step is a step of evaluating T cell proliferation ability using the expression of the gene as an index.

[5] 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.
(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 (II') SEQ ID NOS: 5, 7, 15, 27, and 44 A 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 in the amino acid sequence according to any one of SEQ ID NO: A gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence of any one of 5, 7, 15, 27 and 44.

[6] 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.
(I') a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, and 27; (II') set forth in any one of SEQ ID NOs: 5, 15, and 27; Any of gene (III') SEQ ID NOS: 5, 15, and 27 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 or a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in one.

[7] A reagent for use in the method according to any one of [2] to [6], wherein at least one gene selected from the group consisting of the following genes (I) to (III) A T cell evaluation reagent containing a probe molecule that specifically binds to an expression product.
(I) a gene encoding a polypeptide comprising the amino acid sequence set forth in 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

[8] 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.

[9] 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 reagent of [7] or [8], which is a gene.
(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 (II') SEQ ID NOS: 5, 7, 15, 27, and 44 A 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 in the amino acid sequence according to any one of SEQ ID NO: A gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence of any one of 5, 7, 15, 27 and 44.

[10] 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 reagent according to any one of [7] to [9], which is a gene.
(I') a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, and 27; (II') set forth in any one of SEQ ID NOs: 5, 15, and 27; Any of gene (III') SEQ ID NOS: 5, 15, and 27 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 or a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in one.

ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the T-cell evaluation method which can evaluate the quality of T-cell by a new index, and the reagent for T-cell evaluation used for it.

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.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will now be described in detail with reference to its preferred embodiments.

<Method for evaluating T cells>
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").
(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

[T cells]
In the present invention, the origin of T cells is not particularly limited. depleted humans or animals, or those suffering from malignancies, infectious diseases or autoimmune diseases). Furthermore, T cells differentiated from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); T cells differentiated from somatic stem cells such as hematopoietic stem cells; It may be a T cell.

In the case of T cells collected from humans, for example, T cells are isolated from peripheral blood mononuclear cells (PBMC: Peripheral Blood Mononuclear Cells) isolated from peripheral blood using a conventionally known method or a commercially available kit. It can be isolated. In addition, the T cells according to the present invention may be naive T cells, antigen-stimulated memory T cells or effector T cells. Effector T cells may be any of helper T cells, killer T cells, regulatory T cells, and natural killer T (NK/T) cells.

In addition, in the present invention, "T cells" include T cells that have not been artificially genetically modified (hereinafter sometimes referred to as "unmodified T cells"), as well as artificial genes in the unmodified T cells. Also included are "genetically modified T cells" that have been modified. In the present invention, "artificial genetic modification" includes modification of T cell functions by gene transfer to T cells or gene editing of T cells. The artificial genetic modification may be modification of the gene itself possessed by T cells, deletion of the gene itself possessed by T cells, or modification resulting from the introduction of a foreign gene. A combination of two or more types may also be used.

The artificial genetic modification method includes conventionally known methods and methods based thereon, and is not particularly limited. Gene introduction into T cells includes, for example, the gene itself (DNA, mRNA, miRNA, antagomir, ODN (oligodeoxyribonucleotide), etc.) that modifies the function of T cells, or a vector into which the gene is inserted (lentiviral vector , γ- or α-retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, equine encephalitis viral vectors, etc.) into T cells. Genome editing of T cells using specific nucleases (meganuclease, zinc finger nuclease, TALEN, PPR (Pentatricpeptide repeat), CRISPR-Cas, etc.). In the present invention, the artificial genetic modification method may be one of these methods alone or a combination of two or more of them.

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)
In the detection step according to the method of the present invention, the expression of at least one gene selected from the group consisting of genes (I) to (III) below is detected in T cells.
(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

Here, in the group consisting of the genes (I) to (III), each of the genes (I), (II), and (III) is independently of one kind. may be two or more.

The polypeptide encoded by any one of the genes (I) to (III) is preferably contained in the T cell-derived secretome. In the present invention, the term "secretome" refers to 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.

In the present invention, 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 number and GenBank Accession No. of each amino acid sequence for the amino acid sequences described 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. (GenBank No.); indicates the abbreviation of the protein (protein abbreviation) corresponding to the polypeptide consisting of each amino acid sequence. Tables 6 to 10 below also show the official name (full name) and alternative names, if any, for each protein abbreviation listed in Tables 1 to 5.

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.

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. 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 principal component loading of the first principal component are expressed in cells with low PC1 score, That is, it was found to be expressed in low-quality T cells. Genes encoding polypeptides consisting of amino acid sequences represented by SEQ ID NOS: 1 to 2, shown in Tables 1 and 2, among genes expressed in T cells after TCR stimulation, contribute to the first principal component is high (specifically, the absolute value of the principal component loading with respect to the first principal component of the expression level of the gene is 0.9 or more), and is a gene that encodes a transmembrane protein, and is shown in Tables 3 to 5 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. Therefore, these have a high contribution rate to the first principal component (specifically, the absolute value of the principal component loading with respect to the first principal component of the gene expression level is 0.9 or more), SEQ ID NOS: 1 to 77 Expression of a gene encoding a polypeptide containing an amino acid sequence represented by any one of can be used as an index for evaluating the quality of T cells.

In the present invention, homologous genes (e.g., counterpart genes in organisms other than humans) of "(I) a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 77" It can also be the target gene. In addition, since the nucleotide sequence of a gene can be mutated in nature (that is, non-artificially) due to mutation thereof, etc., such natural mutants can also be used as the target gene in the present invention. can. Furthermore, in the current state of the art, for example, when the genetic information of (I) is obtained, a person skilled in the art can modify the nucleotide sequence and modify the function, although it is different from the amino acid sequence that codes for it. Since proteins can also be prepared, such variants may also be used as the target gene.

Therefore, in the aspect of the target gene according to the present invention, "(II) one or more amino acid residues are substituted, deleted, or inserted in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 77. , and/or a gene encoding a polypeptide comprising an added amino acid sequence". Here, the term "amino acid sequence in which amino acid residues are substituted, deleted, inserted, and/or added" means an amino acid sequence in which amino acid residues are substituted, deleted, inserted, or added, or It shows that the amino acid sequence is a combination of two or more of Further, "plurality" means, for example, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 is preferably an integer of, but not limited to. One or more amino acid residues is preferably 1 to 10 amino acid residues, more preferably 1 to 5, still more preferably 1 to 3, particularly preferably 2 or less. is.

Furthermore, in the current state of the art, for example, when the genetic information of (I) above is obtained, a person skilled in the art can perform hybridization techniques (Southern, EM, J. Med. Mol. Biol., 98:503-517, 1975) and polymerase chain reaction (PCR) technology (Saiki, RK, et al. Science, 230: 1350-1354, 1985, Saiki, RK et al.

Therefore, in embodiments of the target gene according to the present invention, "(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. The term "stringent conditions" means that the washing operation of the membrane after hybridization is performed at a high temperature in a low-salt solution. 2×SSC) in 0.5% (w/v) SDS solution at 60° C. for 20 minutes. In addition, 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 Biosciences). The more stringent the hybridization conditions, the more highly homologous DNA can be expected to be isolated. However, these conditions are only examples, and the necessary stringency can be achieved by appropriately combining DNA concentration, DNA length, hybridization reaction time, and the like.

Furthermore, for example, a protein encoded by a homologous gene obtained by such a method usually has a high degree of identity with the amino acid sequence set forth in 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). In addition, the 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).

In the present invention, 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 one type alone or two or more types.

In addition, as 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.

(Detection of gene expression)
In the present invention, "detecting gene expression" includes both detection of the presence or absence of gene expression and detection of the degree of expression. In the present invention, the expression of the target gene is detected. The gene expression level can be grasped as an absolute amount or as a relative amount. When grasping the relative amount, for example, it can be judged by comparing with the gene expression level of a prepared standard sample. A "standard sample" refers to a sample for which it has been specified in advance whether or not the target gene is expressed, and if so, the amount of expression. For example, T cells that have been previously identified as having good or bad quality can be used as the standard sample.

In the present invention, the term "gene expression" includes 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.

In eukaryotic cells, in the process of gene transcription, a reaction (splicing) occurs in which introns in the pre-mRNA are removed and the exons before and after are recombined (splicing). 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.

For detection of gene expression in the present invention, a known technique or a technique based thereon can be used as appropriate. Among them, as 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')) Preferably, the expression product of a gene (ie, said target gene) is detected using a probe molecule that specifically binds to said expression product.

[Detection at transcription level (detection of target polynucleotide)]
In detection at the transcriptional level, the expression product of the target gene is mRNA transcribed from the target gene (hereinafter sometimes referred to as "target polynucleotide"). The target polynucleotide also includes cDNA using the mRNA as a template. Detection methods in this case include, for example, Northern blotting, dot blotting, and RNase protection assay using oligonucleotide probes designed to hybridize to appropriate positions in the base sequence of the target polynucleotide as the probe molecule. , DNA microarray analysis, in situ hybridization, and the like to detect the target polynucleotide.

In this case, for example, as the oligonucleotide probe, one labeled with a labeling substance is used, a signal corresponding to the labeling substance is detected, and the detected signal amount is the amount of the target polynucleotide (i.e., expression level of the target gene). Examples of labeling substances at this time include FITC (fluorescein isothiocyanate), FAM (fluorescein amidite), DEAC (7-(diethylamino)coumarin), R6G (rhodamine 6G), Texas Red, Cy5, and BODIPY FL (trade name). enzymes such as β-D-glucosidase, luciferase, HRP (horseradish peroxidase); radioactive isotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²³ I; biotin, streptavidin, etc. luminescence substances such as luminol, luciferin, and lucigenin.

In the present invention, the "signal" includes coloration (coloration), reflected light, luminescence, quenching, fluorescence, radiation from a radioactive isotope, and the like, and in addition to those that can be confirmed with the naked eye, depending on the type of signal It also includes those that can be confirmed by measuring methods and equipment.

Further, for example, using an oligonucleotide primer designed to sandwich an appropriate position in the target polynucleotide as the probe molecule, 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.

In this case, for example, a signal (fluorescence) obtained by intercalating the obtained amplification product with a fluorescent dye (e.g., ethidium bromide, SYBR Green (trade name), SYTO63 (trade name), etc.) is detected. Then, the detected signal amount (fluorescence intensity) can be used as the amount of the target polynucleotide (that is, the expression level of the target gene). Moreover, detection may be performed in combination with the oligonucleotide probe (double dye probe method, etc.). Furthermore, the amount of the target polynucleotide may be determined by directly subjecting a sample containing the target polynucleotide to a sequencer for analysis.

Such oligonucleotide probes and oligonucleotide primers can be designed by those skilled in the art based on the sequence of the target polynucleotide by known methods or methods based thereon. In addition, the length of the oligonucleotide probe and oligonucleotide primer are each independently preferably at least 15 bases, usually 15 to 100 bases, preferably 17 to 30 bases, more preferably 20-25 bases. The oligonucleotide probes and oligonucleotide primers can be synthesized by, for example, a commercially available oligonucleotide synthesizer. In addition, it may not consist only of natural nucleotides (deoxyribonucleotides and / or ribonucleotides), for example, PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), ENA (registered trademark, 2'- O,4'-C-Ethylene-bridged nucleic acid) may be partially or wholly composed of non-natural nucleotides.

[Detection at translation level (detection of target polypeptide)]
In translational level detection, the expression product of the target gene is a polypeptide encoded by the target gene (hereinafter sometimes referred to as "target polypeptide"). More specifically, it is a polypeptide encoded by any one of the genes (I) to (III), that is, 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 A polypeptide comprising:

In the group consisting of the polypeptides (i) to (iii), the polypeptide (i), the polypeptide (ii), and the polypeptide (iii) are each independently one type. or two or more.

Among these, the 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.
(i') 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') 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.

In the group consisting of the polypeptides (i') to (iii'), the polypeptide (i'), the polypeptide (ii'), and the polypeptide (iii') each independently One type may be used alone, or two or more types may be used.

Moreover, in the present invention, these target polypeptides to be detected are preferably the above secretomes (T cell-derived secretomes).

Examples of detection methods for detection at the translation level include detection using anti-polypeptide antibodies and/or aptamers, and detection using labeled amino acids.

<Detection by anti-polypeptide antibody and/or aptamer>
Detection methods for detection at the translation level include, for example, antibodies that specifically bind to the target polypeptide (hereinafter sometimes referred to as "anti-polypeptide antibodies") and/or aptamers (hereinafter , In some cases, simply referred to as "aptamer"), immune cell staining method, imaging cytometry, flow cytometry, ELISA (Enzyme-Linked Immuno Sorbent Assay) method, radioimmunoassay, immunoprecipitation method, immunoblotting (Western blotting method etc.), methods of detection using antibodies such as antibody arrays and in vivo imaging (immunological techniques); methods of detection using aptamers instead of the antibodies. In addition, the immunological method 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.

In the present invention, an "antibody" may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody. "Antibody" also includes all classes and subclasses of immunoglobulins. A "functional fragment" of an antibody refers to a portion (partial fragment) of an antibody that specifically recognizes the polypeptide encoded by the target gene in the present invention. Specifically, Fab, Fab′, F(ab′) ₂ , variable region fragment (Fv), disulfide-bonded Fv, single-chain Fv (scFv), sc(Fv) ₂ , diabody, multispecific antibodies, short-chain antibodies, and polymers thereof.

If the anti-polypeptide antibody according to the present invention is a polyclonal antibody, an immunized animal is immunized with an antigen (target polypeptide, its partial peptide, or cells expressing these, etc.), and the antiserum is obtained by conventional means ( Salting out, centrifugation, dialysis, column chromatography, etc.) can be appropriately purified and obtained. Monoclonal antibodies can also be produced by the hybridoma method or recombinant DNA method. 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, PJ 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)).

In this case, the anti-polypeptide antibody and the aptamer are each labeled with a labeling substance, the signal corresponding to the labeling substance is detected, and the amount of the detected signal is calculated as the amount of the target polypeptide. (that is, the expression level of the target gene). The labeling substance at this time is not particularly limited as long as it can bind to the anti-polypeptide antibody or aptamer and can be detected by a chemical or optical method. ), fluorescent substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC), enzymes such as peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP) and alkaline phosphatase, and radioactive substances. .

When the anti-polypeptide antibody and/or aptamer is used, a method of using a secondary antibody bound with the labeling substance, or a method of using a polymer binding the secondary antibody and the labeling substance. Indirect detection methods such as can also be used. Here, the "secondary antibody" is an antibody that exhibits specific binding to the anti-polypeptide antibody and/or aptamer. For example, when the anti-polypeptide antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. Labeled secondary antibodies that can be used for antibodies derived from various species such as rabbits, goats, and mice are commercially available. Antibodies can be selected and used. In place of the secondary antibody, protein G (for example, derived from Streptococcus), protein A (for example, derived from Staphylococcus aureus), protein L (derived from Peptostreptococcus magnus), etc., bound with a labeling substance can also be used. be.

<Detection with labeled amino acids>
Other detection methods for detection at the translation level include, for example, a method of culturing T cells in a medium supplemented with a labeled amino acid and detecting a target polypeptide labeled with the labeled amino acid. As such a method,
a labeling step of culturing T cells in a T cell labeling medium containing a labeled amino acid in which the amino acid is labeled instead of at least one essential amino acid for T cell proliferation and containing a common γ chain family cytokine;
a detection step of detecting the T-cell-derived polypeptide labeled with the labeled amino acid (hereinafter sometimes referred to as "detection step A");
is preferred.

Here, the term “T cell labeling medium” refers to a medium containing at least a labeling amino acid for culturing and labeling T cells. In addition, “essential amino acids for T cell proliferation (hereinafter, sometimes simply referred to as “essential amino acids”)” refers to amino acids essential for T cell proliferation, more specifically, valine, isoleucine, leucine, methionine, lysine, phenylalanine, tryptophan, threonine, histidine.

The T cell labeling medium contains a labeled amino acid in which the amino acid is labeled instead of at least one of the essential amino acids, that is, does not contain at least one of the essential amino acids and the essential amino acid that is not contained Contains labeled amino acids, where the amino acids are labeled amino acids. "Containing no essential amino acids" means that the T cell labeling medium does not substantially contain the essential amino acids. More specifically, the content of the essential amino acids in the T cell labeling medium is is 1×10 ⁻³ mmol/L or less, preferably less than 1×10 ⁻⁴ mmol/L. The essential amino acids excluded from the T cell labeling medium (that is, not substantially contained in the T cell labeling medium) may be any one of the above or a combination of two or more of them. Among them, methionine is preferred from the viewpoint of efficient incorporation into the protein synthesis pathway.

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.

Examples of such labeled amino acids include isotope-labeled amino acids in which some of the atoms of the amino acid are substituted with pulse stable isotopes (e.g., ² H, ¹³ C, ¹⁵ N); Examples include structural analogs of amino acids modified by modifying groups such as groups, and corresponding to the essential amino acids, any one of them or a combination of two or more of them good.

For example, when the T cell labeling medium does not contain methionine among the essential amino acids, the labeling amino acid contained in the T cell labeling medium instead of methionine may be, for example, one of the atoms constituting methionine. ² H-labeled methionine, ¹³ C-labeled methionine, and ¹⁵ N-labeled methionine, which are isotopically-labeled methionines in which the moiety is replaced with the stable isotope; L-azidohomoalanine (AHA), which is a structural analogue of methionine , L-homopropargylglycine (HPG: L-Homopropargylglycine), L-homoallylglycine (HAG: L-Homoallylglycine), any one of these, even a combination of two or more good.

In the T cell labeling medium, the content of the labeled amino acids (the total content of the labeled amino acids when there are two or more labeled amino acids; the same shall apply hereinafter) includes the T cell uptake efficiency, the culture efficiency, the T cell It can be determined as appropriate in consideration of the toxicity etc., and is not particularly limited. Preferably, it is 0.001 to 0.02 mmol/L. More specifically, for example, when the labeled amino acid is AHA, it is preferably 0.0005 to 0.03 mmol/L, more preferably 0.001 to 0.03 mmol/L. More preferably 0.002 to 0.03 mmol/L, even more preferably 0.005 to 0.02 mmol/L. When the content of the labeled amino acid exceeds the upper limit, the cells tend to weaken or die, whereas when the content is less than the lower limit, the polypeptide labeled with the labeled amino acid is secreted or exposed outside the cell. As a result, the number of cells to be detected tends to decrease, and the detection accuracy of the polypeptide tends to decrease.

The T cell labeling medium further contains common γ chain family cytokines. By containing this, it becomes possible to obtain cells expressing the polypeptide labeled with the labeled amino acid with high efficiency. A "common γ chain family cytokine" refers to a cytokine that acts through a receptor containing a common γ chain (γ subunit). More specifically, interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 15 (IL-15) , and interleukin 21 (IL-21), any one of which may be used in combination. Among them, interleukin 7, interleukin 15, interleukin 21, and combinations of two or more of these are more preferable from the viewpoint of better maintenance of cell survival and proliferation.

In the T cell labeling medium, the content of the common γ-chain family cytokine (when there are two or more common γ-chain family cytokines, the total content thereof; the same shall apply hereinafter) is determined according to the properties of T cells, etc. Although it is not limited to this, it is preferably 1 to 1000 ng/mL, more preferably 5 to 100 ng/mL, and 5 to 20 ng/mL. It is even more preferable to have When the content of the common γ-chain family cytokine exceeds the upper limit, the effect of containing the common γ-chain family cytokine tends to decrease or cell proliferation tends to be inhibited. The number of cells that secrete or extracellularly expose a polypeptide labeled with a labeled amino acid tends to decrease, resulting in a decrease in the detection accuracy of the polypeptide.

In order to remove the essential amino acids, the T cell labeling medium preferably does not contain serum or, if it does contain serum, is dialyzed serum from which the essential amino acids have been removed. In addition, the T cell labeling medium may also contain cytoprotective additives such as serum albumin and ITS (insulin-transferrin-sodium selenite); scaffolding agents such as ECM (extracellular matrix) components. may be These T cell labeling media or their basal media are not particularly limited as long as they do not contain the desired essential amino acids, and are DMEM (Dulbecco's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute Medium), αMEM (α-modified basal medium such as Eagle minimum essential medium); AIM V ^TM medium (manufactured by Thermo Fisher Scientific) which is a serum-free medium exclusively for T cells; PRIME-XV T cell Expansion XSFM (manufactured by Fujifilm Wako Pure Chemical) or a commercially available medium for T cells can be used as appropriate.

In the labeling step, T cells are cultured in the T cell labeling medium. As a result, the labeled amino acid is taken up by T cells, and a polypeptide labeled with the labeled amino acid (T cell-derived polypeptide) is secreted from the T cells or exposed outside the cells. The culture method in the labeling step is not particularly limited, and known T cell proliferation culture conditions can be appropriately adopted, and the characteristics and concentration of T cells can be taken into consideration and adjusted as appropriate. For example, the culture temperature is 35 to 37.5° C., preferably 36 to 37° C., and the culture time is 2 to 48 hours, preferably 8 to 24 hours. The culture apparatus for the labeling step is not particularly limited, and examples thereof include plates, dishes, columns, flasks, culture bags, and the like. Ejection means (sensors, valves, pumps, tanks, etc.) may also be provided.

In the detection step A, the T cell-derived polypeptide that has been labeled with the labeling amino acid and has been secreted or exposed outside the cell is detected. The detection step A may be performed simultaneously (in real time) with the labeling step. As a detection method in the detection step A, a detection method suitable for the type of the labeled amino acid can be appropriately employed. Such a detection method is not particularly limited, and a known method or a method based thereon can be appropriately employed. When the labeled amino acid is an isotope-labeled amino acid or a structural analogue of an amino acid, for example, the medium after the labeling step or during the labeling step (that is, during the culture) is subjected to gas isotope ratio mass spectrometry, By performing mass spectrometry such as infrared spectroscopy, liquid chromatography mass spectrometry (LC-MS/MS), a signal corresponding to the labeled amino acid is detected, and the amount of the detected signal is the amount of the target polypeptide (i.e. , expression level of the target gene).

Moreover, when the labeled amino acid is a structural analogue of an amino acid, it can be detected by staining the modified group specifically. For example, when the modifying group is an azide group (such as AHA), the azide group can be stained with a fluorescent reagent such as Click-iT ^™ Alexa Fluor 488 sDIBO Alkyne (manufactured by Thermo Fisher Scientific). Using fluorescence microscopy, flow cytometry, imaging cytometry, etc., a signal (color development, fluorescence, etc.) corresponding to the labeled amino acid is detected, and the amount of the detected signal is the amount of the target polypeptide (i.e., the amount of the target gene). expression level).

Other detection methods for detection at the translation level include, for example, a method of recognizing and detecting the sugar chain of the target polypeptide. Such methods include, for example, methods using commercially available reagents such as Click-iT ^™ (manufactured by Thermo Fisher Scientific). The detection method for detection at the translation level may be used in combination with other methods as appropriate.

In the present invention, detection of the expression of the gene is preferably detection at the translation level (detection of the target polypeptide) from the viewpoint of convenience. Methods that detect polypeptides are preferred.

[Evaluation process]
In the evaluation step according to the method of the present invention, the quality of T cells is evaluated using the presence or absence or the expression level of the target gene detected in the detection step as an index. The quality of T cells to be evaluated in the present invention includes proliferative ability and cancer cytotoxic activity. Among them, the method of the present invention is preferable as a method for evaluating the proliferative ability of T cells. More specifically, the term "proliferative ability" indicates the ability to activate T cells and increase cells having cytotoxic activity in vitro or in vivo by TCR stimulation or antigen stimulation. The proliferative ability of T cells can be confirmed, for example, by stimulating 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.

In the present invention, among the target genes shown in Tables 1 to 5 detected in the detection step, 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. On the other hand, 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. For example, in 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. For example, 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).

<T cell evaluation reagent, T cell evaluation kit>
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").
(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

In 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. Examples of probe molecules related to the reagent of the present invention include oligonucleotide probes, oligonucleotide primers, anti-polypeptide antibodies, and aptamers mentioned in the method of the present invention described above. in the method of the present invention. The probe molecule may be one of these or a combination of two or more of them. Among them, the anti-polypeptide antibody and/or the aptamer are preferable, and the anti-polypeptide antibody is more preferable.

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. In addition to the probe molecule, the kit of the present invention also contains a buffer, a solution for dilution or suspension such as physiological saline; a reagent for extracting and/or purifying DNA or protein; a blocking agent; a chelating agent; The labeling substance; the fluorescent dye; reagents necessary for the detection step; reagents such as pH adjusters; standard samples; instructions for use;

EXAMPLES The present invention will be described in more detail based on examples below, but the present invention is not limited to the following examples.

1. Evaluation of quality (proliferation ability) of T cells (1) 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 αMEM (α modified Eagle minimal essential medium)) at 1×10 ⁶ cells/well, respectively. sown.

(2) 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.

(3) Each T cell after stimulation on day 14 of culture was counted using a hemocytometer, and the following formula:
The fold proliferation of each T cell was calculated by multiplying fold=number of T cells after 14 days culture after TCR stimulation/number of T cells before TCR stimulation. The results are shown together in Table 11 below. From the value of the proliferation rate, T cells with the same proliferation rate of 20 or more (T cells of cell Nos. 1 to 12 in Table 11) are classified as "high-quality T cells", and T cells with the same proliferation rate of less than 20 are classified as "high-quality T cells". The cells (T cells of Cell Nos. 13-24 in Table 11) were classified as "low quality T cells" respectively.

2. Transcriptome analysis of T cells (1) 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) 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.

(2) Cells on day 0 (day0), day 6 (day6), and day 14 (day14) after TCR stimulation were collected, and total RNA was first extracted using RNeasy Micro Kit (manufactured by QIAGEN). Extracted. Next, cDNA was synthesized and amplified from 10 ng of the extracted total RNA using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (manufactured by Clontech).

(3) Using 1 ng of cDNA obtained in (2), library preparation was performed using Nextera XT DNA Library Preparation Kit (manufactured by Illumina) and Nextera XT v2 Index Kit Set A (manufactured by Illumina). , Next-seq500 (manufactured by Illumina), read length: 75 bp, by performing sequence analysis under the conditions of single end read, more than 5 million reads of genes per sample were decoded.

(4) The gene (sequence data) decoded in (3) was mapped to the human genome sequence using TopHat2 (JOHNS HOPKINS University) and Bowtie2 (JOHNS HOPKINS University). For human genome sequence information and human gene information, BUILD GRCh38 published by NCBI (National Center for Biological Information) was used. For the mapped genes, the expression level of each gene was determined in units of FPKM (Fragments Per Kilobase of exon per Million reads mapped) from the number of reads of each gene decoded by Cufflinks (University of Washington).

3. Secretome analysis of T cells (1) For T cells (TCR unstimulated) 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, 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. .

(2) 1×10 ⁶ cells were collected from each of the cells 6 days after TCR stimulation, and 15% (w/v) dialyzed FBS, 4 mmol/L L-glutamine, ITS (insulin-transferrin-sodium selenite) supplement. (×1), suspended in αMEM (-Met) medium (α-modified Eagle minimal essential medium, methionine-free) supplemented with 50 μg/mL ascorbic acid, 5 ng/mL IL-7, and 5 ng/mL IL-15. 0.00625 mmol/L L- _{azidohomoalanine} (AHA), 15% (w/v) dialyzed FBS, 4 mmol/L L-glutamine, ITS Using αMEM (-Met) medium supplemented with (insulin-transferrin-sodium selenite) supplement (x1), 50 μg/mL ascorbic acid, 5 ng/mL IL-7, and 5 ng/mL IL-15, 37 °C and 5% _CO2 for 24 hours.

(3) After culturing, 0.4 mL of the culture supernatant was collected, and the AHA-labeled protein (secretome) secreted into the culture supernatant was collected using Click-iT ^™ Protein Enrichment Kit, for click chemistry capture of azide-. A modified protein (manufactured by Thermo Fisher Scientific) was used to capture the azide group of the AHA on alkyne agarose resin for extraction.

(4) The protein extracted in (3) was analyzed by LC-MS/MS (Ultimate 3000 RSLCnano and Q Exactive, both manufactured by Thermo Fisher Scientific). The amino acid sequences of the extracted proteins were extracted from the analyzed data using Proteome Discoverer software (ver.2.2, manufactured by Thermo Fisher Scientific).

(5) Using Mascot software (ver.2.5, manufactured by Matrix Science), by matching with a database constructed by combining the amino acid sequences described in (A) to (C) below, from the determined amino acid sequence protein was identified. The identification of significant proteins was performed by adjusting the identification score threshold so that the false discovery rate (FDR) was 1%. Identified proteins were quantified using Label Free Quantification (LFQ) in Proteome Discoverer 2.2.
(A) Homo sapiens-derived protein entries registered in SwissProt (http://www.uniprot.org/) version 2018_11 (20413 in total);
(B) protein entries from bovine (Bos taurus) registered in SwissProt (http://www.uniprot.org/) version 2018_11 (5992 in total);
(C) Amino acid sequences of human and non-bovine protein contaminants obtained from The Global Proteome Machine Organization (http://www.thegpm.org/crap/index.html) (46 in total).

4. Principal component analysis (PCA)
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. A principal component analysis (PCA) was performed on the data of all 37,912 genes combining these (a) to (d) in each T cell (23 types: cell Nos. 1 to 21, 23, 24). In addition, the cell no. 22 T cells were not included in the results because day 14 cells were not obtained in the transcriptome analysis of 2 above.

A scatter plot obtained with the PCA is shown in FIG. From FIG. 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).

From Table 12 and FIG. 2, 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. In PCA, since the coefficient of the principal component score and the value of the principal component loading are almost proportional, 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. Therefore, genes with a high principal component loading value of the first principal component are expressed in cells with a high PC1 score, i.e., high-quality T cells, and genes with a low principal component loading of the first principal component are expressed in cells with a high PC1 score. was found to be expressed in cells with low cytotoxicity, ie, low quality T cells.

Therefore, from the PCA, the principal component loading of the first principal component was determined for each gene expression level obtained in (a) to (d) above. Among all 37,912 genes 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. In addition, 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.

5. Correlation between T cell quality (proliferative ability) and marker protein expression (part 1)
As a result of “4. LGALS8 (nucleotide sequence number 121) was selected, and the correlation between the expression levels of proteins translated from these genes and the quality of T cells was confirmed.

(1) Cell sample preparation before TCR stimulation (day 0) (1-1) 10 types of T cell lines (cell Nos. 5, 10, 11, 13, 15, 16, 18, 19, 22, 24) was thawed and added to 96-well plate medium A (αMEM supplemented with 15% (w/v) FBS (manufactured by BioWest), 5 ng/mL IL-7, and 5 ng/mL IL-15), respectively. Seeded at 10 ⁶ cells/well.
(1-2) The seeded cells were suspended in 1 mL of Phosphate Buffered Saline (PBS) (-) and freeze-thawed three times, or dissolved by adding 1 mL of Lysis buffer 3 (manufactured by Mybiosource). was defined as "pre-stimulation cell sample (day 0)".

(2) Sample preparation of cells (day 2) after TCR stimulation (2-1) 11 types of iPS-T cell lines (cell Nos. 5, 10, 11, 13, 16, 18-20, 22- 24) was thawed and seeded onto medium A of a 96-well plate at 1×10 ⁶ cells/well.
(2-2) Using T cell receptor (TCR) stimulation magnetic beads (Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (manufactured by Thermo Fisher Scientific)), the TCR of the seeded T cells is induced. After the stimulation, the cells were cultured under conditions of 5% CO ₂ and 37° C. for 2 days while appropriately replacing the medium with medium A.
(2-3) The cultured iPS-T cells were recovered (collected amount: 0.7×10 ⁷ to 1×10 ⁷ cells).
(2-4) The collected cells were lysed by the method described in (1-2), and this was used as the "post-stimulation cell sample (day 2)".

(3) Protein expression level measurement For the pre-stimulation cell sample (day0) prepared in (1) and the post-stimulation cell sample (day2) prepared in (2), using the following commercially available kit, each by ELISA method Protein expression level measurement was performed.
ITGB7 (amino acid sequence number 7): Enzyme-linked Immunosorbent Assay Kit For Integrin Beta 7 (ITGb7) (manufactured by Mybiosource)
BMPR2 (amino acid SEQ ID NO: 15): Human Bone Morphogenetic Protein Receptor II (BMPR-II) ELISA Kit (manufactured by Mybiosource)
TP53I3 (amino acid sequence number 27): TP53I3 elisa kit (manufactured by Mybiosource)
LGALS8 (amino acid sequence number 44): Human Galectin 8 ELISA Kit (manufactured by Mybiosource).

(4) Correlation Coefficient Calculation For the four proteins (ITGB7, BMPR2, TP53I3, and LGALS8), correlation coefficients between the proliferation potential (logarithmic value) and the protein expression level (logarithmic value) were calculated. The results are shown in Table 13 below.

6. Correlation between T cell quality (proliferative ability) and marker protein expression (part 2)
As a result of “4. Correlation with quality was confirmed.

(1) Obtaining 0.5×10 ⁷ to 1×10 ⁷ cells of 18 kinds of T cell lines (Cell Nos. 5 and 8 to 24 shown in Table 11); Cells were lysed by the method described in (1-2) to obtain a “pre-stimulation cell sample (day 0)”.

(2) After aligning the pre-stimulation cell sample (day 0) prepared in (1) so that the amount of protein is 30 μg, add it to an antibody array (Signaling Explorer Antibody Array, manufactured by Full Moon BioSystems) and follow the attached protocol. ALCAM protein expression level measurement was performed according to.

(3) 5. Correlation coefficients were calculated in the same manner as in (4). The results are shown in Table 14 below.

In Tables 13 and 14, when the absolute value of the correlation coefficient was 0.5 or more, it was determined that there was a "correlation". As shown in Tables 13 and 14, in the pre-stimulation cell sample (day 0), ALCAM (amino acid SEQ ID NO: 5) and TP53I3 (amino acid SEQ ID NO: 27) confirmed the correlation between proliferation ability and protein expression level. This suggests that ALCAM and TP53I3 are markers that can assess the quality of T cells at steady state. On the other hand, in the post-stimulation cell sample (day 2), ITGB7 (amino acid sequence number 7), BMPR2 (amino acid sequence number 15), and LGALS8 (amino acid sequence number 44) were confirmed to have a correlation between proliferation ability and protein expression level. Therefore, 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. Among these, 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.

ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the T-cell evaluation method which can evaluate the quality of T-cell by a new index, and the reagent for T-cell evaluation used for it.

Claims (10)

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;
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 detecting step is a step of detecting the expression product using a probe molecule that specifically binds to the expression product of at least one gene selected from the group consisting of the genes (I) to (III). A method according to claim 1. The expression product is a polypeptide encoded by any one of the genes (I) to (III), and the probe molecule is an antibody that specifically binds to the polypeptide and/or or an aptamer. 4. The method according to any one of claims 1 to 3, wherein the evaluation step is a step of evaluating T cell proliferation ability using expression of the gene as an index. 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 claims 1 to 4.
(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 (II') SEQ ID NOS: 5, 7, 15, 27, and 44 A 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 in the amino acid sequence according to any one of SEQ ID NO: A gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence of any one of 5, 7, 15, 27 and 44. 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 claims 1 to 5.
(I') a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, and 27; (II') set forth in any one of SEQ ID NOs: 5, 15, and 27; Any of gene (III') SEQ ID NOS: 5, 15, and 27 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 or a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in one. A reagent for use in the method according to any one of claims 2 to 6, which is specific for the expression product of at least one gene selected from the group consisting of the following genes (I) to (III) T cell evaluation reagent containing a probe molecule that binds to
(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 expression product is a polypeptide encoded by any one of the genes (I) to (III), and the probe molecule is an antibody that specifically binds to the polypeptide and/or or an aptamer, the reagent of claim 7. 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') , a reagent according to claim 7 or 8.
(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 (II') SEQ ID NOS: 5, 7, 15, 27, and 44 A 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 in the amino acid sequence according to any one of SEQ ID NO: A gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence of any one of 5, 7, 15, 27 and 44. 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 reagent according to any one of claims 7-9.
(I') a gene encoding a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5, 15, and 27; (II') set forth in any one of SEQ ID NOs: 5, 15, and 27; Any of gene (III') SEQ ID NOS: 5, 15, and 27 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 or a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in one.

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