JP2023064787A - Quality evaluation method of t-cell, and reagent used in the method - Google Patents

Abstract

To provide a quality evaluation method of a T-cell which is capable of evaluating the quality of a T-cell by a new index.SOLUTION: A quality 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 sequence number 1. (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 sequence number 1. (iii) a gene encoding polypeptide including an amino acid sequence having 70% or more of identity with the amino acid sequence described in sequence number 1.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a T cell quality evaluation method and a reagent used in the method, that is, a T cell quality evaluation method and a T cell quality evaluation reagent used therein.

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 discloses a method of inducing CD8α ⁺ β ⁺ cytotoxic T cells by culturing CD4 ⁺ CD8 ⁺ T cells in a medium containing interleukin 7 and a TCR (T cell receptor) activator. is described.

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, Vol. 439, 2006, p. 682-687

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

The present invention has been made in view of the above-mentioned problems of the prior art, and provides a T cell quality evaluation method capable of evaluating the quality of relatively young T cells using a new index, and a T cell quality evaluation reagent used therein. intended to provide

The present inventors have made intensive studies to solve the above problems. Analysis and transcriptome analysis were performed. Next, from these analysis results, we identified signal transduction pathways involving genes and proteins with large differences in expression levels between high-proliferation T cells and low-proliferation T cells, and identified proteins involved in such signal transduction pathways (CD39 and CD73) were selected as candidate markers for T cell quality assessment. Next, when the selected proteins were examined to see if they could be used as quality evaluation markers for T cells, the expression level of a specific protein (CD39), which is a type of T cell-derived secretome, and the proliferative ability of T cells, We found that there is a correlation between exhaustion. Therefore, the present inventors have found that the quality of relatively young T cells can also be evaluated by using such a protein as a new marker and using this expression level as an index, thus completing the present invention.

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

[1] a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells;
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
A method for assessing T cell quality, comprising:
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.

[2] 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] A reagent for use in the method according to any one of [2] to [4], containing at least one gene selected from the group consisting of the following genes (i) to (iii) A T cell quality assessment reagent containing a probe molecule that specifically binds to an expression product.
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.

[6] The expression product is a polypeptide encoded by any one of the genes (i) to (iii), and the probe molecule specifically binds to the polypeptide The reagent of [5], which is an antibody and/or an aptamer.

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

A graph showing the relationship between the proliferative ability of each T cell (X axis) and the expression level of the gene encoding CD39 or CD73 in the T cell (Y axis) in "Verification of T cell evaluation marker candidates 1" be. (a) is a graph in which the Y-axis is the expression level of the gene encoding CD39, and (b) is a graph in which the Y-axis is the expression level of the gene encoding CD73. In "Verification of Marker Candidates for T Cell Evaluation 2", the expression level of CD45RA in T cells after 1 stimulation treatment and 10 stimulation treatments (Y-axis), and CD45RO, CD39, or PD-1 in the T cells is a graph showing the relationship between the expression level (X-axis) of (a) is a graph when the X axis is the expression level of CD45RO, (b) is a graph when the X axis is the expression level of CD39, and (c) is a graph when the X axis is the expression level of PD-1 It is a graph when it is a quantity.

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 the quality of T cells>
The method for evaluating the quality of T cells of the present invention comprises
a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells;
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
(hereinafter sometimes simply referred to as "the method of the present invention").
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.

[T cells]
In the present invention, the origin of T cells is not particularly limited, and may be animal-derived or human-derived. or from a person or animal suffering from a malignant tumor, an infectious disease, or an autoimmune disease). Furthermore, T cells differentiated from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); T cells differentiated from somatic stem cells such as hematopoietic stem cells; It may be a T cell.

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 encephalopathic viral vectors, etc.) into T cells, and gene editing of T cells includes, for example, , gene editing (genome editing) of T cells using site-specific nucleases (meganucleases, zinc finger nucleases, TALENs, PPR, 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, expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells is detected.
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.

The polypeptide encoded by any one of the genes (i) to (iii) is preferably contained in the T cell-derived secretome. In the present invention, the term "secretome" refers to a protein secreted from a cell or exposed outside the cell through the cell membrane, and includes a secretory protein endogenous to the cell.

In the present invention, the gene whose expression is detected (hereinafter sometimes referred to as "target gene") is human-derived and typically contains "(i) the amino acid sequence set forth in SEQ ID NO: 1 A gene encoding a polypeptide". The amino acid sequence of SEQ ID NO: 1 is CD39, a secretome derived from T cells (UniProt No.: P49961). CD39, also known as SPG64, is also known to have ENTPD1 (EctoNucleoside TriphosPhate Diphosphohydrolase 1) activity, ATPase activity, ADPase activity, and NTPDase-1 activity.

In the present invention, the target gene is a homologous gene (e.g., a counterpart gene in an organism other than human) of "(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1". can also In addition, since the nucleotide sequence of a gene can be mutated in nature (that is, non-artificially) due to mutation thereof, etc., such natural mutants can also be used as the target gene in the present invention. can. Furthermore, in the current state of the art, for example, when the genetic information of (i) above is obtained, a person skilled in the art can modify the nucleotide sequence and modify the function, although it is different from the amino acid sequence that codes for it. Since proteins can also be prepared, such variants may also be used as the target gene.

Therefore, in the aspect of the target gene according to the present invention, "(ii) one or more amino acid residues are substituted, deleted, inserted, and/or added in the amino acid sequence set forth in SEQ ID NO: 1 A gene that encodes a polypeptide comprising an amino acid sequence is also included. Here, the term "amino acid sequence in which amino acid residues are substituted, deleted, inserted, and/or added" means an amino acid sequence in which amino acid residues are substituted, deleted, inserted, or added, or It shows that the amino acid sequence is a combination of two or more of Further, "plurality" means, for example, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 is preferably an integer of, but not limited to. One or more amino acid residues is preferably 1 to 10 amino acid residues, more preferably 1 to 5, still more preferably 1 to 3, particularly preferably 2 or less. is.

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

Accordingly, embodiments of the target gene according to the present invention also include "(iv) a nucleotide sequence that hybridizes under stringent conditions with the complementary strand of the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:1". Hybridization reactions are typically performed under stringent conditions to isolate homologous genes. The term "stringent conditions" means that the washing operation of the membrane after hybridization is performed at a high temperature in a low-salt solution. trisodium citrate, 150 mM sodium chloride), 0.5% SDS solution at 60° C. for 20 minutes. Hybridization can be performed, for example, according to the method described in the instruction manual attached to the known ECL direct DNA/RNA labeling/detection system (manufactured by GE Healthcare Biosciences). The more stringent the hybridization conditions, the more highly homologous DNA can be expected to be isolated. However, the above conditions are only examples, and the necessary stringency can be achieved by appropriately combining DNA concentration, DNA length, hybridization reaction time, and the like.

Furthermore, for example, a protein encoded by a homologous gene obtained by such a method usually has a high degree of identity with the amino acid sequence set forth in SEQ ID NO:1. Therefore, the target gene aspect of the present invention also includes "(iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO: 1". Amino acid sequence identity can be determined, for example, using the BLASTP program (Altschul et al., J. Mol. Biol., 215, 1990, p. 403-410). In addition, the identity with the amino acid sequence set forth in SEQ ID NO: 1 is typically preferably 70% or more, more preferably 75% or more, 80% or more, 85% or more, 90% or more, 95% or more (eg, 96% or more, 97% or more, 98% or more, 99% or more).

(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). , which produces a variety of mature mRNAs. In turn, various proteins are translated thereby. Various mRNAs and proteins produced by 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, the detection step according to the method of the present invention includes binding specifically to the expression product of at least one gene selected from the group consisting of the genes (i) to (iii) (that is, the target gene). Preferably, the expression product is detected using a probe molecule that

[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 fluorescent substances such as FITC (fluorescein isothiocyanate), FAM (fluorescein amidite), DEAC (7-(diethylamino)coumarin), R6G (rhodamine 6G), TexRed, Cy5, and BODIPY FL; enzymes such as β-D-glucosidase, luciferase, HRP (horseradish peroxidase); radioactive isotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹²³ I; affinity substances such as biotin and streptavidin; Luminescent substances such as luminol, luciferin, and lucigenin are included.

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, the group consisting of the following polypeptides (i') to (iii') A polypeptide selected from
(i') a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 (ii') in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or added (iii') A polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.

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

<Detection by anti-polypeptide antibody and/or aptamer>
Detection methods for detection at the translation level include, for example, antibodies that specifically bind to the target polypeptide (hereinafter sometimes referred to as "anti-polypeptide antibodies") and/or aptamers (hereinafter , In some cases, simply referred to as "aptamer"), immune cell staining method, imaging cytometry, flow cytometry, ELISA (Enzyme-Linked Immuno Sorbent Assay) method, radioimmunoassay, immunoprecipitation method, immunoblotting (Western blotting method etc.), methods of detection using antibodies such as antibody arrays and in vivo imaging (immunological techniques); methods of detection using aptamers instead of the antibodies. In addition, the immunological method may be performed automatically using an enzyme immunoassay device such as AIA-900 or AIA-CL2400 (both manufactured by Tosoh Corporation) after preparing necessary reagents.

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

If the anti-polypeptide antibody according to the present invention is a polyclonal antibody, an immunized animal is immunized with an antigen (target polypeptide, its partial peptide, or cells expressing these, etc.), and the antiserum is obtained by conventional means ( Salting out, centrifugation, dialysis, column chromatography, etc.) can be appropriately purified and obtained. Monoclonal antibodies can also be produced by the hybridoma method or recombinant DNA method. Hybridoma methods include, for example, Kohler and Milstein's method (Kohler & Milstein, Nature, 256:495 (1975)). Recombinant DNA methods include, for example, DNA encoding the anti-polypeptide antibody. It is cloned from B cells or the like, incorporated into an appropriate vector, introduced into host cells (mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.), and the anti-polypeptide antibody is produced as a recombinant antibody. (For example, 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 a of detecting the T cell-derived polypeptide labeled with the labeled amino acid;
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, substantially not contained in the T cell labeling medium) may be any one of the above or a combination of two or more of them. Among them, methionine is preferred from the viewpoint of efficient incorporation into the protein synthesis pathway.

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 the essential amino acid methionine, the labeling amino acid contained in the T cell labeling medium instead of methionine may be, for example, ² H-labeled methionine, ¹³ C-labeled methionine, and ¹⁵ N-labeled methionine, which are isotopically-labeled methionines substituted with the stable isotope; L-azidohomoalanine (AHA), which are structural analogues of methionine; -homopropargylglycine (HPG: L-Homopropargylglycine), L-homoallylglycine (HAG: L-Homoallylglycine), any one of which may be used or a combination of two or more thereof may be used.

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 growth.

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

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 above 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 indicator. The quality of T cells to be evaluated in the present invention includes proliferative ability and cancer cytotoxic activity. Among them, the method of the present invention is preferable as a method for evaluating the proliferative ability of T cells. More specifically, the term "proliferative ability" indicates the ability to activate T cells and increase cells having cytotoxic activity in vitro or in vivo by TCR stimulation or antigen stimulation. The proliferative ability of T cells can be confirmed, for example, by stimulating T cells with TCR-stimulating magnetic beads by the method shown in the Examples below, and checking the ratio of the number of cells before stimulation to the number of cells 14 days after stimulation. However, it is not limited to this.

In the present invention, the lower the expression level of the target gene detected in the detection step, the higher the growth ability, that is, the better the quality. It can be evaluated as having low growth ability, ie, poor quality. The evaluation criteria for the quality of the T cells may be appropriately set according to the purpose, and are not particularly limited. For example, in the detection step, if even a little expression of the target gene is not detected according to the expression level of the target gene (transcription level or translation level of the target gene), that is, the target polynucleotide or the target polypeptide If even a little is not detected, the proliferative ability of the T cells may be evaluated as high, and if the expression level of the target gene above a certain threshold is not detected, the proliferative ability may be evaluated as high. Further, the degree of T cell proliferation ability may be evaluated according to the expression level of the target gene. For example, with respect to the expression level of the target gene detected in a standard sample such as T cells that have been specified in advance to have low proliferative ability, it is below a certain value, or below the average expression level of the target gene. A signal amount of 4 SD or less, 3 SD or less, or 2 SD or less may be evaluated as having a high proliferative capacity, ie, good quality.

<T cell quality evaluation reagent, T cell evaluation kit>
The T cell quality evaluation reagent of the present invention is a reagent (composition) for use in the T cell quality evaluation method of the present invention, and is selected from the group consisting of the following genes (i) to (iii): is a composition (hereinafter sometimes simply referred to as "reagent of the present invention") containing a probe molecule that specifically binds to the expression product of at least one gene.
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1.

In the reagent of the present invention, the genes (i) to (iii) and expression products of the genes are as described in the method of the present invention, including preferred embodiments thereof. Examples of probe molecules related to the reagent of the present invention include oligonucleotide probes, oligonucleotide primers, anti-polypeptide antibodies, and aptamers mentioned in the method of the present invention described above. in the method of the present invention. The probe molecule may be one of these or a combination of two or more of them. Among them, the anti-polypeptide antibody and/or the aptamer are preferable, and the anti-polypeptide antibody is more preferable.

The reagent of the present invention may be liquid or solid such as powder. Additional ingredients may be included.

The T cell evaluation kit of the present invention is a kit for use in the T cell quality evaluation method of the present invention, and includes at least one gene selected from the group consisting of the genes (i) to (iii) above. (hereinafter, sometimes simply referred to as "the kit of the present invention").

The probe molecule according to the kit of the present invention is preferably the reagent of the present invention. 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.

<Selection of marker candidates for T cell evaluation>
1. Secretome analysis of high-proliferation T cells and low-proliferation T cells (1) As high-proliferation T cells and low-proliferation T cells, the following cells were used, respectively.
High-proliferation T cells (1 type): T cell receptors (TCR) of naive T cells (CD45RA ⁺ , CCR7 ⁺ ) collected from human peripheral blood mononuclear cells were treated with Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (manufactured by Thermo Fisher Scientific) (hereinafter referred to as "TCR stimulation magnetic beads") was used for stimulation, medium A (15% (w/v) fetal bovine serum (FBS, BioWest), 5 ng/ The cells were cultured for 14 days under conditions of 37° C. and 5% CO ₂ while appropriately replacing the medium with αMEM (α-modified Eagle minimum essential) medium supplemented with mL interleukin 7 and 5 ng/mL interleukin 15. T cells with a proliferation rate of 81 times that before stimulation;
Low-proliferation T cells (5 types): TCR of iPS cell-derived T cells was stimulated using the TCR-stimulating magnetic beads, and medium A was changed as appropriate for 14 days at 37°C and 5% _CO2 . As a result of culturing, T cells whose proliferation rate is 20 times or less before stimulation.

(2) The TCR of the high-proliferation T cells and low-proliferation T cells of (1) above is stimulated using the TCR-stimulating magnetic beads, and medium A is exchanged as appropriate under conditions of 5% CO ₂ and 37°C. was cultured for 6 days.

(3) Collect 1×10 ⁶ cells of each T cell cultured in (2) above, add 15% (w/v) dialyzed FBS, 4 mmol/L L-glutamine, ITS (insulin-transferrin-sodium selenite ) in αMEM (-Met) medium (α-modified Eagle minimal essential medium, no methionine) supplemented with supplements (×1), 50 μg/mL ascorbic acid, 5 ng/mL IL-7, and 5 ng/mL IL-15 After suspending and culturing for 1 hour at 37° C., 5% CO ₂ , 0.00625 mM L-azidohomoalanine (AHA), 15% (w/v) dialyzed FBS, 4 mM L-glutamine, ITS (× 1), cultured for 24 hours at 37° C., 5% CO ₂ in αMEM (-Met) medium supplemented with 50 μg/mL ascorbic acid, 5 ng/mL IL-7, and 5 ng/mL IL-15. After culturing, 0.4 mL of the culture supernatant was collected, and AHA secreted into the culture supernatant was extracted using Click-iT ^™ Protein Enrichment Kit, for click chemistry capture of azide-modified protein (manufactured by Thermo Fisher Scientific). The labeled protein (secretome) was extracted by chaptering the azide groups of AHA onto alkyne agarose resin using click chemistry.

(4) The proteins extracted in (3) above were 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 extracted amino acid sequence protein was identified. The identification of significant proteins was performed by adjusting the identification score threshold so that the false discovery rate (FDR) was 1%. Identified proteins were quantified using Label Free Quantification (LFQ) in Proteome Discoverer 2.2.
(A) Homo sapiens-derived protein entries registered in SwissProt (http://www.uniprot.org/) version 2018_11 (20413 in total);
(B) protein entries from bovine (Bos taurus) registered in SwissProt (http://www.uniprot.org/) version 2018_11 (5992 in total);
(C) Amino acid sequences of human and non-bovine protein contaminants obtained from The Global Proteome Machine Organization (http://www.thegpm.org/crap/index.html) (46 in total).

2. Transcriptome analysis of high-proliferation T cells and low-proliferation T cells (1) The T cells stimulated and cultured in (2) of 1 above were collected, and total RNA was extracted using RNeasy Micro Kit (manufactured by QIAGEN). Extracted. cDNA was synthesized and amplified from 10 ng of total RNA using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (manufactured by Clontech).

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

(3) The genes (sequence data) decoded in (2) above were mapped to the human genome sequence using TopHat2 (JOHNS HOPKINS University) and Bowtie2 (JOHNS HOPKINS University). For human genome sequence information and human gene information, BUILD GRCh38 published by NCBI (National Center for Biological Information) was used. For the mapped genes, the expression level 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. Pathway analysis (1) Secretome analysis results obtained in 1 (5) above (quantitative values of identified proteins) and transcriptome analysis results obtained in 2 above (3) (expression level for each mapped gene ) were integrated, and the high-proliferation T cells and the low-proliferation T-cell group were compared, and the following conditions: secretome analysis results of unique peptide 2 or more, and transcriptome analysis results of expression ratio 0.67 Signals to which proteins encoded by genes whose expression levels differ significantly between high-proliferation T cells and low-proliferation T cells by KeyMolnet analysis using the correlation search method, narrowed down to less than or 1.5 or more Transduction pathways (signaling pathways with high scores calculated by the following formula; in the following formula, molecules belonging to pathways indicate proteins belonging to the signaling pathways). The score is a value indicating the overall degree of involvement calculated by the following formula based on a statistical method (hypergeometric distribution), and the higher the value, the more significant the signal transduction pathway.

Table 1 below shows the 1st to 10th signaling pathways and their scores (scores) in descending order of scores obtained by pathway analysis. From the pathways shown in Table 1, the adenosine signaling pathway at position 5 and the P2Y signaling pathway at position 9 were selected and described in Trends in Molecular Medicine, Vol. 19, 2013, p. 355-367, CD39 and CD73, which are proteins involved in these signaling pathways, were selected as candidate markers for quality evaluation of T cells.

<Verification of marker candidate for T cell evaluation 1>
It was verified whether CD39 and CD73 selected in <Selection of T cell evaluation marker candidates> can be used as T cell quality evaluation markers.

(1) The following cells were used as T cells (22 types) with different proliferative potentials, which is one of the evaluation items of T cell quality.
Primary T cells (9 types): T cells that have been stimulated with different culture conditions or stimulation times to naïve CTL-derived T cells, and that have shown different proliferative abilities;
iPS cell-derived T cells (13 types): T cells derived from different iPS cells and having different proliferative potentials.

(2) The TCR of the primary T cells or iPS cell-derived T cells of (1) above is stimulated using the TCR-stimulating magnetic beads, and the medium is replaced with medium A as appropriate under conditions of 5% CO ₂ and 37°C. for 14 days. The cultured T cells were collected, and transcriptome analysis was performed in the same manner as in 2 of <Selection of Marker Candidates for Evaluation of T Cells> above to determine the expression level of the gene encoding CD39 or CD73.

FIG. 1 shows a graph in which the proliferative capacity (fold expansion) of each T cell is plotted on the X axis and the expression level of the gene encoding CD39 or CD73 in the T cells (CD39 expression or CD73 expression) is plotted on the Y axis. . The proliferative ability of each T cell is the ratio of the number of cells before stimulation to the number of cells 14 days after stimulation (number of cells after 14 days/stimulation cell number). Expression levels of the gene encoding CD39 varied based on differences in proliferative capacity. Specifically, the expression level of the gene encoding CD39 was higher in T cells with lower proliferation ability (Fig. 1(a), ^R2 = 0.5085). From this, it was confirmed that CD39 can be used as a marker for evaluating the proliferation ability of T cells (for example, an exhaustion marker). CD39 is represented by the amino acid sequence shown in SEQ ID NO:1. On the other hand, the expression level of the gene encoding CD73 hardly changes due to differences in proliferation ability (Fig. 1(b), ^R2 = 0.0068), and is not suitable as a marker for evaluating T cell proliferation ability. It was confirmed (CD73: Comparative Example).

<Verification of T cell evaluation marker candidate 2>
The expression times of CD39 selected in <Selection of T cell evaluation marker candidates> and PD-1, which is a known exhaustion marker, were compared.

(1) iPS cell-derived T cells exhibiting naive traits (CD45RA ⁺ , CCR7 ⁺ ) were stimulated with the mitogen PHA (Phytohemagglutinin) and a peripheral blood mononuclear cell feeder, and medium A was exchanged as appropriate. , 37° C., 5% CO ₂ for 14 days, and stimulation treatment was repeated 10 times. The expression levels of CD39, CD45RA, CD45RO, and PD-1 on the cell surface after one stimulation treatment and after ten stimulation treatments were measured by flow cytometry.

The results of plotting the expression levels of CD45RA in each T cell after 1 stimulation treatment and 10 stimulation treatments on the Y axis and the expression levels of CD45RO, CD39, or PD-1 in the T cells on the X axis, respectively. Shown in FIG. After 10 stimulation treatments compared to after 1 stimulation treatment, the expression level of CD45RA, a marker for naive cells, was decreased and the expression level of CD45RO was increased, and T cell differentiation progressed. It was confirmed (Fig. 2(a)). The CD39 expression level was elevated by repeated TCR stimulation, and showed a negative correlation with the CD45RA expression level (Fig. 2(b)). On the other hand, the expression level of PD-1 showed a slight increase even after 10 stimulation treatments. This suggests that CD39 can be used as an exhaustion marker that is expressed earlier than PD-1, which is a conventionally known exhaustion marker.

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

Claims (6)

a detection step of detecting expression of at least one gene selected from the group consisting of the following genes (i) to (iii) in T cells;
an evaluation step of evaluating the quality of T cells using the expression of the gene as an index;
A method for assessing T cell quality, comprising:
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1. 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. A reagent for use in the T cell quality evaluation method according to any one of claims 2 to 4, wherein at least one gene selected from the group consisting of the following genes (i) to (iii) A T cell quality assessment reagent containing a probe molecule that specifically binds to an expression product.
(i) a gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (ii) in the amino acid sequence set forth in SEQ ID NO: 1, one or more amino acid residues are substituted, deleted, inserted, and/or or a gene encoding a polypeptide comprising the added amino acid sequence (iii) a gene encoding a polypeptide comprising an amino acid sequence having 70% or more identity with the amino acid sequence set forth in SEQ ID NO:1. The expression product is a polypeptide encoded by any one of the genes (i) to (iii), and the probe molecule is an antibody that specifically binds to the polypeptide and/or or an aptamer, the reagent of claim 5.

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