JP2009011236A - Method for analyzing/identifying t-cell antigen receptor gene at one cell level - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for efficiently analyzing/identifying one human T-cell antigen receptor (TCR) gene, and to provide a transformed human T-cell or the like manifesting the human TCR originated from the one T-cell. <P>SOLUTION: A CTL cell considered to produce the TCR to gp100<SB>209-217</SB>peptide amplified in a melanoma patient administered with a dendritic cell vaccine is doubly stained by an FITC-labeled anti-CD8 antibody and a PE-labeled gp100-HLA-A2 tetramer, and the doubly stained CTL cell is fractionated by one cell by using a micromesh device or the like. The whole RNA is extracted from the separated one human CTL cell, and the antigen receptor gene of the one human T-cell is analyzed and identified. A naive T-cell is transformed by incorporating the identified TCR gene. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for analyzing and identifying an antigen receptor gene of one human T cell, a method for preparing a recombinant vector capable of expressing an antigen receptor derived from one human T cell, and one human T cell. The present invention relates to a transformed human T cell in which a recombinant vector capable of expressing an antigen receptor derived from is introduced into a naive T cell and the antigen receptor derived from one human T cell is expressed.

  Malignant melanoma is a cancer in which immunotherapy using interleukin 2 or the like is relatively effective (see, for example, Non-Patent Document 1), but in vivo antitumor effects include cytotoxicity that reacts with tumor cells. It has been shown that T cells (Cytotoxic T lymphocyte: CTL) are important (see, for example, Non-Patent Document 2). After immunotherapy, tumor-reactive T cells are activated, and T cell receptors (TCR) on T cells are degraded by tumor cell proteins presented on the surface of tumor cells by HLA (human leukocyte antigen) molecules. Recognize the peptide (tumor antigen) produced and damage tumor cells. Furthermore, it is considered that various cytokines are secreted and other immune cells such as macrophages are induced and activated to cause tumor rejection in vivo. Tumor-reactive T cells recognize not only autologous tumor cells but also common tumor antigens that react to tumor cells from other patients who share HLA, and unique antigens that react only to autologous tumor cells There is a case.

  In order to elucidate the immune response mechanism against human tumor cells and develop new immunotherapy, it is important to identify tumor antigens recognized by these tumor-reactive T cells. When tumor-reactive T cells have been established, it is possible to isolate a malignant melanoma antigen using a functional cDNA expression cloning method utilizing the reactivity. For example, it is possible to isolate a malignant melanoma antigen that can be used for the prevention, diagnosis, and treatment of melanoma using tumor-reactive CTLs from patients with metastatic melanoma and tumor cell lines that express HLA-A2. One of these is MART1, which has been isolated and identified by a similar method. Representative examples to date include pigment cell (melanocyte) tissue-specific proteins such as MART1 and gp100, MAGE, BAGE, GAGE, NY-ESO-I and the like that are observed in testes in various tumors and normal tissues, etc. CT antigens (Cancer-Testis antigen), β-catenin produced by abnormal gene of tumor cells, CDK4 (cyclin dependent kinase 4), p15, GnT-V and other mutant peptide antigens have been isolated (for example, non- (See Patent Document 3).

In addition, cytomegalovirus (CMV) infection is not a serious problem in normal individuals who are in a normal immune state, but is associated with severe interstitial pneumonia, especially in an immunocompromised state such as after organ transplantation. It is a serious disease that can cause symptoms. At present, the clinical symptoms of CMV infection are alleviated by the administration of antiviral agents, but antiviral agents that can be a radical countermeasure are not yet known. So far, studies on HLA-A2-restricted epitopes mainly derived from CMVpp65 protein have been conducted, and induction of specific CTL cells using CMVpp65 _495-503 peptide and evaluation of CTL clones using tetramer reagents have been conducted. Has been done. However, not much knowledge has been obtained regarding the induction response of CTL cells to CMV in patients with advanced cancer, and nothing is known about the TCLβ chain gene of CTL cells specific for CMV infection.

  T cells recognize MHC molecule / antigen peptide complexes by TCR and bind to target cells such as antigen presenting cells (APCs), but CTLs only bind to MHC class I molecule / antigen peptide complexes. It binds (MHC class I restriction), and helper T (Th) cells are considered to be able to bind only to MHC class II molecule / antigen peptide complexes (MHC class II restriction). In addition, MHC class I molecules are expressed in most nucleated cells, but MHC class II molecules have special functions related to the elimination of foreign substances such as B cells, macrophages, Kupffer cells, activated T4 lymphocytes and defense against infection. It is expressed only in cells with For this reason, Th cells can bind to dendritic cells, B cells, activated T cells, etc. expressing MHC class II molecules, but cannot bind directly to other tumor cells or virus-infected cells. However, MHC class II-restricted CD4-positive CD8-negative T cells into which a CTL-derived TCR gene, which is made MHC class I-restricted by genetic manipulation, reacts with APC pulsed with the corresponding antigen in a CD8-independent manner. It was shown to express a TCR that can be activated and bind to the corresponding antigen. In addition, it has been reported that tumors can be specifically damaged by introducing a TCR gene derived from tumor antigen-specific CTL into peripheral blood lymphocytes having nonspecific antitumor activity (for example, non-tumor activity). (See Patent Document 4).

  On the other hand, it is a membrane surface protein that exists on the surface of T cells, and two types of heterodimers consisting of α and β chains and heterodimers consisting of γ and δ chains have been identified as TCRs responsible for antigen recognition. Has been. Like the immunoglobulin gene, the TCR gene is composed of a variable region (V: variable), a diversity region (D: diversity), and a joining region (J: joining). It is composed of a C domain composed of a constant region (C: constant) including a region, a transmembrane region, and an intracellular region. Since these amino acid sequences differ from one T cell to another, TCR is an antigen recognition molecule and at the same time a mark for individual T cells. This diversity of TCR is generated by the rearrangement of the acquired TCR gene, and TCRβ and δ chains are known to reconstitute VDJ, and TCRα and γ chains are known to reconstitute VJ. Yes.

The early differentiation of T cells is closely related to the rearrangement of the TCR α and β chain genes as in B cells. Reconstruction of TCRβ chain gene DN-T cells ^{(double negative: CD4 - 8 -} ) of the start at step, complexes of successful cell β chain protein and alternate α chain reconstruction of β chain gene (preTα) That is, the pre-T cell receptor is expressed on the cell surface. Then, the expression of CD4 and CD8 molecules was induced and transferred to DP-T cells (double positive: CD4 ⁺ 8 ⁺ ), and the T cells that became DP succeeded in reconstitution of the α chain gene of TCR. It is known that only the cultured cells can express TCR on the surface.

  Regarding the gene structure of the TCR β chain, according to the human genome gene sequence information database, the TCR β chain gene consists of about 30 subgroups (about 70 types of genes), 2 types of D genes, 14 types of genes. It is also known that there are two variable regions (CDR1, CDR2) in Vβ, consisting of the J gene and two types of C genes. Among these, Vβ, Dβ, and Jβ are selected and bonded to each other to constitute a DNA encoding the TCRβ chain. At this time, random base insertions and deletions occur at the Vβ-Dβ-Jβ binding site, and infinite diversity is produced. This Vβ-Dβ-Jβ binding site is called CDR3 (third complementarity determining region) or junction region, and is the most polymorphic portion of TCR. Therefore, even if each gene configuration is the same, it is very rare that the same sequence including the linking portion is obtained.

Japanese National Patent Publication No. 10-505481 JAMA 271, 907, 1994 Microbiol.Immunol. 42, 803, 1998 Immunol Res 16, 313, 1997 J. Immunol., 171: 3287-3295, 2003

  In recent years, abnormal clones of immune cells in the pathology of cancer and autoimmune diseases have attracted attention, but considering the dynamic adaptation mechanism of immunity, it can be easily imagined that the functional characteristics of individual cells are different. Even from the viewpoint of immune response, TCR and antibody gene mutations start with a single cell, and in the study of immune cell function, research at the single cell level is no longer inevitable. is there. In particular, the dynamics of individual immune cells under the pathological condition of cancer has not yet been studied in detail, and can be an important research subject in considering new treatments. The object of the present invention is to position the gene analysis of mRNA derived from one T cell as the purpose of future research and development, and to efficiently analyze and identify the antigen receptor gene of one human T cell. Another object of the present invention is to provide transformed human T cells that express human T cell antigen receptors derived from these T cells.

Since TCR molecules play an important role in T cell-mediated immune responses and immune mechanisms, analysis of these immune responses and immune mechanisms requires obtaining individual TCR molecules. However, when membrane surface proteins are purified from cells, only about 10 μg can be obtained from 10 ¹⁰ cells. Moreover, in order to sort out one type of TCR due to the diversity of TCRs, it is essential to establish T cell lines having respective TCRs and to isolate TCRs from each. However, since the TCR repertoire of T cells reaches 10 ¹⁰ types, it is substantially extremely difficult to establish 10 ¹⁰ types of T cell lines.

The present inventors are conducting a clinical trial of a dendritic cell vaccine treated with HLA-A2 or A24 peptide cocktail for metastatic melanoma cases. At this time, using T cells derived from a melanoma patient treated with a dendritic cell vaccine treated with gp100 _209-217 peptide (hereinafter sometimes referred to as gp100 peptide) having HLA-A2 restriction, Melanoma gp100 peptide specific TCR was analyzed and identified. That is, CTL cells considered to produce TCR against gp100 peptide amplified in melanoma patients who received dendritic cell vaccine were treated with FITC-labeled anti-CD8 antibody and PE-labeled gp100-HLA-A2 tetramer (gp100 peptide and HLA -Double-stained CTL cells using a micro-channel device equipped with a substrate having a plurality of micropores by double-staining by linking 4 complexes of -A2 and further labeled with PE) After the cells are sorted and total RNA is extracted from one isolated human CTL cell, it is found that the antigen receptor gene of one human T cell can be analyzed and identified, and the present invention is completed. It came to.

That is, the present invention includes [1] the following steps (A), (B), (C), (D), (E), and (F): Cellular antigen receptor gene analysis / identification method (A) Collecting peripheral blood mononuclear cells obtained from humans with confirmed immune responses to specific antigens or antigenic epitopes recognized by cytotoxic T cells or helper T cells Process;
(B) T cells in peripheral blood mononuclear cells using the specific antigen or antigen epitope labeled with a labeling substance and an antibody capable of recognizing human T cells labeled with a labeling substance different from the labeling substance Double labeling;
(C) a step of sorting T cells double-labeled with a labeling substance one by one;
(D) a step of extracting total RNA from one cell and synthesizing cDNA by a reverse transcription reaction;
(E) A step of amplifying a human T cell antigen receptor gene fragment by performing a PCR reaction using a synthesized cDNA as a template and a primer pair specific to human T cell antigen receptor;
(F) a step of analyzing and determining the base sequence of the amplified gene fragment;
[2] In the step of collecting peripheral blood mononuclear cells, an immune response against the cancer-specific antigen peptide has been confirmed by administration of a dendritic cell vaccine treated with the cancer-specific antigen peptide. The method for analysis and identification according to [1] above, wherein peripheral blood mononuclear cells obtained from cancer patients are collected.

The present invention also provides a cancer-specific antigen peptide, human histocompatibility as the specific antigen or antigen epitope labeled with a labeling substance in the step of [3] double labeling T cells in peripheral blood mononuclear cells. The analysis / identification method according to [1] or [2] above, wherein a complex comprising an antigen (HLA) protein and a labeling substance is used, or [4] T cells in peripheral blood mononuclear cells. In the double labeling step, the analysis / identification method according to any one of the above [1] to [3], wherein an anti-CD8 antibody or an anti-CD4 antibody is used as an antibody capable of recognizing human T cells, [5] In the step of sorting T cells one by one, a microchannel device including a substrate having a plurality of micropores capable of capturing one cell is used. 4] [6] The following (A), (B), (C), (D), (E), and (G) steps are provided in order, Method for preparing a recombinant vector capable of expressing an antigen receptor derived from human T cells (A) Obtained from a human who has been confirmed to have an immune response to a specific antigen or antigen epitope recognized by cytotoxic T cells or helper T cells Collecting peripheral blood mononuclear cells;
(B) T cells in peripheral blood mononuclear cells using the specific antigen or antigen epitope labeled with a labeling substance and an antibody capable of recognizing human T cells labeled with a labeling substance different from the labeling substance Double labeling;
(C) a step of sorting T cells double-labeled with a labeling substance one by one;
(D) a step of extracting total RNA from one cell and synthesizing cDNA by a reverse transcription reaction;
(E) A step of amplifying a human T cell antigen receptor gene fragment by performing a PCR reaction using a synthesized cDNA as a template and a primer pair specific to human T cell antigen receptor;
(G) a step of incorporating the amplified gene fragment into an expression vector;
About.

  Furthermore, the present invention provides [7] a step of collecting peripheral blood mononuclear cells, whereby an immune response to the cancer-specific antigen peptide is confirmed by administration of a dendritic cell vaccine treated with the cancer-specific antigen peptide. In the preparation method of the above-mentioned [6], which comprises collecting peripheral blood mononuclear cells obtained from a cancer patient, and [8] a step of double labeling T cells in peripheral blood mononuclear cells [6], wherein a complex comprising a cancer-specific antigen peptide, a human histocompatibility antigen (HLA) protein, and a labeling substance is used as the specific antigen or antigenic epitope labeled with a labeling substance [6] Alternatively, an anti-CD8 antibody or an anti-CD4 antibody is used as an antibody capable of recognizing human T cells in the preparation method according to [7] and [9] a step of double labeling T cells in peripheral blood mononuclear cells. This In the preparation method according to any one of [6] to [8] above, and [10] a step of sorting T cells one by one, a plurality of micropores capable of capturing one cell are provided. A preparation method according to any one of [6] to [9] above, or [11] a preparation method according to any one of [6] to [10], wherein a microchannel device having a substrate is used. Or a recombinant vector obtained by the preparation method according to any one of [6] to [10] above, and an antigen derived from one human T cell. It relates to transformed human T cells that express the receptor.

  According to the present invention, it is possible to analyze and identify a functional TCR at a single T cell level, which has been impossible in the past from the viewpoint of tumor immunology, and to monitor an immune response. This is likely to lead to the development of groundbreaking fundamental technologies. In addition, since it becomes possible to comprehensively analyze and identify TCRs for specific immune epitopes and tumor antigens, future tailor-made medical care and diagnosis of cancer will be possible.

As a method for analyzing and identifying the antigen receptor gene of one human T cell of the present invention, the following steps (A), (B), (C), (D), (E) and (F) are sequentially performed. The method is not particularly limited as long as it is a prepared method, and a method for preparing a recombinant vector capable of expressing an antigen receptor derived from one human T cell of the present invention is the following step (A). , (B), (C), (D), (E), and (G) are not particularly limited as long as the method is sequentially provided. And (G) are preferably provided with a step (F).
(A) A step of collecting peripheral blood mononuclear cells obtained from a human whose immune response to a specific antigen or antigen epitope recognized by CTL or Th cell is confirmed;
(B) T cells in peripheral blood mononuclear cells using the specific antigen or antigen epitope labeled with a labeling substance and an antibody capable of recognizing human T cells labeled with a labeling substance different from the labeling substance Double labeling;
(C) a step of sorting T cells double-labeled with a labeling substance one by one;
(D) a step of extracting total RNA from one cell and synthesizing cDNA by a reverse transcription reaction;
(E) A step of amplifying a human T cell antigen receptor gene fragment by performing a PCR reaction using a synthesized cDNA as a template and a primer pair specific to human T cell antigen receptor;
(F) a step of analyzing and determining the base sequence of the amplified gene fragment;
(G) a step of incorporating the amplified gene fragment into an expression vector;

  The specific antigen or antigen epitope recognized by CTL in the above step (A) is a fragment of a virus particle that is captured by an MHC class I molecule and propagated on a cell surface that has been infected with a cancer-specific antigen peptide or virus. The specific antigen or antigen epitope recognized by the Th cell is captured by the MHC class II molecule, such as a macrophage or a Kupffer cell. If foreign antigens such as bacteria that have invaded the body and food allergen incorporated into the body can be expressed on the cell surface of the cell, it can be expressed as an antigenic peptide cleaved into small peptide fragments by enzymes such as proteases in the cell. There is no particular limitation.

In the step (A), it is particularly important to increase the number of T cells that specifically recognize specific antigens or antigen epitopes recognized by these CTLs or Th cells. For example, administration of a dendritic cell vaccine treated with a cancer-specific antigen peptide, preferably an HLA-A24 or HLA-A2-restricted cancer-specific antigen peptide captured by MHC class I molecules, By using peripheral blood obtained from a cancer patient whose immune response to a specific antigen peptide has been confirmed, the TCR-producing CTL that specifically recognizes the cancer-specific antigen peptide can be increased. Examples of such HLA-A2-restricted cancer-specific antigen peptides include gp100 _209-217 peptide derived from melanoma cell-specific antigen gp100. When a dendritic cell vaccine is usually produced from a cancer patient, monocyte cells collected from peripheral blood using a component blood collection device are cultured with cytokine for 7 days, and then a peptide cocktail containing gp100 (25 μg / mL for each peptide) ) And KLH (25 μg / mL), and finally a dendritic cell vaccine is obtained. In the ongoing Phase II trial, 1-5 × 10 ⁷ dendritic cells are first administered 4 times, and if there are no obvious adverse events, the dendritic cell vaccine is administered up to 10 times. . Peripheral blood mononuclear cells obtained from a cancer patient to whom a dendritic cell vaccine has been administered, multiple times using dendritic cells and T2 cells (expressing HLA-A ^* 0201 gene) treated with gp100 peptide Stimulation is preferable for increasing the number of CTL cells having a TCR that specifically recognizes the gp100 peptide in advance. In this step (A), it is extremely important that the target TCR-producing CTL is 10% or more, preferably 20%, more preferably 40% or more of all T cells. That is, this step (A) is obtained from a cancer patient whose immune response to the cancer-specific antigen peptide has been confirmed by administration of a dendritic cell vaccine treated with the cancer-specific antigen peptide. It is extremely important that the TCR-producing CTL is a step of collecting peripheral blood mononuclear cells containing 10% or more, preferably 20%, more preferably 40% or more of all T cells.

  Examples of the labeling substance in the step (B) include fluorescent substances such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), tetramethylrhodamine isothiocyanate (TRITC), and chemiluminescent substances such as luminol, isoluminol, and acridinium derivatives. , Biotin, and magnetic beads. Labeling with a labeling substance can be performed by a conventional method, and for example, Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, New York can be referred to. Further, as an antibody capable of recognizing human T cells in the step (B), an anti-human CD8 antibody capable of recognizing CTL, and an anti-human CD4 antibody and an anti-human CD28 antibody capable of recognizing Th cells are preferably exemplified. be able to. For example, in step (B), PE-labeled cancer-specific antigen peptide-HLA-A2 tetramer is used as a cancer-specific antigen peptide labeled with a labeling substance, and human T cells labeled with different labeling substances are recognized. For example, when using a FITC-labeled anti-human CD8 antibody as a possible antibody, labeling is preferably performed under conditions that do not cause the cell membrane-bound TCR of the cancer antigen-specific TCR-producing CTLs to fall off.

  In the examination so far, T cells contained in human peripheral blood are considered to be about 20 to 30% of all cells, and cytotoxic T (CTL) cells positive for CD8 are considered to be about 20%, Only 5% of the cells are present. Among them, the number of CTL cells having a TCR recognizing gp100 is still smaller (0.01-0.05% of all cells), and it is considered difficult to detect by a usual method. However, in the present invention, in the step (A), T cells of cancer patients who have been immunized with a dendritic cell vaccine treated with a cancer-specific antigen peptide and have already increased antibody titer are used. Can be detected. Thus, in the present invention, the step (A) of collecting peripheral blood mononuclear cells obtained from a patient to which a dendritic cell vaccine previously treated with a cancer-specific antigen peptide has been administered is extremely important. .

    In the step (C), T cells that are double-labeled with a labeling substance and express TCR that recognizes a cancer-specific antigen peptide on the cell membrane are collected one by one. In order to sort T cells one by one, an appropriate method is used according to the type of labeling substance used. For example, when a fluorescent substance is used as a labeling substance, a microchannel device including a substrate having a plurality of micropores capable of capturing one cell due to its shape characteristics can be used (see Examples). . By this microchannel device, a large number of T cells can be easily and simultaneously sorted. In addition, by using the microchannel device, it is possible to avoid the influence of contaminants such as particulate matter other than cells, and to provide a more sensitive analysis / identification method. In this microchannel device, T cells are captured in each micropore provided in the substrate.

As the substrate, for example, a microchip used as a component of a microorganism separation apparatus (see Japanese Patent Application Laid-Open No. 2007-89566) already developed by the present inventor can be used for cell separation in the present invention. is there.
Here, the diameter of the micropores may be appropriately selected according to the size of the cells to be sorted, and the diameter of the opening is preferably 1 to 3 μm in order to capture efficiently.
As means for capturing the target cells in the micropores, for example, the sample water suction means disclosed in JP-A-2007-89566 can be used for capturing cells in the present invention. .
Further, the material of the substrate is bPET (black polyethylene terephthalate), and any of the methods used in the microchip for a microorganism separator can be applied as the method for processing the micropores. bPET is superior to other materials in that it has less autofluorescence and therefore has less background at the time of cell fluorescence observation and can clearly observe cells. Moreover, the hole diameter and shape of the micropores formed in the bPET substrate by the above processing method are more uniform than those of metal substrate materials including, for example, a stainless steel substrate, and as a result, efficient cell supplementation can be performed.

Furthermore, the substrate can be surface-treated with a surfactant such as 10% Pluronic F-127 (Invitrogen) for the purpose of reducing cell damage (to facilitate cell loading). .
In addition, when a fluorescent substance is used as a labeling substance, it is possible to sort cells one by one by flow cytometry (single cell sorter) using fluorescence as an index. Flow cytometry enables efficient and highly accurate cell separation. In addition, even when biotin is employed as the labeling substance, cells can be sorted one by one using a binding reaction with avidin. Similarly, when magnet beads are employed, good separation using magnets is possible. Furthermore, it is possible to sort one cell at a time using a micromanipulator.

  In the above step (D), total RNA is extracted from one cancer antigen-specific TCR-producing CTL sorted by cell, and cDNA is synthesized by a reverse transcription reaction. Isolation of total RNA, isolation and purification of mRNA, cDNA acquisition, and cloning thereof are all conventional methods (eg, Molecular Cloning: A laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989). )).

  In the step (E), a human T cell antigen receptor gene fragment is amplified by a PCR reaction using a synthesized cDNA as a template and a primer pair specific to the human T cell antigen receptor gene. The primer pair specific to the human T cell antigen receptor gene is particularly limited as long as it is specific to each sequence of the α chain gene, β chain gene, γ chain gene or δ chain gene of the human T cell antigen receptor. However, for example, as a primer pair specific to the human T cell antigen receptor β chain gene, a primer pair comprising a V region sequence shown in SEQ ID NO: 2 and a C region sequence shown in SEQ ID NO: 3 is used. (See FIG. 5). The base sequence of the amplified gene fragment is analyzed and determined by a conventional method in the above step (F).

  One T cell-derived antigen receptor gene can be prepared using the gene fragment amplified in the above step (E). For example, a human TCR α chain gene can be prepared by a PCR reaction using a primer pair specific to the human TCR α chain gene, and a human TCR β chain can be prepared by a PCR reaction using a primer pair specific to the human TCR β chain gene. Genes can be prepared. In addition, a human TCR γ chain gene can be prepared by a PCR reaction using a primer pair specific to the human TCR γ chain gene, and a human TCR δ chain can be prepared by a PCR reaction using a primer pair specific to the human TCR δ chain gene. Genes can be prepared. These prepared human TCR genes can be subcloned and amplified. When genomic DNA is used as a template, effective amplification cannot be expected because exons are separated.

  In the above step (G), the human TCR α chain gene, human TCR β chain gene, human TCR γ chain gene or human TCR δ chain gene, which is the gene fragment amplified in step (E), is expressed using an expression vector, Transformed human T cells expressing antigen receptors derived from individual human T cells can be produced. The expression vector is not particularly limited as long as it is suitable for expression of the TCR gene, but is preferably one that can be expressed in animal cells. The recombinant vector of the present invention can be constructed by appropriately integrating the TCR gene into an expression vector. Such an expression vector is preferably one that can replicate autonomously in the host cell, or one that can be integrated into the host cell chromosome, and a control sequence such as a promoter, enhancer, or terminator at a position where the TCR gene can be expressed. In addition, a selection marker gene can be introduced. Introduction of a TCR antibody gene into an expression vector can be performed by a known method using a restriction enzyme and DNA ligase (for example, see Molecular Cloning, Third Edition, 1.84, Cold Spring Harbor Laboratory Press, New York). .

  Examples of expression vectors for animal cells include pCR2.1-TA (Invitrogen), pEGFP-C1 (Clontech), pGBT-9 (Clontech), pcDNAI (Funakoshi), pcDM8 (Funakoshi). PAGE107 (Cytotechnology, 3, 133, 1990), pCDM8 (Nature, 329, 840, 1987), pcDNAI / AmP (Invitrogen), pREP4 (Invitrogen), pAGE103 (J. Blochem., 101) 1307, 1987), plasmid vectors such as pAGE210, and adenoviral vectors used for transient expression in all cells (other than blood cells) including unsorted cells (Science, 252, 431-434, 1991) And retroviral vectors used for long-term expression in dividing cells (Microbiology and Immunology, 158, 1-23, 1992) and non-pathogenic, non-dividing cells that can be used for long-term expression. Roh-associated viral vectors (Curr. Top. Microbiol. Immunol., 158, 97-129, 1992) other, can be exemplified SV40 viral vectors, EB virus vector, a viral vector such as papilloma virus vectors. Examples of promoters for animal cells include cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter and the like. Can do.

  The human TCR α chain gene and human TCR β chain gene, or the human TCR γ chain gene and human TCR δ chain gene are usually inserted into separate expression vectors, respectively, and these two recombinant vectors co-transform naive T cells serving as hosts. It is preferable that the TCR α chain and the TCR β chain, or the TCR γ chain and the TCR δ chain are expressed in the same cell. Examples of a method for transforming naive T cells with a recombinant vector include the lipofectin method, the electroporation method, the calcium phosphate method, and the like. In this way, a transformed human T cell expressing an antigen receptor derived from one human T cell can be produced.

  The naive T cells into which the above-described recombinant vector of the present invention has been introduced in vitro are diseases to which adoptive immunotherapy or the like can be applied (the TCR β chain protein encoded by the TCR β chain gene incorporated in the recombinant vector exhibits specificity). It is useful as a therapeutic composition for a disease that specifically expresses an antigenic peptide. In particular, the base sequence of the TCR β chain gene of the present invention incorporated in the recombinant vector is represented by the base sequence represented by SEQ ID NO: 4 (gp100 peptide-specific TCR β chain gene), the bases represented by SEQ ID NOs: 5 and 6 Sequence (MAGE1-A24 peptide-specific TCRβ chain gene), base sequence represented by SEQ ID NO: 7 (MAGE3-A24 peptide-specific TCRβ chain gene), base sequence represented by SEQ ID NO: 8 (MART1-A2 peptide-specific Naive T cells are useful as a composition for melanoma treatment that can be suitably used for adoptive immunotherapy and the like, and the TCR β chain gene of the present invention incorporated in the recombinant vector. The nucleotide sequence is represented by SEQ ID NO: 9 to 11 (CMVpp65-A24 peptide specific TCR β chain gene), SEQ ID NO: 12. If the nucleotide sequence of (CMVpp65-A2 peptide-specific TCRβ chain gene), naive T cells are useful as CMV infection therapeutic composition can be suitably used in adoptive immunotherapy, or the like.

When used as an immunotherapeutic agent such as adoptive immunotherapy, it is preferable to purify a transformed human T cell clone. As a method for this, a method of mixing and centrifuging in a phosphate buffer or the like, a specific gravity centrifugation using a separating agent The method etc. can be used. The dose of the composition for treating diseases such as melanoma and CMV infection is preferably 10 ⁶ to 10 ¹² cells as transformed human T cells per one time. The dosage form is preferably a liquid such as an injection or infusion, and an injection or infusion in which transformed human T cells are dispersed in a physiological saline solution containing human serum albumin at 0.01 to 5%. More preferred. As the administration method, intravenous drip or injection into veins, arteries, and the like is preferable. The amount of liquid to be administered varies depending on the administration method, the place to administer, etc., but is preferably 50 to 500 cc, and it is preferable that the above-mentioned transformed lymphocytes are included in this liquid amount. The frequency of administration is preferably 1 time / day to 1 time / month, and the frequency of administration can be at least once, preferably 5 times or more.

  Cancer produced in the body of an individual patient by analyzing and identifying the cancer-specific TCR gene of the cancer patient by the method for analyzing and identifying the TCR gene derived from one human T cell of the present invention Information on the overall picture of the type of antigen-specific TCR can be obtained. In addition, by using the method for preparing a recombinant vector capable of expressing an antigen receptor derived from one human T cell of the present invention, a transformed human that expresses a cancer antigen-specific TCR on the membrane surface as described above. A large amount of T cells can be obtained, and a tailor-made individual patient diagnosis and treatment becomes possible.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Collecting peripheral blood mononuclear cells]
Peripheral blood mononuclear cells were collected from patients with metastatic melanoma who received vaccine treatment with the melanoma cell specific antigen peptide gp100. A dendritic cell vaccine treated with HLA-A2-restricted gp100 _209-217 peptide was administered to metastatic melanoma patients four times, and the immune response reaction was confirmed. The amino acid sequence of the gp100 _209-217 peptide is shown in SEQ ID NO: 1. Peripheral blood mononuclear cells (PBMC) were collected from peripheral blood derived from this patient and used for the following experiments.

[Gp100-A2 tetramer staining result]
Dendritic cells and T2-A2 cells treated with gp100 _209-217 peptide from peripheral blood mononuclear cells from HLA-A2-positive patients who received dendritic cell vaccine administration (HLA-A ^* 0201 gene in T2 cells) 4) was used for the stimulation. T2-A2 cells were obtained from ATCC (cat. CRL-1992). Thereafter, staining was performed with a FITC-labeled anti-CD8 + antibody and a PE-labeled gp100-HLA-A2 tetramer. PE-labeled gp100-HLA-A2 tetramer (manufactured by Beckman Coulter) was obtained from Medical and Biological Research Institute (MBL). The results are shown in FIG. As a result, CD8 + / gp100-HLA-A2 tetramer + cytotoxic T lymphocyte (CTL) cells were amplified to 44.3% of the whole. In contrast, control CD8 + / Flu-MP-A2 tetramer positive cells were 0.04%.

[Results of TCR antibody repatova screening]
The frequency of use of TCR repertoire of gp100-HLA-A2 tetramer positive CTL cells amplified in vivo was analyzed using a monoclonal antibody panel against TCR. The results are shown in Table 1. As a result, a significant increase (36.6%) of cells stained with an antibody against TCRVβ12 (new TRBV name by IMGT) (old name by Wei * et al .; TCRVβ8) was observed. (* Reference: Immunogenetics 40: 27-36, 1994)

[Construction of microchannel device]
Using a CAD-CAMM (computer-aided design-computer aided modeling machine system; PNC-300, manufactured by Roland Corporation), the thickness of the mold for producing the microchannel (depth: 1 mm, width: 1 mm) It was prepared by cutting a 3 mm PMMA (polymethylmethacrylate) substrate. This mold was subjected to ultrasonic cleaning and dried, and then PDMS (poly-dimethylsiloxisane, Sylgard 184; manufactured by Dow Corning) was mixed at 10: 1 with the main agent and the curing agent, injected into the mold, and degassed under reduced pressure. The PDMS site to be bonded to the substrate was a mixture of the main agent and the curing agent in a ratio of 50: 1. This PDMS was cured by heating at 85 ° C. for 20 minutes or more. Plasma treatment was performed on these surfaces for 20 seconds, and PDMS was adhered by bonding immediately after the plasma treatment. Further, silicon tubes (inner diameter 1 mm × outer diameter 3 mm) were connected to the inlet and outlet of the flow path. A black bPET (black polyethylene terephthalate) substrate having a thickness of 38 μm was used as a substrate for processing the substrate. Each micropore has a mortar shape, and the diameter on the side where the pore diameter is small is designed to be 2 μm. Furthermore, the distance between these centers was set to 60 μm, and 25 × 50 total 1250 holes were arranged in an array. Based on this design, a photomask was produced from a glass substrate on which chromium was deposited. Using this mask, a through-hole was processed in the bPET substrate using a laser with a wavelength of 248 nm and a frequency of 150 Hz using an excimer laser micro-processing machine (Optec Micro-Master System, Optec). This bPET substrate was bonded to a slide glass having a through hole having a diameter of 10 mm using an epoxy adhesive. Finally, a bPET substrate was sandwiched by PDMS, and a microchannel device was constructed.

[FITC-labeled anti-CD8 antibody + / PE-labeled gp100-HLA-A2 tetramer + CTL cell staining and sorting results]
CTL cells (purity 99.8%) after MACS sorting using PE-labeled gp100-HLA-A2 tetramer mostly stained positively with a monoclonal antibody (MoAb) that recognizes TCRVβ12 repertoire. The results are shown in FIG. Therefore, a sequence that specifically recognizes TCRVβ12 was used as a primer for PCR amplification. In addition, as shown in FIG. 3, CD8 + / gp100-HLA-A2 tetramer positive CTL cells were selected by a cell sorter and collected on a single cell on a substrate having 25 microwells.

[Confirmation of double-stained CTL cells on bPET substrate by fluorescence microscope]
Individual separation of double-stained CTL cells using a microchannel device was performed as follows. First, the surface for suppressing non-specific adsorption to the channel by applying plasma treatment to the above-mentioned device channel surface for 20 seconds, introducing 10% pluronic F-127 into the channel and incubating for 2 hours Processed. Next, for the purpose of isolating only cells expressing both the CTL marker CD8 and the antigen receptor for the gp100 peptide, FITC-labeled anti-CD8 antibody + / PE-labeled gp100-HLA-A2-tetramer The double-stained CTL cell suspension was introduced into the flow path, and the cells were trapped on the bPET substrate by performing vacuum suction at 2.0 kPa. Cells on the bPET substrate were observed using a fluorescence microscope (BX-51; manufactured by Olympus). By merging the obtained images, CTL stained with both FITC and PE were confirmed. The results are shown in FIG.

[MRNA extraction from one CTL cell]
Cells that were double-stained with FITC-labeled anti-CD8 antibody / PE-labeled gp100-HLA-A2 tetramer (cells that appear yellow in the merged image) were collected. b The cells trapped on the PET substrate were sorted for each cell using a micromipulator with a transfer chip (inner diameter: 20 μm) connected to the tip of the cell tram vario (eppendorf), and the cells were collected in a mini tray (Nalge Nunc International). ), And the presence of one cell was confirmed by microscopic observation. Lysis buffer was added to each well and the cells were lysed by pipetting. Thereafter, mRNA was extracted using μMACS mRNA Isolation Kit (Miltenyi Biotec). The extraction operation was performed according to a standard protocol. In addition, mRNA was similarly extracted from 10 cells and 1000 cells prepared by dilution.

[RT-PCR reaction targeting cancer antigen-specific TCR]
Using mRNA extracted from each of 1 cell, 10 cells, and 1000 cells, RT-PCR targeting a cancer antigen-specific receptor gene (about 950 bp) was performed. The PCR primers used were the sequence TRBV12b specific to the TCRβ chain V region shown in SEQ ID NO: 2 (recognized by MoAb against TCRV · 8) and the sequence TRBC2 specific to the TCRβ chain C region shown in SEQ ID NO: 3. Was used (FIG. 5). 55 cycles of 50 ° C for 30 minutes, 99 ° C for 5 minutes, 5 ° C for 5 minutes, followed by 94 ° C for 2 minutes and 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute Amplification was performed by PCR reaction. Similarly, human GAPDH (226 bp) was amplified by PCR at 40 ° C. for 2 minutes, 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds. Then, mRNA extraction from one T cell and evaluation of PCR amplification were performed by performing agarose gel electrophoresis.

[Extraction and purification of PCR products]
The obtained PCR reaction solution was electrophoresed using a 1.5% agarose gel, and the band was confirmed by ethidium bromide staining. As shown in FIG. 6, amplification of the target 950 bp cancer antigen-specific receptor gene fragment was confirmed in 2 out of 5 samples of 1 cell. The PCR reaction product was extracted and purified from the gel using silica gel particles (QIAEX II® Gel Extraction Kit; manufactured by QIAGEN) and a DNA extraction column (MinElute ^™ Reaction Cleanup Kit; manufactured by QIAGE).

First, the gel of the amplification band part was cut out, transferred to a 1.5 mL sample tube, and the cut out gel was weighed. Converted to 1 mg = 1 μL, 3 times the amount of buffer QX1 (QIAEX II ^™ Gel Extraction Kit; manufactured by QIAGE) and 17.2 μL of QIAEXII Suspension (QIAEX II ^™ Gel Extraction Kit; manufactured by QIAGEN) were added. After thoroughly mixing with Vortex, the mixture was placed on a heat block set at 50 ° C. in advance and subjected to Vortex every 2 minutes for a total of 10 minutes. Subsequently, centrifugation was performed at 10,000 × g for 1 minute at room temperature, and the supernatant was removed. Again, 500 μL of buffer QX1 was added to the precipitate, mixed with vortex, and centrifuged at 10,000 × g for 1 minute at room temperature. The supernatant was removed, and 500 μL of buffer PE (QIAEX II ^™ Gel Extraction Kit; manufactured by QIAGE), to which ethanol had been added in advance, was added to the precipitate, mixed by vortex, and centrifuged at 10,000 × g for 1 minute at room temperature. The supernatant was removed. Again, 500 μL of buffer PE was added to the precipitate, mixed with vortex, centrifuged at 10,000 × g for 1 minute at room temperature, and the supernatant was removed. The sample tube was placed in a clean bench with the lid open for 15 minutes to dry the precipitate. 20 μL of buffer EB (MinElute ^™ Reaction Cleanup Kit; manufactured by QIAGEN) was added to the precipitate, mixed with vortex, and placed at room temperature for 5 minutes. Centrifugation was performed at room temperature and 10000 × g for 1 minute, and the supernatant was collected in another 1.5 mL sample tube, and 20 μL of buffer EB was again added to the precipitate. After mixing with Vortex, it was placed at room temperature for 5 minutes, centrifuged at room temperature and 10,000 × g for 1 minute, and added to the supernatant collected earlier.

To a total of 40 μL of the collected liquid, 300 μL of buffer ERC (MinElute ^™ Reaction Cleanup Kit; manufactured by QIAGE) was added and mixed with vortex. After confirming that the mixed solution is yellow, apply the entire amount to a MinElute column (MinElute ^™ Reaction Cleanup Kit; QIAGEN) set in a 2 mL collection tube (MinElute ^™ Reaction Cleanup Kit; QIAGEN). Centrifugation was performed at 10,000 × g for 1 minute. After discarding the effluent, return the column to the collection tube again, add 750 μL of buffer PE (MinElute ^™ Reaction Cleanup Kit; manufactured by QIAGEN) to which ethanol has been added in advance, and centrifuge at room temperature and 10,000 × g for 1 minute. Went. After discarding the effluent, the column was returned to the collection tube again and centrifuged at room temperature and 22000 × g for 1 minute. The droplets on the column trap were removed with a micropipette and the column was set in a new 1.5 mL sample tube. 10 μL of buffer EB (MinElute ^™ Reaction Cleanup Kit; manufactured by QIAGEN) was added and allowed to stand at room temperature for 1 minute, and then centrifuged at room temperature and 10,000 × g for 1 minute to recover the purified DNA fragment.

[Insertion into pCR2.1-TA plasmid vector]
Plasmid DNA was prepared by inserting the PCR fragment obtained in Example 9 into the pCR2.1-TA plasmid vector. 4 μL of DNA fragment, 1 μL of Salt Solution (TOPO TA Cloning (R) Kit pCR2.1TOPO Vector; manufactured by Invitrogen) and 1 μL of TOPO® Vector (TOPO TA Cloning® Kit pCR2.1) TOPOVector (manufactured by Invitrogen) was mixed on ice. After reacting at room temperature for 5 minutes, it was put back on ice and used for transformation.

[Introduction of plasmid DNA into DH5α competent cells]
DH5α competent cells (Competent high DH5α; manufactured by TOYOBO) were thawed on ice and dispensed in 20 μL aliquots into separate sample tubes. 2 μL of the reaction solution prepared in Example 10 was added and gently mixed at the tip of the chip. After being placed on ice for 30 minutes, it was treated at 42 ° C. for 30 seconds using a heat block. Again, after cooling on ice for 2 minutes, 250 μL of SOC medium was added, and shaking culture was performed at 37 ° C. for 1 hour. During the shaking culture, 0.1 M IPTG (Isopropyl β-D-1-thiogalactopyranoside; manufactured by SIGMA) and 0.1 M X-Gal (5-5) were added to 2 × YT solid medium containing 50 μg / mL kanamycin. Bromo-4-chloro-3-indolyl β-D-galactopyranoside (manufactured by SIGMA) was applied in an amount of 50 μL. 100 μL of the cultured sample was seeded on the prepared solid medium and cultured overnight at 37 ° C.

[Screening by blue-white selection and colony PCR]
After confirming the appearance of colonies on the plate prepared in Example 11, the plate was allowed to stand at 4 ° C. for 5 hours. After marking a white colony, it was lightly picked with the tip of a tip, and lightly rinsed in a 96-well plate containing 50 μL of sterile water. After treatment at 95 ° C. for 5 minutes using a thermal cycler, light centrifugation was performed and 2 μL was placed in a well of a new plate. Subsequently, 1 μL of 10 × PCR buffer II (Mg ⁺ ), 0.8 μL of 2.5 mM dNTP Mix, 6.11 μL of dH ₂ O, 0.05 μL of LA-Taq polymerase (TaKaRa LA-Taq® Hot Start version Manufactured by Takara Bio Inc.), 0.02 μL of 100 μM M13 forward primer, and 0.02 μL of 100 μM M13 reverse primer were added, and the plate was lightly centrifuged. Using a thermal cycler GeneAmp ^(R) PCR System9700, after thermal denaturation of 1 minute at 94 ° C., 10 sec at 94 ° C., 10 sec at 50 ° C., and at reaction conditions and repeating 35 cycles of reaction for 2 minutes at 68 ° C. The PCR reaction was performed. The size of the PCR product in each PCR reaction solution was confirmed by electrophoresis using a 1.5% agarose gel, and a clone in which a band having a size of 1.0 kb was confirmed was selected.

[Liquid culture of positive clones and extraction of plasmid DNA]
According to Example 12, colonies in which insertion of PCR-amplified fragments into the vector was confirmed were selected and subjected to shaking culture at 37 ° C. overnight in 2 × YT liquid medium containing 3.5 mL of 50 μg / mL kanamycin. . 1.8 mL of the cultured sample was placed in a 2 mL sample tube and centrifuged at 1000 × g for 10 minutes. The supernatant was discarded, and 250 μL of buffer A1 (NucleoSpin® Multi-8 Plasmid; manufactured by MACHEREY-NAGEL) was added to the precipitate and mixed by vortexing. Subsequently, 250 μL of buffer A2 was added and mixed by inversion, and then placed at room temperature for 5 minutes to lyse the cells. 350 μL of buffer A3 was added and mixed by inversion, followed by centrifugation at 14000 × g for 10 minutes at 4 ° C. The supernatant was transferred to NucleoSipn ^(R) Plasmid Binding Strips set in a NucleoVac vacuum manifold. The solution was permeated through the silica membrane by aspiration at 400 mbar for 1 minute to bind the DNA. After adding 600 mL of buffer AW and sucking at 400 mbar for 1 minute to permeate the solution, 900 mL of buffer A4 was added and sucking at 400 mbar for 1 minute to permeate the solution to wash the silica membrane. Again, 900 mL of buffer A4 was added and the solution was permeated by aspiration at 400 mbar for 1 minute to wash the silica membrane. The silica membrane was dried by aspiration for 15 minutes at 600 mbar. NucleoSipn ^(R) MN Tube Strips were replaced with NucleoVac vacuum manifold, 120 μL of buffer AE was added to the membrane, placed for 1 minute, and aspirated at 400 mbar for 1 minute to recover plasmid DNA. To 3 μL of the collected plasmid DNA solution, 1 μL of 10 × H buffer, 5 μL of dH ₂ O, and 1 μL of EcoRI (manufactured by TOYOBO) were added and treated at 37 ° C. for 1 hour. Confirm the DNA digestion fragments in the enzyme reaction solution by electrophoresis using 1.5% agarose gel, measure the absorbance of each plasmid sample, calculate the DNA concentration, and prepare a 100 μg / μL plasmid DNA dilution. did.

[Determination of nucleotide sequence by Cycle Sequencing method]
6 μL each of the DNA dilution prepared in Example 13 was added to a 96-well plate placed on ice, 2 wells per clone. Subsequently, 3 μL of 5 × Sequencing Buffer (BigDye® Terminator v3.1 Cycle Sequencing Kit; manufactured by Applied Biosystems) and 2 μL of Cycle Sequencing Mix (BigDye® Terminator v3.1 Cycle Sequencing Kit; Applied Biosystems), 8 μL of dH ₂ O, 1 μL of 3.2 μM primer was added, and lightly centrifuged.

Each clone was subjected to cycle sequencing reaction using two primers, M13 reverse primer and M13 forward primer. Using a thermal cycler GeneAmp ^(R) PCR System9700, after thermal denaturation of 1 minute at 94 ° C., 10 sec at 94 ° C., 5 sec at 50 ° C., and the reaction of 4 minutes at 68 ° C. 25 cycles repeated reaction conditions The cycle sequencing reaction was performed. By the end of the reaction, the wells of MultiScreen ^™ HV-plate (MultiScreen® HV-plate; manufactured by MILLIPORE) were charged with Sephadex G-50 (Sephadex ^™ G-50 Superfine; manufactured by GE Healthcare) and 300 μL of sterile water. In addition, the mixture was allowed to stand at room temperature for 2 hours. After sufficient hydration, centrifugation was performed at 1100 × g for 5 minutes at room temperature, and the effluent was discarded. A new 96-well plate (ASSAY PLATE 96 well round bottom; manufactured by IWAKI) was replaced with the Multiscreen ^™ HV-plate, the whole reaction solution was applied to each well, and the sample was collected by centrifugation at 1100 × g for 5 minutes at room temperature.

  Transfer the purified sample to a 96-well plate (MicroAmp (R) Optical 96-well Reaction Plate; Applied Biosystems) for the entire sequencer, add 17.2 μL of sterilized water to the original well, and wash the well to make the entire volume. Was added to the same sample. Using x3130 / Genetic Analyzer (Applied Biosystems), 8 clones were read for each sample, and multiple alignment analysis of the obtained sequences was performed. Regarding the bases that differed between the clones, the sequence with the larger number of clones having the base was regarded as the correct sequence, and the base sequence of each sample was determined.

  The amplified TCR gene band was excised from the electrophoresis gel, and final DNA sequence data was obtained as described above. The gene sequence of the TCR β chain of 4 clones (TRB1-1, 3-1, 5-1, 6-1) successfully cloned from two types of bands this time is already CD8 + / gp100-HLA-A2 tetramer positive CTL of bulk A complete match with the gene sequence (M8-gp100) obtained from the cells was observed. The gene sequence data (SEQ ID NO: 4) of the TCR β chain of two clones (TRB5-1, 6-1) are aligned with the gene sequence data (SEQ ID NO: 4) of the TCR β chain of M8-gp100, and FIG. 7 shows the TCR β of M8-gp100. The gene sequence data of the chain (SEQ ID NO: 4) is shown in FIG.

Dendritic cells and T2-A2 cells treated with gp100 _209-217 peptide from peripheral blood mononuclear cells from HLA-A2-positive patients who received dendritic cell vaccine administration (HLA-A ^* 0201 gene in T2 cells) Is a diagram showing the results of staining with a FITC-labeled anti-CD8 + antibody and PE-labeled gp100-HLA-A2 tetramer after 4 times of stimulation using a phenotype. It is a figure which shows the result of having dye | stained the CTL cell after MACS sorting using PE label | marker gp100-HLA-A2 tetramer with the monoclonal antibody which recognizes TCRV (beta) 12 repertoire. FIG. 6 shows a sorting profile of CD8 + / gp100-HLA-A2 tetramer positive CTL. It is a figure which shows the result of having dye | stained the CTL cell with the FITC labeled anti-CD8 antibody + / PE labeled gp100-HLA-A2-tetramer captured on the bPET substrate. It is a figure which used the primer sequence TRBV12b specific for TCRβ chain V region and the primer sequence TRBC2 specific for TCRβ chain C region used for RT-PCR. It is a figure which shows the amplification result of the TCR (beta) chain gene of HLA-A2 restriction _{| limiting} gp100 _209-217 peptide specific CTL cell using a PCR primer specific for a TCR (beta) chain V area _{| region} . Gene sequence data (TRB5-1, 6-1) of TCRβ chain obtained from one CTL and gene sequence data (M8-gp100) obtained from bulk CD8 + / gp100-HLA-A2 tetramer positive CTL cells ) Completely matches. It is a figure which shows V area | region, D area | region, J area | region, and C of the gene sequence data (M8-gp100) obtained from CD8 + / gp100-HLA-A2 tetramer positive CTL cell of bulk.

Claims (12)

Analysis of antigen receptor gene of one human T cell characterized by sequentially comprising the following steps (A), (B), (C), (D), (E) and (F) Identification method.
(A) a step of collecting peripheral blood mononuclear cells obtained from a human whose immune response to a specific antigen or antigen epitope recognized by cytotoxic T cells or helper T cells has been confirmed;
(B) T cells in peripheral blood mononuclear cells using the specific antigen or antigen epitope labeled with a labeling substance and an antibody capable of recognizing human T cells labeled with a labeling substance different from the labeling substance Double labeling;
(C) a step of sorting T cells double-labeled with a labeling substance one by one;
(D) a step of extracting total RNA from one cell and synthesizing cDNA by a reverse transcription reaction;
(E) A step of amplifying a human T cell antigen receptor gene fragment by performing a PCR reaction using a synthesized cDNA as a template and a primer pair specific to human T cell antigen receptor;
(F) a step of analyzing and determining the base sequence of the amplified gene fragment; Obtained from cancer patients whose immune response to the cancer-specific antigen peptide has been confirmed by administering a dendritic cell vaccine treated with the cancer-specific antigen peptide in the process of collecting peripheral blood mononuclear cells The analysis / identification method according to claim 1, wherein peripheral blood mononuclear cells are collected. In the step of double labeling T cells in peripheral blood mononuclear cells, as the specific antigen or antigen epitope labeled with a labeling substance, a cancer-specific antigen peptide, a human histocompatibility antigen (HLA) protein, and a label The analysis / identification method according to claim 1 or 2, wherein a complex made of a substance is used. The anti-CD8 antibody or the anti-CD4 antibody is used as an antibody capable of recognizing human T cells in the step of double labeling T cells in peripheral blood mononuclear cells. Analysis and identification method. 5. The microchannel device provided with a substrate having a plurality of micropores capable of capturing one cell is used in the step of sorting T cells one by one. Analysis and identification method. Expressing an antigen receptor derived from one human T cell, characterized by comprising the following steps (A), (B), (C), (D), (E) and (G) in sequence: Possible preparation of recombinant vectors.
(A) a step of collecting peripheral blood mononuclear cells obtained from a human whose immune response to a specific antigen or antigen epitope recognized by cytotoxic T cells or helper T cells is confirmed;
(B) T cells in peripheral blood mononuclear cells using the specific antigen or antigen epitope labeled with a labeling substance and an antibody capable of recognizing human T cells labeled with a labeling substance different from the labeling substance Double labeling;
(C) a step of sorting T cells double-labeled with a labeling substance one by one;
(D) a step of extracting total RNA from one cell and synthesizing cDNA by a reverse transcription reaction;
(E) A step of amplifying a human T cell antigen receptor gene fragment by performing a PCR reaction using a synthesized cDNA as a template and a primer pair specific to human T cell antigen receptor;
(G) a step of incorporating the amplified gene fragment into an expression vector; Obtained from cancer patients whose immune response to the cancer-specific antigen peptide has been confirmed by administering a dendritic cell vaccine treated with the cancer-specific antigen peptide in the process of collecting peripheral blood mononuclear cells The method according to claim 6, wherein peripheral blood mononuclear cells are collected. In the step of double labeling T cells in peripheral blood mononuclear cells, as the specific antigen or antigen epitope labeled with a labeling substance, a cancer-specific antigen peptide, a human histocompatibility antigen (HLA) protein, and a label The preparation method according to claim 6 or 7, wherein a complex comprising a substance is used. The anti-CD8 antibody or the anti-CD4 antibody is used as an antibody capable of recognizing human T cells in the step of double labeling T cells in peripheral blood mononuclear cells. Preparation method. 10. The microchannel device including a substrate having a plurality of micropores capable of capturing one cell is used in the step of sorting T cells one by one. Preparation method. A recombinant vector obtained by the preparation method according to claim 6. A transformed human T cell in which the recombinant vector obtained by the preparation method according to any one of claims 6 to 10 is introduced into a naive T cell and expresses an antigen receptor derived from one human T cell.