JP2007097580A - T CELL RECEPTOR beta CHAIN GENE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a TCR (T cell receptor) β chain, its gene and the like, specifically discriminating MART-1 27-35 (AAGIGILTV) of melanoma-specific peptide. <P>SOLUTION: A peripheral blood mononuclear cell is sampled from a patient, added, at a ratio 1:10 to 1:40, with dendritic cell pulsed with MART-1 A2 peptide, and stimulated twice in the presence of low concentration IL-2. To more increase the ratio of cytotoxic T cell, T2 cell pulsed with MART-1 A2 peptide is added to the cell line at a ratio of 1:10 and the obtained is stimulated. Cloning of MART-1 A2 tetramer-specific cytotoxic T cell is performed by magnetic activated cell sorting of cytotoxic T cell, using monoclonal antibody, DNA sequence of TCR β chain is analyzed and a new TCR β chain specifically discriminating MART-1 is identified. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a T cell receptor β chain protein, a T cell receptor β chain gene, a T cell receptor β chain protein expression vector into which the gene is incorporated, and a transformant into which the expression vector has been introduced.

  Malignant melanoma is a cancer in which immunotherapy using interleukin 2 or the like is relatively effective (see, for example, Non-Patent Document 1). However, 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 self-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, malignant melanoma antigens can be isolated using a functional cDNA expression cloning method utilizing the reactivity. For example, a malignant melanoma antigen that can be used for the prevention, diagnosis, and treatment of melanoma is isolated using tumor-reactive cytotoxic T cells from metastatic melanoma patients and a tumor cell line that expresses HLA-A2. One of these is MART-1, which has been isolated and identified by the same method. Representative examples to date include pigment cell (melanocyte) tissue-specific proteins such as MART-1 and gp100, MAGE, BAGE, GAGE, NY-ESO-, which are expressed in testis in various tumors and normal tissues. Mutant peptide antigens such as CT antigens such as I (Cancer-Testis antigen), β-catenin produced by gene abnormality of tumor cells, CDK4 (cyclin dependent kinase 4), p15, GnT-V have been isolated (for example, Non-Patent Document 3).

  T cells recognize MHC molecule / antigen peptide complexes by T cell receptors and bind to target cells such as antigen presenting cells (APCs), whereas CTL binds only to MHC class I molecule / antigen peptide complexes. (MHC class I-restricted), helper T (Th) cells can only bind to MHC class II molecule / antigen peptide complexes (MHC class II-restricted). MHC class I molecules are expressed in most nucleated cells, but MHC class II molecules are expressed only in a limited number of cells. 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 infected cells. However, MHC class II-restricted CD4-positive CD8-negative T cells into which a CTL-derived T cell receptor gene, which is MHC class I-restricted by genetic manipulation, are introduced in an APC and CD8-independent manner by pulsing the corresponding antigen. It was shown to express a T cell receptor that can be activated in response and bind to the corresponding antigen. It has also been reported that tumors can be specifically injured by introducing a tumor antigen-specific CTL-derived T cell receptor gene into peripheral blood lymphocytes having non-specific antitumor activity (for example, Non-Patent Document 4).

  On the other hand, it is a membrane surface protein present on the surface of T cells, and there are two types of T cell receptors responsible for antigen recognition: heterodimers composed of α and β chains and heterodimers composed of γ and δ chains. Has been identified. The T cell receptor gene, like the immunoglobulin gene, is composed of a variable region (V), a diversity region (D), a joining region (J), and a V domain that produces molecular diversity, and an extracellular region. It is composed of a C domain composed of a constant region (C: constant) including a constant region, a transmembrane region, and an intracellular region. Since these amino acid sequences differ from one T cell to another, the T cell receptor is an antigen recognition molecule and at the same time serves as a mark for individual T cells. This diversity of T cell receptors is generated by the rearrangement of the acquired T cell receptor gene. T cell receptor β and δ chains are VDJ, and T cell receptors α and γ chains are V-J. It is known to reconstruct J.

The initial differentiation of T cells is closely related to the rearrangement of the α- and β-chain genes of the T cell receptor, as in B cells. T cell receptor beta reconstruction chain gene DN-T cells ^{(double negative: CD4 - 8 -} ) is started in step, beta chain gene cells which successfully reconstitution of beta-chain protein with an alternate α-chain (preTα) And the pre-T cell receptor is expressed on the cell surface. Then, the expression of CD4 and CD8 molecules is induced and transferred to DP-T cells (double positive: CD4 ⁺ 8 ⁺ ), and the T cells that become DP then reconstitute the α chain gene of the T cell receptor. It is known that only cells that have succeeded in expressing T cell receptors on the surface.

  Regarding the gene structure of the T cell receptor β chain, according to the human genome gene sequence information database, the T cell receptor β chain gene is composed of about 30 subgroups (the gene itself is about 70 types), the V gene, It is also known that it is composed of a D gene, 14 types of J genes, and 2 types of C genes, and two variable regions (CDR1, CDR2) are present in Vβ. Among these, Vβ, Dβ, and Jβ are selected and bonded one by one to constitute DNA encoding the T cell receptor β 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 the T cell receptor. Since base insertion / deletion occurs, it is very rare that the same sequence including the linking portion is the same even if each gene structure is the same.

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

  The diversity of the T cell repertoire is closely related to the ability to recognize various antigens between T cells, so the function of the T cell receptor repertoire should be elucidated when considering the function, immune response, and immune function of T cells. It is very important to do. Recently, in cancer, autoimmune diseases, allergic diseases, etc., research on T cell receptor repertoire analysis of T cells existing in the affected area has been advanced, and disease-specific T cells are being elucidated. However, the function of the T cell receptor molecule on the T cell is problematic because it is difficult to isolate the T cell receptor molecule, and the details are still clear. It has not been done. An object of the present invention is to provide a T cell receptor β chain and its gene that specifically recognize MART-1 27-35 (AAGIGILTV: hereinafter referred to as MART1-A2 peptide), which is a melanoma specific peptide. is there.

Since T cell receptor molecules play an important role in T cell-mediated immune responses and immune mechanisms, it is necessary to obtain individual T cell receptor molecules for analysis of these immune responses and immune mechanisms. 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 T cell receptor due to the diversity of T cell receptors, it is possible to establish T cell lines having respective T cell receptors and to isolate T cell receptors from each of them. Required. However, the T cell receptor repertoire of T cells is as large as 10 ¹⁰ types, and it is substantially difficult to establish 10 ¹⁰ types of T cell lines.

  MART-1 is a cancer antigen expressed in melanoma and strongly induces an antigen-antibody reaction in metastatic melanoma patients treated with peptide vaccines. MART-1 has an HLA-A2 localized antigen-determining site (MART-1 27-35: AAGIGILTV). By MART-1 HLA-A2 tetramer staining, cytotoxic T specific to MART1-A2 peptide The expression frequency of cells became clear. The present inventors constructed a method for inducing MART-1-specific cytotoxic T cells based on dendritic cells and characterized them using MART1-A2 tetramer staining in 7 cases of HLA0201. It was. Peripheral blood mononuclear cells were collected from the patient, and dendritic cells pulsed with MART1-A2 peptide were added at a ratio of 1:10 to 1:40 and stimulated twice in the presence of low concentration of IL2. In order to further increase the proportion of cytotoxic T cells, T2 cells pulsed with MART1-A2 peptide were further added to the cell line at a ratio of 1:10 for stimulation. The total number of stimulated T cells increased in 2 cases compared to before culture and decreased in 4 cases. The ratio of MART1-A2 tetramer positive cytotoxic T cells was 0.1% before stimulation, increased to 1.3% after stimulation with dendritic cells, and further increased to 30% after stimulation with T2 cells. The absolute number of MART1-A2 tetramer positive cytotoxic T cells increased from 46 times to 619 times before and after dendritic cell stimulation. Cytotoxic T cell assays using a MART-1 specific cytotoxic T cell lineage include tetramer positive cytotoxicity against T2 cells pulsed with MART1-A2 peptide and HLA A0201-positive melanoma cells. It showed a strong killing ability depending on the frequency of T cell generation. In purified MART1-A2 tetramer positive cytotoxic T cells, TCRVb13 and TCRVb16 were used in 3 out of 5 cases for T cell receptor repertoire analysis using CDR3 fragment size analysis. Cloning of MART1-A2 tetramer-specific cytotoxic T cells is carried out by magnetically activated cell sorting of cytotoxic T cells using monoclonal antibodies based on T-cell receptor β-chain specific antibodies. By analyzing the DNA sequence of the β chain, a T cell receptor β chain that specifically recognizes novel MART-1 was identified. The present invention has been completed based on the above findings.

  That is, the present invention relates to (1) a T cell receptor β chain gene comprising the nucleotide sequence represented by SEQ ID NO: 1, and (2) a T cell receptor β into which the T cell receptor β chain gene described in (1) above is incorporated. A recombinant vector capable of expressing a chain protein, (3) a transformant introduced with a recombinant vector capable of expressing the T cell receptor β chain protein described in (2) above, or (4) a recombinant vector introduced. (5) a T cell receptor β chain protein comprising the amino acid sequence represented by SEQ ID NO: 2, (6) an amino acid represented by SEQ ID NO: 2 An antibody specifically recognizing a T cell receptor β chain protein comprising the sequence; and (7) a T cell receptor incorporating a T cell receptor β chain gene comprising the base sequence represented by SEQ ID NO: 1. Melanoma therapeutic compositions recombinant vectors capable of expressing the β-chain protein as an active ingredient the introduced lymphocytes in vitro related.

  According to the present invention, it is useful for elucidating the function, immune response, and immune function of T cells in melanoma. Moreover, there is a possibility that melanoma can be treated by gene therapy or adoptive immunotherapy using the T cell receptor β chain gene of the present invention.

  The gene of the present invention is a T cell receptor β chain gene comprising a nucleotide sequence represented by SEQ ID NO: 1, preferably a nucleotide sequence in which 6 nucleotides (GCAGCC) are added upstream of ATG at the 5 ′ end. The expression vector of the present invention is a recombinant vector capable of expressing a T cell receptor β chain protein into which a T cell receptor β chain gene comprising the base sequence represented by SEQ ID NO: 1 has been incorporated. The body is a transformant into which a recombinant vector capable of expressing a T cell receptor β chain protein into which a T cell receptor β chain gene consisting of the base sequence represented by SEQ ID NO: 1 has been introduced, preferably a transformed human. The protein of the present invention is a T cell receptor β chain protein consisting of the amino acid sequence represented by SEQ ID NO: 2, and the antibody of the present invention is SEQ ID NO: In specifically recognizing serial antibodies to T cell receptor β chain protein consisting of the amino acid sequence represented, preferably a monoclonal antibody.

  The method for obtaining and preparing the gene of the present invention is not particularly limited, and is appropriate based on each information of the base sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 2 disclosed in the present specification. The target gene can be isolated by preparing a suitable probe or primer and screening a human cDNA library in which the gene is predicted to exist, or can be prepared by chemical synthesis according to a conventional method . For example, isolation of total RNA from cells or tissues, isolation and purification of mRNA, acquisition of cDNA, and cloning thereof can be performed according to conventional methods. Methods for screening the gene of the present invention from a cDNA library are commonly used by those skilled in the art, such as the method described in Molecular Cloning: A laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989. Can be mentioned.

  The method for obtaining and preparing the protein of the present invention is not particularly limited, and may be a naturally derived protein, a chemically synthesized protein, or a recombinant protein produced by a gene recombination technique. When a naturally derived protein is obtained, the protein of the present invention can be obtained by appropriately combining protein isolation / purification methods from T cells expressing such protein. When preparing a protein by chemical synthesis, for example, the protein of the present invention can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). it can. In addition, the protein of the present invention can be synthesized using various commercially available peptide synthesizers. When a protein is prepared by a gene recombination technique, the protein of the present invention can be prepared by introducing DNA consisting of a base sequence encoding the protein into a suitable expression system. Among these, preparation by a gene recombination technique that can be prepared in a large amount by a relatively easy operation is preferable.

  For example, when the protein of the present invention is prepared by genetic recombination techniques, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography can be used to recover and purify the protein from cell culture. , Known methods including hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, preferably high performance liquid chromatography. In particular, the column used for affinity chromatography is, for example, a column in which an antibody such as a monoclonal antibody against the protein of the present invention is bound, or when a normal peptide tag is added to the protein of the present invention, By using a column to which an affinity substance is bound, purified products of these proteins can be obtained. In addition, when the protein of the present invention is expressed on the cell membrane, a purified sample can be obtained by carrying out the above purification treatment after allowing the cell membrane degrading enzyme to act.

  In addition, the protein of the present invention can be bound to a marker protein or a peptide tag. The marker protein is not particularly limited as long as it is a conventionally known marker protein. Specific examples include an enzyme such as alkaline phosphatase and HRP, an Fc region of an antibody, and a fluorescent substance such as GFP. As peptide tags in the present invention, conventionally known peptide tags such as epitope tags such as HA, FLAG, and Myc, and affinity tags such as GST, maltose binding protein, biotinylated peptide, oligohistidine, etc. Specific examples can be given. Such a fusion peptide can be prepared by a conventional method. Purification of the protein of the present invention utilizing the affinity between Ni-NTA and His tag, detection of the protein of the present invention, and quantification of an antibody against the protein of the present invention. It is also useful as a research reagent in this field.

  The recombinant vector of the present invention is not particularly limited as long as it contains the gene of the present invention and can express the protein of the present invention, but is preferably a vector that can be expressed in animal cells. The recombinant vector of the present invention can be constructed by appropriately integrating the gene of the present invention into an expression vector. Such an expression vector is preferably one that is capable of autonomous replication in a host cell, or one that can be integrated into the host cell chromosome, and includes a promoter, enhancer, terminator, etc. at a position where the gene of the present invention can be expressed. Those containing a control sequence can be preferably used.

  Examples of expression vectors for animal cells include 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), pAGE210, etc. Can do. 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 transformant of the present invention is not particularly limited as long as it is a host cell into which the above-described recombinant vector of the present invention has been introduced. However, lymphocytes are preferable, and naive T cells are particularly preferable. In particular, a lymphocyte into which a recombinant vector capable of expressing a T cell receptor β chain protein into which a T cell receptor β chain gene consisting of the base sequence represented by SEQ ID NO: 1 has been incorporated, preferably naïve T The cells are useful as a composition for melanoma treatment that can be suitably used for adoptive immunotherapy and the like. When used as an immunotherapeutic agent such as adoptive immunotherapy, it is preferable to purify a transformed T cell clone. As the method, a method of mixing and centrifuging in a phosphate buffer or the like, a specific gravity centrifugation using a separating agent Etc. can be used. The dose of the composition for treating melanoma is preferably 10 ⁶ to 10 ¹² cells as transformed lymphocytes per time. As the dosage form, liquids such as injections and infusions are preferable, and injections or infusions in which transformed lymphocytes are dispersed in physiological saline supplemented with human serum albumin at 0.01 to 5% are more preferable. preferable. 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 it is preferably 50 to 500 cc, and the liquid lymphocyte is preferably 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.

  Specific examples of antibodies that specifically bind to the T cell receptor β chain protein of the present invention include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies and the like. These can be prepared by a conventional method using the T cell receptor β chain protein of the present invention as an antigen, and among them, a monoclonal antibody is more preferable in view of its specificity. An antibody that specifically binds to the T cell receptor β chain protein of the present invention, such as a monoclonal antibody, is useful, for example, in diagnosing melanoma and clarifying the molecular mechanism of the T cell receptor β chain protein of the present invention. .

  The antibody against the T cell receptor β-chain protein of the present invention is administered to an animal (preferably a non-human) animal by expressing the T cell receptor β-chain protein of the present invention or a cell expressing the protein on the membrane surface using a conventional protocol. For example, for the preparation of monoclonal antibodies, the hybridoma method (Nature 256, 495-497, 1975), the trioma method, the human B cell hybridoma method (Immunology) Today 4, 72, 1983) and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) can be used. Specific examples of the T cell receptor β chain protein of the present invention derived from a mouse T cell receptor β chain protein of the present invention are exemplified below. A method for producing a binding monoclonal antibody, that is, an anti-hT cell receptor β-chain mouse monoclonal antibody will be described.

  The anti-hT cell receptor β-chain monoclonal antibody can be produced by culturing an anti-hT cell receptor β-chain monoclonal antibody-producing hybridoma in vivo or in vitro by a conventional method. For example, in an in vivo system, it can be obtained by culturing in the abdominal cavity of a rodent, preferably a mouse or a rat. In an in vitro system, it can be obtained by culturing in an animal cell culture medium. As a medium for culturing a hybridoma in an in vitro system, a cell culture medium such as RPMI 1640 or MEM containing an antibiotic such as streptomycin or penicillin can be exemplified.

  An anti-hT cell receptor β chain monoclonal antibody-producing hybridoma can be obtained by, for example, immunizing a BALB / c mouse using the T cell receptor β chain protein of the present invention or a T cell expressing the protein on the membrane surface. Spleen cells and mouse NS-1 cells (ATCC TIB-18) are fused by a conventional method and screened with an immunofluorescent staining pattern to produce an anti-hT cell receptor β-chain monoclonal antibody-producing hybridoma. . In addition, as a method for separating and purifying the monoclonal antibody, any method can be used as long as it is a method generally used for protein purification, and liquid chromatography such as affinity chromatography can be specifically exemplified. .

  In order to produce a single chain antibody against the T cell receptor β chain protein of the present invention, the method for preparing a single chain antibody (US Pat. No. 4,946,778) can be applied. In order to express a humanized antibody, a clone expressing the T cell receptor β chain protein of the present invention can be isolated and identified using a transgenic mouse or other mammals, or using the above antibody. Alternatively, the polypeptide can be purified by affinity chromatography.

Examples of the anti-hT cell receptor β chain monoclonal antibody of the present invention include fluorescent substances such as FITC (fluorescein isocyanate) or tetramethylrhodamine isocyanate, ¹²⁵ I, ³² P, ¹⁴ C, ³⁵ S or ³ H. By using a fusion protein fused with a radioisotope such as alkaline phosphatase, peroxidase, β-galactosidase or phycoerythrin, or a fluorescent protein such as green fluorescent protein (GFP). Functional analysis of the T cell receptor β chain protein of the invention can be performed. Examples of the immunological measurement method include RIA method, ELISA method, fluorescent antibody method, plaque method, spot method, hemagglutination method, and octalony method.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations. The outline of the experiment in Example 1 is as follows: melanoma patient (HLA-A2 positive) lymphocytes → preparation of dendritic cells treated with melanoma antigen MART1-A2 peptide → MART1-A2 peptide-specific CTL cells by dendritic cell stimulation -> Purification of MART-1-specific CTL by MART1-A2 tetramer by Auto MACS method-> RNA extraction-> Reverse transcription reaction-> Amplification of T cell receptor gene by PCR-> DNA fragment analysis using DNA Analyzer (ABI3100) (T Identification of cell receptor gene) → Amplification and cloning of protein coding region by PCR → Determination of base sequence by ABI3100.

  We used lymphocytes derived from melanoma patients obtained in clinical studies (tumor-specific immunotherapy using dendritic cells for malignant melanoma) conducted with the approval of the National Cancer Center Ethics Review Board. This clinical study is a clinical trial that induces tumor immunity by administering dendritic cells treated with 5 types of tumor-associated antigen peptides subcutaneously to HLA-A2 and A24 patients. In this study, lymphocytes derived from HLA-A2-positive patients were treated with dendritic cells treated with melanoma-specific peptide MART-1 27-35 (MART1-A2 peptide: AAGIGILTV; SEQ ID NO: 3). Stimulation was performed several times. The induced MART1-A2 peptide-specific CTL cells were stained with PE-MART1-A2 tetramer and quantitatively evaluated. The ratio of CTL cells positive for tetramer staining was 38%, indicating cytotoxic activity specific to MART1-A2 peptide. MART1-A2 tetramer positive CTL cells were purified using an auto-magnet cell sorting system. When analysis was performed after dendritic cell stimulation of the MART1-A2 tetramer positive cells and after purification by the MACS method, the number of positive cells was 96% (FIG. 1). These CTL cells were used for subsequent repertoire analysis of the T cell receptor (TCR).

  Extract total RNA from isolated cells, synthesize cDNA by reverse transcription reaction, classify expressed T cell receptor genes by T cell receptor repertoire analysis using PCR method, and based on the analysis results T cell receptor protein code The region gene was cloned. The analysis results using lymphocytes before stimulation with dendritic cells are shown in FIG. 2, and the analysis results using CTL cells after stimulation with dendritic cells and after purification of tetramer positive cells are shown in FIG. A clear convergence was observed for T cell receptor gene expression before and after stimulation with dendritic cells.

  Based on the results of T cell receptor repertoire analysis, PCR amplification was performed using primers (SEQ ID NOs: 4-8) corresponding to four types of genes (BV3, BV12, BV13A, BV13B) (FIG. 4), and each amplified fragment The nucleotide sequence of was analyzed. Repatois analysis showed the use of four types of genes, but base sequence analysis revealed that they were derived from a single gene (FIG. 5). As a result of the analysis, the V gene of this T cell receptor was BV3 (new unified name TRBV28, hereinafter referred to as this unified name), and the other three types of primers cross-reacted with this gene, resulting in amplification. There was found.

Using the sense primer (SEQ ID NO: 9) corresponding to the TRBV28 gene and the antisense primer (SEQ ID NO: 10) corresponding to TRBC1 (C gene), gene amplification of the entire T cell receptor gene protein coding region (SEQ ID NO: 1) This was cloned (FIG. 6). As a result of the nucleotide sequence determination, the structure of the T cell receptor gene was TRBV28 ^* 01-TRBD1-TRBJ1-5 ^* 01-TRBC1. Further, FIG. 6 shows the results of analysis using IMGT ^{* 1} public software (Junction Analysis) for the base sequence of the VDJ junction. Further, the nucleotide sequence and amino acid sequence of the T cell receptor gene were analyzed by the NCBI ^{* 2} homology search program (BLAST). As a result, the base sequence of the partial fragment (271-450, 180 bases) including the full length and the VD-J-C junction region did not completely match. Even when searching for the full-length and the amino acid sequence of a partial fragment (60 amino acids) centered on the VD-J-C junction, there are those with high homology, but the junction region that determines antigen specificity is complete. There were no registrations matching.

The primer sequences used in this study were those described by Jeffry R. Currier and Mary Ann Robinson in the book Current Protocols in Immunology ^{* 3} for the T cell receptor repertoire analysis. Based on the genome sequence listed in the database (NCBI).
* 1 ImMunoGeneTics: 1125985409921_0
* 2 National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/
* 3 Current Protocols in Immunology, John E. Coligan et al. Ed. (2000) 10.28.1-10.28.24. John Wiley & Sons, Inc.

  When the T cell receptor β-chain gene derived from MART1-A2 peptide-specific CTL obtained in Example 1 is introduced into an HLA-A2-positive naive T cell, the receptor protein as the gene product is expressed, and tumor Whether specific cytotoxic activity could be induced was examined. Here, naive T cells are T cells that have not yet encountered an antigen after differentiation in the thymus.

  First, the effect of stimulation with anti-CD3 antibody on the proliferation of HLA-A0201-positive naive T cells was examined for two types of media. Anti-human CD3 antibody was added to GT-T503 (Takara Bio) + 5% human serum (Cambrex) medium or PRMI1640 + 5% human serum medium at various concentrations (0, 1, 2, 5, 10 μg / ml, Janssen Pharma) After naive T cells (derived from melanoma patients registered in clinical trials) were cultured for 5 days, the absorbance at OD 450 nm of the culture solution was measured with an absorptiometer (Narge Nunc International). The results are shown in FIG. As a result, it was confirmed that HLA-A0201-positive naive T cells were stimulated and proliferated depending on the concentration of anti-human CD3 antibody. In addition, since the GT-T503 medium has a better growth support effect than the existing RPMI1640 medium, it was decided to use it in this experiment.

Next, the effect of anti-human CD3 antibody and anti-human CD28 antibody on the proliferation of HLA-A2-positive naive T cells was examined. Naive T cells derived from HLA-A0201-positive melanoma patients are suspended in GT-T503 + 5% human serum medium, and anti-human CD3 antibody alone (5 μg / ml), anti-human CD28 antibody alone (2 μg / ml, BD Pharmingen) or The above lymphocytes were seeded at a concentration of 2 × 10 ⁶ / ml in a 75 cm ² flask coated with anti-human CD3 antibody (2.5 μg / ml) and anti-human CD28 antibody (1 μg / ml) for 2 hours. Then, after culturing for 5 days, the absorbance of the culture solution at OD450 nm was measured with an absorptiometer. The results are shown in FIG. As a result, it was confirmed that the anti-human CD28 antibody promotes the proliferation stimulating effect of naive T cells when used in combination with the anti-human CD3 antibody (2.5 μg / ml) at a final concentration of 1 μg / ml.

Furthermore, naive T cells that have been proliferated and stimulated by the combined use of the above-mentioned anti-human CD3 antibody and anti-human CD28 antibody are collected, and the T cell receptor β chain is collected using the Human T cell Nucleofector kit (Amaxa) according to the instruction manual. Gene transfer was performed by gene electroporation (Nucleofector apparatus, Amaxa). First, 5 × 10 ⁶ lymphocytes after stimulation were suspended in 100 μl of an electroporation buffer, followed by mock (4 μg), GFP expression plasmid (2 μg), and T cell receptor β chain gene expression plasmid ( 2 μg or 4 μg), transfer to a cuvette and set the sample in the Nucleofector device. Electroporation was performed at 23. At that time, the vector used as mock was a GFP gene excised from the pmaxGFP vector attached to Amaxa's Human T cell Nucleofector kit. It was created by using a β-chain gene. After recovering the cells, the cells were transferred to a 12-well plate and cultured in GT-T503 + 5% human serum medium for 48 hours, and then introduced with GFP and T cell receptor β chain gene by flow cytometry (FACSCalibur, BD). It was investigated. The results are shown in FIG. As a result, the GFP gene was efficiently introduced into 54.7% of the whole naive T cells by electroporation. The T cell receptor β chain gene was confirmed to be introduced and expressed in 35.6% of all lymphocytes and 51.5% of CD3 positive T cells at a dose of 4 μg.

Cytotoxic activity of HLA-A2-positive naive T cells expressing MART1-A2 peptide-specific T cell receptor β chain was evaluated against T2 cells (ATCC No. CRL-1992) treated with MART1-A2 peptide. As HLA-A2-positive naive T cells into which the T cell receptor β chain gene was introduced and expressed, cells into which 2 μg or 4 μg of the T cell receptor β chain gene expression plasmid was introduced by the electroporation were used. T2 cells are cells obtained by selecting clones expressing HLA-A2 from T1 cells (immortalized lymphoblast cells) which are hybrids of human T / B cells. The T2 cells were suspended in PBS + 1% human serum albumin solution, MART1-A2 peptide was added to a final concentration of 20 μg / ml, and incubated at 37 ° C. for 2 hours. 1 × 10 ⁵ T2 cells treated with the MART1-A2 peptide were mixed and cultured with naive T cells expressing the same number of the T cell receptor β chains. After stimulation for 24 hours, the culture supernatant was collected, IFN-γ produced by stimulation was quantified, and used as an index of the cytotoxic activity of the naive T cells against T2 cells treated with MART1-A2 peptide.

  As a result, HLA-A2-positive naive T cells expressing a MART1-A2 peptide-specific T cell receptor showed IFN-γ production specifically for T2 cells treated with the MART1-A2 peptide. (FIG. 10).

Furthermore, the cytotoxic activity of HLA-A2 positive naive T cells expressing TART receptor β chain specific to MART1-A2 peptide against various cultured melanoma cells was also evaluated. HLA-A2-positive and MART1-positive melanoma cells (C32: manufactured by ATCC), HLA-A2-positive and MART1-negative as many as 1 × 10 ⁵ HLA-A2-positive naive T cells into which the T cell receptor β chain gene has been introduced and expressed Melanoma cells (RPMI: manufactured by ATCC), HLA-A11 positive and MART1 positive melanoma cells were mixed and cultured with 3 types of melanoma cells. After 24 hours of stimulation, the culture supernatant was collected, and IFN-γ produced by the stimulation was quantified to serve as an index of the cytotoxic activity of the naive T cells against melanoma cells.

  As a result, HLA-A2 positive naive T cells expressing a MART1-A2 peptide-specific T cell receptor produced specific IFN-γ for HLA-A2 positive and MART1 positive melanoma cells C32. Although it showed, it did not show specific IFN-γ production against MART1-negative melanoma cells RPMI and HLA-A11-positive melanoma cells. As a result, melanoma cell C32 was suggested to have tumor-specific cytotoxic activity (FIG. 11).

  It has been confirmed that the MRAT1-A2 peptide-specific T cell receptor β chain gene which has been successfully cloned this time can express a functional T cell receptor by gene transfer into HLA-A2 positive naive T cells. It became clear that it is responsible for cytotoxic activity specific to target cells that are positive and express MART1.

It is a figure which shows the analysis result after dendritic cell stimulation and after purification by MACS method. It is a figure which shows the analysis result using the lymphocyte before stimulation by a dendritic cell. It is a figure which shows the analysis result using the CTL cell after stimulation by a dendritic cell and after purification of a tetramer positive cell. It is a figure which shows the electrophoresis image of four types of genes (BV3, BV12, BV13A, BV13B) amplification fragment. It is a figure which shows the alignment of four types of genes (BV3, BV12, BV13A, BV13B) amplification fragment base sequences. It is a figure which shows the analysis result of the base sequence of a sense primer corresponding to TRBV28 gene, and the antisense primer corresponding to TRBC1 (C gene), and a VDJ junction structure. It is a figure which shows the influence on the proliferation of T cell by a CD3 antibody of GT-T503 culture medium. (◯) indicates RPMI 1640 + 5% HS, and (●) indicates GT-T503 + 5% HS. It is a figure which shows the effect with respect to lymphocyte proliferation of each anti-CD antibody. It is a figure which shows the result of having investigated the introduction | transduction efficiency of the GFP and T cell receptor beta chain gene in the proliferation-stimulated lymphocyte by flow cytometry. Using T2 cells treated with MART1-A2 peptide as target cells, cytotoxic activity of HLA-A2-positive naive T cells expressing a TART receptor β chain specific to MART1-A2 peptide, using INF-γ as an index It is the result of evaluation. It is the result of having evaluated the cytotoxic activity of the HLA-A2 positive naive T cell which expressed the T cell receptor (beta) chain specific to MART1-A2 peptide which made various melanoma cells the target cell, using INF- (gamma) as a parameter | index.

Claims (7)

A T cell receptor β chain gene comprising the base sequence represented by SEQ ID NO: 1. A recombinant vector capable of expressing a T cell receptor β chain protein in which the T cell receptor β chain gene according to claim 1 is incorporated. A transformant into which a recombinant vector capable of expressing the T cell receptor β chain protein according to claim 2 has been introduced. The transformant according to claim 3, which is a lymphocyte into which a recombinant vector has been introduced. A T cell receptor β chain protein consisting of the amino acid sequence represented by SEQ ID NO: 2. An antibody that specifically recognizes a T cell receptor β chain protein consisting of the amino acid sequence represented by SEQ ID NO: 2. For the treatment of melanoma comprising as an active ingredient a lymphocyte introduced in vitro with a recombinant vector capable of expressing a T cell receptor β chain protein in which a T cell receptor β chain gene comprising the base sequence represented by SEQ ID NO: 1 has been incorporated Composition.