JP7138881B2 - T cell receptor and its use - Google Patents

Description

Article 30, Paragraph 2 of the Patent Law applies https://www. congre. co. jp/jca2016/, published on September 22, 2016 The 75th Annual Meeting of the Japanese Cancer Society (Japanese Cancer Society), Abstracts published on the above website

Article 30, Paragraph 2 of the Patent Act applies. Poster exhibited at the 75th Annual Scientific Meeting of the Japanese Cancer Society held on October 6, 2016.

Article 30, Paragraph 2 of the Patent Law applies https://www. congre. co. jp/jca2015/, published on September 18, 2015 The 74th Annual Meeting of the Japanese Cancer Society (Japanese Cancer Society), electronic abstracts published on the above website

Article 30, Paragraph 2 of the Patent Act applies. Poster exhibited at the 74th Annual Scientific Meeting of the Japanese Cancer Society held on October 8, 2015.

Application of Article 30, Paragraph 2 of the Patent Law Issued on October 30, 2015, Abstracts from the 44th Annual Meeting of the Japanese Society for Immunology

Article 30, Paragraph 2 of the Patent Law applies https://www. men-eki. org/meneki_abstract_jimu/abst_search3. do, published on November 10, 2015 The 44th Annual Meeting of the Japanese Society for Immunology, Abstracts published on the above website

Application of Article 30, Paragraph 2 of the Patent Law Poster exhibited at the 44th Annual Meeting of the Japanese Society for Immunology held on November 20, 2015

The present invention provides two types of T cell receptors (T cell receptors, hereinafter referred to as "TCR") that recognize cancer antigens restricted to HLA-A*24:02, and HLA-A*24:02 restricted The present invention relates to the use of the TCR in the detection of cancer antigens that have been detected, cancer therapy, and the like.

In Japan, about 360,000 people die of cancer every year, and about 850,000 people are newly diagnosed with cancer. Standard treatments for cancer include surgery, chemotherapy, and radiotherapy. If there is no improvement after these treatments, palliative care is offered to relieve the various pain associated with cancer. There are almost no other options than moving. Under such circumstances of cancer treatment in Japan, the sight of patients themselves or their families wandering around in search of further treatment methods is called "cancer refugees" and has become a social problem.

In recent years, research on cancer vaccine therapy has been actively conducted to fill the gap between standard treatment and palliative care. Cancer vaccine therapy is a treatment aimed at activating the patient's own immunity to eliminate or regress cancer. ) is also one of the cancer vaccine therapies. Approved for patients with metastatic and hormone-refractory prostate cancer, Provenge is injected into the patient's body after stimulating the patient's peripheral blood with the PAP (prostatic acid phosphatase) protein, which is overexpressed in most prostate cancers. It is a treatment that transfers. Since the success or failure of cancer vaccine therapy depends on the patient's inherent immunity, it is believed that it will be more effective if it can be implemented before the patient's symptoms worsen by receiving various standard therapies. In the future, it is hoped that it will be approved as one of the treatment options and as a treatment that can be used in combination with chemotherapy and radiotherapy.

Cancer vaccine therapy is roughly divided into antigen-nonspecific therapy and antigen-specific therapy. Antigen-nonspecific immunotherapy started in the 1970s using vicivanil, krestin, killed tuberculosis bacteria (BCG-CWS), etc., which have an immunostimulatory action. In the 1980s, immunotherapy using cytokines such as IL-2 and LAK (lymphokine-activated killer cell) therapy, which returns non-specifically amplified T cells to the patient's body, were performed, but both were clearly However, it was not recognized as a medical treatment covered by health insurance because it was unable to produce significant clinical effects.

Cytotoxic T cells (Cytotoxic T Lymphocytes, hereinafter referred to as "CTL") isolated and amplified from patients in 1991 specifically target a cancer antigen called MAGE (melanoma antigen) highly expressed in cancer cells. It has been reported to recognize and kill cancer cells (Non-Patent Document 1). In 1994, a mechanism was reported in which CTL recognizes and kills peptide fragments presented on MHC (major histocompatibility complex, called HLA in humans) on the surface of cancer cells ( Non-Patent Document 2). Subsequently, various HLA-presented peptide fragments were identified by researchers around the world. In 1996, Altman et al. developed an MHC tetramer reagent capable of detecting specific CTLs (Non-Patent Document 3), which until then could only be indirectly verified by chromium release assays. Detection could be verified more directly than the conformation of the TCR. A cancer peptide vaccine therapy for melanoma (malignant melanoma) reported in 1998 achieved surprising clinical results, and cancer immunotherapy was made to bloom at once (Non-Patent Document 4). Thereafter, cancer peptide vaccine therapy in which peptides are inoculated subcutaneously or intradermally has been promoted on a global scale.

Rosenberg et al. reported in 2004 reported that the response rate of cancer vaccine therapy was lower than expected and that the subjects of clinical trials should be considered terminal cancer patients (non-patent literature 5). This is because whether or not CTLs that attack cancer cells in the patient's body after inoculation with a peptide vaccine are amplified as expected depends on the factor of the patient's own immunity. Therefore, it is thought that the response rate will naturally decrease when the immune system is remarkably weakened by chemotherapy or radiotherapy. In patients who received a peptide vaccine, dendritic cells called antigen-presenting cells and macrophages present around the injection site take up the peptide and present it to the MHC on the cell surface, thereby activating T cells and further affiliating them. A step is required to recruit antigen presenting cells to the lymph nodes to activate T cells. In patients whose immunity has been significantly weakened by anticancer drugs or radiotherapy, it is assumed that the number of antigen-presenting cells is reduced and normal functions are lost, which is considered to be one of the reasons for the limited antitumor effect. Therefore, dendritic cell therapy has been performed in which dendritic cells with high antigen-presenting ability are isolated and induced and cultured outside the body and inoculated into patients in a state in which antigens have been presented in advance. However, similar to peptide vaccine therapy, even if this method is used, the effect may be limited in patients with weakened immune systems due to the phenomenon that the amount of T cells in the body is insufficient or not sufficiently activated. It is considered.

For the purpose of compensating for the shortcomings of peptide vaccines and dendritic cell therapy, CTL transfer therapy has been performed in which T cells activated outside the body are returned to the patient's body. Since this method transfers cells into the blood, a dedicated culture facility (CPC, Cell Processing Center) is required. In order to repeat complicated cell culture, the development of automatic culture equipment, for example, is progressing, but there is a need for a very difficult culture technology to efficiently amplify specific CTL outside the body in a short period of time. be.

T cells are complexes of MHC molecules and antigenic peptides presented on the surface of target cells such as antigen-presenting cells, cancer cells, and virus-infected cells (hereinafter referred to as " It acts by recognizing and binding to MHC/peptide complexes (referred to as "MHC/peptide complexes"). CD8-positive T cells bind only to the MHC class I molecule/antigen peptide complex (MHC class I restricted), and CD4-positive T cells can bind only to the MHC class II molecule/antigen peptide complex (MHC class I molecule/antigen peptide complex). class II restricted). MHC class I molecules are expressed in most nucleated cells and platelets, whereas MHC class II molecules are expressed only in a limited number of cells. Therefore, CD4-positive T cells can bind to dendritic cells expressing MHC class II molecules, B cells, activated T cells, and the like, but cannot directly bind to other tumor cells, infected cells, and the like.

It has been reported that the TCR gene derived from cancer antigen-specific CTL can specifically damage cancer by introducing it into peripheral blood lymphocytes (Non-Patent Document 6). Since then, so-called TCR gene therapy, in which TCR genes isolated from specific CTLs are artificially expressed in peripheral lymphocytes to produce artificial CTLs and returned to the patient's body, has been actively attempted ( Non-Patent Document 7). The potent cytotoxic activity of CTL is considered important in anti-tumor immunity, and one of the ways to utilize this is the use of variable region chimeric antigen receptors in which the variable regions of tumor antigen-specific monoclonal antibodies are expressed in CTLs. (Chimeric Antigen Receptor, CAR) gene-modified T cell (CAR-T cell) therapy and the like have been reported. Although the effect is actually largely dependent on the performance of the monoclonal antibody, a certain effect has been obtained against acute lymphocytic leukemia, etc., and regarding further improvement in the future and safe maintenance in vivo It is discussed (Non-Patent Document 8).

Recent advances in research on cancer immunotherapy have been particularly remarkable, and what sparked them was the anti-PD-1 antibody (nivolumab), which is known as an antibody drug that targets immune checkpoints. Anti-PD-1 antibodies release immunosuppression by inhibiting signal transduction through PD-1, which acts as an immunosuppressant, that is, a brake, making it possible to more efficiently induce an immune response against cancer ( Non-Patent Documents 9, 10). In 2013, this achievement topped Breakthrough of the Year in the American scientific journal Science and attracted a great deal of attention. Overturning conventional concepts, it has had such an impact that it is said that immunity cannot be ignored in cancer treatment, and in July 2014, manufacturing and marketing was approved for malignant melanoma in Japan. Currently, clinical trials for lung cancer, renal cancer, etc. are being conducted. In the United States, combined therapy with anti-CTLA-4 antibody (ipiribumab), which is also expected to have an immune checkpoint inhibitory effect, has been shown to have a tumor regression effect. Expectations are also gathering for combined therapy with anti-PD-1 antibody. Currently, 9 types of PD-1 / PD-L1-related antibody drugs are undergoing clinical trials (Non-Patent Documents 11, 12), and more than 60 types of PD-1 for more than 30 types of cancer in Japan and overseas. Clinical trials of single-agent and combination therapy have been initiated.

Since the identification of peptide fragments (CTL epitopes) derived from malignant melanoma antigen MAGE protein by Boon et al. in 1991, many CTL epitopes associated with cancer, infectious diseases, autoimmune diseases and transplantation have been identified. Even CTL epitopes derived from the same protein present different types of peptide fragments depending on the HLA type, and are generally called HLA-restricted CTL epitopes. In recent years, analyzes using next-generation sequencers, which have shown remarkable technological progress, have enabled comprehensive analysis of mutations actually occurring in tumor cells. As a result, it has been reported that CTL recognize this tumor-specific mutant antigen very well and damage it, and it is very effective to target the mutant antigen and activate the immune response against cancers that are prone to mutation. It was suggested that As the ultimate order-made medicine, an individualized medical treatment in which a CTL epitope derived from a mutant antigen unique to an individual patient is selected by a computer algorithm and inoculated has been devised and is already in operation toward commercialization (Non-Patent Document 13). However, since this method does not correspond to cancers with few mutations, it can be said that it is necessary to select CTL epitopes according to the characteristics of cancers.

Under such circumstances, it is very important to select a cancer antigen-derived CTL epitope for a target patient when performing cancer vaccine therapy. CTL epitopes are desirably restricted to HLA types that are widely maintained among races, even among highly diverse HLA types. For example, HLA-A*24:02 is possessed by 60-70% of Japanese, HLA-A*02:01 is possessed by approximately 50% of Westerners, and approximately 20% of Japanese possess it. HLA type, but this is very high frequency considering that many other HLA type frequencies are less than 10%. meaningful to

As to which cancer antigen should be administered to a target patient, it is desirable to be diagnosed before starting the vaccine therapy. For example, in order to perform HLA-A*24:02-restricted survivin-2B peptide vaccine therapy, the HLA type of the target patient must be HLA-A*24:02. The HLA type test is an essential test for organ transplantation and bone marrow transplantation, and is performed in various facilities. In addition, since it is essential for recognition by CTL that an HLA-A*24:02-restricted survivin-2B peptide is presented on the target cancer cell surface, HLA-A*24:02-restricted Demonstration of this is most desirable for survivin-2B peptide vaccine therapy in . However, at present, there is no test method for direct proof, and indirect tests are performed. For example, in the case of survivin-2B, a method of confirming the expression level of survivin-2B mRNA by a quantitative PCR method using a biopsy sample, or an anti-survivin-2B antibody is used to express survivin-2B protein in tissues and cells. and a method of confirming HLA expression using an anti-HLA antibody. Antibodies that specifically recognize the state in which peptides are bound to MHC (hereinafter referred to as "anti-pMHC antibodies") and TCR tetramers using TCR are being studied as tools for direct demonstration. and scTCR (single chain TCR) multimers using single-chain TCR.

With regard to anti-pMHC antibodies, an antibody that specifically recognizes MHC class II peptide-presented peptides was reported in 1989 (Non-Patent Document 14), and in 1997, H- ^2Kb , a mouse MHC class I molecule, was reported. An antibody that recognizes the state in which an OVA (ovalbumin)-derived peptide is presented has been reported (Non-Patent Document 15), and many researchers have started attempts to obtain a similar antibody.

Molecules constituting the MHC/peptide complex are MHC with a molecular weight of about 45 kDa and β2m (β2-microglobulin) with a molecular weight of about 12 kDa in a non-covalent state. About 11 amino acids are bound. It is very difficult to obtain, by the hybridoma method, antibodies that specifically recognize only the differences in the presented peptides, and in fact, only a few types of antibodies have been reported in the last 20 years.

For example, in 1996, there is a report that antibodies against MHC/peptide complexes were obtained by the phage display method using a library derived from immunized mice (Non-Patent Document 16). Subsequently, in 2000, it was reported that an antibody specific to the HLA-A1/MAGE-A1 complex was obtained from a library derived from non-immunized humans (Non-Patent Document 17). Antibodies thus obtained can exhibit reactivity when peptides are artificially bound on the cell surface. However, it is known that it is very difficult to develop an antibody that can exhibit reactivity when an endogenous peptide is presented on the cell surface in a natural state. CTLs are sufficiently activated by binding to several to ten MHC/peptide complexes on the surface of target cells, and the system for detecting them also binds several to ten molecules. The state must be detected, and it has been pointed out that current technology may not be sensitive enough.

A detection system for MHC/peptide complexes using soluble TCR has been considered for a long time (Non-Patent Document 18). Due to the difficulty in obtaining the peptide while maintaining its steric structure and the weak binding force between the TCR and the MHC/peptide complex, its practicality was considered to be low. However, it was reported in 2004 that tetramerization of TCR can be used practically in the same manner as the MHC tetramer reagent synthesis technology (Non-Patent Document 19). In 2006, the TCR protein, which forms a heterodimer of α and β chains, was modified into a single-chain TCR (scTCR) in which Vα, Vβ, and Cβ were linked, and this was multimerized. By doing so, they have succeeded in producing a reagent capable of specifically detecting MHC/peptide complexes (Non-Patent Document 20).

As explained above, the TCR is extremely useful in immunotherapy, and it is extremely important in the art to identify the sequences of the α and β chains that constitute it. However, regarding survivin-2B and PBF known as cancer antigens, no TCR that recognizes these cancer antigens restricted to HLA-A*24:02 has been identified yet.

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The present invention has been made in view of the problems of the prior art, and provides a TCR capable of recognizing a survivin-2B peptide restricted by HLA-A*24:02 and a DNA encoding the TCR. intended to Another object of the present invention is to provide a TCR capable of recognizing a PBF peptide restricted by HLA-A*24:02 and a DNA encoding the TCR.

The frequency of cancer antigen-specific CTLs is very low. Therefore, in order to obtain CTLs having the desired TCR gene, it is necessary to devise methods such as growing and culturing CTLs. In addition, TCR gene sequences are extremely diverse. TCR is a membrane surface protein that exists on the surface of T cells, and two types, a heterodimer consisting of α/β chains and a heterodimer consisting of γ/δ chains, have been identified. The TCR gene is a molecule called a signal sequence, a variable region (V: variable), a diversity region (D: diversity), and a joining region (J: joining), which is cleaved when expressed on the cell membrane surface, like an immunoglobulin gene. and a C domain consisting of a constant region (C: constant) including an extracellular constant region, a transmembrane region, and an intracellular region. Since these amino acid sequences differ from T cell to T cell, the TCR is an antigen recognition molecule as well as a marker for individual T cells. This TCR diversity is produced by rearrangement of acquired TCR genes, and it is known that TCR β and δ chains rearrange VDJ, and TCR α and γ chains rearrange VJ. there is Even just by linking the sequences registered in the IMGT database, there are 7,015 combinations of Vα (115 types), Jα (61 types), and Cα (1 type) for the α chain, and Vβ for the β chain. (146 types), Dβ (3 types), Jβ (16 types), and Cβ (4 types) make up 28,032 combinations. Then, the number of combinations of α-chain and β-chain is 2×10 ⁸ (However, since the diversity of T-cells has many insertions and deletions of bases at the binding site of each region, It is difficult to calculate, and some reports put the diversity at 10 ¹⁵ ). Therefore, in order to identify the correct combination of α-chain and β-chain of the target TCR gene, it is necessary to devise a way to propagate CTL having a single TCR.

Therefore, the present inventors first conducted intensive studies on a method for efficiently proliferating and culturing CTLs specific to survivin-2B peptide or PBF peptide under limiting dilution conditions, and found that each of the peptides is HLA-A*. We succeeded in obtaining a CTL capable of recognizing the state presented at 24:02. In addition, when the cytotoxic activity of CTL specific to each of the obtained cancer antigen peptides was verified, it was found that the corresponding cancer antigen peptide was present in HLA-A*24:02 against a lymphoma sub-cell line. , each exhibited specific cytotoxic activity.

Furthermore, when TCR repertoire analysis of each CTL cell population obtained was performed, the Vβ chain of survivin-2B-specific CTL clones belonged to subgroup Vβ8, and the Vβ chain of PBF-specific CTL clones belonged to Vβ1. found.

Moreover, the present inventors determined the full-length sequences of the TCR α-chain and β-chain by PCR or the like after synthesizing cDNA from the total RNA extracted from each of the obtained CTLs by a reverse transcriptase reaction. Furthermore, by expressing the TCR α chain and β chain in cultured cells and performing analysis using each HLA-A*24:02 tetramer reagent, the corresponding cancer antigen peptides presented by HLA-A*24:02 were identified. The correct α-chain and β-chain combinations that specifically recognize each were identified.

Transformed cell lines expressing the correct combination of α and β chains can be efficiently detected with each HLA-A*24:02 tetramer reagent, while using the transformed cell lines , cells in which each HLA-A*24:02 tetramer reagent and the corresponding cancer antigen peptide were presented to HLA-A*24:02 could be efficiently detected.

In addition, it is generally known that the CD8 molecule is a very important molecule in the binding between HLA molecules presenting cancer antigen peptides and TCRs. However, in the present invention, it was also revealed that the binding of these TCRs obtained to HLA-A*24:02 molecules presenting the corresponding cancer antigen peptides does not require the assistance of CD8. Furthermore, it was shown that multimerization of the TCR is useful for the development of tools for detecting HLA-A*24:02 molecules presenting cancer antigen peptides. In fact, it was also confirmed that the obtained TCRs were multimerized and that the obtained TCR multimers could specifically bind HLA-A*24:02 molecules presented with corresponding cancer antigen peptides.

Therefore, the present invention can recognize TCRs capable of recognizing HLA-A*24:02 in which the survivin-2B peptide is restricted, and HLA-A*24:02 in which the PBF peptide is restricted. The present invention relates to TCRs, DNAs encoding these TCRs, and uses thereof, and more specifically provides the following inventions.
<1> A T-cell receptor having any of the following characteristics (i) to (iii): (i) a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NOS: 1-3 and SEQ ID NOS: 6-8 (ii) a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NO: 4 and a T cell having the amino acid sequence set forth in SEQ ID NO: 9 (iii) consisting of a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NO:5 and a T-cell receptor β-chain protein having the amino acid sequence set forth in SEQ ID NO:10;
<2> The T cell receptor of <1>, which recognizes a survivin-2B peptide restricted by HLA-A*24:02.
<3> T-cell receptor α-chain protein having any of the following characteristics (i) to (iii) (i) having the amino acid sequence set forth in SEQ ID NOS: 1 to 3 (ii) having the amino acid sequence set forth in SEQ ID NO: 4 (iii) having an amino acid sequence (iii) having the amino acid sequence set forth in SEQ ID NO:5;
<4> A T-cell receptor β-chain protein having any of the following characteristics (i) to (iii): (i) having an amino acid sequence set forth in SEQ ID NO: 6 to 8; (ii) having an amino acid sequence set forth in SEQ ID NO: 9 (iii) having an amino acid sequence (iii) having the amino acid sequence set forth in SEQ ID NO:10;
<5> A DNA encoding the protein according to any one of <3> and <4>.
<6> A vector containing the DNA according to <5> in an expressible manner.
<7> A transformed cell into which the DNA of <5> has been introduced.
<8> A transformed cell into which the DNA encoding the protein of <3> and the DNA encoding the protein of <4> have been introduced, wherein the survivin- is restricted to HLA-A*24:02 Transformed cells that can be detected by multimerized molecules that bind the 2B peptide.
<9> The transformed cell of <7> or <8>, which is a lymphocyte.
<10> A pharmaceutical composition for treating survivin-2B-positive cancer, comprising the transformed cell of <9> as an active ingredient.
<11> An antibody that specifically binds to the molecule described in any one of (a) to (c) below.
(a) the T-cell receptor α-chain protein according to <3> (b) the T-cell receptor β-chain protein according to <4> (c) the T-cell receptor <12><1> or <2> A molecule multimerized by binding to the T cell receptor according to 1> or <2>.
<13> An agent for detecting or capturing an HLA-A*24:02-restricted survivin-2B peptide, the agent comprising the molecule according to <12>.
<14> A kit for detecting an HLA-A*24:02-restricted survivin-2B peptide, the kit comprising at least one of the following components (a) to (h).
(a) the T-cell receptor α-chain protein described in <3> (b) the T-cell receptor β-chain protein described in <4> (c) the T-cell receptor β-chain protein described in <1> or <2> (d) <5> DNA according to
(e) the vector described in <6> (f) the transformed cell described in any one of <7> to <9> (g) the antibody described in <11> (h) the molecule described in <12>15> A method for quality control of a molecule multimerized by binding an HLA-A*24:02-restricted survivin-2B peptide, comprising the molecule and any one of <7> to <9> A method comprising the step of confirming reactivity with transformed cells.
<16> A T-cell receptor having any of the following characteristics (i) to (iii): (i) a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NOs: 11-13 and SEQ ID NOs: 16-18 (ii) a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NO: 14 and a T cell having the amino acid sequence set forth in SEQ ID NO: 19 (iii) consisting of a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NO:15 and a T-cell receptor β-chain protein having the amino acid sequence set forth in SEQ ID NO:20;
<17> The T cell receptor of <16>, which recognizes a PBF peptide restricted by HLA-A*24:02.
<18> A T-cell receptor α-chain protein having any of the following characteristics (i) to (iii) (i) having an amino acid sequence set forth in SEQ ID NO: 11 to 13 (ii) set forth in SEQ ID NO: 14 (iii) having an amino acid sequence (iii) having the amino acid sequence set forth in SEQ ID NO: 15;
<19> A T-cell receptor β-chain protein having any of the following characteristics (i) to (iii): (i) having an amino acid sequence set forth in SEQ ID NOS: 16 to 18; (ii) having an amino acid sequence set forth in SEQ ID NO: 19 (iii) having an amino acid sequence (iii) having the amino acid sequence set forth in SEQ ID NO:20;
<20> A DNA encoding the protein of any one of <18> and <19>.
<21> A vector containing the DNA according to <20> in an expressible manner.
<22> A transformed cell into which the DNA of <20> has been introduced.
<23> A transformed cell introduced with the DNA encoding the protein of <18> and the DNA encoding the protein of <19>, wherein the PBF peptide is restricted to HLA-A*24:02 Transformed cells that can be detected by multimerized molecules that bind .
<24> The transformed cell of <22> or <23>, which is a lymphocyte.
<25> A pharmaceutical composition for treating PBF-positive cancer, comprising the transformed cell of <24> as an active ingredient.
<26> An antibody that specifically binds to the molecule described in any one of (a) to (c) below.
(a) the T-cell receptor α-chain protein described in <18> (b) the T-cell receptor β-chain protein described in <19> (c) the T-cell receptor <27><17> or <16> A molecule multimerized by binding to the T cell receptor according to 16> or <17>.
<28> An agent for detecting or capturing an HLA-A*24:02-restricted PBF peptide, the agent comprising the molecule of <27>.
<29> A kit for detecting an HLA-A*24:02-restricted PBF peptide, the kit comprising at least one of the following components (a) to (h).
(a) the T-cell receptor α-chain protein according to <18> (b) the T-cell receptor β-chain protein according to <19> (c) the T-cell receptor β-chain protein according to <16> or the T-cell receptor (d)<17>20> DNA according to
(e) the vector described in <21> (f) the transformed cell described in any one of <22> to <24> (g) the antibody described in <26> (h) the molecule described in <27>30> A method for quality control of a molecule multimerized by binding a PBF peptide restricted by HLA-A*24:02, comprising the molecule and the transformation according to any one of <22> to <24> A method comprising the step of confirming reactivity with cells.

The TCR and its transformed cells of the present invention are useful for setting usage conditions and quality control of HLA-A*24:02 tetramer reagents, and survivin-2B peptides or PBF peptides in biological samples are HLA-A * Useful as a companion diagnostic to confirm whether the cells presented in 24:02 are present. Furthermore, it is also useful as a therapeutic agent for cancer associated with survivin-2B or PBF.

Dot-plot development showing the reactivity of survivin-2B CTL populations to MHC tetrameric reagents. Dot-plot development showing the reactivity of PBF CTL populations to MHC tetrameric reagents. FIG. 4 is a dot-plot development showing the reactivity of the survivin-2B CTL clone ITG-MT3 to MHC tetramer reagents. FIG. 4 is a dot-plot development showing the reactivity of the PBF CTL clone FKS-D11P to MHC tetramer reagents. 1 is a graph showing cytotoxic activity against target cells when ITG-MT3 is used as effector cells. FIG. 2 is a graph showing cytotoxic activity against target cells when FKS-D11P is used as effector cells. FIG. FIG. 3 is a dot-plot development showing the results of staining analysis of HLA-A*24:02 survivin-2B peptide-specific CTL clone ITG-MT3. FIG. 10 is a dot-plot development showing the results of staining analysis of the HLA-A*24:02 PBF peptide-specific CTL clone FKS-D11P. FIG. 2 is a photograph showing the results of development by agarose gel electrophoresis of PCR amplification products of TCRα chain gene and TCRβ chain gene of HLA-A*24:02 survivin-2B peptide-specific CTL clone ITG-MT3. Fig. 10 is a photograph showing the results of development by agarose gel electrophoresis of the PCR amplification products of the TCRα chain gene and TCRβ chain gene of the HLA-A*24:02 PBF peptide-specific CTL clone FKS-D11P. Results of analyzing the correct combination of two types of ITG-MT3-derived TCRα chains (A4 and A13-2) and one type of β-chain (B12-4), and FKS-D11P-derived TCRα chain (A1-2) and the TCRβ chain (B9) are correct combinations. Schematic diagram showing the organization of the TCR α and β chains of the HLA-A*24:02 survivin-2B peptide-specific CTL clone ITG-MT3. Schematic representation of TCR α and β chain organization of HLA-A*24:02 PBF peptide-specific CTL clone FKS-D11P. FIG. 4 is a dot-plot development showing the reactivity of the TCR gene-expressing transformed cell line SMT3S to MHC tetramer reagents. FIG. 4 is a dot-plot development showing the reactivity of the TCR gene-expressing transformed cell line PD11S to MHC tetramer reagents. FIG. 3 is a dot-plot development diagram showing the results of evaluation (addition and recovery test) of MHC tetramer reagents using the TCR gene-expressing transformed cell line SMT3S. FIG. 3 is a dot-plot development diagram showing the results of evaluation (addition and recovery test) of MHC tetramer reagents using the TCR gene-expressing transformed cell line PD11S. FIG. 2 shows the results of evaluation of MHC tetramer reagents using SMT3S (verification of concentration-dependent staining and confirmation of storage stability). FIG. 3 shows the results of evaluation of MHC tetramer reagents using PD11S (verification of concentration-dependent staining and confirmation of storage stability). J . Dot-plot development showing the reactivity of the RT3-T3.5 cell line to MHC tetramer reagents. J . Dot-plot development showing the reactivity of the RT3-T3.5 cell line to MHC tetramer reagents. Dot-plot developments showing the reactivity of SMT3S and PD11S to their respective MHC tetramer reagents in the presence of CD8 antibody clones (T8 or RFT-8). FIG. 11C is a graph showing the ratio of the MHC tetramer positive rate when clone RFT-8 was used when the tetramer positive rate when clone T8 was used was taken as 100% in the results shown in FIG. 11C. FIG. 3 is a dot-plot development diagram showing the results of verifying the reactivity of SMT3J to MHC tetramer reagents. FIG. 10 is a dot-plot development diagram showing the results of verifying the reactivity of PD11J to MHC tetramer reagents. 1 is a graph showing the results of analyzing TCR signaling in SMT3J using luciferase activity as an index. FIG. 10 is a graph showing the results of analysis of TCR signaling in PD11J using luciferase activity as an index. FIG. FIG. 2 shows the amino acid sequence of the TCRα chain of HLA-A*24:02 survivin-2B peptide-specific CTL clone ITG-MT3 (amino acid sequence set forth in SEQ ID NO:22). FIG. 2 shows the amino acid sequence of the TCRα chain of the HLA-A*24:02 PBF peptide-specific CTL clone FKS-D11P (amino acid sequence set forth in SEQ ID NO:26). FIG. 3 shows the amino acid sequence of the TCRβ chain of HLA-A*24:02 survivin-2B peptide-specific CTL clone ITG-MT3 (amino acid sequence set forth in SEQ ID NO:24). FIG. 2 shows the amino acid sequence of the TCR β chain of the HLA-A*24:02 PBF peptide-specific CTL clone FKS-D11P (amino acid sequence set forth in SEQ ID NO:28). Schematic showing constructs for making TCR multimers. FIG. 4 is a histogram diagram showing the reactivity of TCR multimers to cells pulsed with HLA-A*24:02-restricted survivin-2B peptides. FIG. 4 is a graph showing the reactivity of TCR multimers to cells pulsed with HLA-A*24:02 restricted survivin-2B peptides. Histograms showing concentration-dependent reactivity of TCR multimers to cells pulsed with HLA-A*24:02-restricted survivin-2B peptides. FIG. 4 is a graph showing the concentration-dependent reactivity of TCR multimers to cells pulsed with HLA-A*24:02-restricted survivin-2B peptides. FIG. 4 is a histogram diagram showing the pulsed peptide concentration-dependent reactivity of TCR multimers to HLA-A*24:02-restricted PBF peptide-pulsed cells. FIG. 4 is a histogram diagram showing the TCR multimer concentration-dependent reactivity of TCR multimers to cells pulsed with HLA-A*24:02-restricted PBF peptides. Histogram showing inhibition of TCR multimer reactivity to HLA-A*24:02-restricted survivin-2B and PBF peptide-pulsed cells in a blocking assay using an anti-HLA-A24 antibody. It is a diagram. Micrographs showing the reactivity of TCR multimers to cells pulsed with HLA-A*24:02-restricted PBF peptides. 2 is a photomicrograph showing the reactivity of TCR multimers to the HLA-A*24:02-positive osteosarcoma-derived cell line KIKU that naturally expressed PBF.

<TCR, TCRα chain, TCRβ chain>
The present invention provides a T-cell receptor (TCR) and its constituent T-cell receptor α-chain protein (TCRα-chain) and T-cell receptor β-chain protein (TCRβ-chain).

As one aspect of the TCR of the present invention, more specifically,
(i) consisting of a T-cell receptor α-chain protein having the amino acid sequences set forth in SEQ ID NOS: 1-3 and a T-cell receptor β-chain protein having the amino acid sequences set forth in SEQ ID NOS: 6-8 (ii) SEQ ID NOS : consisting of a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NO:4 and a T-cell receptor β-chain protein having the amino acid sequence set forth in SEQ ID NO:9, or (iii) the amino acid sequence set forth in SEQ ID NO:5 and a T-cell receptor β-chain protein having the amino acid sequence set forth in SEQ ID NO: 10. Typical characteristics of the TCR are HLA-A*24: 02-restricted survivin-2B peptide recognition.

Further, as one aspect of the TCR of the present invention, more specifically,
(i) a T-cell receptor α-chain protein having the amino acid sequences set forth in SEQ ID NOS: 11-13 and a T-cell receptor β-chain protein having the amino acid sequences set forth in SEQ ID NOS: 16-18 (ii) SEQ ID NOS : consisting of a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NO: 14 and a T-cell receptor β-chain protein having the amino acid sequence set forth in SEQ ID NO: 19, or (iii) the amino acid sequence set forth in SEQ ID NO: 15 and a T-cell receptor β-chain protein having the amino acid sequence set forth in SEQ ID NO: 20. Typical characteristics of the TCR are HLA-A*24: 02-constrained PBF peptide recognition.

In addition, it is generally known that the CD8 molecule is a very important molecule in the binding between HLA molecules presenting cancer antigen peptides and TCRs. However, as shown in Example 10 below, the TCR of the present invention has an excellent feature that it can recognize the corresponding antigenic peptide restricted by HLA-A*24:02 even in the absence of CD8. have

"HLA-A*24:02" in the present invention is encoded at locus A of class I molecule of human histocompatibility leukocyte antigen (HLA), classified as A24 in serological HLA type, and amino acid mutation It is classified into subtypes associated with HLA-A*24:02, and is the type with the highest frequency of possession in Japanese (the amino acid sequence of HLA-A*24:02 is shown in SEQ ID NO: 32).

"Survivin-2B" is a protein belonging to the IAP family that suppresses apoptosis, and is typically identified by GenBank Accession Number: BAA93676.1 if it is of human origin. A protein consisting of an amino acid sequence. "PBF" is a protein called osteosarcoma-specific antigen papillomavirus binding factor (Papillomavirus Binding Factor), ZNF395. It is a protein consisting of the amino acid sequence specified in 1. It should be understood that in both survivin-2B and PBF, in addition to those having such a typical amino acid sequence, naturally occurring amino acid mutations may also exist.

In addition, the "HLA-A*24:02-restricted survivin-2B peptide" and the "HLA-A*24:02-restricted PBF peptide" in the present invention mean that these peptides are present on the cell surface. and when present as isolated or purified molecules (for example, molecules in which these peptides are bound to multimerized TCRs).

In the present invention, "survivin-2B peptide" means "AYACNTSTL" (SEQ ID NO: 29), and "PBF peptide" means "AYRPVSRNI" (SEQ ID NO: 30). It has been revealed that CTLs induced by these peptide fragments have high cytotoxic activity against cancer cells (Japanese Patent Laid-Open No. 2002-284797, International Publication No. 2004/029248).

The present invention also provides TCRα chain protein and TCRβ chain protein that constitute the TCR. The TCR α chain protein constituting the TCR identified by the present inventors has CDR1 consisting of the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 as the complementarity determining region on the variable region. and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:3. In addition, the TCRα chain protein constituting another TCR identified by the present inventors has CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 11 and SEQ ID NO: 12 as the complementarity determining region on the variable region. and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 13. Accordingly, the present invention provides TCRα chain proteins having the amino acid sequences set forth in SEQ ID NOs: 1-3 or 11-13.

The CDRs in the present invention can be identified by analysis using the method described in the literature (Nucleic Acids Res. 2008 July 1; 36 (Web Server issue): W503-W508) and software provided by IMGT.

The variable regions containing these complementarity determining regions have one or more amino acids (e.g., several amino acids or less, three amino acids or less, two amino acids or less) in the amino acid sequence in the regions other than the complementarity determining regions. may be substituted, deleted, added, etc., but preferably the variable region consists of the amino acid sequence set forth in SEQ ID NO: 4 or 14. Also, the TCRα chain protein of the present invention preferably has the amino acid sequence set forth in SEQ ID NO:5 or 15. The amino acid sequences shown in SEQ ID NOS: 5 and 15 are embodiments in which a constant region is added to the variable region. and the amino acid sequence excluding TM (transmembrane region).

On the other hand, the TCR β chain protein constituting the TCR identified by the present inventors has CDR1 consisting of the amino acid sequence of SEQ ID NO: 6 and the amino acid sequence of SEQ ID NO: 7 as the complementarity determining region on the variable region. It has CDR2 consisting of the sequence and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:8. In addition, the TCR β chain protein constituting another TCR identified by the present inventors has CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 16 and SEQ ID NO: 17 as the complementarity determining region on the variable region. and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:18. Accordingly, the present invention provides TCR beta chain proteins having the amino acid sequences set forth in SEQ ID NOS:6-8 or 16-18.

The variable regions containing these complementarity determining regions have one or more amino acids (e.g., several amino acids or less, three amino acids or less, two amino acids or less) in the amino acid sequence in the regions other than the complementarity determining regions. may be substituted, deleted, added, etc., but preferably the variable region consists of the amino acid sequence set forth in SEQ ID NO: 9 or 19. Also, the TCRα chain protein of the present invention preferably has the amino acid sequence set forth in SEQ ID NO: 10 or 20. The amino acid sequences shown in SEQ ID NOS: 10 and 20 are embodiments in which a constant region is added to the variable region. and the amino acid sequence excluding TM.

<Gene, vector, transformed cell>
The present invention also provides DNA encoding the TCRα chain protein and DNA encoding the TCRβ chain protein. By inserting these DNAs into expression vectors and introducing them into cells, the above TCRα chain protein, the above TCRβ chain protein, or a TCR complex comprising these proteins can be expressed in cells. As vectors for expressing these proteins in cells, the vectors shown in this example can be used, but the vectors are not limited thereto. For example, a vector pIRES (TaKaRa Bio) capable of translating two types of genes from a single mRNA via an IRES, a Mammalian PowerExpress System (Toyobo) vector, and the like can also be used. The vector may be a vector that expressably contains either the DNA encoding the TCRα chain protein or the DNA that encodes the TCRβ chain protein, or may be a vector that expressably contains both. Cells into which the vector is introduced are not particularly limited, and various cells can be used depending on the purpose. Gene transfer into cells can be performed by methods known to those skilled in the art, such as electroporation.

Transformed cells thus prepared can be used, for example, as companion diagnostic agents for peptide vaccine therapy. When performing peptide vaccine therapy, which peptide should be used as a vaccine is important diagnostic information. It is possible to evaluate whether the survivin-2B peptide or PBF peptide (hereinafter also collectively referred to as “cancer antigen peptide”) is presented.

For such purposes, cells modified to express a reporter protein in response to activation of TCR signaling (e.g., Jurkat/MA cells, a Jurkat cell substrain) can be used for the preparation of transformed cells. ) can be used (Int J Cancer, 2002;99(1):7-13). For the same purpose, cells that produce cytokines in response to stimulation, such as Jurkat, HPB-ALL, and HPB-MLT, can also be used as cells for preparing the transformed cells. As a result, it is possible to detect the presence of HLA-A*24:02-restricted cancer antigen peptides on the membrane surface of target cells using cytokine production in transformed cells as an index (J. Immunology, 1984; 133:1123-1128).

In addition, dendritic cell vaccine therapy, in which a peptide is bound to dendritic cells isolated from a patient and inoculated into the patient, has been performed. It can also be used as a reagent for

Another preferred embodiment of the transformed cell of the present invention is a transformed cell that can be detected by a molecule that binds and multimerizes an HLA-A*24:02-restricted cancer antigen peptide.

Molecules multimerized by binding HLA-A*24:02-restricted cancer antigen peptides of the present invention, that is, "multimers by binding HLA-A*24:02-restricted survivin-2B peptides "HLA-A*24:02-restricted PBF peptides" or "multimerized molecules" are described in Science, 1996;274:94-96, US Pat. No. 5,635,363; It can be prepared by the method described in Japanese Patent No. 3506384. Specifically, recombinant proteins of HLA-A*24:02 and β2m and chemically synthesized survivin-2B peptide or PBF peptide are associated by stirring in a folding solution, and HLA-A*24:02 and β2m are combined. and a peptide complex (hereinafter referred to as "monomer"). Subsequently, biotin is bound to one amino acid on the C-terminal side of HLA-A*24:02, which constitutes the monomer, by an enzymatic reaction. Biotinylated monomers can be purified by column chromatography and reacted with avidin to synthesize multimerized molecules. By labeling avidin in advance with a fluorescent substance such as FITC (fluorescein isothiocyanate), PE (phycoerythrin), or APC (allophycocyanin), antigen-specific T cells can be detected using a flow cytometer or fluorescence microscope.

Further, in the present invention, the number of monomers forming a polymerized molecule is not particularly limited, but is usually 2 to 10, preferably 4 to 8, more preferably 4 or 5, especially Four is preferred.

Examples of products of "multimerized molecule by binding HLA-A*24:02-restricted survivin-2B peptide" include, for example, T-Select MHC Tetramer HLA-A*24:02 survivin-2B Tetramer- AYACNTSTL (manufactured by MBL, product code for PE label: TS-M025-1, product code for APC label: TS-M025-2), "HLA-A * 24: 02-restricted PBF peptide As a product of "molecules multimerized by binding M136-1, APC-labeled product code: TS-M136-2).

Cells used for the preparation of such transformed cells include, for example, human leukemia-derived Jurkat mutant strains lacking the TCR β chain, J. et al. Examples include RT3-T3.5 and Sup-T1 lacking the TCRα chain. Transformed cell lines SMT3S and PD11S (see Example 7) prepared using these cells are cell lines with unlimited proliferative potential and are not infective. In addition, since it is not necessary to proliferate them by a complicated and special culture method, they can be distributed as positive control cells for HLA-A*24:02 survivin-2B tetramer reagent and HLA-A*24:02 PBF tetramer reagent, respectively. is also possible. By using SMT3S or PD11S as a positive control cell, the experimenter can check the reactivity of the tetramer reagent at any time, so the accuracy and reliability of the data obtained from clinical specimens are expected to be very high. The cell line is also effective for setting uptake conditions for flow cytometry. When distributing SMT3S and PD11S, it is possible to store and transport them using ordinary liquid nitrogen or dry ice, and Immuno-TROL (registered trademark) Cells (registered trademark), which are human whole blood control cells for flow cytometry. Beckman Coulter) can also be stored and transported under refrigerated conditions when diluted in a suitable solution. It can also be lyophilized for added convenience (US Pat. No. 5,861,311).

Another preferred embodiment of the transformed cell of the present invention is a transformed cell prepared using lymphocytes. For example, it is possible to treat cancer by collecting peripheral blood lymphocytes from humans, introducing genes into them, and returning them to the body. Therefore, the present invention also provides a pharmaceutical composition for treating survivin-2B-positive cancer or PBF-positive cancer, which contains the transformed cell of the present invention as an active ingredient.

Examples of survivin-2B-positive cancers to be treated include lung cancer, stomach cancer, colon cancer, bladder cancer, pancreatic cancer, prostate cancer, breast cancer, renal cell carcinoma, hepatocellular carcinoma, bile duct cancer, head and neck cancer, brain tumor, and the like. epithelial cancer, malignant lymphoma, osteosarcoma, synovial sarcoma, and other non-epithelial cancers, but are not limited thereto. PBF-positive cancers mainly include primary bone sarcoma and soft tissue sarcoma, but high expression of PBF is also observed in lung cancer, stomach cancer, breast cancer, liver cancer, etc. It can also be a promising immunotherapeutic target in cancer.

The cytotoxic activity of the prepared cells can be measured, for example, by IMMUNOCYTO Cytotoxicity Detection Kit (MBL) that labels target cells with CFSE (Dojindo) or by measuring LDH (lactate dehydrogenase) released from target cells. It is also possible to measure using Cytotoxicity Detection Kit (Roche). Alternatively, it can be measured by a chromium release assay that utilizes target cells labeled with ⁵¹ Cr, which is a radioactive isotope.

<Antibodies>
The present invention also provides an antibody that specifically binds to the TCRα chain protein, an antibody that specifically binds to the TCRβ chain protein, and an antibody that specifically binds to a TCR composed of these proteins.

By using these antibodies, it is possible to specifically detect the TCR of the present invention expressed on the cell surface. The antibody of the present invention can be used, for example, to assay the transformed cells and pharmaceutical composition of the present invention. It can also be used in isolation for increasing the purity of the transformed cells and pharmaceutical compositions of the present invention during the process of producing them. It can also be used to quantify the active ingredient in the peripheral blood of patients administered as a pharmaceutical composition.

Antibodies of the invention are preferably monoclonal antibodies. A typical method for producing a monoclonal antibody includes the Kohler and Milstein method (Kohler & Milstein, Nature, 256:495 (1975)). Antibody-producing cells used in the cell fusion step in this method include spleen cells, lymphocytes, and lymphocytes of animals (e.g., mice, rats, hamsters, rabbits, monkeys, and goats) immunized with the antigen TCRα chain protein or TCRβ chain protein. Node cells, peripheral blood leukocytes, and the like. It is also possible to use antibody-producing cells obtained by allowing an antigen to act on the aforementioned cells or lymphocytes previously isolated from non-immunized animals in a medium. Various known cell lines can be used as myeloma cells. Hybridomas are produced, for example, by cell fusion between spleen cells obtained from mice immunized with an antigen and mouse myeloma cells, and subsequent screening can yield hybridomas that produce monoclonal antibodies against the antigen. . Monoclonal antibodies against antigens can be obtained by culturing hybridomas or from ascitic fluid of mammals to which hybridomas have been administered.

DNA encoding the antibody is cloned from hybridomas, B cells, etc., incorporated into an appropriate vector, and introduced into host cells (e.g., mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.). , antibodies can also be produced as recombinant antibodies (eg, Antibody Production: Essential Techniques, 1997 WILEY, Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Eur. J. Biochem. 192:767-790) (195). If a transgenic animal (bovine, goat, sheep, pig, etc.) into which an antibody gene is integrated is produced using transgenic animal production technology, a large amount of monoclonal antibody derived from the antibody gene can be produced from the milk of the transgenic animal. It is also possible to obtain

The antibodies of the invention may be labeled for detection of TCR. Examples of labels that can be used include radioactive substances, fluorescent dyes, chemiluminescent substances, enzymes, coenzymes, and the like. Also, tags may be added for TCR isolation. As tags, for example, magnetic beads, biotin, avidin, etc. can be used.

<TCR Multimer>
The present invention also provides a multimerized molecule that binds the above TCR. Preparation of the molecule can be performed, for example, as follows. DNA encoding the extracellular region of TCR is incorporated into an expression vector to artificially produce recombinant proteins of TCR α and β chains, and the C-terminus of TCR α or β chain is biotinylated using an enzymatic reaction. A biotinylated TCR can be purified by column chromatography and reacted with avidin to prepare a multimerized molecule. Furthermore, as shown in Example 12 below, in order to promote heterodimerization of the TCR α chain and β chain, these proteins may be fused with a motif capable of forming a multimer (such as a leucine zipper region). Also, by labeling avidin with a fluorescent substance such as FITC, PE or APC in advance, cells expressing specific MHC/peptide complexes can be detected using a flow cytometer or fluorescence microscope.

As another preparation method, the extracellular regions of the TCR α chain and β chain can be linked using a short peptide linker and expressed as a single protein (scTCR). Then, it is possible to biotinylate the C-terminal side for multimerization in the same manner as described above, and it is also possible to incorporate it into the variable region of IgG for multimerization.

In the present invention, the number of TCRs forming a multimerized molecule is not particularly limited, but is usually 2 to 10, preferably 4 to 8, more preferably 4 or 5, especially Four is preferred.

Similar to the transformed cells of the present invention, the molecule thus prepared can be used as a companion diagnostic agent for peptide vaccine therapy, or for specific HLA on dendritic cells when performing dendritic cell vaccine therapy. It can be used as a reagent for confirming that the peptide is presented. Furthermore, it can be used as a drug delivery system (DDS) tool by combining with radioisotopes or anticancer drugs for the purpose of cancer treatment. Accordingly, the present invention provides an agent for detecting or capturing an HLA-A*24:02-restricted survivin-2B peptide or an HLA-A*24:02-restricted PBF peptide, Also provided is an agent comprising a molecule of

<Detection kit>
The present invention also provides a kit for detecting HLA-A*24:02-restricted survivin-2B peptide or HLA-A*24:02-restricted PBF peptide, comprising the following (a) provides a kit comprising at least one component of (h).
(a) TCR α chain protein of the present invention (b) TCR β chain protein of the present invention (c) TCR complex of the present invention (d) DNA encoding the protein of (a) or (b)
(e) into a transformed cell (g) into a protein (a) or (b) or a complex of (c) into which the vector (f) (d) DNA that retains the DNA of (d) in an expressible manner has been introduced; A molecule multimerized by binding a complex of specifically binding antibodies (h) and (c) where "HLA-A*24:02-restricted survivin-2B peptide" and "HLA-A*24 :02-restricted PBF peptides", as described above, when these peptides are present on the cell surface and isolated or purified molecules (e.g., multimerized TCR-restricted PBF peptides) It is meant to include both cases where it exists as a molecule). The kit of the present invention may further contain instructions for use of the kit.

<Quality control of MHC tetramer reagent>
The present invention also provides molecules multimerized by binding HLA-A*24:02-restricted survivin-2B peptides or multimers by binding PBF peptides restricted by HLA-A*24:02. A method for quality control of a transformed molecule comprising the step of confirming the reactivity of said molecule with said transformed cell of the present invention. Confirmation of reactivity in the quality control method of the present invention can be carried out, for example, by the methods described in Examples 8 to 11. As a result, when the reactivity is maintained, it can be evaluated that the quality of the molecule is maintained, and when the reactivity is decreased, the quality of the molecule is evaluated as deteriorated. can do. The maintenance of reactivity can be determined using, for example, the maintenance of the positive rate and/or the maintenance of the S/N ratio (signal noise ratio) as an index. The S/N ratio is calculated from the value obtained by dividing the MFI (mean fluorescence intensity) of the positive cell population by the MFI of the negative cell population. In addition, if any of the indices is lowered, it is possible to presume the defective point of the quality and to take measures for improvement.

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

(Example 1) Induction of HLA-A*24:02 survivin-2B peptide-specific CTL line and HLA-A*24:02 PBF peptide-specific CTL line and expansion culture The MLPC method (mixed lymphocyte) for efficiently proliferating and culturing cancer antigen-specific CTL under limiting dilution conditions was repeatedly verified and improved with reference to the reported article by Karaniks et al. (J. Immunol. 2003; 171: 4898-4904) -peptide cultures) were established. The MLPC method is a method of directly adding antigenic peptides to PBMCs and culturing them, and it is thought that memory T cells or memory/effector T cells present in the blood donor's body are stimulated to proliferate. Therefore, the risk of stimulating proliferation of naive T cells by artificial priming, which is a concern when using antigen-presenting cells prepared in vitro, is thought to be low. The present inventors demonstrated the induction of HLA-A*24:02 survivin-2B peptide-specific CTL lines and HLA-A*24:02 PBF peptide-specific CTL lines as follows. It was performed by the method used when performing immunomonitoring using a patient specimen.

CTL medium ( ¹⁰ % human AB serum/ 100 U/mL Penicillin/100 μg/mL Streptomycin/1×GlutaMAX/55 μM 2-Mercaptoethanol/25 mM HEPES/AIM-V), adjusted to a final concentration of 40 μg/mL HLA-A*24:02 survivin-2B Peptide (AYACNTSTL, MBL) or HLA-A*24:02 PBF A24.2 peptide (AYRPVSRNI, MBL) was added, stirred well, and then 1 mL was dispensed into each well of a 24-well plate. After that, it was cultured in a 5% _CO2 incubator at 37°C for 48 hours. After 48 hours, 1 mL of CTL medium containing 100 U/mL IL-2 (Shionogi & Co., Ltd.) was added to continue culturing. The CTL medium was exchanged by aspirating about half of the medium while observing the state of culture and adding 50 U/mL IL-2-added CTL medium. It was changed about once in the first week, and once every 2 to 3 days after 1 week.

10 to 14 days after the start of culture, 70 μL of cell suspension was collected from each well, and HLA-A*24:02 survivin-2B tetramer-AYACNTSTL-PE (hereinafter referred to as HLA-A*24:02 survivin-2B tetramer reagent abbreviated as MBL) or HLA-A*24:02 PBF A24.2 tetramer-AYRPVSRNI-PE (hereinafter abbreviated as HLA-A*24:02 PBF tetramer reagent, MBL). The staining procedure is as follows.

FCM buffer [2% FBS (fetal bovine serum)/0.05% NaN ₃ /PBS] was added to the collected cell suspension, and centrifuged at 400×g for 5 minutes. After removing the supernatant by aspiration, an appropriate amount of FCM buffer was added again, and the mixture was centrifuged at 400×g for 5 minutes, and the supernatant was removed by aspiration. 20 μL of FCM buffer and 10 μL of Clear Back Human FcR blocking reagent (MBL) were added and well stirred, and then allowed to stand at room temperature for 5 minutes. 10 μL of HLA-A*24:02 survivin-2B tetramer reagent or HLA-A*24:02 PBF tetramer reagent was added, gently stirred, and allowed to react in a refrigerator at 2 to 8° C. for 30 minutes. 10 μL of CD8 (clone T8)-FITC (Beckman Coulter) was added and allowed to react for 20 minutes in a refrigerator. An appropriate amount of FCM buffer was added and centrifuged at 400 xg for 5 minutes. The supernatant was carefully discarded, 400 μL of FCM buffer containing 1% dead cell staining reagent 7-AAD (Beckman Coulter) was added to suspend the cells, and cell uptake analysis was performed using a flow cytometer (FACSCalibur, BD biosciences). did. Analytical data were analyzed using CellQuest software (BD biosciences) or FlowJo (Tree Star). As a control for the MHC tetramer reagent, HLA-A*24:02 HIV env tetramer-RYLRDQQLL-PE (MBL, SEQ ID NO: 31) was used for staining to confirm non-specific staining. This reagent is synthesized using HLA-A*24:02 as the MHC and a peptide derived from the HIV (human immunodeficiency virus) envelope as the antigen peptide, and is capable of detecting and quantifying a CTL population specific to this. It is possible. Since HIV prevalence is low in Japan, it is often used as a negative control for MHC tetramer reagents. Since the frequency of CTLs is basically low, it is very important to use such a negative control MHC tetramer reagent as a control when determining the presence or absence of MHC-tetramer reagent-positive cells. As a result of this staining test, a cell population that reacted to the HLA-A*24:02 survivin-2B tetramer reagent and a cell population that reacted to the HLA-A*24:02 PBF tetramer reagent were obtained.

FIG. 1A shows the reactivity of survivin-2B CTL populations to MHC tetramer reagents. FIG. 3 is a dot plot development showing the staining intensity (log scale) of the FITC-labeled anti-CD8 antibody on the X axis and the staining intensity (log scale) of the PE-labeled MHC tetramer reagent on the Y axis. The type of MHC tetramer reagent used is indicated above each dot plot. The upper right quadrant of each dot plot shows the abundance (%) of anti-CD8 antibody-positive and MHC tetramer reagent-positive CTL in viable cells.

Anti-CD8 antibody-positive and HLA-A * 24: 02 survivin-2B tetramer reagent-specific CTL account for 3.93% of the cell population, positive for HLA-A * 24: 02 HIV env tetramer reagent Since the rate was 0.04%, most of the anti-CD8 antibody-positive and HLA-A*24:02 survivin-2B tetramer reagent-positive cell populations were considered to be specific CTL populations.

In addition, FIG. 1B shows the reactivity of the PBF CTL population to the MHC tetramer reagent as in FIG. 1A. The proportion of CTLs positive for anti-CD8 antibody and HLA-A*24:02 PBF tetramer reagent-specific in the cell population was 5.25%, and the positive rate for HLA-A*24:02 HIV env tetramer reagent was Since it was 0.10%, most of the anti-CD8 antibody-positive and HLA-A*24:02 PBF tetramer reagent-positive cell population was considered to be a specific CTL population.

(Example 2) Establishment and expansion culture of HLA-A*24:02 survivin-2B peptide-specific CTL clones and HLA-A*24:02 PBF peptide-specific CTL clones ), stimulated with PHA (phytohemagglutinin), which is often used as a mitogen, and cultured for about one month. Furthermore, peptide-pulsed cells were added to X-ray-irradiated PBMC derived from a healthy subject carrying HLA-A*24:02, and mixed culture was performed to stimulate proliferation, and the culture was continued for 2 weeks.

In addition, as other stimulation proliferation methods, lymphoblastoid cell lines (LCL) obtained by immortalizing human B cells with EB virus are used as antigen-presenting cells, anti-CD3 antibody, PHA, IL- Activated T cells that have been activated with a T cell stimulating agent such as 2 can also be used. It is important that these antigen-presenting cells express HLA-A*24:02 on the cell surface. It can be confirmed by flow cytometry using A24 antibody (MBL) or the like. Alternatively, a method of directly stimulating CTL proliferation by utilizing the CTL proliferation effect of an anti-CD137 antibody without using antigen-presenting cells can also be used (Patent No. 5433825, WO2008/023786).

Next, peptide pulse treatment was performed as follows. After washing the HLA-A*24:02-positive antigen-presenting cells once with 2% FBS/PBS, 1 mL of peptide pulse medium [0.1% HSA (human serum albumin)/55 μM 2-Mercaptoethanol/RPMI 1640]. Alternatively, suspend in AIM-V medium (Thermo Fisher Scientific), add survivin-2B peptide or PBF peptide to a final concentration of 10 μg / mL, stir well, and gently mix at intervals of about 15 minutes Incubated for 1 hour at room temperature. It is believed that this operation causes the peptide to bind to HLA molecules on antigen-presenting cells. Subsequently, an appropriate amount of peptide pulse medium was added, and after well stirring, the mixture was centrifuged at 400 xg for 5 minutes. After centrifugation, the supernatant was carefully discarded. After this washing process was carried out two more times to completely remove excess peptides, the cells were resuspended in an appropriate amount of CTL medium and counted. It is important that these antigen-presenting cells lose their proliferative ability by X-ray irradiation or mitomycin treatment, etc. X-ray irradiation may be performed at the same time as incubating the peptide and antigen-presenting cells at room temperature for 1 hour after mixing. It is possible. Mitomycin treatment is desirably performed before peptide pulse treatment. Peptide-pulsed antigen-presenting cells are used in an amount 1/10 times the number of cells in the cell population that exhibits reactivity with the HLA-A*24:02 survivin-2B tetramer reagent or the HLA-A*24:02 PBF tetramer reagent. ~equal volume was added.

FIG. 2A shows the reactivity of survivin-2B CTL clone ITG-MT3 to MHC tetramer reagents. FIG. 3 is a dot plot development showing the staining intensity (log scale) of the FITC-labeled anti-CD8 antibody on the X axis and the staining intensity (log scale) of the PE-labeled MHC tetramer reagent on the Y axis. The type of MHC tetramer reagent used is indicated above each dot plot. The upper right quadrant of each dot plot shows the abundance (%) of anti-CD8 antibody-positive and MHC tetramer reagent-positive CTL in viable cells.

Anti-CD8 antibody-positive and HLA-A * 24: 02 survivin-2B tetramer reagent-specific CTL account for 87.7% of the cell population, positive for HLA-A * 24: 02 HIV env tetramer reagent With a rate of 0.75%, cloning was considered complete for the anti-CD8 antibody-positive and HLA-A*24:02 survivin-2B tetramer reagent-positive cell population.

FIG. 2B also shows the reactivity of the PBF CTL clone FKS-D11P to the MHC tetramer reagent, as in FIG. 2A.
Anti-CD8 antibody-positive and HLA-A*24:02 PBF tetramer reagent-specific CTL accounted for 98.8% of the cell population, and the positive rate for HLA-A*24:02 HIV env tetramer reagent was Since it was 0.00%, it was considered that the cloning of the anti-CD8 antibody-positive and HLA-A*24:02 PBF tetramer reagent-positive cells was completed.

(Example 3) Confirmation of cytotoxic activity in ITG-MT3 and FKS-D11P As shown in FIG. It was found to be present in a proportion of 87.7%. In addition, as shown in FIG. 2B, it was revealed that FKS-D11P had 98.8% of cells with TCRs that reacted with the HLA-A*24:02 PBF tetramer reagent.

However, since the HLA-A*24:02 tetramer reagent is composed of a ternary complex of artificially synthesized HLA-A*24:02, β2m, and an epitope peptide, It is necessary to confirm whether the TCR can recognize each of the expressed tripartite complexes. Furthermore, whether or not CTLs having each of these TCRs can be activated by the binding of the TCR and the tripartite complex, and can exhibit cytotoxic activity against cells expressing the tripartite complex, each CTL clone This is important for speculating whether lymphocytes into which the α-chain and β-chain of the derived TCR are artificially introduced and expressed function in vivo. Therefore, the cytotoxic activity of HLA-A*24:02-restricted peptide-specific CTL cell populations (ITG-MT3 or FKS-D11P) detectable as HLA-A*24:02 tetramer reagent-positive cell populations was measured. .

The cytotoxic activity was confirmed according to a standard method using ITG-MT3 or FKS-D11P as effector cells and cultured cell C1R-A24 and HLA expression deficient cell line K562 as target cells. C1R-A24 cells are also a lymphoma cell line that expresses HLA-A*24:02 (see Oiso M, et al., Int. J. Cancer 81:387-394, 1999).

Specific experimental methods are shown below. Target cells C1R-A24 and K562 were each suspended in 1 mL of peptide pulse medium, survivin-2B peptide, PBF peptide or HIV peptide was added to a final concentration of 20 μg/mL, stirred well, and then incubated at room temperature. for 1 hour. An excess amount of peptide pulse medium was added, and after well stirring, the mixture was centrifuged at 400×g for 5 minutes. After centrifugation, the supernatant was carefully discarded. This washing process was carried out two more times in order to remove excess peptides, followed by staining with CFSE. The cells were resuspended in an appropriate amount of CTL medium and counted. 5,000 target cells were added per well of a 96-well U-bottom plate, and the amount of effector cells was added to 500-150,000 and placed in a 5% CO ₂ incubator at 37°C. cultured for 5 hours. After culturing, a calcium chloride solution was added at a concentration of 1 mM, the cells were stained with fluorescence-labeled Annexin V, and the dead cell rate of the CFSE-labeled target cells was measured by flow cytometry.

Cytotoxic activity (%) was calculated by the following formula. Cytotoxicity = [(E-T0)/(100-T0)] x 100
E indicates the Annexxin V-positive cell rate in the CFSE-positive cell population when target cells and effector cells are co-cultured, and T0 indicates the Annexxin V-positive cell rate in the CFSE-positive cell population when only target cells are cultured. .

The results are shown in Figures 3A and B. In these drawings, the X-axis shows the ratio of the number of effector cells to the number of target cells (E/Tratio), and the Y-axis shows the cytotoxic activity (%). FIG. 3A shows the results when ITG-MT3 was used as effector cells, and FIG. 3B shows the results when FKS-D11P was used as effector cells. C1R-A24 pulsed with survivin-2B peptide showed 97% cytotoxic activity at E/T ratio = 10, and C1R-A24 pulsed with PBF peptide showed 73.4% at E/T ratio = 1. cytotoxic activity was observed. On the other hand, almost no cytotoxic activity was detected in the case of HIV peptide-pulsed C1R-A24 and K562. From the above, the survivin-2B peptide-specific CTL cell population (ITG-MT3) and the PBF peptide-specific CTL cell population (FKS-D11P) are HLA-A*24 expressed on the membrane surface of target cells: It was clarified that they are cell populations capable of recognizing and killing the survivin-2B peptide or PBF peptide presented in 02. That is, the TCR expressed on the CTL membrane surface recognizes the survivin-2B peptide or PBF peptide presented by HLA-A*24:02 expressed on the membrane surface of the target cell, and recognizes the target cell. It was shown to be a TCR that has the function of transducing killing signals into cells.

(Example 4) TCR β-chain repertoire analysis of survivin-2B-specific CTL clone ITG-MT3 and PBF-specific CTL clone FKS-D11P To avoid possible insertion of mutations, we thought that we should have available information before extracting genetic information. For this purpose, a kit (IOTest beta Mark TCR Vβ Repertoire Kit) that enables repertoire analysis of 24 types of Vβ regions by flow cytometry using an antibody capable of specifically recognizing the Vβ region of the TCRβ chain , Beckman Coulter) was utilized. Antibodies specific to the TCRVβ region included in this kit are Vβ1, Vβ2, Vβ3, Vβ4, Vβ5.1, Vβ5.2, Vβ5.3, Vβ7.1, Vβ7.2, Vβ8, Vβ9, Vβ11, There are 24 types of Vβ12, Vβ13.1, Vβ13.2, Vβ13.6, Vβ14, Vβ16, Vβ17, Vβ18, Vβ20, Vβ21.3, Vβ22, and Vβ23. This kit can detect about 70% of the Vβ region of the TCRβ chain, and can discriminate information only on the Vβ region of the TCRβ chain.

According to the operating procedure of the data sheet, the results of staining analysis of the survivin-2B peptide-specific CTL clone ITG-MT3 are shown in FIG. 4A, and the results of staining analysis of the PBF peptide-specific CTL clone FKS-D11P are shown in FIG. 4B. . This kit contains 8 staining reagents, and 24 types of Vβ regions can be analyzed by staining CTL clones with 8 reagents. One reagent contains an FTIC-labeled anti-Vβ antibody, a PE-labeled anti-Vβ antibody, and an anti-Vβ antibody labeled with both PE and FITC. In the dot plot developments shown in FIGS. 4A and 4B, the X axis indicates the fluorescence intensity (log scale) of the FTIC-labeled anti-Vβ antibody, and the Y axis indicates the fluorescence intensity (log scale) of the PE-labeled anti-Vβ antibody. The lower left area of the quadrant dot plot development shows the cell population that was not stained with either antibody, the upper left area shows the cell population that was stained with PE-labeled anti-Vβ antibody, and the upper right area shows both PE and FITC. shows the cell population stained with anti-Vβ antibody labeled with , and the lower right area shows the cell population stained with FTIC-labeled anti-Vβ antibody. The subgroup name of the Vβ region specifically stained by each anti-Vβ antibody is indicated in the figure. As a result, almost all cells contained in ITG-MT3 were Vβ8, and almost all cells contained in FKS-D11P were Vβ1.

(Example 5) Survivin-2B-specific CTL clone ITG-MT3 and PBF-specific CTL clone FKS-D11P-derived TCR gene cloning -TCR gene cloning-
The present inventors comprehensively analyzed the gene information of TCRs registered in IMGT, and designed a large number of primers so that the full-length α and β chains of all TCRs can be cloned. The advantage of this method is that a full-length sequence can be obtained with a single round of PCR. This method eliminates the need to design primers according to DNA sequence information obtained from PCR products, thus minimizing the possibility of containing mutations. However, the number of primer combinations is enormous, and the number of PCRs that can be verified using a small amount of cDNA is limited. Therefore, PCR was performed using a primer mix obtained by mixing 10 to 11 types of primers that were confirmed not to mutually inhibit the PCR reaction, and PCR was performed using all combinations of primer mixes from which gene products were obtained. , was devised to obtain the full-length sequence. The combination of the primer mix and the setting of the PCR reaction temperature conditions used cDNA prepared from each of Jurkat cells, untreated human PBMC, and T cells expressing a specific Vβ chain, for which a TCR repertoire has already been reported. went. T cells expressing specific Vβ chains were prepared by separating and concentrating human PBMCs using Vβ chain antibodies with an automated magnetic separator.

Total RNA was recovered from the aforementioned ITG-MT3 and FKS-D11P using RNeasy Protect Mini Kit (QIAGEN). Subsequently, cDNA was prepared using Oligo(dT)20 primer according to the manual of SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). In order to obtain the full-length sequence of the TCRα chain, 10 to 11 kinds of comprehensively designed forward primers specific to each TCRα chain were mixed, and 4 kinds of primer mixes and 1 kind of reverse primer designed for the Cα region of the TCRα chain were used. PCR was performed using the cDNA obtained using the primers as a template. As a result, a PCR product was obtained from the ITG-MT3-derived cDNA with three types of primer mix, and a PCR product was obtained with one type of primer mix from the FKS-D11P-derived cDNA. Using individual forward primers contained in each primer mix and one type of reverse primer designed for the Cα region, PCR was performed using the resulting cDNA as a template. The results of developing the amplified PCR product by 1% agarose gel electrophoresis are shown in FIGS. 5A and 5B.

The α chain of ITG-MT3 was subjected to PCR using a total of 32 sets of primers. As a result, as shown in FIG. 5A, amplification of PCR products was confirmed with primers TRAV4, TRAV9-2, and TRAV13-2. Further, as shown in FIG. 5B, the α chain of FKS-D11P was subjected to PCR using a total of 12 sets of primers, and as a result, amplification of PCR products was confirmed with primers TRAV1-1, TRAV1-2, and TRAV8-2.

Next, these PCR products were excised from the agarose gel, purified using MinElute Gel Extraction Kit (QIAGEN), and the gene fragment was inserted into pCR2.1-TOPO using TOPO TA Cloning Kit (Thermo Fisher Scientific). After that, the DNA sequence was analyzed according to the standard method. The deciphered sequence information was subjected to repertoire analysis using the IMGT database.

As a result, two types of ITG-MT3-derived TCRα chains were identified, and the configuration of Vα-Jα was TRAV4/TRAJ27/TRAC (hereinafter referred to as "A4") and TRAV13-2/TRAJ24/TRAC (hereinafter referred to as " A13-2”) (refer to FIG. 14A for “A4”). In the FKS-D11P-derived TCRα chain, the PCR products of TRAV1-1 and TRAV1-2 have the same DNA sequence, and the configuration of Vα-Jα is TRAV1-2/TRAJ42/TRAC (hereinafter referred to as "A1-2"). (see FIG. 14B). The sequence of the PCR product of TRAV8-2 is considered to be a pseudogene because a stop codon was included in the middle of the amino acid substitution.

Next, the TCRβ chain of ITG-MT3 and FKS-D11P was analyzed. The TCRβ chain of ITG-MT3 was Vβ8 from the results of FIG. 4A, so the full-length sequence of the TCRβ chain corresponds to TRBV12-3 or TRBV12-4. Signal sequence-specific forward primers of TRBV12-3 and TRBV12-4 registered in this IMGT were designed.

The TCRβ chain of FKS-D11P was Vβ1 from the results in FIG. 4B, so the full-length sequence of the TCRβ chain corresponds to TRBV9. Therefore, a forward primer specific for the signal sequence of TRBV9 registered in IMGT was designed. PCR was performed using these forward primers and two types of reverse primers (BC1 and BC2) specific to the TCR Cβ region as templates. The results of developing the amplified PCR product by 1% agarose gel electrophoresis are shown in FIGS. 5A and 5B.

As shown in FIG. 5A, PCR products were obtained for both BC1 and BC2 for the TCR β chain of ITG-MT3. In addition, as shown in FIG. 5B, a PCR product was obtained with BC1 for the TCRβ chain of FKS-D11P. All of the PCR products were excised from the agarose gel, purified using MinElute Gel Extraction Kit (QIAGEN), and the gene fragment was inserted into pCR2.1-TOPO using TOPO TA Cloning Kit (Thermo Fisher Scientific). DNA sequences were analyzed according to standard methods. As a result, in the TCRβ chain of ITG-MT3, the four types of PCR products, TRBV12-3/BC1, TRBV12-3/BC2, TRBV12-4/BC1, and TRBV12-4/BC2, all had the same DNA sequence. . As a result of repertoire analysis using the IMGT database, one type of TCRβ chain derived from ITG-MT3 was identified, and the configuration of Vβ/Dβ/Jβ/Cβ was TRBV12-4/TRBD2/TRBJ2-1/TRBC2 (hereinafter , denoted as “B12-4”) (see FIG. 15A). In addition, one type of TCRβ chain of FKS-D11P was identified, and the configuration of Vβ/Dβ/Jβ/Cβ was TRBV9/TRBD1/TRBJ1-1/TRBC1 (hereinafter referred to as “B9”) (FIG. 15B see).

(Example 6) Expression in cultured cells and confirmation of combination of TCR α chain and β chain Correct combination of two TCR α chains (A4 and A13-2) derived from ITG-MT3 and one β chain (B12-4) In order to elucidate , the cDNA was subcloned into mammalian cell expression vectors pcDNA3.1 (Thermo Fisher Scientific) and pEF6/Myc-His (Thermo Fisher Scientific). As a control, an expression vector was similarly constructed using the HLA-A*24:02 WT1-specific TCR α-chain and β-chain cDNAs previously identified by the present inventors (JP 2014-143982, Biomed Res. 2013;34:41-50).

The cultured cell line used for the gene transfer was a Jurkat mutant strain derived from human leukemia, lacking the TCRβ chain. RT3-T3.5, Jurkat/MA, and Sup-T1 lacking the TCRα chain were used. It was confirmed by flow cytometry using an anti-TCR pan α/β antibody (Beckman Coulter) that these three cell lines did not express TCR. Gene transfer was performed by electroporation using Neon (registered trademark) Transfection System (Thermo Fisher Scientific). After static culture for 3 days, a portion of the cell population was collected, stained with HLA-A*24:02 survivin-2B tetramer reagent or HLA-A*24:02 PBF tetramer reagent, and flow cytometered. analyzed.

The results are shown in FIG. The X-axis shows the staining intensity (log scale) of the FITC-labeled anti-CD8 antibody, and the Y-axis shows the staining intensity (log scale) of the PE-labeled MHC tetramer reagent. Cell population transfected with pcDNA3.1 and pEF6/Myc-His (control cells) and cell population transfected with pcDNA3.1-A13-2 and pEF6/Myc-His-B12-4 survivin-2B TCR A13- In 2/B12-4, no HLA-A*24:02 survivin-2B tetramer reagent-positive cell population could be confirmed. On the other hand, the cell population survivin-2B TCR A4/B12-4 transfected with pcDNA3.1-A4 and pEF6/Myc-His-B12-4 has an HLA-A*24:02 survivin-2B tetramer reagent of 24.0 % positive cells were confirmed. In the cell population PBF TCR A1-2/B9 transfected with pcDNA3.1-A1-2 and pEF6/Myc-His-B9, 16.4% positive cells were confirmed with HLA-A*24:02 PBF tetramer reagent. was done.

From the above, the TCR that specifically binds to the HLA-A*24:02 survivin-2B tetramer reagent has a TCRα chain of TRAV4/TRAJ27/TRAC and a TCRβ chain of TRBV12-4/TRBD2/TRBJ2-1/TRBC2. One thing became clear. The TCR that specifically binds to the HLA-A*24:02 PBF tetramer reagent has a TCRα chain of TRAV1-2/TRAJ42/TRAC and a TCRβ chain of TRBV9/TRBD1/TRBJ1-1/TRBC1. became. Schematic representations of the identified TCRs are shown in Figures 7A and B.

(Example 7) Establishment of TCR gene-expressing transformed cells (SMT3S, PD11S) In cell populations survivin-2B TCR A4/B12-4 and PBF TCR A1-2/B9 in which positive images were obtained, resistant drug of each vector was added. G418 (Roche) and blastisidin (Thermo Fisher Scientific) were added to establish drug-resistant TCR gene-expressing transformed cell lines SMT3S and PD11S, respectively. FIG. 8A shows the results of examining the reactivity of SMT3S to MHC tetramer reagents. FIG. 8B shows the results of examining the reactivity of PD11S to MHC tetramer reagents. In these figures, the X-axis shows the staining intensity (log scale) of the FITC-labeled anti-CD8 antibody, and the Y-axis shows the staining intensity (log scale) of the PE-labeled MHC tetramer reagent. The type of MHC tetramer reagent used is indicated above each dot plot. The abundance (%) of anti-CD8 antibody-positive and MHC tetramer reagent-positive cells present in the upper right quadrant of each dot plot is shown.

In SMT3S, 89.6% of the cell population reacted with HLA-A*24:02 survivin-2B tetramer reagent, the mean fluorescence intensity (MFI) was 720, and the coefficient of variation (CV value) was 61.2. . As for PD11S, 95.8% of the cell population reacted to the HLA-A*24:02 PBF tetramer reagent, with an MFI of 379 and a CV value of 42.8. On the other hand, no specific staining was observed with HLA-A*24:02 HIV env tetramer-RYLRDQQLL-PE used as a control for the MHC tetramer reagent.

From the above, it was clarified that the TCR genes introduced into SMT3S and PD11S are translated as TCR proteins having the ability to bind to tetramer reagents, respectively, and are functionally expressed on the respective cell membrane surfaces. .

(Example 8) Evaluation of HLA-A*24:02 tetramer reagent using SMT3S and PD11S (addition recovery test)
Addition and recovery for the purpose of confirming the accuracy of the evaluation of the HLA-A*24:02 survivin-2B tetramer reagent using SMT3S cells and the accuracy of the evaluation of the HLA-A*24:02 PBF tetramer reagent using PD11S cells did the test. The experimental method is to mix SMT3S or PD11S with parental cells into which no TCR gene has been introduced, stain with HLA-A*24:02 tetramer reagent, and measure the positive rate and the positive rate expected from the mixed rate. compared. 20 μL of cell suspension was collected from each of the parent cell lines of SMT3S, PD11S and these transformed cells in culture, and 20 μL of Trypan Blue Stain 0.4% (Thermo Fisher Scientific) was added to the cell suspension, and the cells were viable with a hemocytometer. Cell number was counted. Next, based on the measured number of living cells, SMT3S or PD11S and the parent cell line were mixed, and the abundance ratio of SMT3S or PD11S was 100%, 50%, 25%, 12.5%, 6.3%, 3 .1%, 1.6%, 0.8%, 0.4%, 0% cell populations were prepared. 5×10 ⁵ cells from each cell population were collected in an Eppendorf tube, centrifuged at 400×g for 5 minutes, and the supernatant was carefully discarded. 1 mL of FCM buffer was added, resuspended, centrifuged at 400×g for 5 minutes, and the supernatant was carefully discarded. After adding 20 μL of FCM buffer and 10 μL of Clear Back Human FcR blocking reagent and stirring well, the mixture was allowed to react at room temperature for 5 minutes. 10 μL of HLA-A*24:02 survivin-2B tetramer reagent or HLA-A*24:02 PBF tetramer reagent was added and stirred gently, followed by reaction at 4° C. for 30 minutes. 10 μL of CD8 (clone T8)-FITC was added and reacted at 4° C. for 20 minutes. An appropriate amount of FCM buffer was added and centrifuged at 400 xg for 5 minutes. The supernatant was carefully discarded, 400 μL of FCM buffer containing 1% 7-AAD was added to suspend the cells, and the cells were taken up and analyzed by a flow cytometer. In the analysis, the region selected in the FSC-H / SSC-H dot plot development is R1, and the viable cell region in the FSC-H / 7-AAD dot plot development (i.e., 7-AAD negative cell population) as R2, performed in the "R1 and R2" cell population. The results obtained are shown in FIGS. 9A and B. FIG. In these figures, the X-axis shows the staining intensity (log scale) of the FITC-labeled anti-CD8 antibody, and the Y-axis shows the staining intensity (log scale) of the PE-labeled MHC tetramer reagent. FIG. 9A is the result of staining with HLA-A*24:02 survivin-2B tetramer reagent. The positive rate is shown in the upper right corner of the dot plot developed diagram for each mixing ratio of SMT3S divided into quarters. Since the positive rate was 91.0% when SMT3S was 100%, the positive rate expected from the mixture rate is shown in the upper right parenthesis dividing the dot plot development diagram into four parts. FIG. 9B is the result of staining with HLA-A*24:02 PBF tetramer reagent. Since the positive rate was 92.8% when PD11S was 100%, the positive rate expected from the mixture rate is shown in the upper right parenthesis dividing the dot plot development diagram into four parts. From the results of these addition and recovery tests, it was clarified that the mixed ratio of SMT3S and PD11S was accurately detected as a positive ratio by the HLA-A*24:02 tetramer reagent.

(Example 9) Evaluation of HLA-A*24:02 tetramer reagent using SMT3S and PD11S (verification of concentration-dependent stainability and confirmation of storage stability)
Positive control cells are essential to confirm the storage stability of MHC tetramer reagents used in flow cytometers. It is important that the positive control cells always show the same reactivity to the reagent. A stable transformed cell into which a TCR gene to which a tetramer reagent specifically binds, such as SMT3S or PD11S, is introduced can be said to be an ideal positive control cell. Therefore, the present inventors used SMT3S and PD11S to verify the method of carrying out the storage stability test of the survivin-2B tetramer reagent and the PBF tetramer reagent. The storage stability of the MHC tetramer reagent can be evaluated as the period during which the expected positive rate is maintained by flow cytometry. Adjust the positive rate of SMT3S and PD11S to 5 to 15% respectively, periodically confirm the reactivity by dilution series of the reagent, and analyze the HLA-A*24:02 tetramer reagent positive rate, MFI, and CV value. Verification of storage stability was performed.

FIG. 10A shows test data for the HLA-A*24:02 survivin-2B tetramer reagent using a cell population in which the abundance ratio of SMT3S cells was adjusted to about 10% as an example of the storage stability test. The HLA-A*24:02 survivin-2B tetramer reagent was used so that the concentration in the reaction solution was 10, 5, 2.5, 1.25, 0.625, and 0 μg/mL in terms of the purified biotinylated monomer concentration. adjusted to In FIG. 10A(a), the X axis indicates the fluorescence intensity (log scale) of the FITC-labeled CD8 antibody, and the Y axis indicates the fluorescence intensity (log scale) of the HLA-A*24:02 survivin-2B tetramer reagent. The positive rate and MFI of the MHC tetramer reagent-positive and CD8-positive cell population are shown in the upper right quadrant of each dot plot development. FIG. 10A(b) shows a graph showing the relationship between MFI and reagent concentration, and FIG. 10A(c) shows a graph showing the relationship between CV value and reagent concentration. From this result, the HLA-A*24:02 survivin-2B tetramer reagent can detect positive cells even at a concentration of 0.625 μg / mL, but the MFI gradually decreases at concentrations of 2.5 μg / mL or less as the detection sensitivity. The decreased and increased CV values suggested that 2.5 μg/mL could be set at the borderline of acceptable detection sensitivity.

FIG. 10B shows test data of the HLA-A*24:02 PBF tetramer reagent using a cell population in which the proportion of PD11S cells was adjusted to about 14% as an example of the storage stability test. The HLA-A*24:02 PBF tetramer reagent was adjusted so that the concentration in the reaction solution was 10, 5, 2.5, 1.25, 0.625, and 0 μg/mL in terms of purified biotinylated monomer concentration. did. In FIG. 10B(a), the X axis indicates the fluorescence intensity (log scale) of the FITC-labeled CD8 antibody, and the Y axis indicates the fluorescence intensity (log scale) of the HLA-A*24:02 PBF tetramer reagent. The positive rate and MFI of the MHC tetramer reagent-positive and CD8-positive cell population are shown in the upper right quadrant of each dot plot development. FIG. 10B(b) shows a graph showing the relationship between MFI and reagent concentration, and FIG. 10B(c) shows a graph showing the relationship between CV value and reagent concentration. From this result, the HLA-A * 24:02 PBF tetramer reagent can detect positive cells even at a concentration of 0.625 μg / mL, but the detection sensitivity is lower than the concentration of 1.25 μg / mL MFI and CV Values also gradually increased, suggesting that 1.25 μg/mL can be set as a borderline acceptable range for detection sensitivity.

From the above, by verifying the reactivity over time using stably transformed cells into which a TCR gene to which the tetramer reagent specifically binds, such as SMT3S and PD11S, the storage stability of the MHC tetramer reagent was confirmed. It was shown that it is possible to conduct a sex test.

(Example 10) Expression of TCR genes (survivin-2B TCR A4/B12-4, PBF TCR A1-2/B9) and study on CD8 dependence of tetramer reagent RT3-T3.5 is a cell line that does not express the CD8 molecule that assists MHC-TCR binding. If a TCR gene is introduced into this cell line and reactivity is confirmed with a tetramer reagent, it can be proved that the TCR does not require the assistance of CD8 molecules when binding to MHC molecules. Obtaining a TCR that is less affected by CD8 molecules upon binding to MHC molecules is very important for making the obtained TCR gene into a tool such as a TCR tetramer or a TCR multimer. Regarding the TCR gene sequences survivin-2B TCR A4/B12-4 and PBF TCR A1-2/B9, J. et al. These TCR genes were introduced into RT3-T3.5 and their expression was confirmed. As a result, as shown in FIGS. 11A and B, both survivin-2B TCR A4/B12-4 and PBF TCR A1-2/B9 were confirmed to react with each tetramer reagent.

As described above, the CD8 molecule is a very important molecule in the interaction between the tetramer reagent and TCR, and it is a well-known fact that the reactivity of the tetramer reagent is affected by the type of clone from which the CD8 antibody is derived ( Mathew C. et al., J. Immunol. Jul 2011;187:654-663). It is also difficult to predict in advance whether the obtained TCR will function in a CD8-independent manner (see TV Moore et al., Cancer Immunol Immunother, May 2009;58(5):719-28). . Therefore, CD8 antibody clone T8 (Beckman
Coulter) and CD8 antibody clone RFT-8 (SouthernBiotech), which is known to act as an inhibitor, were used to confirm whether there was a difference in the tetramer staining of the TCR obtained this time. did. The results are shown in FIG. 11C. For staining, the cell populations established this time, each having an abundance ratio of SMT3S and PD11S adjusted to about 10%, were used.

As a control, the WT1 TCR gene expression transformed cell line SK37 was used (see Watanabe K, et al., Biomed Res. 34(1):41-50, 2013). The WT1 TCR expressed in this cell line has been shown to require the assistance of CD8 molecules in its reaction with the corresponding tetramer reagent. In addition, FIG. 11D shows the ratio of the tetramer positive rate when clone RFT-8 is used when the tetramer positive rate when clone T8 is used is taken as 100%.

As shown in FIG. 11C, in SMT3S and PD11S, reaction with each tetramer reagent was confirmed when using either clone T8 or clone RFT-8. In SK37, reactivity with the tetramer reagent was confirmed when clone T8 was used, but reactivity with the tetramer reagent was not observed when clone RFT-8 was used.

In addition, as is clear from the results shown in FIG. 11D, in SMT3S and PD11S, 92.3% and 90.8%, respectively, even when RFT-8 was used, and more than 90% of tetramer-positive cells when T8 was used. detected, but only 5.1% was detected in SK37.

These findings reveal that survivin-2B TCR A4/B12-4 and PBF TCR A1-2/B9 do not require CD8 assistance when binding to HLA-A*24:02 molecules. , also demonstrated that these multimerized TCRs function as useful tools in the detection of MHC/peptide complexes.

(Example 11) Detection of cancer cells or antigen-presenting cells using TCR α and β chains 1
It is very difficult to determine whether the target peptide fragment is presented on HLA, and research on anti-HLA/peptide complex antibodies, TCR multimers, etc. is being actively conducted. Clinical trials of more than 200 types of cancer peptide vaccine therapy are currently underway around the world. Detection of cancer antigen proteins by tissue staining using antibodies is used as a main analysis method. However, CTLs that specifically recognize cancer do not kill cancer cells by recognizing the cancer antigen protein itself, but the cancer antigen protein is degraded in cancer cells, and its peptide fragment binds to HLA and binds to the cell membrane surface. Recognizes and kills cancer cells when presented above. That is, it recognizes the state in which HLA and peptide form a complex on the cancer cell membrane surface. A reagent that can detect the state in which HLA and a peptide form a complex can be an essential analytical tool for selecting an appropriate cancer antigen for cancer immunotherapy. The inventors investigated whether the TCR gene identified by the present invention could be used as such an analysis tool.

Jurkat/MA cells, a Jurkat cell substrain, are transfected with a vector in which a reporter gene is integrated downstream of the transcription factor NF-AT that is activated by TCR signals. (Int J Cancer, 2002;99(1):7-13). In this example, a gene-introduced cell line stably expressing the specific TCR shown in Example 6 was established in Jurkat/MA. It was verified whether it is possible to detect that the target peptide is presented on the surface HLA.

First, the survivin-2B TCR gene (A4/B12-4) or the PBF TCR gene (A1-2/B9) was transfected into Jurkat/MA cells in the same manner as in Example 7 to form stable transformants, SMT3J and PD11J was established respectively. The results of confirming the specificity of the established cell lines are shown in FIGS. 12A and 12B. In these figures, the X-axis shows the staining intensity (log scale) of the FITC-labeled anti-CD8 antibody, and the Y-axis shows the staining intensity (log scale) of the PE-labeled MHC tetramer reagent. The type of MHC tetramer reagent used is indicated above each dot plot. The percentage of existing anti-CD8 antibody-positive and MHC tetramer reagent-positive cells in living cells (%) is shown in the upper right quadrant of each dot plot.

In SMT3J, 89.2% of the cell population reacted to the HLA-A*24:02 survivin-2B tetramer reagent, with an MFI of 266 and a CV value of 65.1. As for PD11S, 93.2% of the cell population reacted to the HLA-A*24:02 PBF tetramer reagent, with an MFI of 756 and a CV value of 37.2. On the other hand, no specific staining was observed with HLA-A*24:02 HIV env tetramer-RYLRDQQLL-PE used as a control for the MHC tetramer reagent. This result shows that SMT3J binds to the artificially produced complex of HLA-A*24:02, survivin-2B peptide (AYACNTSTL) and β2m, and HLA-A*24:02, HIV env peptide (RYLRDQQLL) and β2m It shows that it does not bind to the complex of Similarly, PD11J binds to a complex of HLA-A*24:02, PBF peptide (AYRPVSRNI), and β2m that is artificially prepared, and a complex of HLA-A*24:02, HIV env peptide (RYLRDQQLL), and β2m. indicates that it does not bind to

Next, it was verified whether or not it also reacts with a complex of HLA-A*24:02, β2m, surivin-2B peptide or PBF peptide, which are actually expressed on the cell membrane surface. That is, using SMT3J and PD11J established as described above, signal transduction via NF-AT existing downstream from TCR was detected using the activity of luciferase as an index. Experiments were performed as follows. As target cells, K562 or C1R-A24 pulsed with various peptides were used. K562 was pulsed with survivin-2B peptide or PBF peptide at a concentration of 10 μg/mL, and HIV peptide was pulsed with C1R-A24 at a concentration of 10 μg/mL. C1R-A24 cells were pulsed with survivin-2B peptide or PBF peptide at concentrations of 0.1, 1, 10 and 100 μg/mL. 1×10 ⁵ target cells and the same amount of SMT3J cells or PD11J cells were mixed in 200 μL of medium in a U-bottom 96-well plate and incubated in a 37° C. 5% CO ₂ incubator for 24 hours. After culturing, the U-bottomed 96-well plate was centrifuged at 400×g for 5 minutes, the supernatant was discarded, the cells were washed twice with 200 μL of PBS, suspended in Cell Culture Lysis Reagent (Promega), and frozen once. melting was performed. The supernatant was recovered by centrifugation, and luciferase activity was measured using Luciferase Assay Reagent (Promega). The results are shown in Figures 13A and B. In these figures, target cell type and pulsed peptide concentration are plotted on the X-axis, and luciferase activity is plotted on the Y-axis.

As shown in FIG. 13A, SMT3J cells recognized survivin-2B peptide-pulsed C1R-A24 cells, and activity increased in a peptide concentration-dependent manner. In addition, as shown in FIG. 13B, it was revealed that PD11J cells recognize PBF peptide-pulsed C1R-A24 cells, and activity increases in a peptide concentration-dependent manner.

The above results show that even if the TCR identified in the present invention is used and the same analysis is performed on a patient-derived biopsy sample, the epitope is found in HLA-A*24:02 of the cell population contained in the sample. It means that it can be determined whether or not it is presented.

(Example 12) Preparation of TCR multimers and detection of cancer cells or antigen-presenting cells using TCR α and β chains 2
Example 11 describes a method for detecting antigen-presenting cells or cancer cells expressing a target HLA-peptide complex using a stably transformed cell line expressing a cancer antigen-specific TCR gene. Indicated. However, in the method using a stably transformed cell line expressing a specific TCR gene, it is always necessary to maintain the culture of the stably transformed cell line under good conditions. In addition, in stably transformed cell lines, repeated passages may cause unpredictable phenomena such as loss of genes, and it is not always possible to guarantee that stable experimental systems can be carried out at multiple facilities. Therefore, it is desirable to be able to substitute an artificially synthesized reagent such as an MHC tetramer reagent that reacts with a specific TCR.

Multimerization of the MHC tetramer reagent made it possible to detect binding to TCR. In addition, it is considered that the TCR as a monomer also has a low ability to bind to the complex of MHC (HLA) and peptide. Therefore, the present inventors artificially produced a TCR protein using a TCR gene in the same manner as in the production of an MHC tetramer reagent, and used this protein to study the conversion of a TCR multimer into a reagent. That is, the purpose of this example is to prepare soluble TCR multimers (TCR multimers) based on the TCR sequences obtained as described above, and to prepare HLA and peptides presented on the cell surface by the TCR multimers. It is to verify whether the complex of is detectable.

(1) Preparation of expression constructs encoding soluble TCRs As shown in FIG. 16, first, the leucine zipper region of Fos and the A nucleotide sequence encoding a 6×His-tagged polypeptide was designed. Nucleotides encoding a polypeptide in which a Jun leucine zipper region and a biotin binding site (BirA recognition site) are added via a linker to the C-terminal side of the extracellular region of the ITG-MT3 TCR β chain or FKS-D11P TCR β chain. I also designed an array.

In addition, Chemistry and Biology Vol. 27, No. 8 “Leucine Zipper: A New Structure Unique to Transcription Regulator”, TCR α chain to which these tags are added by utilizing the fact that the leucine zipper regions of Fos and Jun bind to each other and β chain in cultured cells, it is possible to dimerize them to produce a soluble TCR.

Next, based on the nucleotide sequence designed above, cDNA was chemically synthesized at Integrated DNA Technologies. Then, the above cDNAs were subcloned into mammalian cell expression vectors pXC17.4 and pXC18.4 (Lonza), respectively, to obtain pXC17.4-survivin-2B TCRα and pXC18.4-survivin-2B TCRβ. Subsequently, the survivin-2B TCRβ gene fragment obtained by cleaving pXC18.4-survivin-2B TCRβ with restriction enzymes Not I and Sal I is incorporated into pXC17.4-survivin-2B TCRα cleaved with the same restriction enzymes. generated a double gene vector of pXC-survivin-2B TCRαβ. A pXC-PBF TCRαβ double gene vector was constructed in the same manner.

(2) Preparation of TCR multimer Next, the expression vector was introduced into a cultured cell line in order to express the soluble TCR designed as described above. The cultured cell line used for gene transfer was Chinese Hamster CHO-K1SV GSKO (Lonza). CHO-K1SV GSKO is a deletion mutant strain of glutamine synthase (GS) in CHO-K1SV cells, and by using it in combination with the above expression vector (pXC17.4) encoding GS, the synthase system can be used as an index. (GS Gene Expression System (registered trademark)). Gene transfer was performed by electroporation using Neon Transfection System (Thermo Fisher Scientific). After gene transfer was performed in this manner, cells expressing soluble TCR were monocloned by limiting dilution to obtain stable transformed cell lines that highly express soluble TCR. The expression of soluble TCR was confirmed by ELISA using an anti-His-tag antibody.

The TCR secreted from the cultured cells into the culture supernatant was purified using Ni Sepharose Excel (GE Healthcare) using the 6×His-tag added to the α-chain of the soluble TCR. Next, biotin was added to the biotin-binding site added to the C-terminal side of the β chain of soluble TCR using BirA enzyme. The biotinylated soluble TCR was purified by column chromatography, and the dye-labeled streptavidin and biotinylated soluble TCR were mixed at a molar ratio of 1:4 to obtain a TCR multimer.

(Example 13) Evaluation of TCR multimer To evaluate the stainability of the TCR multimer obtained as described above, cells forming a complex between HLA-A*24:02 and survivin-2B peptide were first prepared. gone. As antigen-presenting cells, the above-described C1R-A24 expressing HLA-A*24:02 was used, and the cells were mixed with survivin-2B peptide at a final concentration of 10 μg / mL, or with HIV peptide at the same concentration as a negative control. Peptides were pulsed to HLA-A*24:02 by mixing and culturing in a 37° C. 5% CO ₂ incubator for 24 hours.

The TCR multimer prepared as described above was added to an appropriate amount of peptide-pulsed C1R-A24 cells so as to have a final concentration of 20 μg/mL in terms of soluble TCR monomer concentration, and mixed gently. After standing at 4°C for 30 minutes, 2 µL of HLA-A24-FITC antibody (MBL) was added and left at 4°C for 30 minutes. After adding 1.5 mL of FCM buffer and stirring, the cells were centrifuged at 3,000 rpm for 5 minutes, the supernatant was aspirated and discarded, and the cells were resuspended in 400 μL of FCM buffer and analyzed with a flow cytometer within 24 hours. The results are shown in Figures 17A and B.

In addition, FIG. 17A is represented by a histogram diagram in which the X-axis indicates the fluorescence for the TCR multimer in log scale and the Y-axis indicates the number of cells. , gray histograms show HIV peptide-pulsed cells, black histograms show survivin-2B peptide-pulsed cells. FIG. 17B is a bar graph showing the MFI of each cell population, the MFIs in DMSO-only, HIV peptide-pulsed and survivin-2B peptide-pulsed cells were 0.76, 0.68 and 0.68, respectively. was 2.02.

In addition, FIGS. 18A and 18B show the results of analyzing the concentration dependence of TCR multimers by the same method as described above. The final concentration of the TCR multimer added to the appropriate amount of peptide-pulsed C1R-A24 cells was 0.3 μg/mL, 1.2 μg/mL, 5 μg/mL, in terms of the soluble TCR monomer concentration. These five conditions were examined at 20 μg/mL and 80 μg/mL. FIG. 18A is a histogram plot with log scale fluorescence for TCR multimers on the X-axis and cell number on the Y-axis, gray histograms for HIV peptide-pulsed cells, black histograms for survivin- Cells pulsed with 2B peptide are shown. In addition, the MFI of each cell population was calculated, and the signal in the survivin-2B peptide-pulsed cell population and the noise in the HIV peptide-pulsed cell population were converted to the soluble TCR monomer concentration of the TCR multimer. The signal/noise ratio (S/N ratio) at the final concentration was calculated. The results obtained are shown in FIG. 18B. In FIG. 18B, the X-axis represents the final concentration of the TCR multimer (concentration in terms of soluble TCR monomer), and the Y-axis represents the S/N ratio. Final concentrations of TCR multimers: S/N ratios at 0.3 μg/mL, 1.2 μg/mL, 5 μg/mL, 20 μg/mL and 80 μg/mL respectively are 1.042, 1.464, 2.267, 3 .533 and 5.549.

As shown in Figures 17A, 17B, 18A and 18B, an increase in MFI was observed only when the survivin-2B peptide was pulsed (see especially Figure 17B), with an increase in S/N ratio dependent on the final concentration of TCR multimers. (See in particular FIG. 18B). Thus, the HLA-A*24:02-survivin-2B-specific TCR multimers produced in Example 12 were specific for the complex of HLA-A*24:02 and the survivin-2B peptide presented thereby. It was found that they reacted positively.

(Example 14) Evaluation of HLA-A*24:02-PBF-specific TCR multimers To evaluate the reactivity of HLA-A*24:02-PBF-specific TCR multimers, HLA-A*24:02 Preparations of cells that form complexes with peptides were performed. As antigen-presenting cells, C1R-A24 expressing HLA-A*24:02 was used, and the cells were treated with PBF peptide at final concentrations of 0 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL. HLA-A*24:02 was pulsed with peptides by mixing with and culturing in a 37° C. 5% CO ₂ incubator for 24 hours.

To an appropriate amount of peptide-pulsed C1R-A24 cells, TCR multimer was added to a final concentration of 20 μg/mL in terms of soluble TCR monomer concentration, gently mixed, and allowed to stand at 4° C. for 30 minutes. did. After adding 1.5 mL of FCM buffer and stirring, the cells were centrifuged at 3,000 rpm for 5 minutes, the supernatant was aspirated and discarded, and the cells were resuspended in 400 μL of FCM buffer and analyzed with a flow cytometer within 24 hours. The results obtained are shown in FIG. 19A. In addition, FIG. 19A is represented by a histogram diagram in which the X-axis indicates the fluorescence against the TCR multimer in log scale and the Y-axis indicates the number of cells, and the numerical values in the figure indicate the MFI. In addition, in order to analyze the concentration dependence of HLA-A*24:02-PBF-specific TCR multimers, soluble TCR multimers were added to an appropriate amount of peptide-pulsed C1R-A24 cells at a final concentration of 10 μg/mL. Converted to the TCR monomer concentration, the final concentrations were added to 0 μg/mL, 2 μg/mL, 6 μg/mL and 20 μg/mL, gently mixed, and allowed to stand at 4° C. for 30 minutes. After adding 1.5 mL of FCM buffer and stirring, the cells were centrifuged at 3,000 rpm for 5 minutes, the supernatant was aspirated and discarded, and the cells were resuspended in 400 μL of FCM buffer and analyzed with a flow cytometer within 24 hours. The results obtained are shown in FIG. 19B. In addition, FIG. 19B is represented by a histogram diagram in which the X-axis indicates the fluorescence against the TCR multimer in log scale and the Y-axis indicates the number of cells, and the numerical values in the figure indicate the MFI.

As shown in FIGS. 19A and 19B, an increase in MFI was observed only when the PBF peptide was pulsed, and the MFI increased in a concentration-dependent manner of the pulsed PBF peptide (see FIG. 19A in particular), indicating that the final concentration of the TCR multimer A dependent increase in MFI was observed (see FIG. 19B in particular). Therefore, the HLA-A*24:02-PBF-specific TCR multimer prepared in Example 12 specifically reacted with the complex of HLA-A*24:02 and the PBF peptide presented thereby. It was confirmed that

(Example 15) Blocking assay with anti-HLA-A24 antibody C1R-A24 cells are a lymphoma cell line expressing HLA-A*24:02, but also HLA-A*02:01 *35:03, HLA-C*04:01. Therefore, when an anti-HLA-A24 antibody is bound to a complex of HLA-A24 and an epitope peptide for the purpose of confirming the specificity for HLA-A*24:02, a change occurs in the reactivity of the TCR multimer. It was confirmed.

Using C1R-A24 as antigen-presenting cells, mixing the cells with survivin-2B peptide or PBF peptide at a final concentration of 10 μg/mL, and culturing in a 5% CO ₂ incubator at 37° C. for 24 hours, HLA-A * Peptides were pulsed to 24:02. Anti-HLA-A24 antibody clone C7709A2 (Immunity, 1997; 6(2): 199- 208) or as a negative control, an isotype control antibody was added at a concentration of 0.1 mg/mL, mixed gently, and blocked at 4°C for 1 hour. After adding 1.5 mL of FCM buffer and stirring, the cells were centrifuged at 3,000 rpm for 5 minutes, and the supernatant was aspirated and discarded. Let stand for a minute. After adding 1.5 mL of FCM buffer and stirring, the cells were centrifuged at 3,000 rpm for 5 minutes, the supernatant was aspirated and discarded, and the cells were resuspended in 400 μL of FCM buffer and analyzed with a flow cytometer within 24 hours. The results obtained are shown in FIG. In FIG. 20, white histograms show cells blocked with an isotype control antibody, and gray histograms show cells blocked with an anti-HLA-A24 antibody.

As shown in FIG. 20, when the MFIs when blocked with an isotype control antibody and when blocked with an anti-HLA-A24 antibody were compared, the HLA-A*24:02-survivin-2B-specific TCR multimer The MFI of HLA-A*24:02-PBF-specific TCR multimers decreased from 982 to 11.3. Therefore, all of the TCR multimers produced in Example 12 specifically reacted with the complex of HLA-A*24:02 and the survivin-2B peptide or PBF peptide presented thereby. confirmed.

(Example 16) Observation with a Fluorescence Microscope Of the TCR multimers produced above, the HLA-A*24:02-PBF-specific TCR multimer with high reactivity was observed with a fluorescence microscope. As antigen-presenting cells, C1R-A24 expressing HLA-A*24:02 was used, and the cells were treated with PBF peptide at a final concentration of 0 μg/mL, 0.1 μg/mL, or 10 μg/mL or HIV peptide at 10 μg/mL. Peptides were pulsed to HLA-A*24:02 by mixing and culturing in a 37° C. 5% CO ₂ incubator for 24 hours. To an appropriate amount of peptide-pulsed C1R-A24 cells, TCR multimer was added to a final concentration of 20 μg/mL in terms of soluble TCR monomer concentration, gently mixed, and allowed to stand at 4° C. for 30 minutes. did. After adding 1.5 mL of FCM buffer and stirring, centrifuge at 3,000 rpm for 5 minutes. After aspirating and discarding the supernatant, resuspend the cells in FCM buffer, 100 μL of which is placed in a 3 cm GLASS BASE DISH (IWAKI). aliquoted. These cells were observed with an HS all-in-one BZ9000 fluorescence microscope (KEYENCE). The results obtained are shown in FIG. 21A. In FIG. 21A, "TCR-multimer" indicates the result of detection of fluorescence signals derived from the labeling dye of the TCR multimer (the same applies to FIG. 21B).

As shown in FIG. 21A, cell surface-specific signals were observed in cells pulsed with 0.1 μg/mL and 10 μg/mL PBF peptide. No specific signal was observed in cells pulsed with 10 μg/mL HIV peptide.

Next, HLA-A*24:02-positive osteosarcoma-derived cell line KIKU (see Cancer Res. 1988;48(8):2273-2279) known to express PBF. - Staining with A*24:02-PBF-specific TCR multimers was performed. A TCR multimer was added to an appropriate amount of KIKU to a final concentration of 20 μg/mL in terms of soluble TCR monomer concentration, mixed gently, and allowed to stand at 4° C. for 30 minutes. After adding 1.5 mL of FCM buffer and stirring, centrifuge at 3,000 rpm for 5 minutes. After aspirating and discarding the supernatant, resuspend the cells in FCM buffer, 100 μL of which is placed in a 3 cm GLASS BASE DISH (IWAKI). aliquoted. These cells were observed with an HS all-in-one BZ9000 fluorescence microscope (KEYENCE). The results obtained are shown in FIG. 21B.

As shown in FIG. 21B, a cell surface-specific signal was observed for KIKU. KIKU was stained by HLA-A*24:02-PBF-specific TCR multimers without PBF peptide pulse, suggesting that PBF expressed in KIKU is degraded by proteasomes and converted to HLA-A* as PBF peptide. It was confirmed that it bound to 24:02 and was presented on the KIKU cell surface.

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it is possible to recognize the state in which the survivin-2B peptide is presented to HLA-A*24:02 and the state in which the PBF peptide is presented to HLA-A*24:02. can. Cells expressing the TCR of the present invention and molecules obtained by multimerizing the TCR of the present invention can be used as companion diagnostic agents when peptide vaccine therapy is performed, and when dendritic cell vaccine therapy is performed, dendritic cell HLA It can be used as a reagent for confirming whether or not the peptide of interest is presented in the It can also be used as a tool for delivering drugs to cancer cells. Furthermore, it can be used for quality control of MHC tetramer reagents. Therefore, the present invention can greatly contribute mainly to the medical field and related research fields.

SEQ ID NO: 1
<223> CDR1 of ITG-MT3 TCRα chain
SEQ ID NO: 2
<223> CDR2 of ITG-MT3 TCRα chain
SEQ ID NO: 3
<223> CDR3 of ITG-MT3 TCRα chain
SEQ ID NO: 4
<223> ITG-MT3 TCRα chain variable region SEQ ID NO: 5
<223> Soluble ITG-MT3 TCR α chain SEQ ID NO: 6
<223> CDR1 of ITG-MT3 TCR beta chain
SEQ ID NO: 7
<223> CDR2 of ITG-MT3 TCR beta chain
SEQ ID NO: 8
<223> CDR3 of ITG-MT3 TCR beta chain
SEQ ID NO:9
<223> ITG-MT3 TCR beta chain variable region SEQ ID NO: 10
<223> Soluble ITG-MT3 TCR beta chain SEQ ID NO: 11
<223> CDR1 of FKS-D11P TCRα chain
SEQ ID NO: 12
<223> CDR2 of FKS-D11P TCRα chain
SEQ ID NO: 13
<223> CDR3 of FKS-D11P TCRα chain
SEQ ID NO: 14
<223> FKS-D11P TCR α chain variable region SEQ ID NO: 15
<223> Soluble FKS-D11P TCR α chain SEQ ID NO: 16
<223> CDR1 of FKS-D11P TCR beta chain
SEQ ID NO: 17
<223> CDR2 of FKS-D11P TCR beta chain
SEQ ID NO: 18
<223> CDR3 of FKS-D11P TCR beta chain
SEQ ID NO: 19
<223> FKS-D11P TCR beta chain variable region SEQ ID NO: 20
<223> Soluble FKS-D11P TCR beta chain SEQ ID NO: 21
<223> ITG-MT3 TCR α chain SEQ ID NO: 23
<223> ITG-MT3 TCR beta chain SEQ ID NO: 25
<223> FKS-D11P TCR α chain SEQ ID NO: 27
<223> FKS-D11P TCR beta chain SEQ ID NO: 29
<223> HLA-A*24:02-restricted survivin-2B peptide SEQ ID NO: 30
<223> HLA-A*24:02 restricted PBF peptide SEQ ID NO:31
<223> HLA-A*24:02 restricted HIV env peptide SEQ ID NO:32
<223> HLA-A*24:02

Claims (15)

(i) a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NOS: 11-13 and a T-cell receptor α-chain protein set forth in SEQ ID NOS: 16-18, having any of the following characteristics (i) to (iii): (ii) a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NO: 14 and a T-cell receptor β-chain having the amino acid sequence set forth in SEQ ID NO: 19; (iii) consisting of a T-cell receptor α-chain protein having the amino acid sequence set forth in SEQ ID NO:15 and a T-cell receptor β-chain protein having the amino acid sequence set forth in SEQ ID NO:20; 2. The T cell receptor of claim 1, which recognizes a PBF peptide restricted to HLA-A*24:02. A T-cell receptor α-chain protein having any of the following characteristics (i) to (iii): (i) having the amino acid sequence set forth in SEQ ID NOS: 11 to 13; (ii) having the amino acid sequence set forth in SEQ ID NO: 14; (iii) having the amino acid sequence set forth in SEQ ID NO: 15; A T-cell receptor β-chain protein having any of the following characteristics (i) to (iii): (i) having an amino acid sequence set forth in SEQ ID NOS: 16 to 18 (ii) having an amino acid sequence set forth in SEQ ID NO: 19 (iii) having the amino acid sequence set forth in SEQ ID NO:20; A DNA encoding the protein according to any one of claims 3 and 4. A vector containing the DNA according to claim 5 in an expressible manner. Transformed cells obtained by introducing the DNA according to claim 5 into lymphocytes. A lymphocyte into which the DNA encoding the protein according to claim 3 and the DNA encoding the protein according to claim 4 have been introduced, wherein the PBF peptide bound to HLA-A*24:02 is bound. Transformed cells that can be detected by multimerizing molecules. Lymphocytes into which the DNA is introduced are described in Jurkat, Jurkat/MA, HPB-ALL, HPB-MLT, J. Am. 9. The transformed cell according to claim 7 or 8, which is RT3-T3.5 or Sup-T1. A pharmaceutical composition for treating PBF-positive cancer, comprising the transformed cell according to any one of claims 7 to 9 as an active ingredient. An antibody that specifically binds to a molecule according to any one of (a) to (c) below.
(a) the T-cell receptor α-chain protein according to claim 3; (b) the T-cell receptor β-chain protein according to claim 4; and (c) the T-cell receptor according to claim 1 or 2. A molecule multimerized by binding the T cell receptor according to claim 1 or 2. An agent for detecting or capturing HLA-A*24:02 restricted PBF peptides, said agent comprising a molecule according to claim 12. A kit for detecting HLA-A*24:02-restricted PBF peptides, the kit comprising at least one of the following components (a) to (e).
(a) the T cell receptor of claim 1 or 2; (b) a DNA encoding the protein of claim 3 and a DNA encoding the protein of claim 4; or the protein of claim 3. and a DNA encoding the protein of claim 4
(c) a vector that expressably contains the DNA encoding the protein of claim 3 and a vector that expressably contains the DNA that encodes the protein of claim 4, or the protein of claim 3 and the vector (d) lymphocyte containing the DNA encoding the protein of claim 4 in an expressible manner, the DNA encoding the protein of claim 3 and the DNA encoding the protein of claim 4 a transformed cell into which it has been introduced (e) the molecule of claim 12 A method for quality control of a molecule multimerized by binding an HLA-A*24:02-restricted PBF peptide, wherein the molecule reacts with the transformed cell according to any one of claims 7 to 9. When the reactivity is maintained, it is evaluated that the quality of the molecule is maintained, and when the reactivity is decreased, the quality of the molecule is evaluated A method including the step of assessing as degraded.

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