T-cell receptor for recognizing complex of HSP105-derived peptide and MHC class i molecule, and method for using same

Abstract

The present invention addresses the problem of identifying a TCR that targets a suitable tumor antigen and enabling a new immunotherapy for cancer. The present invention provides: a TCR gene for recognizing a complex of an HSP105-derived peptide and a human MHC class I molecule; and a method for using the TCR gene.

Description

T cell receptor that recognizes a complex of HSP105-derived peptide and MHC class I molecule, and method of using the same

[0001] The present invention relates to a T cell receptor that recognizes a complex of an HSP105-derived peptide and an MHC class I molecule, and uses thereof.

[0002] Even after being diagnosed with cancer and undergoing curative surgery, many patients do not survive for more than 10 years. In Japan, 1 million people are diagnosed with cancer each year, and 400,000 die from it. Immunotherapy is attracting attention as an alternative to surgical treatment for cancer. If new immunotherapies such as vaccines and TCR / CAR-T cell therapy are developed as cancer treatments, it is expected that many of the 1 million patients diagnosed each year will be eligible for treatment.

[0003] Tumor-specific vaccine therapy, using epitope peptides derived from tumor-associated antigens, involves administering vaccine antigens to induce tumor-specific immune responses, promoting the proliferation and activation of antigen-specific cytotoxic T cells (CTLs). These antigens then damage cancer cells presenting the corresponding cancer antigens, suppressing tumor growth in cancer patients. However, peptide vaccines often require several months to induce sufficient CTLs for therapeutic efficacy in vivo, and immunosuppression can occur due to the presence of immunosuppressive mechanisms. As a result, vaccine-based cancer therapy has been limited or ineffective in some cases. Furthermore, it is generally difficult to predict the number of CTLs induced by peptide vaccines, making it difficult to predict therapeutic efficacy.

[0004] T cell receptors (TCRs) are antigen receptors for members of the immunoglobulin superfamily expressed on T cells. T cells recognize antigen peptides presented on the MHC (HLA in humans) of antigen-presenting cells through TCRs. This induces T cell proliferation and differentiation, leading to the production of cytokines (e.g., interferon-γ) or the secretion of cytotoxic substances (perforin and granzymes). As a result, the proliferation of cancer cells and other cells expressing the target antigen is suppressed.

[0005] TCR-T cell therapy is an immunotherapy in which TCR genes that specifically recognize antigens presented by HLA on cancer cells are introduced into T cells collected from the patient's peripheral blood, the genetically modified T cells are expanded, and then reintroduced into the patient. This immunotherapy has been shown to have a certain tumor growth suppression effect. Therefore, if cancer antigen-specific TCRs can be identified, TCR-T cell therapy will enable cancer immunotherapy. The TCR-T cells required for treatment can be easily expanded by culture, and faster and more effective therapeutic effects can be expected compared to peptide vaccines. Furthermore, since the number of activated CTLs is known in TCR-T cell therapy, the therapeutic effect can be easily predicted. However, no TCR-T cell therapy has yet emerged that is promising as a treatment for solid cancers.

[0006] International Publication WO2023 / 224096

[0007] To develop a TCR-T cell therapy that is suitable for many patients, it is necessary to identify antigens expressed on target cancer cells and TCRs that recognize such antigens. Common cancer antigens, which are expressed in a high frequency of cancers but not in most normal organs, are useful targets for TCR-T cell therapy. An urgent task is to select antigens highly specific to cancer cells and identify TCRs with appropriate affinity for such antigens, so as to have tumor growth-suppressing effects while avoiding adverse events such as off-target effects on normal tissues. Identifying a TCR that targets a suitable tumor antigen would enable the development of T cell therapy (TCR-T cell therapy) transfected with that TCR, enabling new immunotherapy for solid tumors in other patients with the same HLA type.

[0008] The present inventors have discovered a novel antigenic peptide for HSP105 (Heat Shock Protein 105), which has been confirmed to be highly expressed in a variety of cancers, including colorectal cancer, esophageal cancer, pharyngeal cancer, pancreatic cancer, breast cancer, and melanoma (International Publication No. WO2023 / 224096). This novel epitope peptide, which induces an immune response, is expected to be a useful target antigen for TCR-T cell therapy.

[0009] An object of the present invention is to provide a TCR that recognizes a complex of an HSP105-derived peptide and a human MHC class I molecule, for use in the treatment of a wide range of cancers, and a method for using the same. Another object of the present invention is to provide a means for treating cancer using the above-mentioned T cell receptor (TCR).

[0010] As a result of intensive research to achieve the above object, the present inventors established an HSP105-derived peptide-specific CTL clone and identified a TCR gene from the CTL clone that recognizes a complex between an HSP105-derived peptide and a human MHC class I molecule. The inventors then introduced the TCR gene into T cells collected from the peripheral blood of a subject, and found that the resulting HSP105-TCR-T cells recognized a cell line presenting the HSP105 peptide, secreted interferon-γ (IFN-γ) in an antigen-specific manner, and possessed cytotoxic activity. Furthermore, the inventors found that administration of the HSP105-TCR-T cells can suppress tumor growth in vivo. Furthermore, the inventors have found that by using a circular nucleic acid without a stop codon when introducing the above-mentioned TCR gene into T cells, expression of HSP105-derived peptide-specific TCR (HSP105-TCR) in HSP105-TCR-T cells is sustained for a long period of time after gene introduction, and that HSP105-TCR-T cells that recognize cell lines presenting HSP105 peptides and secrete IFNγ in an antigen-specific manner can be present for a long period of time. Furthermore, the inventors have found that administration of HSP105-TCR-T cells into which the TCR gene has been introduced using a circular nucleic acid can also suppress tumor growth in vivo. The present invention was completed based on the above findings.

[0011] That is, the present invention provides the following: <1> A T cell receptor that recognizes a complex of an amino acid sequence RLMNNDMTAV, which is a partial peptide derived from HSP105, and a human MHC class I molecule. <2> The T cell receptor according to <1>, comprising a T cell receptor α chain including an α chain CDR3 specified by the amino acid sequence set forth in SEQ ID NO: 1, or an α chain CDR3 functionally equivalent thereto, and a T cell receptor β chain including a β chain CDR3 specified by the amino acid sequence set forth in SEQ ID NO: 2, or a β chain CDR3 functionally equivalent thereto. <3> The T cell receptor according to <2>, comprising a T cell receptor α chain comprising an α chain CDR1 specified by the amino acid sequence of SEQ ID NO: 3 or an α chain CDR1 functionally equivalent thereto, and an α chain CDR2 specified by the amino acid sequence of SEQ ID NO: 4 or an α chain CDR2 functionally equivalent thereto, and a T cell receptor β chain comprising a β chain CDR1 specified by the amino acid sequence of SEQ ID NO: 5 or a β chain CDR1 functionally equivalent thereto, and a β chain CDR2 specified by the amino acid sequence of SEQ ID NO: 6 or a β chain CDR2 functionally equivalent thereto. <4> The T cell receptor according to <1>, comprising a T cell receptor α chain specified by the amino acid sequence of SEQ ID NO: 7 and a T cell receptor β chain specified by the amino acid sequence of SEQ ID NO: 8. <5> A combination of nucleic acids comprising a nucleic acid encoding a T cell receptor α chain specified by the amino acid sequence of SEQ ID NO: 7 and a nucleic acid encoding a T cell receptor β chain specified by the amino acid sequence of SEQ ID NO: 8. <6> The combination of nucleic acids according to <5>, wherein the combination of nucleic acids is contained in parallel in a single circular nucleic acid that does not contain a stop codon. <7> The combination of nucleic acids according to <5>, wherein the nucleic acid encoding the T cell receptor α chain is DNA specified by the base sequence of SEQ ID NO: 9, and the nucleic acid encoding the T cell receptor β chain is DNA specified by the base sequence of SEQ ID NO: 10. <8> The combination of nucleic acids according to <5>, wherein the nucleic acid encoding the T cell receptor α chain is RNA specified by the base sequence of SEQ ID NO: 11, and the nucleic acid encoding the T cell receptor β chain is RNA specified by the base sequence of SEQ ID NO: 12. <9> A vector comprising the combination of nucleic acids according to <5>.<10> A cell transfected with the combination of nucleic acids according to <5>. <11> A cell transfected with the vector according to <9>. <12> The cell according to <10> or <11>, wherein the cell is a T cell, a NK cell, an invariant NK cell, a NKT cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell. <13> The cell according to <10> or <11>, wherein the cell is a T cell that does not have an endogenous T cell receptor. <14> The T cell that does not have an endogenous T cell receptor is CD3. － CD8 ＋ <15> A cancer therapeutic agent comprising the T cell receptor according to any one of <1> to <4>, a combination of nucleic acids according to any one of <5> to <8>, the vector according to <9>, or the cell according to <10> or <11>.

[0012] By using the T cell receptor (HSP105-TCR) according to the present invention that recognizes a complex of an HSP105-derived peptide and an MHC class I molecule, it is possible to produce pharmaceuticals or regenerative medicine products targeting cancers that express HSP105, such as colorectal cancer.

[0013] Figure 1 shows the results of ex vivo immune monitoring for screening HLA-A2-restricted HSP105-specific T cells, in which antigen-specific IFN-γ production of peripheral blood mononuclear cells (PBMCs) collected from a patient was evaluated by ELISPOT assay. Figure 2 shows the results of in vitro immune monitoring for screening HLA-A2-restricted HSP105-specific T cells, in which antigen-specific IFN-γ production of CD8-positive T cells isolated from PBMCs after antigen stimulation was evaluated by ELISPOT assay. Figure 3 shows the process for sorting HLA-A2-restricted HSP105-specific T cells. PBMCs collected from a patient's lymph node tumor specimen after peptide vaccination were evaluated for antigen-specific IFN-γ production by ELISPOT assay, followed by sorting of CD107a-expressing HLA-A2-restricted HSP105-specific T cells by flow cytometry. Figure 4 shows the results of an ELISPOT assay to evaluate IFN-γ production of the established HLA-A2-restricted HSP105-specific T cell clone. Figure 5 shows the results of an in vitro cytotoxic activity test of the established HLA-A2-restricted HSP105-specific T cell clone. Figure 6 shows the results of a CD107a assay to confirm the antigen-specific immune response of the established HLA-A2-restricted HSP105-specific T cell clone. Figure 7 shows the amino acid sequences of the α-chain and β-chain of the T cell receptor (TCR) of the established HLA-A2-restricted HSP105-specific T cells. Figure 8 shows the DNA sequences of the α-chain and β-chain of the T cell receptor (TCR) identified from the amplification product of the established HLA-A2-restricted HSP105-specific T cell clone. Figure 9 shows the results of confirming the expression of HSP105-derived peptide-specific TCR in HSP105-TCR-T cells by flow cytometry. Figure 10 shows the results of evaluating antigen-specific IFN-γ production by HSP105-TCR-T cells by ELISPOT assay. Figure 11 shows the results of evaluating the cytotoxic response of HSP105-TCR-T cells by CD107a assay. Figure 12 shows the results of evaluating the cytotoxic response of HSP105-TCR-T cells by CD107a assay. Figure 13 shows the results of an in vitro cytotoxic activity test of HSP105-TCR-T cells.Figure 14 shows the results of an in vivo study using NSG mice administered HSP105-TCR-T cells. Figure 15 shows the results of flow cytometry confirming the expression of HSP105-derived peptide-specific TCR (HSP105-TCR) in HSP105-TCR-T cells transfected with linear mRNA (Liner mRNA) or circular mRNA (Circulating mRNA). For comparison, the results of KO-T cells (EP only), which were subjected to EP without mRNA transfection, are also shown. Figure 16 shows the results of an ELISPOT assay evaluating antigen-specific IFN-γ production by HSP105-TCR-T cells 1 day or 7 days after EP. Non mRNA refers to KO-T cells that underwent EP alone, L mRNA refers to HSP105-TCR-T cells transfected with linear mRNA, and C mRNA refers to HSP105-TCR-T cells transfected with circular mRNA. Figure 17 shows the results of an in vivo study using NSG mice, in which HSP105-TCR-T cells transfected with circular mRNA were administered. Figure 18 shows a schematic map of the linear mRNA used to transfect the HSP105-TCR gene. Figure 19 shows a schematic map of the circular mRNA used to transfect the HSP105-TCR gene. Figure 20 shows the first half of an example of a linear nucleic acid sequence containing a TCR α chain coding sequence and a TCR β chain coding sequence. Figure 21 shows the second half of an example of a linear nucleic acid sequence containing a TCR α chain coding sequence and a TCR β chain coding sequence. Figure 22 shows the front of an example of a circular nucleic acid sequence containing a TCR α chain coding sequence and a TCR β chain coding sequence, Figure 23 shows the middle of an example of a circular nucleic acid sequence containing a TCR α chain coding sequence and a TCR β chain coding sequence, and Figure 24 shows the rear of an example of a circular nucleic acid sequence containing a TCR α chain coding sequence and a TCR β chain coding sequence.

[0014] 1. Definitions The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides. These terms apply not only to naturally occurring nucleic acids such as DNA and RNA, but also to artificial nucleic acids that have been modified in the nucleobase, sugar, or phosphate diester moiety that constitutes natural nucleic acids and have a chemical structure different from that of natural nucleic acids.

[0015] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to polymers of naturally occurring amino acids as well as polymers containing non-naturally occurring amino acid residues. Non-naturally occurring amino acids include amino acid analogs and amino acid mimetics.

[0016] As used herein, the term "amino acid" refers to naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are post-translationally modified in cells. The term "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid (an alpha carbon attached to a hydrogen, a carboxy group, an amino group, and an R group) but has a modified R group or a modified backbone. The term "amino acid mimetic" refers to a compound that has a different structure from a typical amino acid but functions similarly to an amino acid. Amino acids can be either L- or D-amino acids, although the peptides of the present invention are preferably polymers of L-amino acids.

[0017] The term "HSP" refers to a group of proteins called heat shock proteins (HSPs). HSPs are classified into several families according to their molecular weight. The term "HSP105" refers to a type of HSP family protein and one of the stress proteins highly conserved in various organisms. HSP105 is a cancer antigen that is highly expressed in various cancers such as colorectal cancer, pancreatic cancer, pharyngeal cancer, esophageal cancer, and breast cancer. HSP105 is encoded by the HSP105 gene, and two types, HSP105α and HSP105β, are produced by alternative splicing.

[0018] The term "antigenic peptide" refers to a peptide fragment obtained by intracellular degradation of an endogenous antigen or an antigenic protein taken up into a cell into several amino acid residues by the proteasome. These antigenic peptides are presented on the cell surface by MHC and induce an immune response. The term "HSP105-derived peptide" refers to an antigenic peptide derived from HSP105α, and is a peptide fragment constituting HSP105α. It is an HLA class I-binding peptide and specifically binds to HLA-A2 (A2 group). When expressed on the cell surface together with HLA, it is presented to cytotoxic T lymphocytes (CTLs). In one example, the HSP105-derived peptide is preferably a peptide consisting of the amino acid sequence RLMNNDMTAV.

[0019] The terms "Major Histocompatibility Complex," "MHC," and "HLA" are used interchangeably herein to refer to MHC class I molecules. The term "MHC class I" refers to a dimer of the MHC class I α chain and β2 microglobulin chain.

[0020] The term "HLA-A2" refers to an HLA-A2 type, including subtypes such as HLA-A*02:01, HLA-A*02:06, and HLA-A*02:07. In the context of a subject or patient, the phrase "a subject (or patient) who is HLA-A2" refers to the subject or patient homozygously or heterozygously possessing the HLA-A2 antigen gene as an MHC class I molecule, and the HLA-A2 antigen being expressed as an HLA antigen in the cells of the subject or patient. In one example, the patient is preferably HLA-A*02:01.

[0021] The term "peptide-MHC complex" refers to an MHC molecule (MHC class I) to which a peptide is bound, with the bound peptide being successfully accommodated in a depression present in the peptide-accommodating groove of the MHC. In one example, the MHC molecule can be a membrane-bound protein expressed on the cell surface. In another example, the MHC molecule can be a soluble protein lacking a transmembrane or cytoplasmic region.

[0022] The terms "cytotoxic T lymphocytes," "cytotoxic T cells," and "CTLs" are used interchangeably herein and refer to a subgroup of T lymphocytes that recognize antigenic peptides presented on MHC class I molecules on the surface of target cells and exhibit antigenic peptide-specific cytotoxicity. Upon recognizing an antigenic peptide, CTLs secrete cytotoxic granules such as perforin and granzymes, and induce apoptosis in the target cells.

[0023] The terms "T cell receptor" and "TCR" are used interchangeably and refer to a protein molecule consisting of a heterodimer of an α chain and a β chain. T cells recognize peptides presented on MHC molecules through TCR. As a result, T cell proliferation, differentiation, production of cytokines (e.g., IFN-γ), and secretion of cytotoxic substances (e.g., perforin and granzymes) are induced.

[0024] 2. T Cell Receptor The present invention provides a T cell receptor (herein sometimes referred to as "TCR of the present invention" or "HSP105-derived peptide-specific TCR") that recognizes a complex of the amino acid sequence RLMNDMTAV, which is a partial peptide derived from HSP105, and a human MHC class I molecule. The TCR of the present invention can have antigen specificity for a peptide consisting of the amino acid sequence RLMNDMTAV. In other words, the TCR of the present invention can have HLA-A2-restricted binding specificity for an HSP105-derived peptide.

[0025] The TCR of the present invention is composed of α and β chains that form subunits, and is composed of an extracellular region containing an immunoglobulin-like domain (Ig domain), a transmembrane region, and an intracellular region. The α chain is encoded by the TCR-α gene, and the β chain is encoded by the TCR-β gene. The TCR-α gene includes the Vα gene, the Jα gene, and the Cα gene, while the TCR-β gene includes the Vβ gene, the Dβ gene, the Jβ gene, and the Cβ gene.

[0026] TCRs contain a constant region (C region) and a variable region (V region). The variable region consists of an extracellular region formed from the N-terminal side of the Ig domain, while the constant region consists of an extracellular region formed from the C-terminal side of the Ig domain, a transmembrane region, and an intracellular region. Herein, the constant region of the α chain of a TCR may be referred to as the "α chain constant region," and the constant region of the β chain of a TCR may be referred to as the "β chain constant region." As used herein, with respect to a TCR, the term "extracellular domain" refers to the portion of the TCR chain located outside the cell, the term "transmembrane domain" refers to the portion of the TCR chain embedded in the plasma membrane of a cell, and the term "cytoplasmic domain" refers to the portion of the TCR chain located in the cytoplasm of a cell.

[0027] As used herein, the term "costimulatory signaling domain" refers to the intracellular portion of a costimulatory molecule that is involved in mediating intracellular signaling events.

[0028] The constant region (C region) is encoded by the Cα gene of the α chain TCR-α gene, and the constant region of the β chain is encoded by the Cβ gene of the TCR-β gene.

[0029] In the variable region (V region), TCR genes undergo gene rearrangement. In the variable region of the α chain, Vα and Jα genes of the TCR-α genes are rearranged, and in the variable region of the β chain, Vβ, Dβ, and Jβ genes of the TCR-β genes are rearranged. Herein, the variable region of the TCR α chain may be referred to as the "α chain variable region," and the variable region of the TCR β chain may be referred to as the "β chain variable region."

[0030] Furthermore, the variable region of a TCR contains regions known as complementarity-determining regions (CDRs) (sometimes referred to as "hypervariable regions") that directly interact with the MHC-peptide complex. CDRs are regions deeply involved in specific binding to MHC-restricted antigens and include CDR1, CDR2, and CDR3. Since CDR3 is thought to be in direct contact with peptides, the amino acid sequence of CDR3 is particularly important in determining the antigen recognition specificity of a TCR. Furthermore, it is believed that diverse variations arise in CDR3 due to random insertions and deletions of bases during gene rearrangement. Herein, a CDR present in the variable region of the α chain of a TCR may be referred to as an "α chain CDR," and a CDR present in the variable region of the β chain of a TCR may be referred to as a "β chain CDR."

[0031] The variable region of a TCR also contains regions called "framework regions (FR)." A framework region refers to a region that is part of the TCR variable region but not part of the CDRs. That is, a TCR variable region is generally composed of framework regions (FR) 1, 2, 3, and 4, and complementarity-determining regions (CDR) 1, 2, and 3, with CDR1, CDR2, and CDR3 being arranged via FR1, FR2, FR3, and FR4 of the framework regions (FR). The region between the N-terminus of the variable region and CDR1 is defined as FR1, the region between CDR1 and CDR2 as FR2, the region between CDR2 and CDR3 as FR3, and the region between CDR3 and the C-terminus of the variable region as FR4.

[0032] In genetically modified T cell therapy (sometimes referred to herein as "TCR-T cell therapy"), when preparing T cells that recognize antigen peptides presented on MHC class I molecules of target cancer cells and secrete cytotoxic substances, a nucleic acid encoding the amino acid sequence of a TCR identified from T cells that recognize the HSP105-derived peptide shown herein can be genetically transferred into immune cells collected from a patient, thereby inducing an immune response against target cancer cells that express HSP105. In particular, the amino acid sequence in the CDR3 of the TCR shown herein is important for antigen-specific immune responses in TCR-T cell therapy.

[0033] In one example, the present invention relates to a T cell receptor comprising a TCR α chain having an α chain CDR3 specified by the amino acid sequence set forth in SEQ ID NO: 1, and a TCR β chain having a β chain CDR3 specified by the amino acid sequence set forth in SEQ ID NO: 2.

[0034] Generally, altering one, two, or more amino acids in a polypeptide may not affect the function of the polypeptide. It is also known that, in some cases, desired functions of the original polypeptide may be enhanced. Even polypeptides composed of amino acid sequences altered by substitution, deletion, insertion, addition, or the like of one, two, or several amino acid residues may retain the biological activity of the original polypeptide. In particular, changes between amino acids that maintain similar structural and / or chemical properties are readily tolerated. It is well known in the art that altering polypeptides by "conservative substitution," which is the substitution of functionally similar amino acids, can produce functionally equivalent modified polypeptides.

[0035] In one example, the CDR3 of the present invention includes an α-chain CDR3 or β-chain CDR3 containing modified amino acids, more preferably by conservative substitution, of one, two, or several amino acids relative to the amino acid sequence shown in SEQ ID NO: 1 or 2, and conferring HLA-A2-restricted binding specificity to HSP105-derived peptides to the TCR. Herein, such a CDR3 may be referred to as an α-chain CDR3 or β-chain CDR3 "functionally equivalent" to the α-chain CDR3 or β-chain CDR3 consisting of the original amino acid sequence. However, modifications are not limited to these, and non-conservative modifications may also be included as long as the modified α-chain CDR3 or β-chain CDR3 retains the function of the original α-chain CDR3 or β-chain CDR3.

[0036] The present invention provides an HSP105-derived peptide-specific T cell receptor comprising a T cell receptor α chain comprising an α chain CDR3 specified by the amino acid sequence set forth in SEQ ID NO: 1, or an α chain CDR3 functionally equivalent thereto, and a T cell receptor β chain comprising a β chain CDR3 specified by the amino acid sequence set forth in SEQ ID NO: 2, or a β chain CDR3 functionally equivalent thereto.

[0037] In one example, the present invention is preferably a T cell receptor in which the TCR α chain is a T cell receptor α chain further comprising an α chain CDR1 specified by the amino acid sequence set forth in SEQ ID NO: 3 and an α chain CDR2 specified by the amino acid sequence set forth in SEQ ID NO: 4, and the TCR β chain is a T cell receptor β chain further comprising a β chain CDR1 specified by the amino acid sequence set forth in SEQ ID NO: 5 and a β chain CDR2 specified by the amino acid sequence set forth in SEQ ID NO: 6.

[0038] In one example, the CDR1 of the present invention includes an α-chain CDR1 or β-chain CDR1 containing modified amino acids, more preferably by conservative substitution, selected from the group consisting of substitution, deletion, insertion, and addition of one, two, or several amino acids relative to the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5, and which confers HLA-A2-restricted binding specificity to HSP105-derived peptides to the TCR. Herein, such an α-chain CDR1 or β-chain CDR1 may be referred to as an α-chain CDR1 or β-chain CDR1 "functionally equivalent" to the α-chain CDR1 or β-chain CDR1 consisting of the original amino acid sequence. However, modifications are not limited to these, and the modified α-chain CDR1 or β-chain CDR1 may also include non-conservative modifications as long as it retains the function of the original α-chain CDR1 or β-chain CDR1.

[0039] In one example, the CDR2 of the present invention includes an α chain CDR2 or β chain CDR2 containing modified amino acids, more preferably modified by conservative substitution, of one, two, or several amino acids relative to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:6, and which confers HLA-A2-restricted binding specificity to HSP105-derived peptides to the TCR. Herein, such an α chain CDR2 or β chain CDR2 may be referred to as an α chain CDR2 or β chain CDR2 that is "functionally equivalent" to the α chain CDR2 or β chain CDR2 consisting of the original amino acid sequence. However, modifications are not limited to these, and non-conservative modifications may also be included as long as the modified α chain CDR2 or β chain CDR2 retains the function of the original α chain CDR2 or β chain CDR2.

[0040] In one example, the present invention relates to a T cell receptor comprising a T cell receptor α chain comprising an α chain CDR3 specified by the amino acid sequence of SEQ ID NO: 1 or an α chain CDR3 functionally equivalent thereto, and a T cell receptor β chain comprising a β chain CDR3 specified by the amino acid sequence of SEQ ID NO: 2 or a β chain CDR3 functionally equivalent thereto, and preferably further comprising a T cell receptor α chain comprising an α chain CDR1 specified by the amino acid sequence of SEQ ID NO: 3 or an α chain CDR1 functionally equivalent thereto, and an α chain CDR2 specified by the amino acid sequence of SEQ ID NO: 4 or an α chain CDR2 functionally equivalent thereto, and a T cell receptor β chain comprising a β chain CDR1 specified by the amino acid sequence of SEQ ID NO: 5 or a β chain CDR1 functionally equivalent thereto, and a β chain CDR2 specified by the amino acid sequence of SEQ ID NO: 6 or a β chain CDR2 functionally equivalent thereto.

[0041] In one example, the present invention is preferably a T cell receptor comprising an α chain variable region containing the α chain variable region identified by the amino acid sequence set forth in SEQ ID NO: 13 or a mutated sequence thereof, and a β chain variable region containing the amino acid sequence set forth in SEQ ID NO: 14 or a mutated sequence thereof.

[0042] With regard to the variable region of the TCR of the present invention, a mutant sequence of the amino acid sequence set forth in SEQ ID NO: 13 or 14 preferably has an identity of 90% or more, more preferably 95% or more, even more preferably 98% or more, and still more preferably 99% or more to the amino acid sequence set forth in SEQ ID NO: 13 or 14. The mutation position is not particularly limited, but is preferably a site other than CDRs 1 to 3, i.e., a framework region.

[0043] In one example, the amino acid sequence of the constant region of the present invention is not particularly limited as long as it retains the function of the constant region (e.g., the function of penetrating the cell membrane and exposing the variable region on the cell surface), but it is preferable that the T cell receptor comprises an α chain constant region comprising the amino acid sequence of SEQ ID NO: 15 or a mutated sequence thereof, and a β chain constant region comprising the amino acid sequence of SEQ ID NO: 16 or a mutated sequence thereof.

[0044] In one example, for the constant region of the TCR of the present invention, a mutant sequence of the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16 has an identity of preferably 70% or more, more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more to the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16.

[0045] By gene transfer, the TCR of the present invention forms a dimer of TCRα chain and TCRβ chain proteins, which forms a complex with CD3 in the host cell, is expressed on the surface of the host cell, and binds to a complex of HSP105 peptide and MHC class I molecule to activate the host cell. However, it is also possible to add modifications such as connecting the extracellular domains of the TCRα chain and TCRβ chain of the present invention with a linker, replacing the transmembrane domain with any transmembrane domain, or replacing the intracellular domain with the cytoplasmic domain of a known CAR or the like.

[0046] In one example, the transmembrane domain of the TCR of the present invention may be any other transmembrane domain as long as it retains the function of a TCR. Examples of other transmembrane domains include transmembrane domains made of artificially constructed polypeptides, transmembrane domains of CD28, CD3ε, CD8α, CD3, CD4, 4-1BB, and the like, or modified domains thereof.

[0047] The cytoplasmic domain of the TCR of the present invention is not particularly limited as long as it retains the function of a TCR, and may be any other cytoplasmic domain. Examples of other cytoplasmic domains include a cytoplasmic domain consisting of an artificially constructed polypeptide, a cytoplasmic domain of CD3, or a modified domain thereof. It may also contain a costimulatory signaling domain. For example, it may contain costimulatory signaling domains such as OX40, 4-1BB, GITR, CD27, CD278, CD28, and JAK2 / 3.

[0048] The TCR of the present invention may contain a linker between each domain or in place of the constant region, such as a GGS linker, a GS linker, or a GGG linker.

[0049] In one example, the present invention is preferably a T cell receptor specific for an HSP105-derived peptide, which comprises a T cell receptor α chain specified by the amino acid sequence set forth in SEQ ID NO: 7 and a T cell receptor β chain specified by the amino acid sequence set forth in SEQ ID NO: 8.

[0050] The TCR of the present invention may be tagged with a known peptide tag, signal sequence, or labeling substance, as long as it can recognize an HSP105-derived peptide. Examples of peptide tags that can be added include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, and PA tag. Examples of signal sequences include nuclear export signals (NES) and nuclear localization signals (NLS). Examples of labeling substances that can be added include fluorescent proteins such as GFP, gold particles, and the like.

[0051] Furthermore, the present invention provides a nucleic acid encoding a TCR alpha chain, a nucleic acid encoding a TCR beta chain, and a combination of nucleic acids encoding a TCR alpha chain and a TCR beta chain. In one example, the present invention provides a nucleic acid combination comprising a nucleic acid encoding a T cell receptor alpha chain defined by the amino acid sequence set forth in SEQ ID NO:7 and a nucleic acid encoding a T cell receptor beta chain defined by the amino acid sequence set forth in SEQ ID NO:8.

[0052] In one example, a preferred combination of nucleic acids is one in which the nucleic acid encoding the T cell receptor α chain is DNA specified by the base sequence set forth in SEQ ID NO: 9, and the nucleic acid encoding the T cell receptor β chain is DNA specified by the base sequence set forth in SEQ ID NO: 10. The DNA specified by the base sequence set forth in SEQ ID NO: 9 and / or the DNA specified by the base sequence set forth in SEQ ID NO: 10 may contain a modified artificial nucleic acid.

[0053] In one example, a preferred combination of nucleic acids is one in which the nucleic acid encoding the T cell receptor α chain is RNA specified by the base sequence set forth in SEQ ID NO: 11, and the nucleic acid encoding the T cell receptor β chain is RNA specified by the base sequence set forth in SEQ ID NO: 12. The RNA specified by the base sequence set forth in SEQ ID NO: 11 and / or the RNA specified by the base sequence set forth in SEQ ID NO: 12 may include a modified artificial nucleic acid. In one example, the RNA is preferably one produced using modified UTP in which uridine is substituted with N1-methylpseudouridine.

[0054] The nucleic acids encoding the TCR α chain and the TCR β chain may be separate nucleic acids, or may be a single nucleic acid encoding both the TCR α chain and the TCR β chain. In this specification, one or more of these nucleic acids may be referred to as the "nucleic acid of the present invention."

[0055] In one example, when the nucleic acid of the present invention is a single nucleic acid containing both nucleic acids encoding a TCR α chain and a TCR β chain, the TCR α chain and the TCR β chain are expressed as a single polypeptide, but can be linked by inserting a linker sequence between the region encoding the TCR α chain and the region encoding the TCR β chain, for example, a P2A sequence, so that the chain is cleaved after expression.

[0056] In one example, when the nucleic acid of the present invention is a single nucleic acid containing both nucleic acids encoding a TCRα chain and a TCRβ chain, the TCRα chain and the TCRβ chain may be linear nucleic acids or circular nucleic acids without a stop codon. From the viewpoint of enabling highly efficient protein synthesis, the nucleic acid of the present invention is preferably a circular nucleic acid without a stop codon. In one example, from the viewpoint of enabling long-term expression of the TCRα chain and the TCRβ chain in the introduced cell after introducing the nucleic acids encoding the TCRα chain and the TCRβ chain into the cell as described below, the nucleic acid of the present invention is preferably a circular single nucleic acid containing both nucleic acids encoding the TCRα chain and the TCRβ chain. In one example, from the viewpoint that after nucleic acids encoding the TCRα chain and the TCRβ chain are introduced into cells as described below, the introduced cells can recognize a cell line that presents an HSP105 peptide and secrete IFNγ in an antigen-specific manner for a long period of time, the nucleic acid of the present invention is preferably a single circular nucleic acid that contains both nucleic acids encoding the TCRα chain and the TCRβ chain and does not contain a stop codon.

[0057] In one example, the present invention provides a nucleic acid encoding a TCR alpha chain, a nucleic acid encoding a TCR beta chain, and a combination of nucleic acids encoding a TCR alpha chain and a TCR beta chain, which are contained juxtaposed in a single circular nucleic acid that does not contain a stop codon.

[0058] The nucleic acid of the present invention may contain other sequences as long as it is capable of expressing the TCR of the present invention. For example, such other sequences include a secretory signal peptide coding sequence, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, an origin of replication, a reporter protein (e.g., a fluorescent protein), a drug resistance gene coding sequence, etc. In addition, in one example, when suppressing the expression of an endogenous TCR, the nucleic acid may contain a sequence encoding a short RNA (e.g., siRNA, miRNA, etc.) that suppresses the expression of the endogenous TCR.

[0059] In one example, the nucleic acid of the present invention can be codon-optimized to suit the cell into which the nucleic acid is introduced (e.g., bacteria, fungi, plants, or animals). In addition, the nucleic acid can appropriately contain regulatory sequences specific to the cell into which the nucleic acid is introduced, such as initiation and termination codons for transcription and translation. When the nucleic acid of the present invention is a circular nucleic acid, it does not need to contain a termination codon.

[0060] In one example, the nucleic acid of the present invention may be a modified nucleic acid. For example, the nucleic acid of the present invention may be mRNA produced using modified UTP containing N1-methylpseudouridine. Generally, modified UTP in which the uridine of UTP (uridine triphosphate) is substituted with N1-methylpseudouridine can be used as a raw material for RNA synthesis. It is known that mRNA produced using this N1-methylpseudouridine reduces inflammatory responses and improves protein synthesis efficiency compared to normal mRNA. In addition, it is also possible to suppress degradation of the nucleic acid after gene transfer into host cells or reduce cytotoxicity by appropriately modifying the nucleic acid base, sugar, phosphate diester moiety, etc.

[0061] In one example, the nucleic acid of the present invention may be the linear mRNA shown in SEQ ID NO: 18 or the circular mRNA shown in SEQ ID NO: 19, or a modified mRNA thereof (e.g., an mRNA containing N1-methylpseudouridine).

[0062] The present invention provides vectors comprising a nucleic acid encoding a TCR α chain, a vector comprising a nucleic acid encoding a TCR β chain, and a vector comprising a combination of nucleic acids encoding a TCR α chain and a TCR β chain. In one example, the present invention provides a vector comprising a combination of nucleic acids, including a nucleic acid encoding a T cell receptor α chain defined by the amino acid sequence set forth in SEQ ID NO:7 and a nucleic acid encoding a T cell receptor β chain defined by the amino acid sequence set forth in SEQ ID NO:8.

[0063] Preferably, the nucleic acid encoding the T cell receptor α chain is a DNA specified by the base sequence set forth in SEQ ID NO: 9, and the nucleic acid encoding the T cell receptor β chain is a DNA specified by the base sequence set forth in SEQ ID NO: 10.

[0064] The DNA specified by the base sequence set forth in SEQ ID NO: 9 and / or the DNA specified by the base sequence set forth in SEQ ID NO: 10 may contain a modified artificial nucleic acid.

[0065] Preferably, the nucleic acid encoding the T cell receptor α chain is RNA specified by the base sequence set forth in SEQ ID NO: 11, and the nucleic acid encoding the T cell receptor β chain is RNA specified by the base sequence set forth in SEQ ID NO: 12.

[0066] The RNA specified by the base sequence of SEQ ID NO: 11 and / or the RNA specified by the base sequence of SEQ ID NO: 12 may include a modified artificial nucleic acid. In one example, the RNA is preferably produced using modified UTP in which uridine is substituted with N1-methylpseudouridine.

[0067] The nucleic acids encoding the TCR α chain and the TCR β chain may be separate nucleic acids that are incorporated into separate vectors, or a single nucleic acid encoding both the TCR α chain and the TCR β chain may be incorporated into a single vector. In this specification, one or more of these vectors may be referred to as the "vector of the present invention."

[0068] In one example, the nucleic acid of the present invention can be incorporated into an appropriate vector, which is well known in the art, including, but not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral (AVV) vectors, Sendai viral vectors, and plasmid vectors.

[0069] In one example, the recombinant expression vector of the present invention can include regulatory sequences, such as transcription and translation initiation and termination codons, that are specific for the cell (e.g., bacteria, fungi, plants, or animals) into which the vector will be introduced.

[0070] In one example, the recombinant expression vector of the present invention may contain other sequences as long as they enable expression of the TCR of the present invention in a host cell. In one example, the other sequences may include promoters such as a lacZ promoter, a T7 promoter, a murine stem cell virus (MSCV), a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, a CAG promoter, or an EF1α promoter.

[0071] Other examples include a secretory signal peptide coding sequence, an enhancer sequence, a repressor sequence, an insulator sequence, an origin of replication, a reporter protein (e.g., a fluorescent protein), a drug resistance gene coding sequence, etc. In addition, in one example, when suppressing the expression of an endogenous TCR, the sequence may include a sequence encoding a short RNA (e.g., siRNA, miRNA, etc.) that suppresses the expression of an endogenous TCR.

[0072] The vectors of the present invention may be designed to transiently express or stably express the TCRs of the present invention. Furthermore, the recombinant expression vectors may be designed to constitutively express or inducibly express the TCRs of the present invention. Furthermore, the recombinant expression vectors may be designed to contain a suicide gene.

[0073] The vector of the present invention may be added with a sequence encoding a protein tag, a signal sequence, or a labeling substance, such as a sequence encoding biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, nuclear export signal (NES), nuclear localization signal (NLS), GFP, or other known sequences.

[0074] 3. Cells transfected with a TCR gene In one example, the present invention provides cells transfected with the nucleic acid of the present invention or the vector of the present invention (herein, such cells may be referred to as "cells of the present invention"). The cells of the present invention can be obtained by introducing the nucleic acid of the present invention or the vector of the present invention into a host cell. The cells of the present invention express a TCR specific for an HSP105-derived peptide.

[0075] In one example, the present invention provides a cell transfected with a combination of nucleic acids, including a nucleic acid encoding a T cell receptor alpha chain identified by the amino acid sequence set forth in SEQ ID NO:7 and a nucleic acid encoding a T cell receptor beta chain identified by the amino acid sequence set forth in SEQ ID NO:8.

[0076] In one example, the present invention provides a cell transfected with a vector comprising a combination of nucleic acids, the combination comprising a nucleic acid encoding a T cell receptor alpha chain identified by the amino acid sequence set forth in SEQ ID NO:7 and a nucleic acid encoding a T cell receptor beta chain identified by the amino acid sequence set forth in SEQ ID NO:8.

[0077] The cells of the present invention may be cells into which separate nucleic acids encoding the TCRα chain and the TCRβ chain have been transfected, or may be cells into which a single nucleic acid encoding both the TCRα chain and the TCRβ chain has been transfected. The cells of the present invention may be cells into which multiple vectors have been transfected, each of which contains separate nucleic acids encoding the TCRα chain and the TCRβ chain, or may be cells into which a single vector has been transfected with a single nucleic acid encoding both the TCRα chain and the TCRβ chain.

[0078] The nucleic acid of the present invention or the vector of the present invention can be introduced into host cells and target tissues using methods known to those skilled in the art. Introduction into host cells can be performed using, for example, electroporation, lipofection, microinjection, biolistics, calcium chloride, calcium phosphate, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, etc. In one example, electroporation is preferred.

[0079] The host cells into which the nucleic acid or vector of the present invention is introduced are not particularly limited and may be either prokaryotic or eukaryotic. Examples of eukaryotic cells that can be used include animal cells, plant cells, insect cells, and fungal cells.

[0080] In one example, when amplifying or replicating a vector of the present invention, the host cell may be a prokaryotic cell such as E. coli, e.g., a DH5α cell. In one example, when producing a recombinant T cell receptor or a polypeptide thereof, the host cell is preferably an animal cell, more preferably a mammalian cell, and even more preferably a human cell.

[0081] In one example, the TCR of the present invention, the nucleic acid of the present invention, or the vector of the present invention can be introduced into a host cell for in vivo or ex vivo gene therapy.

[0082] In one example, when producing a cell preparation for use in immunotherapy or the like, the host cells used are peripheral blood mononuclear cells (PBMCs) and peripheral blood lymphocytes (PBLs), cells prepared from pluripotent stem cells, embryonic stem (ES) cells, or induced pluripotent stem (iPS) cells. Peripheral blood mononuclear cells (PBMCs), more specifically, αβ T cells and their subsets, CD8 T cells, contained in PBMCs and PBLs, are used. ＋ T cells, CD4 ＋ T cells, CD4 ＋ CD8 － T cells, CD4 － CD8 ＋ T cells, CD4 ＋ CD8 ＋ T cells, CD3 ＋ CD8 － T cells, CD3 － CD8 ＋ T cells, CD3 ＋ CD8 ＋These include T cells, T cells prepared from pluripotent cells, αβ T cells, γδ T cells, B cells, dendritic cells, NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), and groups of these cells derived from pluripotent stem cells, embryonic stem (ES) cells, or induced pluripotent stem (iPS) cells. In one example, when pluripotent stem cells are used as host cells, the nucleic acid or vector of the present invention may be transfected into ES cells or iPS cells, followed by differentiation into immune cells such as T cells under appropriate culture conditions known to those skilled in the art. Alternatively, the nucleic acid or vector of the present invention may be transfected into pluripotent stem cells under appropriate culture conditions known to those skilled in the art, followed by differentiation into immune cells such as T cells.

[0083] In one example, the present invention relates to a cell transfected with a combination of nucleic acids comprising a nucleic acid encoding a T cell receptor α chain identified by the amino acid sequence set forth in SEQ ID NO: 7 and a nucleic acid encoding a T cell receptor β chain identified by the amino acid sequence set forth in SEQ ID NO: 8, and the cell is preferably a T cell, an NK cell, an invariant NK cell, an NKT cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell.

[0084] In a further example, the present invention relates to a cell transfected with a vector comprising a combination of nucleic acids, the combination comprising a nucleic acid encoding a T cell receptor α chain identified by the amino acid sequence set forth in SEQ ID NO: 7 and a nucleic acid encoding a T cell receptor β chain identified by the amino acid sequence set forth in SEQ ID NO: 8, and the cell is preferably a T cell, an NK cell, an invariant NK cell, an NKT cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell.

[0085] In one embodiment, the host cell is preferably a T cell, and more preferably a T cell that does not have an endogenous T cell receptor. In one example, the T cell that does not have an endogenous T cell receptor is a CD3 － CD8 ＋ Even more preferably, it is a T cell.

[0086] In one example, to prevent mispairing between the four TCR chains (the endogenous TCR α and β chains in the host cell and the genetically introduced TCR α and TCR β chains of the present invention) and the resulting unintended generation of new antigen specificities, it is preferable to knock out or knock down the endogenous TCR chains in the host cell before introducing the nucleic acid or vector of the present invention. That is, T cells lacking endogenous T cell receptors can be produced by knocking out or knocking down the expression of endogenous TCR chains. For example, before introducing the TCR gene of the present invention, the endogenous TCR in the host cell can be knocked out using known techniques such as the CRISPR-Cas9 system. Alternatively, the endogenous TCR can be knocked down using known techniques such as siRNA, shRNA, miRNA, or antisense nucleic acid. Host cells in which the endogenous TCR has been knocked out or knocked down can be selected and sorted using known methods, such as flow cytometry or immunomagnetic methods.

[0087] "T cells that do not have endogenous T cell receptors" refer to cells in which the expression level of endogenous T cell receptors on the cell surface is 10% or less, preferably 5% or less, and more preferably 3% or less, compared to T cells before the endogenous T cell receptor is knocked out or knocked down. The expression level of endogenous T cell receptors on the cell surface can be measured by flow cytometry, ELISA, or the like.

[0088] In one example, T cells of a host in which endogenous TCR has been knocked out can be selected by recovering a CD3-negative fraction using magnetic beads to which an anti-CD3 antibody has been immobilized. In one example, cytotoxic T cells can be selected from the recovered T cells in which endogenous TCR has been knocked out by a known method, for example, by recovering a CD8-positive fraction using magnetic beads to which an anti-CD8 antibody has been immobilized. That is, cytotoxic T cells in which endogenous TCR has been knocked out can be selected from peripheral blood mononuclear cells of a subject and used as host cells.

[0089] In one example, by introducing the nucleic acid of the present invention into a T cell in which the endogenous TCR has been knocked out, a heterodimer can be formed by combining the α chain and β chain of the TCR of the present invention. As a result, an HSP105 peptide-specific TCR can be expressed in the host cell while avoiding adverse events such as off-target effects. In one example, the host cell can express the HSP105 peptide-specific TCR for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after introduction of the TCR gene.

[0090] In one example, the cells of the present invention can be a population containing a host cell comprising a nucleic acid of the present invention or a vector of the present invention and at least one other cell, such as a non-transfected host cell (e.g., a T cell, a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc.).

[0091] In one example, host cells expressing the TCR of the present invention, such as cytotoxic T cells, can be isolated and purified from such a cell population using methods known to those skilled in the art, such as flow cytometry or immunomagnetic methods.

[0092] In one example, the cells of the present invention can be isolated by co-culturing T2 cells (a lymphoblastoid-derived cell line expressing HLA-A2) to which an HSP105-derived peptide has been added, and single-sorting cells expressing CD8 and the degranulation marker CD107a using flow cytometry.

[0093] In one example, the cell of the present invention can be a clonal population of cells composed of a single host cell containing the nucleic acid of the present invention or the vector of the present invention. In one example, the cell of the present invention can be a clonal population of cytotoxic T cells introduced with the TCR gene of the present invention.

[0094] In one example, cytotoxic T cells genetically introduced with the TCR of the present invention (sometimes referred to herein as "HSP105-TCR-T cells") can recognize HLA-A*02:01. The HSP105-TCR-T cells of the present invention recognize antigen-presenting cells that present HSP105-derived peptides bound to MHC molecules, and can have HLA-A2-restricted binding specificity for HSP105-derived peptides. The HLA-A2-restricted HSP105-derived peptide-specific immune responsiveness of HSP105-TCR-T cells can be evaluated by measuring the amount of interferon-γ secreted and / or cytotoxic activity, according to the Examples described below.

[0095] In one example, the HSP105-TCR-T cells of the present invention can recognize a peptide-MHC complex between an HSP105-derived peptide and an MHC class I molecule in the variable region of the TCR chain of the present invention, and can induce interferon-γ secretion and / or cytotoxicity against cells having such a peptide-MHC complex. In one example, the HSP105-TCR-T cells of the present invention can recognize cells expressing HSP105, which is a target antigen, and can induce interferon-γ secretion and / or cytotoxicity against such cells or tissues. In one example, the HSP105-TCR-T cells of the present invention can recognize a peptide-MHC complex or cells expressing HSP105 and induce interferon-γ secretion and / or cytotoxicity at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after TCR gene introduction.

[0096] The HSP105-TCR-T cells of the present invention can be cultured according to known methods, without particular limitation. For example, when animal cells are used as host cells, culture media such as DMEM, MEM, RPMI 1640, and IMDM can be used. The culture medium may be serum-free, or human serum or fetal bovine serum (FBS) can be used as a supplement. The culture medium may be feeder-free, or feeder cells may be used. Growth factors may be added to promote cell proliferation. Cell culture can usually be performed at pH 6-8, approximately 30-40°C, for approximately 15-200 hours. Medium replacement and agitation can be performed as needed. The cells of the present invention can be subcultured as needed, or can be cryopreserved.

[0097] In one example, when human serum is used, serum from a subject to be treated may be used. When feeder cells are used, feeder cells inactivated by irradiating peripheral blood mononuclear cells may be used. When a growth factor or a T cell stimulating antibody is used to increase the cell survival rate and / or proliferation rate, for example, IL-2, IL-15, IL-7, anti-CD3 antibody, anti-CD2 antibody, or the like may be used.

[0098] 4. Genetically Modified T Cell Therapy (TCR-T Cell Therapy) The T cell receptors, nucleic acids, vectors, cells, or pharmaceutical compositions containing them of the present invention can be used in methods for treating cancer.

[0099] Genetically modified T cell therapy (TCR-T cell therapy) is a type of adoptive immunotherapy in which T cells are collected from a living body or the like, activated by antigen stimulation or genetically modified outside the body, and the T cells that have cytotoxic activity against target cells are proliferated and then transplanted back into the patient. The T cells to be activated or modified may be T cells collected from the patient himself or herself or from another person, or may be pluripotent stem cells such as ES cells or iPS cells, or may be immune cells differentiated from pluripotent stem cells.

[0100] In one example, the T cell receptor of the present invention, any nucleic acid or vector comprising a nucleic acid encoding a TCR of the present invention, or any host cell comprising any of them, can be used in genetically modified T cell therapy (TCR-T cell therapy) for the treatment of cancer.

[0101] The subjects of treatment include humans and non-human mammals, such as humans or experimental animals such as mice. In one example, the subject may be a subject with cancer or a solid tumor, in which treatment to inhibit cell proliferation or tumor growth is required.

[0102] "Cancer" includes carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant lymphoma, such as squamous cell carcinoma, lung cancer, lung adenocarcinoma, peritoneal cancer, digestive cancer, stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, anal carcinoma, penile carcinoma, and head and neck cancer.

[0103] The term "solid tumor" refers to a mass of cells (tumor mass) formed in a specific tissue or organ through uncontrolled cell division without the control of the cell cycle, whether benign or malignant. Examples of solid tumors include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0104] Examples of cancers or solid tumors to be treated include, but are not limited to, colon cancer, esophageal cancer, pharyngeal cancer, pancreatic cancer, breast cancer, melanoma, hepatocellular carcinoma, etc. These cancers or solid tumors express HSP105.

[0105] In one example, the HSP105-TCR-T cells of the present invention are HLA-A2 restricted, recognize HSP105-derived peptides, and exhibit cytokine secretion and / or cytotoxic activity. In one example, the HSP105-TCR-T cells of the present invention recognize cancer cells that express HSP105, and exhibit cytokine secretion and / or cytotoxic activity against cancer cells that express HSP105. In one example, the HSP105-TCR-T cells can suppress the growth of tumors containing cells that express HSP105.

[0106] In one example, a nucleic acid encoding the TCR of the present invention can be introduced into cytotoxic T cells (CTLs) collected from a patient, and the patient's CTLs can be genetically engineered to express the TCR of the present invention. In one example, the genetically engineered T cells can be cultured and expanded, and then administered back to the patient. The genetically engineered T cells express the TCR of the present invention, can recognize peptide-MHC complexes of HSP105-derived peptides and MHC molecules, and can secrete cytokines and / or exhibit cytotoxic activity against target cancer cells that express HSP105. As a result, patients who receive the HSP105-TCR-T cells of the present invention can experience suppression of cell proliferation or tumor expansion in cancer or solid tumors.

[0107] In one example, peripheral blood mononuclear cells (PBMCs) are separated from peripheral blood collected from a patient by centrifugation, and endogenous TCRs of the host cells can be knocked out using known techniques such as the CRISPR-Cas9 system. Furthermore, cytotoxic T cells in which the patient's endogenous TCR has been knocked out can be fractionated using known methods such as flow cytometry or immunomagnetic methods. For example, the CD3-negative fraction can be collected using magnetic beads immobilized with an anti-CD3 antibody, and the CD8-positive fraction can be collected using magnetic beads immobilized with an anti-CD8 antibody, thereby selecting and isolating cytotoxic T cells in which the patient's endogenous TCR has been knocked out.

[0108] In one example, by introducing the nucleic acid of the present invention into cytotoxic T cells in which the patient's endogenous TCR has been knocked out, it is possible to cause the patient-derived cytotoxic T cells to express an HSP105-derived peptide-specific TCR consisting of a combination of the α chain and β chain of the TCR of the present invention. The HSP105-TCR-T cells administered to the patient can recognize target cells expressing HSP105, such as cancer cells, and exhibit cytokine secretion and / or cytotoxic activity against target cancer cells expressing HSP105.

[0109] In one example, genetically modified T cell therapy can be administered in conjunction with other cancer treatments, such as chemotherapy, radiation therapy, surgery, and immunological checkpoint inhibitors, including, for example, anti-cytotoxic T-lymphocyte-associated antigen (CTLA-4) antibodies, anti-programmed cell death (PD)-1 antibodies, and anti-PD-ligand-1 (PD-L1) antibodies.

[0110] 5. Cancer Therapeutic Agents The present invention provides cancer therapeutic agents comprising a T cell receptor that recognizes a complex of an amino acid sequence RLMNNDMTAV, which is a partial peptide derived from HSP105, and a human MHC class I molecule, a combination of nucleic acids comprising a nucleic acid encoding a T cell receptor α chain specified by the amino acid sequence of SEQ ID NO:7 and a nucleic acid encoding a T cell receptor β chain specified by the amino acid sequence of SEQ ID NO:8, or a vector comprising said combination of nucleic acids, or a cell transfected with said combination of nucleic acids or vector.

[0111] In one example, the cancer therapeutic agent of the present invention contains the TCR of the present invention, the nucleic acid of the present invention, the vector of the present invention, or the cell of the present invention as an active ingredient, and may also contain a pharmaceutically acceptable carrier or other ingredients, and the present invention provides such a formulated pharmaceutical composition. Herein, the above-mentioned cancer therapeutic agent may be referred to as the "cancer therapeutic agent of the present invention" or, interchangeably, as the "pharmaceutical composition of the present invention."

[0112] The cancer therapeutic agent of the present invention can be administered to a patient as a cell preparation for treating cancer or solid tumors, comprising cells expressing the TCR of the present invention. In one example, when the pharmaceutical composition is a cell preparation, the following components may be included as pharmaceutically acceptable carriers or other components: An isotonic carrier may include normal saline, phosphate buffered saline (PBS), NORMOSOL R electrolyte solution, PLASMA-LYTE A, dextrose, or Ringer's lactate. A lipophilic solvent may include a fatty oil such as sesame oil, a synthetic fatty acid ester such as ethyl oleate or a triglyceride, or a liposome. A component for imparting viscosity to the suspension may include sodium carboxymethylcellulose, sorbitol, or dextran. A component for protecting cells may include, for example, dimethyl sulfoxide (DMSO) or serum albumin. A component for activating, proliferating, or differentiating cells may include, for example, vitamins, cytokines, growth factors, or steroids. A component for preventing bacterial contamination may include, for example, antibiotics.

[0113] Examples of cancers or solid tumors to be treated include, but are not limited to, colon cancer, esophageal cancer, pharyngeal cancer, pancreatic cancer, breast cancer, melanoma, hepatocellular carcinoma, and the like.

[0114] The dose of the cancer therapeutic agent of the present invention to be administered is desirably an amount sufficient to induce an immune response in the subject. In one example, the dose is determined by a known method based on the efficacy of the cancer therapeutic agent of the present invention and the condition and weight of the subject (e.g., human), taking into consideration the presence, nature, and extent of adverse side effects. The immune response in the subject can be confirmed, for example, by measuring the volume of the target tumor and monitoring the inhibition or reduction of tumor growth, or by culturing peripheral blood mononuclear cells collected from a tumor specimen of the subject and monitoring cytokine secretion or the expression of the degranulation marker CD107a. In one example, when the active ingredient of the cancer therapeutic agent is one or more cells, the dose per administration is 1 x 10 ４ pieces ~ 1×10 １０The dosage for inducing a short-term or long-term immune response in a subject can be determined by assays well known in the art, for example, a model in which cancer cells are transplanted into immunodeficient mice to form tumor masses, and then gene-transduced immune cells are administered.

[0115] The administration method of the cancer therapeutic agent of the present invention is not particularly limited, and can be, for example, intravenous injection, intraarterial injection, intraportal vein injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or intratumoral administration. In one example, intravenous injection is preferred. Systemic administration or local administration is also possible. Local administration can be performed by direct injection into the target tissue, organ, or tumor. A single administration or multiple repeated administrations can be performed. In the case of multiple administrations, the administration form and dosage can be changed appropriately for each administration. In one example, a second administration can be performed 7 days after the first administration. Generally, cells are prepared as, for example, an injectable liquid solution or suspension and can be injected intravenously, intradermally, or subcutaneously.

[0116] The cancer therapeutic agent of the present invention can be manufactured by known methods such as mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing.

[0117] In one example, a T cell receptor linked to an effector therapeutic agent can be used as an active ingredient. For example, the T cell receptor of the present invention can be used to target HSP105-expressing cancer cells of a subject and deliver an effector molecule to induce cancer cell damage or apoptosis.

[0118] In one example, the T cell receptor of the present invention can be used to screen subjects for whom the cancer therapeutic agent of the present invention is effective. For example, a cell or tissue sample from a subject suspected of having colon cancer, esophageal cancer, pharyngeal cancer, pancreatic cancer, breast cancer, melanoma, or hepatocellular carcinoma, which express HSP105, can be contacted with a T cell receptor labeled with a detectable label, such as gold particles, to form a complex, which can then be detected. Subjects for whom a complex is detected may be subjects for whom the cancer therapeutic agent of the present invention is effective.

[0119] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0120] <Screening of HLA-A2-restricted HSP105-specific T cells> Patients with HLA-A2 haplotypes who received 3 mg of HSP105 peptide vaccine were screened for T cells reactive to HSP105 peptides. Peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood collected from the patients by centrifugation. 5 x 10 PBMCs were collected. ５ Cells / well were applied to a 96-well flat-bottom plate and cultured in RPMI 1640 (10% FBS) medium containing 10 μg / ml of an HSP105-derived peptide (amino acid sequence A2-7: RLMNNDMTAV (SEQ ID NO: 17), sometimes referred to herein as "A2-7" or "HSP105 A2-7"). Ex vivo immune monitoring was performed every two weeks by ELISPOT assay to monitor secreted IFN-γ (Figure 1). As a negative control, 10 μg / ml of an HIV (human immunodeficiency virus)-derived peptide was added instead of the HSP105-derived peptide. As a positive control, 50 ng / ml of phorbol myristate acetate (PMA) and 2 μg / ml of ionomycin were added instead of the HSP105-derived peptide. The difference between the number of spots in the group to which the HSP105-derived peptide was added and the number of spots in the negative control group that were monitored is shown in FIG. 1 as the number of HSP105-derived peptide-specific spots.

[0121] Each peptide was further added to the PBMCs at 10 μg / ml, and the PBMCs were stimulated and cultured for 2 weeks in a culture medium containing IL-2 50 IU / ml and IL-15 10 ng / ml. CD8-positive T cells were isolated using microbeads and cultured at 1 × 10 ５ The target cells were T2 cells (lymphoblastoid cell line expressing HLA-A2) pulsed with HSP105-derived peptides and exogenously bound to the target cells, and the cells were suspended in 5 × 10 cells / well. ４ The cells were used in a 100% RT-PCR assay. Figure 2 shows the results of immune monitoring evaluation of antigen stimulation-dependent IFN-γ production in vitro. The difference in the number of spots in the monitored HSP105-derived peptide-added group and the number of spots in the negative control group is also shown in Figure 2 as the number of HSP105-derived peptide-specific spots. From these immune monitoring experiments, subjects in which IFN-γ production dependent on HSP105-derived peptide antigen stimulation was observed were identified.

[0122] <Establishment of HLA-A2-restricted HSP105-specific T cell clones> Patients with the HLA-A2 haplotype who exhibited IFN-γ production dependent on HSP105-derived peptide antigen stimulation were administered 3 mg of a peptide vaccine containing HSP105-derived peptides. Peripheral blood mononuclear cells (PBMCs) were isolated from lymph node tumor specimens from the patients after administration. These PBMCs were co-cultured for 20 hours with target cells (T2 cells) exogenously bound to HSP105-derived peptides, as described above, and antigen-stimulation-dependent IFN-γ production was confirmed by ELISPOT assay (Figure 3). Furthermore, to select T cells expressing TCR genes reactive to HSP105-derived peptides from the patient's peripheral blood, peripheral blood from patients who had received a cancer peptide vaccine containing HSP105-derived peptides was stimulated in the presence of antigen. After stimulation, the cells were again co-cultured with T2 cells exogenously bound to HSP105-derived peptides, and T cells that recognized the antigen and showed degranulation were detected by a CD107a assay (a degranulation evaluation method utilizing the fact that CD107a molecules, which are normally present intracellularly, are exposed to the cell surface when degranulation occurs). CD107a-positive CD8-positive T cells shown in the bold frame in Figure 3 were isolated by single sorting and cultured in AIM-V (10% ABS, IL-2 100 / mL, IL-15 10 ng / mL) to establish T cell clones (Figure 3).

[0123] <Evaluation of IFN-γ production of HLA-A2-restricted HSP105-specific T cell clones> Each established T cell clone was co-cultured for 20 hours with target cells (T2 cells) exogenously bound to HSP105-derived peptides, the HSP105- liver cancer cell line HepG2, and the HSP105+ colon cancer cell line SW620, and antigen stimulation-dependent IFN-γ production was evaluated by ELISPOT assay. Antigen specificity was evaluated using cells bound to HIV-derived peptides as a negative control. Effector cells were used at 1 x 10 ５ cells / well, target cells were 5 x 10 ４As shown in Figure 4, the established HLA-A2-restricted HSP105-specific T cell clone produced IFN-γ when co-cultured with HLA-A2 T2 cells in the presence of peptide, and furthermore, it recognized the HSP105+ colon cancer cell line SW620 and produced IFN-γ.

[0124] <Cytotoxic activity of HLA-A2-restricted HSP105-specific T cell clone> T2 cells bound to exogenous peptides were used as target cells and labeled with a fluorescent dye (calcein AM). 1 × 10 target cells were ４ and 3 × 10 HLA-A2-restricted HSP105-specific T cell clones (effector cells). ４ pieces (ET ratio = 3:1), 10 x 10 ４ (ET ratio = 10:1), 30 x 10 ４ The cells were co-cultured with calcein AM (ET ratio = 30:1) for 4 hours, and fluorescence was measured at 0 and 4 hours after the start of co-culture using a Terascan VPC (Minerva Tech). Cytotoxic activity against target cells was assessed using the decay of fluorescence at 0 and 4 hours as an indicator. Calcein AM emits strong fluorescence when present within target cells, but upon target cell injury, intracellular calcein AM is released into the culture medium, weakening the fluorescence. The difference in fluorescence between 0 and 4 hours allowed the calculation of the percentage of cytotoxic activity. Antigen specificity was assessed using cells bound to an HIV-derived peptide (HIVA2) as a negative control. Cytotoxic activity was calculated using the following formula: For spontaneous release, a group seeded with only calcein AM-labeled target cells was used; for maximum release, a group in which target cells were lysed with NP40 and all calcein AM leaked was used.

[0125] The formula for calculating cytotoxic activity is shown below: Cytotoxic activity (%) = (difference in fluorescence intensity of co-culture group between 0 hours and 4 hours - difference in fluorescence intensity of spontaneous release between 0 hours and 4 hours) / (difference in maximum fluorescence intensity of free release between 0 hours and 4 hours - difference in fluorescence intensity of spontaneous release between 0 hours and 4 hours) x 100. As shown in Figure 5, the HLA-A2-restricted HSP105-specific T cell clones exhibited cytotoxic activity against T2 cells exogenously bound to HSP105 peptide in an E:T ratio-dependent manner.

[0126] <Confirmation of cytotoxic response of HLA-A2-restricted HSP105-specific T cell clones by CD107a assay> HLA-A2-restricted HSP105-specific T cell clones were cultured at 10 × 10 ４ and 1 × 10 cells / ml of T2 cells supplemented with HSP105 peptide, T2 cells supplemented with HIV peptide, HSP105+ (positive) colon cancer cell line SW620, or HSP105- (negative) liver cancer cell line HepG2. ４ The HLA-A2-restricted HSP105-specific T cell clones were co-cultured in RPMI medium containing 10% FBS and 1% penicillin, streptomycin, and L-glutamine. After 4 hours, the expression of CD107a in the HLA-A2-restricted HSP105-specific T cell clones was analyzed by flow cytometry. The horizontal axis represents CD8, which identifies the HLA-A2-restricted HSP105-specific T cell clones. The vertical axis represents CD107a, with dots moving upward as the T cells recognize cancer cells. As a result, as shown in Figure 6, it was confirmed that the expression of CD107a in the HLA-A2-restricted HSP105-specific T cell clones increased only in the group co-cultured with T2 containing HSP105 peptide and the HSP105+ colon cancer cell line SW620, which endogenously expresses HSP105 (boxed). On the other hand, in T2 cells and the HSP105-liver cancer cell line HepG2 to which HIV peptides had been added, CD107a expression did not increase, confirming that an antigen-specific cytotoxic response was induced.

[0127] <Identification of the TCR gene of HLA-A2-restricted HSP105-specific T cell clone> The established HLA-A2-restricted HSP105-specific T cell clone was subjected to full-length TCR gene sequence analysis using a capillary sequencer. The amino acid sequence and gene sequence of the HLA-A2-restricted T cell receptor (HSP105-TCR) that recognizes an HSP105-derived peptide (amino acid sequence RLMNNDMTAV) isolated from the HLA-A2-restricted HSP105-specific T cell clone are shown below. The amino acid sequence of the HLA-A2-restricted HSP105-specific T cell receptor is also shown in Figure 7. Figure 8 shows the gene sequence of the T cell receptor identified from the amplification product of the HLA-A2-restricted HSP105-specific T cell clone.

[0128] SEQ ID NO: 1: TCR α chain CDR3 of HLA-A2-restricted HSP105-specific T cell clone (amino acid sequence): CAYRSPVSGTYKYIF

[0129] SEQ ID NO: 2: TCR β chain CDR3 of HLA-A2-restricted HSP105-specific T cell clone (amino acid sequence): CASSLRGNTGELFF

[0130] SEQ ID NO: 3: TCRα chain CDR1 (amino acid sequence) of HLA-A2-restricted HSP105-specific T cell clone: ​​TSESDYY

[0131] SEQ ID NO: 4: TCR α chain CDR2 of HLA-A2 restricted HSP105 specific T cell clone (amino acid sequence): QEAYKQQN

[0132] SEQ ID NO: 5: TCR β chain CDR1 (amino acid sequence) of HLA-A2-restricted HSP105-specific T cell clone: ​​SEHNR

[0133] SEQ ID NO: 6: TCR β chain CDR2 (amino acid sequence) of HLA-A2-restricted HSP105-specific T cell clone: ​​FQNEAQ

[0134] SEQ ID NO: 7: Full length TCR α chain of HLA-A2 restricted HSP105 specific T cell clone (amino acid sequence): MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRSPVSGTYKYIFGTGTRLKVLANI QNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDF ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLKVAGFNLLMTLRLWSS

[0135] SEQ ID NO: 8: Full-length TCR β chain of HLA-A2-restricted HSP105-specific T cell clone (amino acid sequence): MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLRGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSAEISH TQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0136] Accession No. 9: Full-length TCR α chain (DNA sequence) of HLA-A2-restricted HSP105-specific T cell clone: ATGGCCATGCCCTGGCTTCCTGTGGGCTTGTTGATCTCCACCTGTCTTGAAATTTTAGCATGGCTCTGAGACAGTCACTCTAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACCCTGACCTGAGCTGCACATATGACACCAATGAGAGTGATTATTATTTATCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATCTCGTTATTCCGCCAAGAAGCTTATAAGCAAACAGAAATGCAACAGAGAAATCGTTTTCTCTGTGAAACTTCCAGAAAAGCAGCCAAAATCCTTCAGTCTCAAGATCTCTAGACTCTACAGCTGGGGGGATGCCGCAGATGTATTTTCTGTGCTTATAGGAGCCCTGTATCAGGAACCTACAAAAATACATCTTTGGAAACAGGCACCAGGCCTGAAGGTTTTTAGCAAAATATCCAGAACCTGACCCCTGCCGTGTACCAGCTGAGAGACTCTAAAAACCAGTGACAAGTCCTGTCTGCCATTTCCACCGATTTTGATTTCTCAAAACAAAATGTGTCCACAAAAGTAAGGATTTCTGATGTGTATATTCACAGACAAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAAACAGTGCTGTGGCCCTGGAGCAAACAAATCTGACTTTGCATGTGCAATACGCCCTTCAAACAAACAGCATTTATTCAGAAGACACCTTCTTCCCCCAGCCCCAGAAAAGTTCCTGTGATGTCCAAGCTGGTCGAGAAAAAGCTTTGAAAAACAGATACGAAACCTAAAAACTTTAAAAAACTGTCCAGTGATTGGGTTCCGAAATCCTCCCTCCCTGAAAAGTGGCCGGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCTAGCTGAA

[0137] Accession Number 10: Full-length TCRβ chain (DNA sequence) of HLA-A2-restricted HSP105-specific T cell clone: ATGGGCACCAGCCCTCTCTGCTGGATGGCCCTGTGTCTCCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCCCAGGACCCCAGACACAAGATCACAAAGAGGAGGACAGAGATGTAACTTTCCAGGTGTGATCCAAATTTCTGAACACAACCGCCCTTTATTGGTTACCGACAGACCCCTGGGGGCAGGGCCCCAGAGTTTCTGACTTACTTCCAGAAATGAAGCTCAACTAGAAAAAATCAAGGCTGCTCTAGTGATCGGTTCCTCTGCAGAGAGGGCCAAGGGATCTTTCTCCACCTTGGAATCCAGCGCACAGAGCAGGGGGGGACTCCGGCCATGTATCTCTGTGCCAGCAGCTTAAGGGGAAACACCGGGGGAGCTGTTTTTTTGGAGAAGGCTCTAGGCAGACCGTACTGGAGGACCAAAAAACGTGTTCCCACCCCGAGGTCCGCTGTGTTTGAGCCATCAGAAGCAGAGAGATCTCCCCACACCCAAAAGGCCACACTGGGTGTGCCCTGGCCACAGGCCTCTACCCCCGACCACGTGGAGCTGAGCTGGGTGGGGTAATGGGGAAGGAGGGCGTGCGCACAGTGGGGGTTCAGCACAGACCCCGCAGCCCCCTCAAGGAGCAGCCCCGCCCCCTCAATGACTCCTAGATACTGCCCTGAGCAGCCGCCCTGAGGGTCTCCGGCCACCTTCTGGCAACACCCTCCGCAACACCCTCCGCTGTCAATGTCCAGTTCTACGGGGCTCTCCGGAGAAATGACGAGTGGACCCTAGGGATAGGGCCAAACCTGTCCACCCAGATCGTCAGCGCCGAGGGCCCTGGGGGTAGAGCAGAGACTGTGGCTTCCACCTCCGAGTCTTACCAGCAAGGGGTTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTAATGCCGTGCTGGTCAGTGCCCCTCGTGCTGATGGCCATGGTCAATGAGAAAGGATTCCAGAGGGCTAG

[0138] Accession No. 11: Full-length TCR α-chain (RNA sequence) of HLA-A2-restricted HSP105-specific T cell clone: AUGGC AUGCCCUGGCUUC CUGUGGGCA CUU GUGAUCUCCACCUGUCUUGAAUUUA GCAUGGCUC AGA CAGUCACUCA GUCUCAA CCA GAG AUGUCUGUGCAGGA GGCA GAG ACCGUGACCCUGAGCUGCA CAUAUGACACCAGUGAGAGUGA UUAAUUAUUUAUUCUGGUACAAGCAGCCUCCCA GCAGGCA GAG AUGAUCUC GUUAUUC GCCAAGAAGCUUAAGA CAACAGA AUGCAA CAGA GAAUC GUUUUCUCUGUGAA CUUCCAGA AAGCAGCCAAAUCCUUCA GUCUCAAGAUCUCAGA CUCACA GCUGGGGGG AUGCCGCG AUGUAUUU CUGUGC UUAUAGGAGCCCU GUAUCAGGA ACCUACAAAUA CACAUCUUU GGAACAGGCAC CAGGCUGAAGGUUUUUAGCAAAUAUCCAGA ACCCUGACC CUGCCGUGUACCA GCUGAGA GACUCUAAAUCCAGUGA CAAGU CUGUGCUGCCU AUUCA CCGAUUUUGA UUCUCAAA CAA AUGUGUCACA AAGUAAGG AUAUCUGAUGUGUAUAUCA CAGA CAAAAACUGUGCUAGACAUGAGGU CUAUAGGACUCUAAGAGCAA CAGU GCU GUGGCCUGGA GCAACAAAUCUGACUUU GCAUGUGCAAACGCCUUCAA CAA CAGCAUUAAUUC CAGA AGACAC CUUCUUC CCCCA GC CCCAGA AAGUUCCUGUGAUGUCAAGCUGGUCGAGA AAGCUUUUGAAACAGAUA CGA AACCUAAA CUUUUCAAAACCUGUCAGUGA UUGGGUUCCGA AUCUCUCCUCCUGAAAGUGGCCGGGUUUUAUCUGCUC AUGA CGCUGCGGGCUGUGGUC CAGC

[0139] Accession Number 12: Full-length TCRβ chain (RNA sequence) of HLA-A2-restricted HSP105-specific T cell clone: AUGGGCACCAGCCUCCUCUGCUGGAUGGCCCUGUGUCUCCUGGGGGCAGAUCAACGCAGAUACUGGAGUCUCCCAGGAUCCCCAGACACAAGAUCAACAAGAGGGGGACAGAAUGUAAACUUUCAUGUGUGAUCAAUUUUCUGAACACAAACCGCCUUUAUUGGUACCGACAGACCUGGGGGCAGGGGCCCCAGAGUUUUCUGACUUACUUCUCCAGAAUGAAGCUCAAACUAGAAAAAAUCAAGGCCUGCUCAGUGAUCUGGUUCUCUCUGCAGAGAGGGCCUAAGGGAUCUUUUCUCCACCUUGGAGAUCUCCAGCGCACAGAGCAGGGGCGACUCGGCCAUUGUAUCUCUGUGGCCAGCAGCUUAAGGGGAAACACCUGGGGAGCUGUUUUUUUGGAGAAGGCCUCUAGGCUGACCGAUACUGGAGGGACCUGAAAAAACGUGUUCUCCCACCUCGAGGGUCUGCUGUGUUUGAGCCAUUCAGAAGCAGAGAUUCUCCACCACACCAAAAGGCCACACUGGUGUGUGCCUGGCCACAGGCUUUCUACCUCCGACCACGUGGAGCUGAGCUGGUGGUGGAUGGGGAACGGAGGGUGGCACAGUGGGGGUCAGCACAGACCUGCGAGCCCCUCAAGGAGCAGCCCCGCCCCUCAAUGACUCCAGAUACUGCCUGAGCAGCCGCCUGAGGGUCUCUCGGCCACCUUUCUGGCCAGAACCCCCCGCAACACCUUCCGCCUGUCAAGUCUCCAGUUUCUACGGGGCUCUCUCGGAGAAUGACGAGUGGACCUCAGGAUAGGGCCAAAACCUGUCACCUCCAGAUCGUCAUCGCCGAGGGCCUGGGGGUAGAGCAGACUGUGGGCUUUCACCUCCGAGUCUUAACCCAGCAAGGGGGUCCUGUGUCUGGCCACCUACCUUCUAGAGAUUCUUGCUGAGGGAACGGCCACCCUUGUAUUGCCGUGCUGGUUCAGUGGCCCUCUGUGCUGAUGGCCAUUGGUUCAAGAGAAAGGAUUUCCAGAGGGCC

[0140] SEQ ID NO: 13: TCR alpha chain variable region of HLA-A2 restricted HSP105 specific T cell clone (amino acid sequence): MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCAYRSPVSGTYKYIFGTGTRLKVLA

[0141] SEQ ID NO: 14: TCR β chain variable region of HLA-A2 restricted HSP105 specific T cell clone (amino acid sequence): MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLRGNTGELFFGEGSRLTVL

[0142] SEQ ID NO: 15: TCR α chain constant region of HLA-A2 restricted HSP105 specific T cell clone (amino acid sequence): NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0143] SEQ ID NO: 16: TCR β chain constant region of HLA-A2 restricted HSP105 specific T cell clone (amino acid sequence): EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0144] <Preparation of HSP105-TCR-T Cells> Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood collected from donors by centrifugation. They were cultured for one week in AIM-V medium containing 5% human AB serum, 50 ng / mL OKT3 (anti-CD3 antibody), and 600 U / mL interleukin-2 (IL-2) at 5% CO2 and 37°C. To prevent the HSP105-TCR to be introduced from binding to endogenous TCR, the α chain (TRA) and β chain (TRB) of the endogenous TCR of the PBMCs were knocked out using the Alt-R (registered trademark) CRISPR-Cas9 System (Integrated DNA Technologies). After an additional week of culture, the CD3-negative fraction was collected using magnetic beads immobilized with anti-CD3 antibodies to select TCR-knockout T cells. From the collected cell population, the CD8-positive fraction was selected using magnetic beads immobilized with anti-CD8 antibodies to select killer T cells. TCR-knockout killer T cells (KO-T cells) were cultured for one week in AIM-V medium containing 5% human AB serum and 600 U / mL IL-2, suspended in Cellbanker® 1 (Nippon Zenyaku Kogyo Co., Ltd.), and stored frozen at -80°C. When conducting various tests, frozen KO-T cells were thawed and cultured for 7 to 10 days in AIM-V medium containing 5% human AB serum and 600 U / mL IL-2. Then, mRNA encoding HSP105-TCR was introduced by electroporation (EP) using MaxCyte® GT (MaxCyte). The mRNA encoding HSP105-TCR was designed so that the RNA sequences shown in SEQ ID NOs: 11 and 12 were linked in parallel via a P2A sequence, substituting N1-methylpseudouridine for uridine, to form a single linear mRNA. Specifically, mRNA was used in which all "U"s in the RNA sequence shown in SEQ ID NO: 18 were replaced with N1-methylpseudouridine. The transfected cells were then cultured overnight in AIM-V medium containing 5% human AB serum and 600 U / mL IL-2, and used in each test.As shown in Figure 9, expression of the introduced TCR was confirmed by flow cytometry (FCM) Canto (registered trademark) (Becton Dickinson). It was confirmed that KO-T cells that underwent only EP without mRNA introduction did not express CD3 or TCR-αβ. On the other hand, it was confirmed that KO-T cells introduced with HSP105-TCR mRNA (HSP105-TCR-T cells) expressed CD3 and TCR-αβ, and that expression was maintained up to day 4 after EP.

[0145]

[0146] <Evaluation of IFN-γ production by HSP105-TCR-T cells> An ELISPOT assay was performed to confirm whether HSP105-TCR-T cells recognize cell lines presenting HSP105 peptides and secrete interferon-γ (IFN-γ). IFN-γ is a cytokine that contributes to the killing of cancer cells by suppressing cancer cell proliferation and activating surrounding T cells. The lymphoblast-derived cell line T2 expresses HLA-A2 but does not express HSP105. The human colon adenocarcinoma-derived cell line SW620 and the cervical cancer-derived cell line CaSki express both HLA-2 and HSP105, and therefore present HLA-A2 and HSP105-derived peptides on the cell surface. KO-T cells or HSP105-TCR-T cells treated with EP alone were co-cultured with T2, T2 treated with an HSP105-derived peptide (RLMNDMTAV), SW620, or CaSki for 20 hours, and IFNγ secretion into the culture supernatant was confirmed by ELISPOT assay (Figure 10). KO-T cells or HSP105-TCR-T cells treated with EP alone stimulated with PMA / Ionomycin (PI) served as positive controls. IFN-γ secretion into the culture supernatant was detected as red dots. In the positive control, PI stimulation, the entire well was stained red, while the negative control, T cell-only wells, remained white. It was confirmed that IFN-γ was not secreted in the co-culture groups of T2 cells and KO-T cells or HSP105-TCR-T cells treated with EP alone. In the group in which HSP105 peptide was added to T2 to artificially create an HSP105-HLA-A2 complex, IFN-γ was secreted in the group co-cultured with HSP105-TCR-T cells. Furthermore, in the group co-cultured with SW620 and CaSki, only HSP105-TCR-T cells were found to secrete IFN-γ (Fig. 10, boxed).

[0147] <Evaluation of Cytotoxic Response of HSP105-TCR-T Cells by CD107a Assay> HSP105-TCR-T cells were co-cultured with various target cells, and a CD107a assay was performed. CD107a is a protein present on the membrane surface of lysosomes that encapsulate perforin and granzymes, cytokines that kill cancer cells. When HSP105-TCR-T cells recognize the complex of HSP105 and MHC class I on the surface of cancer cells, lysosomes encapsulating perforin and granzymes migrate to the cell membrane surface, and perforin and granzymes are secreted extracellularly, damaging the cancer cells. At this time, CD107a present in the lysosomes migrates to the cell surface. Therefore, the cytotoxic activity of HSP105-TCR-T cells against cancer cells can be evaluated using CD107a expression as an indicator. 10 × 10 KO-T cells or HSP105-TCR-T cells treated with EP alone ４ 1 × 10 T2, T2 supplemented with HSP105 peptide, or T2 supplemented with GPC3 (Glypican-3) peptide ４ The target cells were co-cultured in RPMI medium containing 10% FBS and 1% penicillin, streptomycin, and L-glutamine. Target cells were stained with CFSE (carboxyfluorescein succinimidyl ester) before the start of co-culture. After 4 hours, CD107a expression was analyzed by FCM in KO-T cells or HSP105-TCR-T cells that had undergone EP alone (Figure 11). The horizontal axis represents CFSE, distinguishing T cells that were not stained with CFSE. The vertical axis represents CD107a, with dots moving upward as T cells recognize cancer cells. The results confirmed that CD107a expression increased only in HSP105-TCR-T cells co-cultured with T2 containing HSP105 peptide (boxed in Figure 11). On the other hand, the expression of CD107a did not increase when T2 cells were cultured alone or in co-culture with T2 cells supplemented with GPC3 peptide, confirming that this immune response was antigen-specific.

[0148] <Evaluation of Cytotoxic Response of HSP105-TCR-T Cells by CD107a Assay> HSP105-TCR-T cells were cocultured with SW620, CaSki, or the liver cancer-derived cell line HepG2 using the same procedure as in the cytotoxic response test described above, and a CD107a assay was performed ( Figure 12 ). CD107a expression was observed in HSP105-TCR-T cells cocultured with HSP105-expressing SW620 or CaSki, confirming that HSP105-TCR-T cells recognize the HSP105 peptide and HLA-A2 complex ( Figure 12 , bold frame). HepG2 has been reported not to express HSP105, and was used as a negative control in this study. However, slight CD107a expression was observed in the group cocultured with HSP105-TCR-T cells ( Figure 12 , thin frame). Therefore, immunostaining of HepG2 was performed, and HSP105 expression was observed, confirming that the reaction of HSP105-TCR-T cells was not incorrect.

[0149] <Confirmation of cytotoxic activity of HSP105-TCR-T cells> Calcein AM-labeled T2, HSP105 peptide-added T2, SW620, or CaSki (Target cells) were cultured at 1 × 10 ４ 1 × 10 cells, and 1 × 10 KO-T cells or HSP105-TCR-T cells (effector cells) that had undergone EP alone. ４ pieces (ET ratio = 1:1), 3 x 10 ４ pieces (ET ratio = 3:1), 10 x 10 ４ Cells were co-cultured at a ratio of 10:1 (ET ratio = 10:1), and fluorescence was measured using a Terascan VPC (Minervatec) at 0 and 4 hours after the start of co-culture (Figure 13). Calcein AM emits strong fluorescence when present within the target cells; however, when the target cells are damaged by KO-T cells or HSP105-TCR-T cells, the intracellular calcein AM is released into the culture medium, weakening the fluorescence. The difference in fluorescence between 0 and 4 hours allowed the calculation of the percentage of cytotoxic activity. Cytotoxic activity was calculated using the following formula. For spontaneous release, a group seeded with only calcein AM-labeled target cells was used. For maximum release, a group in which target cells were lysed with NP40 and all calcein AM had leaked out was used.

[0150] The formula for calculating cytotoxic activity is as follows: Cytotoxic activity (%) = (difference in fluorescence intensity of co-culture group between 0 hours and 4 hours - difference in fluorescence intensity of spontaneously released cells between 0 hours and 4 hours) / (difference in maximum fluorescence intensity of spontaneously released cells between 0 hours and 4 hours - difference in fluorescence intensity of spontaneously released cells between 0 hours and 4 hours) x 100

[0151] As shown in Figure 13, almost no cytotoxic activity was observed in the group in which T2 was co-cultured with KO-T cells or HSP105-TCR-T cells that had undergone only EP. When comparing groups in which T2, SW620, or CaSki cells supplemented with HSP105 peptide were co-cultured with KO-T cells or HSP105-TCR-T cells that had undergone only EP, higher cytotoxic activity was observed in the group co-cultured with HSP105-TCR-T cells. Furthermore, a tendency for cytotoxic activity to increase with increasing ET ratio was confirmed.

[0152] <In vivo test to confirm the tumor growth suppression effect of HSP105-TCR-T cells> Fifteen NSG mice were divided into three groups, and SW620 was administered at 1 x 10 ６ 1 × 10 KO-T cells and PBS (phosphate buffered saline). ７ or 1 x 10 HSP105-TCR-T cells ７ A 100 μL cell suspension containing a mixture of SW620 and HSP105-TCR-T cells was administered intradermally, and tumor diameter was measured every 2 to 3 days using a scale ( FIG. 14 ). Tumor growth was suppressed in the group administered a mixture of SW620 and HSP105-TCR-T cells compared to the groups administered a mixture of SW620 and PBS or KO-T cells. Furthermore, no tumor masses were observed in two of the five NSG mice in the group administered a mixture of SW620 and HSP105-TCR-T cells from the start of administration until day 19.

[0153] <Comparison of Linear mRNA and Circular mRNA> KO-T cells that had been frozen and stored in the above <Preparation of HSP105-TCR-T Cells> were thawed and cultured for 7 to 10 days in AIM-V medium containing 5% human AB serum and 600 U / mL IL-2. mRNA encoding HSP105-TCR was introduced into the cultured KO-T cells by electroporation (EP). Two types of mRNA were used: the linear mRNA used in the previous examples (sometimes referred to herein as Liner mRNA or L mRNA) and a circular mRNA lacking a stop codon (sometimes referred to herein as Circulating mRNA or C mRNA). The circular mRNA without a stop codon was designed by substituting N1-methylpseudouridine for uridine in the RNA sequences shown in SEQ ID NO:11 and SEQ ID NO:12, linking them in parallel via a P2A sequence, and forming a circular mRNA. That is, a circular mRNA was used in which all "U"s in the RNA sequence shown in SEQ ID NO:19 were replaced with N1-methylpseudouridine. KO-T cells containing no mRNA were prepared as a negative control. Cells transfected with linear or circular mRNA were maintained and cultured in AIM-V medium containing 5% human AB serum and 600 U / mL IL-2, and used for testing within 8 days after transfection.

[0154]

[0155] As shown in Figure 15, it was confirmed that KO-T cells that underwent only EP without mRNA transfection did not express either CD3 or TCR-αβ. On the other hand, it was confirmed that KO-T cells (HSP105-TCR-T cells) transfected with HSP105-TCR mRNA expressed CD3 and TCR-αβ. In particular, in the cell group transfected with cyclic mRNA, expression peaked around 4 days after EP, and expression continued for up to 7 days.

[0156] ELISPOT assays were performed using KO-T cells or HSP105-TCR-T cells 1 or 7 days after EP, co-cultured with T2, T2 supplemented with an HSP105-derived peptide (RLMNDMTAV), or SW620, as described above in <Evaluation of IFN-γ production by HSP105-TCR-T cells>. As shown in the upper panel of Figure 16, a large number of T cells producing antigen-specific IFN-γ were confirmed in HSP105-TCR-T cells 1 day after mRNA transfection by EP, compared with the negative control KO-T cells. It was confirmed that the number of IFN-γ-producing cells was equivalent between HSP105-TCR-T cells transfected with linear and circular mRNA. As shown in the lower panel of Figure 16, it was confirmed that the HSP105-TCR-T cells 7 days after EP contained antigen-specific IFNγ-producing T cells compared to the negative control. Furthermore, it was confirmed that the HSP105-TCR-T cells transfected with circular mRNA contained more antigen-specific IFNγ-producing cells than the HSP105-TCR-T cells transfected with linear mRNA.

[0157] <In vivo test to confirm the tumor growth suppression effect of HSP105-TCR-T cells> Sixteen NSG mice were divided into two groups, and SW620 was administered at 1 x 10 ６ 1 × 10 KO-T cells were administered intradermally in 50 μL of a cell suspension in PBS (phosphate buffered saline). ７ 1 x 10 HSP105-TCR-T cells into which the TCR gene of 1 or 10 circular mRNA was introduced by the EP method ７50 μL of cell suspension containing each of the 100 cells was administered into or near the tumor. Tumor diameter was measured using a scale every 2-3 days after tumor administration to confirm the antitumor effect. As shown in Figure 17, tumor growth was suppressed in the HSP105-TCR-T cell administration group compared to the KO-T cell administration group. (p<0.0001: Two-way ANOVA)

[0158] The HSP105-TCR-T cells of the present invention have immune responsiveness specific to HLA-A2-restricted HSP105-derived peptides. The features described herein enable rapid modification of T cells derived from a subject, T cells derived from another subject, or T cells derived from pluripotent stem cells, and enable rapid and easy production of modified T cells that secrete cytokines and have cytotoxic activity against cancer cells expressing HSP105, providing an excellent cancer therapeutic agent.

Claims

1. A T cell receptor that recognizes a complex of a partial peptide derived from HSP105, the amino acid sequence RLMNDMTAV (SEQ ID NO: 17), and a human MHC class I molecule.

2. The T cell receptor according to claim 1, comprising a T cell receptor α chain containing an α chain CDR3 specified by the amino acid sequence set forth in SEQ ID NO: 1, or an α chain CDR3 functionally equivalent thereto, and a T cell receptor β chain containing a β chain CDR3 specified by the amino acid sequence set forth in SEQ ID NO: 2, or a β chain CDR3 functionally equivalent thereto.

3. The T cell receptor according to claim 2, comprising a T cell receptor α chain comprising an α chain CDR1 specified by the amino acid sequence set forth in SEQ ID NO: 3, or an α chain CDR1 functionally equivalent thereto, and an α chain CDR2 specified by the amino acid sequence set forth in SEQ ID NO: 4, or an α chain CDR2 functionally equivalent thereto, and a T cell receptor β chain comprising a β chain CDR1 specified by the amino acid sequence set forth in SEQ ID NO: 5, or a β chain CDR1 functionally equivalent thereto, and a β chain CDR2 specified by the amino acid sequence set forth in SEQ ID NO: 6, or a β chain CDR2 functionally equivalent thereto.

4. The T cell receptor according to claim 1, comprising a T cell receptor α chain identified by the amino acid sequence set forth in SEQ ID NO: 7 and a T cell receptor β chain identified by the amino acid sequence set forth in SEQ ID NO:

8.

5. A combination of nucleic acids comprising a nucleic acid encoding a T cell receptor alpha chain identified by the amino acid sequence set forth in SEQ ID NO: 7 and a nucleic acid encoding a T cell receptor beta chain identified by the amino acid sequence set forth in SEQ ID NO:

8.

6. The combination of nucleic acids according to claim 5, wherein the combination of nucleic acids is contained in parallel in a single circular nucleic acid that does not contain a stop codon.

7. The nucleic acid combination described in claim 5, wherein the nucleic acid encoding the T cell receptor alpha chain is DNA specified by the base sequence set forth in SEQ ID NO: 9, and the nucleic acid encoding the T cell receptor beta chain is DNA specified by the base sequence set forth in SEQ ID NO:

10.

8. A combination of nucleic acids according to claim 5, wherein the nucleic acid encoding the T cell receptor alpha chain is RNA specified by the base sequence set forth in SEQ ID NO: 11, and the nucleic acid encoding the T cell receptor beta chain is RNA specified by the base sequence set forth in SEQ ID NO:

12.

9. A vector comprising the nucleic acid combination according to claim 5.

10. A cell transfected with the nucleic acid combination described in claim 5.

11. A cell into which the vector according to claim 9 has been introduced.

12. The cell of claim 10 or 11, wherein the cell is a T cell, an NK cell, an invariant NK cell, an NKT cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell.

13. The cell of claim 10 or 11, wherein the cell is a T cell that does not have an endogenous T cell receptor.

14. T cells that do not have endogenous T cell receptors are CD3 － CD8 ＋ The cell of claim 13, which is a T cell.

15. A cancer therapeutic agent comprising a T cell receptor according to any one of claims 1 to 4, a combination of nucleic acids according to any one of claims 5 to 8, a vector according to claim 9, or a cell according to claim 10 or 11.

Images