Peptide-specific tcr derived from HLA-a02-restricted glypican-3

Abstract

The present invention provides a T cell receptor or a fragment thereof which is capable of binding to a peptide having the amino acid sequence represented by SEQ ID NO: 20 or a complex of the peptide and HLA-A02, wherein the peptide includes the amino acid sequences for CDR1 to CDR3 which are respectively represented by SEQ ID No. 1 to SEQ ID No. 3 as complementarity determining regions in an α-chain and the amino acid sequences for CDR1 to CDR3 which are respectively represented by SEQ ID No. 7 to SEQ ID No. 9 as complementarity determining regions in a β-chain, or includes the amino acid sequences for CDR1 to CDR3 which are respectively represented by SEQ ID No. 4 to SEQ ID No. 6 as complementarity determining regions in the α-chain and the amino acid sequences for CDR1 to CDR3 which are respectively represented by SEQ ID No. 10 to SEQ ID No. 12 as complementarity determining regions in the β-chain.

Description

HLA-A02-restricted glypican 3-derived peptide-specific TCR

[0001] The present invention relates to a novel T cell receptor that is HLA-A02 restricted and specific for a glypican 3-derived peptide.

[0002] Primary liver cancer, primarily hepatocellular carcinoma (HCC), is the fifth most common cancer in Japan, but it has a very poor prognosis and a very high mortality rate. One of the main reasons for this poor prognosis is the limited treatment options for advanced HCC. Patients with advanced HCC can only undergo symptomatic treatment, such as local resection or administration of the multikinase inhibitor sorafenib. However, sorafenib has a low response rate and a high incidence of side effects, especially in elderly patients. Therefore, there is a need to develop new treatments that minimize the risk of side effects and improve the survival rate of patients with advanced HCC.

[0003] Immunotherapy is considered to be one of the most effective treatments for HCC. For example, glypican 3 (GPC3) is particularly overexpressed in HCC and is associated with poor prognosis, making it an ideal target for cancer immunotherapy for HCC. Furthermore, immunotherapies for HCC using GPC3-specific antibodies and human chimeric antigen receptors (CARs) targeting GPC3 have been reported (Patent Document 1). Furthermore, HLA-A02-restricted GPC3 ３６７－３７５ Peptide-specific T cell receptors (TCRs) have also been reported (Patent Documents 2 and 3).

[0004] TCR is a receptor used by T cells to recognize antigens, and is composed of a dimer of α and β chains, or γ and δ chains. TCR forms a complex with CD3 molecules on the surface of T cells, recognizes antigens, and transmits stimulatory signals to T cells. Each TCR chain has a variable region and a constant region. The constant region has a short cytoplasmic portion that penetrates the cell membrane, while the variable region exists extracellularly and binds to the antigen-HLA (MHC) complex. The variable region contains three regions called complementarity-determining regions (CDRs), which bind to the antigen-HLA (MHC) complex. The three CDRs are called CDR1, CDR2, and CDR3, respectively.

[0005] T cells undergo a differentiation process to become mature T cells that express either CD4 or CD8. Of these, CD8-positive T cells are also called cytotoxic T cells (CTLs), and they recognize cancer cells or virus-infected cells via TCR and kill them. CD8 binds to MHC (Major Histocompatibility Gene Complex), which can strengthen the binding between TCR and antigen-HLA (MHC) complexes.

[0006] International Publication No. WO 2013 / 070468 International Publication No. WO 2015 / 173112 International Publication No. WO 2018 / 143454

[0007] An objective of the present invention is to provide a novel T cell receptor (TCR) that specifically recognizes glypican 3 (GPC3), and to provide a pharmaceutical agent for preventing or treating cancers or tumors expressing GPC3, using the TCR (e.g., using cytotoxic T cells containing the TCR).

[0008] The present inventors have identified a GPC3 peptide (GPC3 １４４－１５２ CTL clones were established from peripheral blood mononuclear cells (PBMCs) derived from patients who had been inoculated with the HLA-A02-restricted GPC3 peptide, and the TCR sequences of specific CTL clones were decoded. １４４－１５２ The present inventors have found that the TCR is peptide-specific, exhibits CD8-independent cytotoxic activity, and is responsive to GPC3-expressing cancer cells, and have completed the present invention.

[0009] That is, the present invention provides the following: [1] A T cell receptor (TCR) or a fragment thereof, comprising, as the α-chain complementarity-determining regions, the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, and as the β-chain complementarity-determining regions, the amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, or comprising, as the α-chain complementarity-determining regions, the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, and as the β-chain complementarity-determining regions, the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, wherein the TCR or fragment thereof is capable of binding to a complex of HLA-A02 and a peptide having the amino acid sequence shown in SEQ ID NO: 20.[2] An α-chain variable region comprises: the amino acid sequence shown in SEQ ID NO: 13; the amino acid sequence shown in SEQ ID NO: 13 in which one or several amino acids have been deleted, substituted or added; or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 13; and a β-chain variable region comprises: the amino acid sequence shown in SEQ ID NO: 15; the amino acid sequence shown in SEQ ID NO: 15 in which one or several amino acids have been deleted, substituted or added; or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 15; or an α-chain variable region comprises: the amino acid sequence shown in SEQ ID NO: 14; the amino acid sequence shown in SEQ ID NO: 14 in which one or several amino acids have been deleted, substituted or added; or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 14; and a β-chain variable region comprises: the amino acid sequence shown in SEQ ID NO: 16; the amino acid sequence shown in SEQ ID NO: 16 in which one or several amino acids have been deleted, substituted or added; The TCR or a fragment thereof according to [1], comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 16.[3] The full-length amino acid sequence of the α chain comprises: the amino acid sequence shown in SEQ ID NO: 21; the amino acid sequence shown in SEQ ID NO: 21 in which one or several amino acids have been deleted, substituted or added; or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 21; and the full-length amino acid sequence of the β chain comprises: the amino acid sequence shown in SEQ ID NO: 23; the amino acid sequence shown in SEQ ID NO: 23 in which one or several amino acids have been deleted, substituted or added; or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 23; or the full-length amino acid sequence of the α chain comprises: the amino acid sequence shown in SEQ ID NO: 22; the amino acid sequence shown in SEQ ID NO: 22 in which one or several amino acids have been deleted, substituted or added; or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 22; and the full-length amino acid sequence of the β chain comprises: the amino acid sequence shown in SEQ ID NO: 24; The TCR or fragment thereof according to [1] or [2], comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 24. [4] A nucleic acid encoding the TCR or fragment thereof according to any of [1] to [3]. [5] A vector comprising the nucleic acid according to [4]. [6] A cell comprising the nucleic acid according to [4] or the vector according to [5]. [7] The cell according to [6], wherein the cell is a T cell. [8] The cell according to [7], wherein the cell has CD8-independent cytotoxic activity. [9] A method for producing the cell according to any of [6] to [8], comprising a step of introducing the nucleic acid according to [4] or the vector according to [5] into a cell.

[10] A pharmaceutical comprising the cell according to any of [6] to [8].

[11] The pharmaceutical according to

[10] , for use in the prevention or treatment of cancer.

[12] An agent for killing a cell expressing glypican 3, comprising the cell according to any one of [6] to [8].

[13] The cell according to any one of [6] to [8] for use in the prevention or treatment of cancer.

[14] Use of the cell according to any one of [6] to [8] for the production of a preventive or therapeutic agent for cancer.

[0010] The T cell receptor of the present invention is a GPC3 peptide (GPC3 １４４－１５２ The nucleic acid encoding the T cell receptor is an HLA-A02-restricted T cell receptor that has the ability to bind to a GPC3 peptide or a complex of the peptide and an HLA-A molecule (HLA-A02). Furthermore, the nucleic acid encoding the T cell receptor can confer cytotoxic activity to T cells against cells that present an HLA-A molecule and a GPC3 peptide, and is therefore useful for the prevention or treatment of cancers or tumors that express GPC3.

[0011] FIG. 1 shows GPC3 １４４－１５２ Anti-CD3 antibody, anti-CD8 antibody, and GPC3 to measure peptide specificity １４４－１５２Figure 2 shows the results of flow cytometry analysis using a dextramer staining assay for HLA-A*02:01. Figure 2 shows the results of an ELISPOT assay for interferon-gamma (IFN-γ) to measure antigen-specific cytokine production. Figure 3 shows the results of flow cytometry analysis using an anti-CD8 antibody and anti-CD107a antibody to measure antigen-specific cytotoxic activity. Figure 4 shows the results of a cytotoxicity test in which cells were co-cultured with calcein AM-labeled target cells to measure antigen-specific cytotoxicity, and the cytotoxicity rate was calculated. Figure 5 shows the configuration of pMu1-GPC3-siTCR for constructing a plasmid for producing a retroviral vector. Figure 6 shows the results of dextramer staining and flow cytometry analysis of CD8-positive cells after anti-CD8 antibody blocking of siTCR-T. Figure 7 shows the results of dextramer staining and flow cytometry analysis of CD8-negative cells after anti-CD8 antibody blocking of siTCR-T. Figure 8 shows the results of measuring the cytotoxic activity of siTCR-T using the xCelligence real-time cell analysis system, showing the number of target cells (cell index) over time. Figure 9 shows the results of measuring the in vivo cytotoxic activity of siTCR-T. In the figure, the vertical axis indicates tumor volume, and the horizontal axis indicates the number of days after co-injection of siTCR-T and tumor cells. #63 to #68, #70, #71, #73, and #74 indicate individual mouse numbers. Figure 10 shows (a) the test scheme and (b) the test results for evaluating the in vivo antitumor activity of siTCR-T. In graph (b), the vertical axis indicates tumor volume, and the horizontal axis indicates the number of days after tumor cell injectio. #80 to #83, #86, #87, #89, #91, #96, #100, #201, and #202 indicate individual mouse numbers. Figure 11 is a photograph showing the condition of a tumor excised 15 days after tumor cell transplantation in an in vivo antitumor activity evaluation test of siTCR-T. Figure 12 shows the results of flow cytometry analysis of the 5B6 siTCR-T administration group in an in vivo antitumor activity evaluation test of siTCR-T.

[0012] (Detailed Description of the Invention) 1. T Cell Receptor The present invention relates to GPC3 １４４－１５２The present invention provides a T cell receptor (also called TCR) capable of binding to a peptide or a complex of the peptide and HLA-A02, and a fragment thereof. １４４－１５２ The TCR of the present invention is a peptide or a fragment thereof capable of binding to a complex of the peptide and HLA-A02. In the present specification, the above-mentioned TCR and its fragment may be collectively abbreviated as "TCR of the present invention." The TCR of the present invention may be isolated. The TCR of the present invention is characterized by its ability to recognize and bind to the complex in a CD8-independent manner.

[0013] In the present invention, "T cell receptor (TCR)" refers to a receptor composed of a dimer of TCR chains (α chain, β chain), which recognizes an antigen or the antigen-HLA (human leukocyte antigen) (MHC; major histocompatibility complex) complex and transmits a stimulatory signal to a T cell. Each TCR chain is composed of a variable region and a constant region, and the variable region contains three complementarity-determining regions (CDR1, CDR2, CDR3). The TCRs of the present invention include not only TCRs in which the α chain and β chain of the TCR constitute a heterodimer, but also TCRs in which the chain constitutes a homodimer. Furthermore, the TCRs of the present invention also include TCRs in which the constant region is partially or entirely deleted, TCRs with recombinant amino acid sequences, soluble TCRs, and the like.

[0014] In the present invention, the term "soluble TCR" refers to a TCR that has been solubilized by chemical modification of the TCR, binding to an Fc receptor, or removal of the transmembrane domain, and the term "soluble" refers to a TCR that has been solubilized, for example, in phosphate-buffered saline (PBS) (KCl 2.7 mM, KH ２ P.O. ４ 1.5 mM, NaCl 137 mM and Na ２ P.O. ４"Soluble TCR" refers to the property that a soluble TCR exists as a monodisperse heterodimer in a solution containing 8 mM TCR and 8 mM IgG, pH 7.1-7.5, and that 90% or more of the TCR remains as a monodisperse heterodimer after incubation at 25°C for 1 hour. To enhance stability, a disulfide bond may be artificially introduced between the constant regions of each chain. Such soluble TCRs can be prepared according to known methods, such as those described in International Publication No. 2004 / 074322 and Boulter et al., Clin Exp Immunol, 2005, 142(3):454-460. When using a soluble TCR, its concentration is not particularly limited as long as the TCR can bind to an antigen or the antigen-HLA complex; however, for example, when used in in vitro tests, a concentration of 40 μg / ml or more is preferred.

[0015] In the present invention, "GPC3 １４４－１５２ peptide" or "HLA-A02-restricted GPC3 １４４－１５２ The term "peptide" refers to a peptide fragment of GPC3 consisting of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing. In a preferred embodiment, the TCR of the present invention is a GPC3 １４４－１５２ It can specifically recognize and bind to a complex of a peptide and HLA-A02. １４４－１５２ The "GPC3 peptide" may be abbreviated as "GPC3 peptide."

[0016] The ability of the TCR of the present invention to specifically recognize and bind to the above-mentioned complex can be confirmed by known methods, and suitable methods include, for example, a dextramer assay using an HLA-A02 molecule and a GPC3 peptide, or an ELISPOT (Enzyme-Linked ImmunoSpot) assay. By performing an ELISPOT assay, it can be confirmed that a T cell expressing the TCR on its cell surface recognizes a target cell via the TCR and that the signal is transmitted into the cell.

[0017] As used herein, the term "capable of binding" means "having an ability to bind" and refers to the ability to form a non-covalent complex with one or more other molecules. Examples of complexes of the present invention include a complex of a GPC3 peptide and an HLA molecule (e.g., HLA-A02), or a complex of a GPC3 peptide and a TCR. Another example of a complex of the present invention is a complex of a TCR and a GPC3 peptide that itself forms a complex with an HLA. Various methods and assays for determining binding ability are known in the art.

[0018] In the present invention, "isolated" means that a particular component (eg, a TCR) has been identified, separated, or recovered from a component of its natural environment.

[0019] In the present invention, "one or several amino acids" refers to, for example, 1, 2, 3, 4, or 5 amino acids (e.g., 1 to 4 amino acids, 1 to 3 amino acids, or 1 to 2 amino acids). For example, in the context of a CDR region of a TCR, "one or several" preferably refers to 1, 2, or 3 amino acids. In the context of a TCR variable region or TCR, "one or several" preferably refers to 1 to 5, 1 to 4, or 1 to 3, particularly 1, 2, or 3 amino acids.

[0020] In the present invention, "percent identity" means, for example, 90% or greater identity (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater). Amino acid sequence identity can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) under the following conditions: expectancy=10; gap allowed; matrix=BLOSUM62; filtering=OFF. It is understood that to determine % identity, the entire length of the sequence of the present invention is compared to another sequence. In other words, % identity in the present invention excludes comparing a short fragment (e.g., 1-3 amino acids) of the sequence of the present invention to another sequence, or vice versa.

[0021] In one embodiment of the present invention, the α chain of the TCR of the present invention comprises, as a complementarity determining region, each of the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOS: 1 to 3, respectively, or each of the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOS: 4 to 6, respectively, and the β chain of the TCR of the present invention comprises, as a complementarity determining region, each of the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOS: 7 to 9, respectively, or each of the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOS: 10 to 12, respectively. １４４－１５２As long as the TCR has the ability to bind to a peptide or a complex of the peptide and HLA-A02, one to several (e.g., two or three) amino acids may be deleted, substituted, or added. In a preferred embodiment, the TCR of the present invention comprises a TCR α chain comprising the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOS: 1 to 3, respectively, and a TCR β chain comprising the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOS: 7 to 9, respectively, and the α chain and β chain of the TCR form a heterodimer. In another preferred embodiment, the TCR of the present invention comprises a TCR α chain comprising the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOS: 4 to 6, respectively, and a TCR β chain comprising the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOS: 10 to 12, respectively, and the α chain and β chain of the TCR form a heterodimer.

[0022] In another embodiment of the present invention, the TCR of the present invention comprises a TCR α chain comprising a CDR3 as set forth in SEQ ID NO: 3 and a TCR β chain comprising a CDR3 as set forth in SEQ ID NO: 9, and the α chain and β chain of the TCR form a heterodimer. Preferably, this heterodimer comprises GPC3 １４４－１５２ It has the ability to bind to a peptide or a complex of said peptide and HLA-A02.

[0023] In yet another embodiment of the present invention, the α chain of the TCR of the present invention preferably comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 13, or the TCR of the present invention comprises GPC3 １４４－１５２ The α chain of the TCR of the present invention preferably comprises a variable region comprising an amino acid sequence in which one or several (e.g., 2, 3, 4, or 5) amino acids have been deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) identity with the amino acid sequence shown in SEQ ID NO: 13, provided that the TCR can bind to the peptide or a complex of the peptide and HLA-A02. Alternatively, the α chain of the TCR of the present invention preferably comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 14, or the TCR of the present invention preferably comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 15. １４４－１５２The variable region includes an amino acid sequence in which one or several (e.g., 2, 3, 4, or 5) amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) identity to the amino acid sequence of SEQ ID NO: 14, provided that the variable region can bind to the peptide or a complex of the peptide and HLA-A02. The variable region preferably includes each of the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOs: 1 to 3, respectively, or each of the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOs: 4 to 6, respectively. The identity of the amino acid sequences can be calculated as described above. The identity of the following amino acid sequences can also be calculated in a similar manner.

[0024] Furthermore, the β chain of the TCR of the present invention preferably comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 15, or the TCR of the present invention preferably comprises GPC3 １４４－１５２ The TCR of the present invention preferably comprises a variable region comprising an amino acid sequence in which one or several (e.g., 2, 3, 4, or 5) amino acids have been deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) identity to the amino acid sequence shown in SEQ ID NO: 15, provided that the variable region can bind to the peptide or a complex of the peptide and HLA-A02. Alternatively, the β chain of the TCR of the present invention preferably comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 16, or a TCR comprising the variable region of the β chain preferably comprises a variable region comprising the amino acid sequence shown in SEQ ID NO: 16. １４４－１５２Provided that it can bind to a peptide or a complex of said peptide and HLA-A02, it comprises a variable region comprising an amino acid sequence in which one or several (e.g., 2, 3, 4, or 5) amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) identity to the amino acid sequence of SEQ ID NO: 16. The variable region preferably comprises each of the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOs: 7 to 9, respectively, or each of the amino acid sequences of CDR1 to CDR3 shown in SEQ ID NOs: 10 to 12, respectively.

[0025] In a preferred embodiment, the TCR of the present invention comprises a TCR α chain comprising the amino acid sequence shown in SEQ ID NO: 13, and a TCR β chain comprising the amino acid sequence shown in SEQ ID NO: 15, and the α chain and β chain of the TCR form a heterodimer. In another preferred embodiment, the TCR of the present invention comprises a TCR α chain comprising the amino acid sequence shown in SEQ ID NO: 14, and a TCR β chain comprising the amino acid sequence shown in SEQ ID NO: 16, and the α chain and β chain of the TCR form a heterodimer.

[0026] Furthermore, the α chain of the TCR of the present invention preferably comprises a constant region comprising the amino acid sequence set forth in SEQ ID NO: 17, or, provided that the TCR of the present invention can transmit a stimulatory signal to T cells, it preferably comprises a constant region comprising an amino acid sequence in which one or several (e.g., 2, 3, 4, or 5) amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) identity to the amino acid sequence set forth in SEQ ID NO: 17. In a specific embodiment of the present invention, the constant region of the α chain comprises the amino acid sequence set forth in SEQ ID NO: 17. Furthermore, the β chain of the TCR of the present invention preferably comprises a constant region comprising the amino acid sequence set forth in SEQ ID NO: 18, or, provided that the TCR of the present invention can transmit a stimulatory signal to a T cell, preferably comprises a constant region comprising an amino acid sequence in which one or several (e.g., 2, 3, 4, or 5) amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) identity to the amino acid sequence set forth in SEQ ID NO: 18. In a specific embodiment of the present invention, the constant region of the β chain comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0027] Furthermore, the constant region of the TCR α-chain or β-chain of the present invention is preferably modified in the constant region of the TCR α-chain or β-chain of the CTL clone from which it is derived, such that, for example, a specific amino acid residue in the constant region of the TCR of the CTL clone is substituted with a cysteine ​​residue (e.g., substitution of threonine at position 48 in the constant region of the TCR α-chain with cysteine, or substitution of serine at position 57 in the constant region of the TCR β-chain of the CTL clone with cysteine), thereby enhancing the efficiency of dimer formation via disulfide bonds between the α-chain and the β-chain, but this modification is not limited to this.

[0028] Examples of the α chain of the TCR of the present invention having the above-mentioned variable region and constant region include, but are not limited to, polypeptides comprising the amino acid sequence shown in SEQ ID NO: 21 or 22. Examples of the β chain of the TCR of the present invention having the above-mentioned variable region and constant region include, but are not limited to, polypeptides comprising the amino acid sequence shown in SEQ ID NO: 23 or 24. Furthermore, in the amino acid sequence shown in any of SEQ ID NOs: 21 to 24, preferably, the TCR comprising the α chain or β chain is GPC3 １４４－１５２ The present invention also provides suitable use of TCR α- or β-chains, provided that they can bind to a peptide or a complex of the peptide and HLA-A02. These include amino acid sequences in which one or several (e.g., 2, 3, 4, or 5) amino acids have been deleted, substituted, or added, or amino acid sequences that share 90% or more (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more) identity with the amino acid sequence. Preferred TCRs of the present invention include heterodimers composed of an α-chain represented by SEQ ID NO: 21 and a β-chain represented by SEQ ID NO: 23 (herein, this heterodimer may be referred to as TCR5B6), and heterodimers composed of an α-chain represented by SEQ ID NO: 22 and a β-chain represented by SEQ ID NO: 24 (herein, this heterodimer may be referred to as TCR5B4).

[0029] The TCR of the present invention can be produced by genetic engineering using the nucleic acid or vector of the present invention described below. For example, both a nucleic acid encoding the α chain and a nucleic acid encoding the β chain of the TCR of the present invention can be introduced into a cell to express the TCR α chain and β chain polypeptides, thereby allowing the cell to express the TCR of the present invention, and the TCR can then be isolated by a method known per se.

[0030] 2. Nucleic Acids of the Present Invention The present invention provides nucleic acids (hereinafter abbreviated as "nucleic acids of the present invention") encoding the above-described TCRs of the present invention or fragments thereof. The nucleic acids of the present invention may be isolated.

[0031] The nucleic acid of the present invention may be any of a nucleic acid encoding a TCR α chain or a fragment thereof, a nucleic acid encoding a TCR β chain or a fragment thereof, and a nucleic acid encoding both a TCR α chain and a β chain or fragments thereof.

[0032] The present invention also relates to nucleic acids encoding any one or more of the CDRs, variable regions and / or constant regions described herein.

[0033] The present invention also encompasses nucleic acids that can hybridize to the complement of any of the nucleic acids defined herein under stringent conditions. Stringent conditions may be those generally known in the art. In a preferred embodiment, the hybridizable nucleic acid encodes an amino acid sequence of a CDR, variable region, or constant region that has the function described herein. Specifically, the hybridizable nucleic acid is a nucleic acid that encodes a TCR comprising the amino acid sequence, wherein the TCR is a TCR comprising: a) GPC3 １４４－１５２ The antibody encodes an amino acid sequence capable of binding to a peptide or a complex of the peptide and HLA-A02.

[0034] The nucleic acid encoding the α chain of the TCR of the present invention may be any nucleic acid that encodes the α chain of the TCR defined above, and examples thereof include a nucleic acid that encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 21 or 22. The nucleic acid encoding the β chain of the TCR of the present invention may be any nucleic acid that encodes the β chain of the TCR defined above, and examples thereof include a nucleic acid that encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 23 or 24.

[0035] The nucleic acid of the present invention may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. When the nucleic acid is RNA, T in the sequence listing for the RNA sequence should be read as U. The nucleic acid of the present invention may contain natural nucleotides, modified nucleotides, nucleotide analogs, or mixtures thereof, as long as it is capable of expressing a polypeptide in vitro or in a cell.

[0036] The nucleic acid of the present invention can be constructed by a method known per se. For example, a DNA encoding the full-length or a part of the TCR of the present invention can be constructed by chemically synthesizing a DNA strand based on the amino acid sequence or nucleic acid sequence of the TCR or a fragment thereof listed in the Sequence Listing, or by connecting synthesized, partially overlapping short oligo-DNA strands using PCR or Gibson Assembly.

[0037] 3. Expression Vector Comprising the Nucleic Acid of the Present Invention The nucleic acid of the present invention can be incorporated into an expression vector. Thus, the present invention provides an expression vector comprising any of the above-described nucleic acids of the present invention (hereinafter abbreviated as "the vector of the present invention").

[0038] The vector of the present invention may be a vector that is not integrated into the genome of a target cell. In one embodiment, a vector that is not integrated into the genome may replicate autonomously or non-autonomously outside the genome of a target cell. The vector may exist in multiple copies outside the genome of a target cell. In a further embodiment of the present invention, the vector is integrated into the genome of a target cell. In a preferred embodiment, the vector is integrated into a random or predetermined location in the genome of a target cell.

[0039] Examples of promoters that can be used in the vectors of the present invention include the EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MMLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, TCR Vα gene promoter, and TCR Vβ gene promoter. Of these, the EF1α promoter, CAG promoter, MMLV LTR, CMV promoter, and SRα promoter are preferred.

[0040] In addition to the promoter, the vector of the present invention may optionally contain transcriptional and translational regulatory sequences, a ribosome binding site, an enhancer, a replication origin, a poly(A) addition signal, a selection marker gene, etc. Examples of the selection marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, and a puromycin resistance gene.

[0041] In one embodiment of the present invention, an expression vector containing a nucleic acid encoding the α chain and a nucleic acid encoding the β chain of the TCR of the present invention described above can be introduced into a target cell, thereby forming a heterodimer of the TCR α chain and the β chain within the target cell or on the cell surface. In this case, the nucleic acid encoding the TCR α chain and the nucleic acid encoding the β chain may be incorporated into separate expression vectors, or may be incorporated into a single expression vector. When incorporated into a single expression vector, these two types of nucleic acids are preferably incorporated via a sequence that enables polycistronic expression. The use of a sequence that enables polycistronic expression enables more efficient expression of multiple genes incorporated into a single expression vector. Examples of sequences that enable polycistronic expression include the T2A sequence of Thosea asigna virus, the P2A sequence of porcine teschovirus (PLoS ONE, 3, e2532, 2008), the F2A sequence of foot-and-mouth disease virus (Stem Cells, 25, 1707, 2007), the E2A sequence of equine rhinitis A virus (Scientific Reports, 7, 2193, 2017), and an internal ribosome entry site (IRES) (U.S. Patent No. 4,937,190).

[0042] The expression vector that can be used in the present invention is not particularly limited as long as it can express TCR for a period of time sufficient for the prevention or treatment of disease when introduced into cells, and examples thereof include viral vectors and plasmid vectors. Examples of viral vectors include retroviral vectors (including lentiviral vectors and pseudotype vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, and episomal vectors. A transposon expression system (PiggyBac system) may also be used. Examples of plasmid vectors include animal cell expression plasmids (e.g., pa1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo).

[0043] 4. Cells containing the nucleic acid or vector of the present invention When the nucleic acid or vector of the present invention is introduced into a cell and a TCR or a fragment thereof is present on the cell surface, the cell expresses HLA-A02-restricted GPC3 against a target cell. １４４－１５２ The nucleic acid of the present invention may have specific cytotoxic activity. Therefore, the present invention provides cells containing the nucleic acid or vector of the present invention (in other words, cells having the nucleic acid or vector of the present invention) (hereinafter abbreviated as "cells of the present invention"). Here, the nucleic acid of the present invention is preferably introduced into a desired cell in the form of a vector of the present invention. The present invention also encompasses introducing the nucleic acid of the present invention into a host genome by genome editing (e.g., CRISPR system, TALEN system, etc.). A preferred embodiment of the cells of the present invention includes, but is not limited to, cells into which both a nucleic acid encoding a TCRα chain and a nucleic acid encoding a TCRβ chain have been introduced. Whether the cells of the present invention have cytotoxic activity can be confirmed by known methods, and a suitable method includes, for example, measuring cytotoxic activity against HLA-A02-positive target cells using the xCelligence real-time cell analysis system (Agilent Technologies). In a preferred embodiment, the cells of the present invention are mammalian cells, more preferably human cells.

[0044] Cells into which the nucleic acid or expression vector of the present invention can be introduced include, for example, lymphocytes and lymphocyte precursor cells, including pluripotent stem cells. In the present invention, "lymphocyte" refers to one of the subtypes of white blood cells in the immune system of vertebrates, including T cells, B cells, and natural killer cells (NK cells). Because T cell receptors play an important role in antigen recognition by T cells, T cells are preferred as cells into which the nucleic acid or vector of the present invention can be introduced. In the present invention, "T cell" refers to a type of white blood cell found in lymphoid organs or peripheral blood, and is a classification of lymphocytes characterized by differentiation and maturation mainly in the thymus and expression of T cell receptors (TCR). Examples of T cells that can be used in the present invention include CD8-positive cells, such as cytotoxic T cells (CTLs), and CD4-positive cells, such as helper T cells, regulatory T cells, and effector T cells. Cytotoxic T cells are preferred. CD4 / CD8-positive cells are also included in the T cell category. T cells expressing the TCR of the present invention can be obtained by introducing the nucleic acid or vector of the present invention into T cells collected from a living body.

[0045] The cells of the present invention (e.g., cytotoxic T cells) have, in addition to the TCR genes that the cells naturally have, an exogenous TCR gene derived from the nucleic acid or vector of the present invention. In this respect, the cells of the present invention differ from cells collected from a living body.

[0046] The lymphocytes can be collected from, for example, peripheral blood, bone marrow, and umbilical cord blood of a human or non-human mammal. When the TCR gene-introduced cells of the present invention are used to treat a disease such as cancer, the cell population is preferably collected from the subject or a donor whose HLA type matches that of the subject. A preferred subject or donor is a human.

[0047] The method for introducing the nucleic acid or vector of the present invention into cells is not particularly limited, and known methods can be used. When introducing a nucleic acid or a plasmid vector, for example, calcium phosphate coprecipitation, PEG, electroporation, microinjection, lipofection, etc. can be used. For example, methods described in Cell Engineering, Special Issue 8, New Cell Engineering Experimental Protocols, pp. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, p. 456 (1973), Folia Pharmacol. Jpn., Vol. 119 (No. 6), pp. 345-351 (2002), etc. can be used. When a viral vector is used, the nucleic acid of the present invention can be introduced into cells by introducing the nucleic acid into appropriate packaging cells (e.g., Plat-E cells) or complementation cell lines (e.g., 293T cells), recovering the viral vector produced in the culture supernatant, and infecting the cells with the vector by a method appropriate for each viral vector. For example, specific means for using retroviral vectors as vectors are disclosed in WO 2007 / 69666, Cell, 126, 663-676 (2006), and Cell, 131, 861-872 (2007), among others. In particular, when a retroviral vector is used, highly efficient gene transfer into various cells is possible by using RetroNectin (registered trademark, manufactured by Takara Bio Inc.), a recombinant fibronectin fragment.

[0048] The nucleic acid of the present invention may also be directly introduced into cells in the form of RNA and used to express TCR in the cells. As a method for introducing RNA, known methods can be used, and for example, lipofection and electroporation can be preferably used.

[0049] When the nucleic acid of the present invention is introduced into a T cell, the expression of the endogenous TCRα and TCRβ chains naturally expressed by the T cell may be suppressed by siRNA, from the viewpoint of increasing the expression of the TCR of the present invention, suppressing the appearance of mispaired TCRs, or suppressing non-autoreactivity. When the nucleic acid is applied to the method, in order to avoid the effect of the siRNA on the TCR of the present invention, it is preferable to use a nucleotide sequence (codon-altered sequence) that differs from the nucleotide sequence corresponding to the RNA on which the siRNA acts to suppress the expression of the endogenous TCRα and TCRβ chains. These methods are described, for example, in WO 2008 / 153029. The nucleotide sequence can be prepared by introducing silent mutations into a nucleic acid encoding a naturally occurring TCR or by chemically synthesizing an artificially designed nucleic acid. Alternatively, to avoid mispairing with the endogenous TCR chain, part or all of the constant region of the nucleic acid encoding the TCR of the present invention may be replaced with a constant region derived from an animal other than a human, such as a mouse.

[0050] 5. Method for Producing the Cell of the Present Invention The present invention also provides a method for producing the cell of the present invention (hereinafter abbreviated as "the method of the present invention"), which comprises the step of introducing the nucleic acid or vector of the present invention into a cell. The cell into which the nucleic acid or vector of the present invention is introduced, the introduction method, etc. are as described in 4.

[0051] In one embodiment of the method of the present invention, there is provided a method for producing cells, which comprises the step of transducing a retrovirus containing the nucleic acid or vector of the present invention into human peripheral blood mononuclear cells (PBMCs). After the transfection step, the cells may be optionally cultured.

[0052] In the production method of the present invention, the medium used for culturing cells during or after the above-mentioned introduction step is not particularly limited, and a medium used for culturing animal cells can be prepared as a basal medium. Examples of basal media include Iscove's Modified Dulbecco's Medium (IMDM), Medium 199, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12, RPMI 1640, Fischer's Medium, Neurobasal Medium (Life Technologies), and mixtures thereof. The medium may contain serum or may be serum-free. If necessary, the basal medium may contain, for example, vitamin C (e.g., ascorbic acid), albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, cytokines, etc.

[0053] In the present invention, "vitamin C" refers to L-ascorbic acid and its derivatives, and "L-ascorbic acid derivatives" refers to those that become vitamin C through an enzymatic reaction in vivo. Examples of ascorbic acid derivatives used in the present invention include vitamin C phosphate, ascorbyl glucoside, ascorbyl ethyl, vitamin C ester, ascorbyl tetrahexyldecanoate, ascorbyl stearate, and ascorbic acid-2-phosphate-6-palmitate. Vitamin C phosphate is preferred, and examples include L-ascorbate phosphates such as sodium L-ascorbate phosphate and magnesium L-ascorbate phosphate.

[0054] The culture temperature conditions are not particularly limited, but are, for example, approximately 37°C to 42°C, preferably approximately 37 to 39°C. Furthermore, those skilled in the art can appropriately determine the culture period while monitoring the cell count, etc. The number of days is not particularly limited as long as the cells of the present invention are obtained, but may be, for example, 1 day or more, 2 days or more, 3 days or more, 5 days or more, 6 days or more, 7 days or more, 10 days or more, 14 days or more, or 21 days or more, preferably 7 days. Long culture periods are not usually a problem in the production method of the present invention, but for example, 10 days or less is preferred, and 7 days or less is more preferred. Culture may also be performed under hypoxic conditions, and examples of hypoxic conditions in the present invention include oxygen concentrations of 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, or 5% or less.

[0055] An anti-CD3 antibody may be used in the production method of the present invention. The anti-CD3 antibody is not particularly limited as long as it specifically recognizes CD3, and examples include antibodies produced from the OKT3 clone. The anti-CD3 antibody may be bound to magnetic beads or the like and added to the cell culture medium. Alternatively, instead of adding the anti-CD3 antibody to the medium, cells (e.g., T lymphocytes) may be stimulated by culturing them for a certain period of time on a culture vessel having an anti-CD3 antibody bound to its surface. Such cell stimulation with the anti-CD3 antibody may be performed, for example, before or during the introduction of the nucleic acid or vector of the present invention into the cells. The concentration of the anti-CD3 antibody in the medium is preferably 10 ng / ml to 1000 ng / ml (e.g., 10 ng / ml, 50 ng / ml, 100 ng / ml, 200 ng / ml, 300 ng / ml, 400 ng / ml, 500 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1000 ng / ml), with 500 ng / ml being particularly preferred. Concentrations of other antibodies can also be appropriately determined by those skilled in the art based on the culture conditions, etc.

[0056] 6. Pharmaceuticals Comprising the Nucleic Acid, Vector, or Cell of the Present Invention The present invention provides pharmaceuticals (hereinafter abbreviated as "pharmaceuticals of the present invention") comprising the nucleic acid, vector, or cell of the present invention as an active ingredient. The cells containing the nucleic acid of the present invention bind to HLA-A02 molecules and GPC3１４４－１５２ The nucleic acid, vector, or cell of the present invention can exhibit cytotoxic activity against cells that present the peptide. Therefore, a pharmaceutical comprising the nucleic acid, vector, or cell of the present invention can be used for the prevention or treatment of diseases in which GPC3 is expressed, and can be administered to mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, and humans), preferably humans. Diseases in which GPC3 is expressed include, but are not limited to, cancers and tumors that express GPC3. Therefore, in a preferred embodiment of the present invention, an anticancer agent for the prevention or treatment of cancers and tumors that express GPC3 is provided.

[0057] Such cancers and tumors that express GPC3 are described, for example, in "Daniel Baumhoer et al., Am J. Clin Pathol, 2008, 129, 899-906". Specific examples include, but are not limited to, liver cancer (e.g., hepatocellular carcinoma), ovarian cancer (e.g., ovarian clear cell adenocarcinoma), childhood cancer (e.g., hepatoblastoma), lung cancer (e.g., squamous cell carcinoma, small cell lung carcinoma), testicular cancer (e.g., non-seminomatous germ cell tumor), soft tissue tumor (e.g., liposarcoma, malignant fibrous histiocytoma), uterine cancer (e.g., cervical intraepithelial neoplasia, cervical squamous cell carcinoma), melanoma, adrenal tumor (e.g., adrenal adenoma), neural tumor (e.g., schwannoma), gastric cancer (e.g., gastric adenocarcinoma), kidney cancer (e.g., Grawitz tumor), breast cancer (e.g., invasive lobular carcinoma, mucinous carcinoma), thyroid cancer (e.g., medullary carcinoma), laryngeal cancer (e.g., squamous cell carcinoma), and bladder cancer (e.g., invasive transitional cell carcinoma). Among these, liver cancer, ovarian cancer, childhood cancer, and lung cancer are preferred from the viewpoint of GPC3 expression level, and liver cancer, particularly hepatocellular carcinoma, is preferred.

[0058] When a nucleic acid or vector is used as the active ingredient of the pharmaceutical of the present invention, it is preferable to prepare a pharmaceutical composition by mixing it with a known pharmaceutically acceptable carrier (including excipients, diluents, bulking agents, binders, lubricants, flow aids, disintegrants, surfactants, etc.) or conventional additives. Excipients are well known to those skilled in the art, and examples include phosphate-buffered saline (e.g., 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), aqueous solutions containing mineral acid salts such as hydrochloride, hydrobromide, phosphate, and sulfate, physiological saline, solutions such as glycol or ethanol, and salts of organic acids such as acetate, propionate, malonate, and benzoate. In addition, auxiliary agents such as wetting agents or emulsifiers, and pH buffers may also be used. Furthermore, formulation auxiliary agents such as suspending agents, preservatives, stabilizers, and dispersing agents may also be used. The pharmaceutical composition may also be in a dry form for reconstitution with an appropriate sterile liquid prior to use. The pharmaceutical composition can be administered systemically or locally, orally or parenterally, depending on the preparation form (oral preparations such as tablets, pills, capsules, powders, granules, syrups, emulsions, and suspensions; parenteral preparations such as injections, infusions, topical preparations, and suppositories). When administered parenterally, it can be administered intravenously, intradermally, subcutaneously, rectally, transdermally, or the like. When used in the form of an injection, an acceptable buffer, solubilizer, isotonic agent, etc. can also be added.

[0059] When the active ingredient is a nucleic acid, the dosage is, for example, in the range of 0.001 mg to 10 mg of the nucleic acid per kg of body weight per administration. For example, when administered to a human patient, the dosage is in the range of 0.001 to 50 mg for a patient weighing 60 kg. When the active ingredient is a viral vector particle, the dosage is, for example, about 1 x 10 viral titer per administration for a subject weighing 60 kg. ３ pfu ~ 1 x 10 １５ The above dosages are merely examples, and can be appropriately selected depending on the type of nucleic acid or vector used, the administration route, and the age, weight, and symptoms of the recipient or patient.

[0060] When the cells of the present invention are used as the active ingredient of the pharmaceutical of the present invention, the cells may be cultured and / or stimulated using an appropriate medium and / or stimulatory molecules before administration to a subject. Examples of stimulatory molecules include, but are not limited to, cytokines, appropriate proteins, and other components. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, and IFN-γ, and IL-2 is preferably used. The concentration of IL-2 in the medium is not particularly limited, but is preferably 0.01 to 1 x 10 ５ U / mL, more preferably 1 to 1 x 10 ４ U / mL. Examples of suitable proteins include CD3 ligand, CD28 ligand, and anti-IL-4 antibody. In addition, lymphocyte stimulating factors such as lectin can also be added. Furthermore, serum or plasma may be added to the medium. The amount of these to be added to the medium is not particularly limited, but examples include 0% by volume to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage. For example, the serum or plasma concentration can be gradually reduced. The serum or plasma may be derived from either autologous or non-autologous sources, but from the viewpoint of safety, autologous sources are preferred.

[0061] In the present invention, a pharmaceutical containing the cells of the present invention as an active ingredient is preferably administered parenterally to a subject. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage is appropriately selected depending on the condition, weight, age, etc. of the subject, but typically, the number of cells is 1 x 10 per administration for a subject weighing 60 kg. ６ ~1 x 10 １０ Preferably, 1×10 ７ ~1 x 10 ９ 5×10 ７ ~5 x 10 ８The pharmaceutical composition of the present invention is administered so that the number of cells reaches 100. It may be administered once or multiple times. The pharmaceutical composition of the present invention can be in a known form suitable for parenteral administration, such as an injection or infusion. The pharmaceutical composition of the present invention may contain a pharmacologically acceptable excipient, as appropriate. Examples of pharmacologically acceptable excipients include those described above. The pharmaceutical composition of the present invention may contain physiological saline, phosphate-buffered saline (PBS), a culture medium, etc., in order to stably maintain the cells. Examples of culture media include, but are not limited to, RPMI, AIM-V, and X-VIVO10. Furthermore, the pharmaceutical composition may contain a pharmaceutically acceptable carrier (e.g., human serum albumin), a preservative, etc., for the purpose of stabilization.

[0062] Furthermore, the cells of the present invention can kill cells that express GPC3, and therefore can be used as a killing agent for cells that express GPC3. Such killing agents can be prepared and used in the same manner as the above-mentioned pharmaceuticals.

[0063] The TCRs of the present invention can also be used as fusion proteins, for example, by combining a TCR with a single-chain antibody fragment (scFv) of an anti-CD3 antibody (or a similar antibody fragment that binds to T cells and activates T cell responses). Such fusion proteins may contain artificial disulfide bonds between the constant regions of the two TCR chain polypeptides to produce stable, soluble, high-affinity TCRs. Preferably, the scFv of the fusion protein is fused to the constant region of the TCR β chain. Such fusion proteins are described, for example, in U.S. Pat. No. 7,569,664, Liddy et al., Nat. Med. 18:908-7 (2012), Oates and Jakobsen, OncoImmunology 2:e22891 (2013), and elsewhere.

[0064] When introduced into the body, such a fusion protein binds to cells expressing GPC3 through specific recognition by TCR, and the scFv binds to CD3 present on the cell surface of cytotoxic T cells, thereby damaging the GPC3-expressing cells. Therefore, pharmaceuticals containing the fusion protein or a nucleic acid encoding this protein can be used for the prevention or treatment of diseases expressing GPC3, similar to pharmaceuticals containing the nucleic acid or cells of the present invention. When used as a pharmaceutical, it can be prepared in the same manner as described above.

[0065] The sequence numbers in the sequence listing of this specification represent the following sequences. (SEQ ID NO: 1) Amino acid sequence of CDR1 of TCR5B6 α chain DRGSQS (SEQ ID NO: 2) Amino acid sequence of CDR2 of TCR5B6 α chain IYSNGD (SEQ ID NO: 3) Amino acid sequence of CDR3 of TCR5B6 α chain CAVKYGNQFYF (SEQ ID NO: 4) Amino acid sequence of CDR1 of TCR5B4 α chain DRGSQS (SEQ ID NO: 5) Amino acid sequence of CDR2 of TCR5B4 α chain IYSNGD (SEQ ID NO: 6) Amino acid sequence of CDR3 of TCR5B4 α chain CAVNFVVTGGGNKLTF (SEQ ID NO: 7) Amino acid sequence of CDR1 of TCR5B6 β chain MNHEY (SEQ ID NO: 8) Amino acid sequence of CDR2 of TCR5B6 β chain SVGEGT (SEQ ID NO: 9) Amino acid sequence of CDR3 of TCR5B6 β chain CASSYSTLRNQPQHF (SEQ ID NO: 10) Amino acid sequence of CDR1 of TCR5B4 β chain SGHNT (SEQ ID NO: 11) Amino acid sequence of CDR2 of TCR5B4 β chain YYREEE (SEQ ID NO: 12) Amino acid sequence of CDR3 of TCR5B4 β chain CASSYRAAEQYF

[0066] (SEQ ID NO: 13) Amino acid sequence of the variable region of the TCR5B6 α chain MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKYGNQFYFGTGTSLTVIP (SEQ ID NO: 14) Amino acid sequence of the variable region of the TCR5B4 α chain MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNFVVTGGGNKLTFGTGTQLKVEL (SEQ ID NO: 15) Amino acid sequence of the variable region of the TCR5B6 β chain MSLGLLCCAAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYSTLRNQPQHFGDGTRLSIL (SEQ ID NO: 16) Amino acid sequence of the variable region of the TCR5B4 β chain MGPGLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQSGHNTVSWYQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYLCASSYRAAEQYFGPGTRLTVT (SEQ ID NO: 17) Amino acid sequence of the constant region of the TCR α chain NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 18) Amino acid sequence of the constant region of TCR5B6 β chainEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 19) Amino acid sequence of the constant region of TCR5B4 β chain: EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0067] (SEQ ID NO: 20) GPC3 １４４－１５２ Peptide amino acid sequence FVGEFFTDV

[0068] (SEQ ID NO: 21) Full-length amino acid sequence of TCR5B6 α chain MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKYGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 22) Full-length amino acid sequence of TCR5B4 α chain MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNFVVTGGNKLTFGTGTQLKVELNI QNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0069] (SEQ ID NO: 23) Full-length amino acid sequence of TCR5B6 β chain MSLGLLCCAAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYSTLRNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 24) Full-length amino acid sequence of TCR5B4 β chain MGPGLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQSGHNTVSWYQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQFPNYSSELNVNALELDDSALYLCASSYRAAEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0070] (Accession number: 25) Base sequence of the variable region of the TCR5B6 α-chain ATGATGAAGAGCCTGAGAGTGCTGCTGGTGATCCTGTGGCTGCAGCTGAGCTGGGTGTGGAGCCAGCAGAAGGAGGTGGAGCAGAATTCTGGCCCTCTGAGCGTGCCTGAAGGAGCCATTGCCAGCCTGAATTGCACCTACAGCGACAGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCGAGCTGATCATGTTCATCTACAGCAACGGCGACAAGGAGGACGGCAGATTCACCGCCCAGCTGAACAAGGCCAGCCAGTACGTGAGCCTGCTGATCAGAGACAGCCAGCCTAGCGATAGCGCCACCTATCTGTGCGCCGTGAAGTACGGCAACCAGTTCTACTTCGGCACCGGCACCAGCCTGACCGTGATCCCC (Accession number: 26) Base sequence of the variable region of the TCR5B4 α-chain ATGATGAAGAGCCTGAGAGTGCTGCTGGTGATCCTGTGGCTGCAGCTGAGCTGGGTGTGGAGCCAGCAGAAGGAGGTGGAGCAGAATAGCGGCCCACTGAGCGTGCCAGAAGGCGCTATCGCCAGCCTGAATTGCACCTACAGCGACAGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCGAGCTGATCATGTTCATCTACAGCAACGGCGACAAGGAGGACGGCAGATTCACCGCCCAGCTGAACAAGGCCAGCCAGTACGTGAGCCTGCTGATCAGAGACAGCCAGCCCAGCGATAGCGCCACCTACCTGTGTGCCGTGAACTTCGTGGTGACCGGCGGCGGCAACAAACTGACCTTTGGCACCGGCACCCAGCTGAAGGTGGAGCTG

[0071] (SEQ ID NO: 27) Nucleotide sequence of the variable region of the TCR5B6 β chain: ATGAGCCTGGGCCTGCTGTGTTGCGCCGCCTTTAGCCTGCTGTGGGCTGGACCAGTGAATGCCGGCGTGACACAGACCCCAAAGTTCAGAGTGCTGAAGACCGGCCAGAGCATGACCCTGCTGTGCGCCCAGGACATGAACCACGAGTATATGTATTGGTACAGACAGGACCCCGGCATGGGCCTGAGACTGATCCACTACAGCGTGGGAGAGGGCACAACAGCCAAAGGCGAGGTGCCTGATGGCTACAATGTGAGCAGACTGAAGAAGCAGAATTTCCTGCTGGGACTGGAGTCTGCCGCCCCTAGCCAGACAAGCGTGTACTTTTGCGCCAGCAGCTACAGCACCCTGAGAAACCAGCCCCAGCACTTCGGCGACGGCACCAGACTGAGCATCCTG (SEQ ID NO: 28) Nucleotide sequence of the variable region of the TCR5B4 β chain ATGGGCCCCGGCCTGCTGTGTTGGGTGCTGCTGTGTCTGCTGGGCGCCGGCTCTGTGGAAACAGGCGTGACCCAGTCTCCTACCCACCTGATCAAGACCAGAGGCCAGCAGGTGACCCTGAGATGCAGCAGCCAGAGGCGGCCACAACACCGTGTCTTGGTATCAGCAGGCTCTGGGCCAGGGCCCTCAGTTCATC TTCCAGTACTACAGAGAGGAGGAGAACGGCAGAGGCAACTTCCCCCCCAGATTCAGCGGCCTGCAGTTCCCCAACTACAGCAGCGAGCTGAACGTGAACGCCCTGGAGCTGGACGATAGCGCCCTGTACCTGTGTGCCAGCAGCTACAGAGCCGCCGAGCAGTACTTCGGCCCTGGCACAAGACTGACCGTGACC

[0072] The present invention will be explained in more detail below by way of examples, but these are merely illustrative and the present invention is not limited to these examples.

[0073] Example 1 (1) Antigen Administration and Acquisition of CTL Clones In a non-random, open-label phase 1 clinical trial of glypican-3 (GPC3) peptide in pediatric cancer patients [Study name: Phase I clinical trial of HLA-A24- and -A2-binding Glypican-3 (GPC3)-derived peptide vaccine therapy for various pediatric cancers, UMIN study ID: 000006357], an antigen (HLA-A*02:01-restricted GPC3) synthesized in accordance with the guidelines of Good Manufacturing Practice was administered to HLA-A2-positive pediatric hepatoblastoma patients. １４４－１５２ Peptide (hereinafter referred to as "GPC3 １４４－１５２ The vaccine was an emulsion of IgG1-1000-10000 (SEQ ID NO: 20: FVGEFFTDV; American Peptide) and incomplete Freund's adjuvant (IFA; Montanide ISA-51VG; Seppic Pharmaceuticals), and administered intradermally every two weeks (body weight < 20 kg: 1.5 mg; > 20 kg: 3.0 mg). CTL clones 5B6 and 5B4 were established from peripheral blood mononuclear cells (PBMCs) obtained after vaccination by the following method.

[0074] (2) Isolation of PBMCs and Establishment of CTL Bulk PBMCs were isolated by centrifuging the peripheral blood (10 mL) through a Ficoll-Paque gradient. The isolated PBMCs (2 x 10 ６ 10 μg / mL of GPC3 １４４－１５２ The cells were cultured for 14 days in AIM-V medium supplemented with the peptide, 10% human AB serum, 50 IU / ml recombinant human interleukin-2 (IL-2), and 10 ng / ml recombinant human interleukin-15 (IL-15) to establish a CTL bulk.

[0075] (3) Establishment of CTL clones GPC3 obtained in (2) above １４４－１５２ Peptide-reactive CD8 ＋CD8-positive and GPC3 dextramer-positive cells were isolated from the CTL bulk using a FACSAria cell sorter. The CD8-specific antibody used for isolation was GPC3 (ProImmune). １４４－１５２ / HLA-A*02:01 Dextramers were purchased from Immudex.

[0076] Each of the cells obtained by the above isolation was seeded in a 96-well plate (1 cell / well), and irradiated (100 Gy) non-autologous PBMCs (8 x 10 per well) were cultured in AIM-V culture medium supplemented with 10% human AB serum, IL-2 (100 U / mL), IL-15 (10 ng / mL), and phytohemagglutinin-P (PHA) (1 μg / mL). ４ The CTL clones 5B6 and 5B4 were established by stimulation for 14-21 days using these cells as feeder cells.

[0077] Test Example 1 Dextramer analysis GPC3 of established CTL clones 5B6 and 5B4 １４４－１５２ To measure peptide specificity, anti-CD3 antibody, anti-CD8 antibody, and GPC3 １４４－１５２ / HLA-A*02:01 Dextramer staining was performed and measured using a flow cytometer. The results are shown in Figure 1.

[0078] As a result, CTL clones 5B6 and 5B4 were 99.9% CD3-positive, CD8-positive, and GPC3-positive. １４４－１５２ / HLA-A*02:01 Dextramer positive.

[0079] Test Example 2 ELISPOT Assay To measure the antigen-specific cytokine production ability of the established CTL clones 5B6 and 5B4, an interferon-gamma (IFN-γ) ELISPOT assay was performed. CTL clones 5B6 and 5B4 (1 x 10 per well) were used. ５ ) at 37°C and 5% CO ２In the presence of α-glucan, the cells were cultured for 20 hours with a cancer cell line (SK-Hep-1 / hGPC3) in which GPC3 was forcibly expressed or its mock control cancer cell line (SK-Hep-1 / vec). As a positive control, T2 cells (HLA-A*02:01 positive, TAP negative) were cultured with GPC3. １４４－１５２ As a negative control, T2 cells pulsed with an HIV peptide were used. The results are shown in Figure 2.

[0080] As a result, each CTL clone used in this test was GPC3 １４４－１５２ It was found that the cells responded only to cancer cells pulsed with the peptide and cancer cells expressing GPC3, and had the ability to produce IFN-γ.

[0081] Test Example 3: CD107a Analysis CD107a analysis was performed to measure the antigen-specific cytotoxic activity of the established CTL clones 5B6 and 5B4. CTL clones 5B6 and 5B4 were each co-cultured with target cells for 3.5 hours, stained with anti-CD8 antibody and anti-CD107a antibody, and measured with a flow cytometer. A cancer cell line (SK-Hep-1 / hGPC3) in which GPC3 was forcibly expressed or its mock control cancer cell line (SK-Hep-1 / vec) was used as the target cells. As a positive control, T2 cells (HLA-A*02:01 positive, TAP negative) were co-cultured with GPC3. １４４－１５２ As a negative control, T2 cells pulsed with an HIV peptide were used. The results are shown in Figure 3.

[0082] As a result, each CTL clone used in this test was GPC3 １４４－１５２ It was found that the cells respond only to cancer cells pulsed with the peptide and cancer cells expressing GPC3, and have the ability to produce cytotoxic granules such as perforin and granzymes.

[0083] Test Example 4: Cytotoxicity Test A cytotoxicity test was performed to measure the antigen-specific cytotoxicity of the established CTL clones 5B6 and 5B4. CTL clones 5B6 and 5B4 were each co-cultured with target cells labeled with calcein AM. As target cells, a cancer cell line (SK-Hep-1 / hGPC3) in which GPC3 was forcibly expressed or its mock control cancer cell line (SK-Hep-1 / vec) was used, and the cells were co-cultured overnight. As a positive control, T2 cells (HLA-A*02:01 positive, TAP negative) were co-cultured with GPC3. １４４－１５２ The CTL clones were pulsed with the HIV peptide and co-cultured for 3 hours with T2 cells pulsed with the HIV peptide as a negative control. The cytotoxicity rate (%) for each CTL clone was calculated using the following formula. The results are shown in Figure 4.

[0084]

[0085] As a result, CTL clones 5B6 and 5B4 were found to be GPC3 １４４－１５２ It was shown that the peptide exhibited cytotoxicity only against cancer cells pulsed with the peptide and cancer cells expressing GPC3.

[0086] Test Example 5: TCR Sequence Decoding The TCR sequences of CTL clones 5B6 and 5B4 were analyzed using the following method. Total RNA from T cells was extracted using the RNeasy Mini Kit (QIAGEN). First-strand cDNA was synthesized using SuperScript III reverse transcriptase (ThermoFisher Scientific) and oligo dT primers (Invitrogen), and the cDNA was amplified by PCR. This was subjected to repertoire analysis and preliminary sequence analysis using a next-generation sequencer (MiSeq, Illumina). Subsequently, primers for the V region (5' untranslated region) and C region (immediately preceding the poly(A) addition signal in the 3' untranslated region) were designed based on the partial sequence data from the repertoire analysis results, and amplified by PCR. The PCR-amplified fragment was column purified and then reacted with sequencing primers using BigDye Terminator V3.1 Cycle Sequencing Kit (ThermoFisher Scientific).

[0087] After column purification, the TCR sequences of 5B6 and 5B4 were determined using an ABI capillary sequencer. The sequences of the complementarity-determining regions (CDRs) and variable regions (V regions) of each chain were assigned the sequence numbers shown in Table 1.

[0088]

[0089] Example 2: Construction of a Plasmid for Retroviral Vector Production First, pMu1-MC was prepared according to the method described in WO 2012 / 157742. pMu1-MC is a retroviral vector plasmid having, from the 5' end, a 5' LTR (long terminal repeat) derived from MMLV (Moloney murine leukemia virus), an SD (splice donor) sequence derived from MMLV, a ψ (packaging signal) sequence derived from MMLV, a sequence for transcribing four types of single-stranded RNA that form a stem-loop structure, an SA (splice acceptor) sequence derived from the human EF1α gene, and an MMLV-derived 3' LTR, with the U3 region of the 3' LTR replaced with a sequence derived from MSCV (murine stem cell virus).

[0090] Next, the base sequences of the variable and constant regions of the GPC3-specific TCR α chain and β chain derived from the 5B6 and 5B4 clones were codon-optimized and then artificially synthesized. Using In-Fusion (registered trademark) HD ​​Cloning Kit (Takara Bio Inc.), the following sequences were inserted into pMu1-MC in this order: TCR β chain variable region, TCR β chain constant region (CTL clone 5B6: SEQ ID NO: 18, CTL clone 5B4: SEQ ID NO: 19), T2A, TCR α chain variable region, and TCR α chain constant region (SEQ ID NO: 17). These sequences were then named pMu1-5B6-GPC3-siTCR and pMu1-5B4-GPC3-siTCR, respectively, with the arrangement shown in FIG. 5. The amino acid sequence of the full-length TCRα chain of CTL clone 5B6 is shown in SEQ ID NO: 21, the amino acid sequence of the full-length TCRα chain of CTL clone 5B4 is shown in SEQ ID NO: 22, the amino acid sequence of the full-length TCRβ chain of CTL clone 5B6 is shown in SEQ ID NO: 23, and the amino acid sequence of the full-length TCRβ chain of CTL clone 5B4 is shown in SEQ ID NO: 24.

[0091] Example 3 Preparation of Retrovirus Solution Transformants were obtained by transforming Escherichia coli HST08 with pMu1-5B6-GPC3-siTCR and pMu1-5B4-GPC3-siTCR prepared in Example 2. The plasmid DNA contained in these transformants was purified using NucleoSpin (registered trademark) Plasmid Midi (Machlinergel GmbH) and subjected to the following procedure as DNA for transfection.

[0092] The prepared transfection DNA and the pGP and pE-eco vectors contained in the Retrovirus Packaging Kit Eco (Takara Bio) were transfected into 293T cells. This procedure was performed according to the kit's product protocol. Supernatants containing the ecotropic virus were obtained from each of the resulting transduced cells and filtered through a 0.45 μm filter (Milex HV, Millipore). PG13 cells (ATCC CRL-10686) were infected with the ecotropic virus using a polybrene method. The culture supernatants of the resulting cells were collected and filtered through a 0.45 μm filter to produce retrovirus solutions Mu1-5B6-GPC3-siTCR and Mu1-5B4-GPC3-siTCR, which were used in the following examples.

[0093] Example 4 Infection of human PBMCs with a GPC3-specific siTCR-expressing retroviral vector PBMCs derived from a healthy subject (Charles River Laboratories Cell Solutions) were costimulated with an anti-CD3 antibody (clone OKT3) (Takara Bio Inc.) and RetroNectin (registered trademark, Takara Bio Inc.), and then incubated. Three days later, using RetroNectin-coated plates, the cells were infected with each of the retrovirus solutions prepared in Example 3 (Mu1-5B6-GPC3-siTCR, Mu1-5B4-GPC3-siTCR) and, as a control, a retrovirus solution containing a GPC3-specific TCR gene obtained previously (Mu1-Pt16-GPC3-siTCR), and the cells were infected again the following day to prepare TCR-expressing T cells (5B6 siTCR-T, 5B4 siTCR-T, Pt16 siTCR-T). Cells 3, 6, and 7 days after virus infection were used in later examples.

[0094] Example 5 Dextramer staining and flow cytometry analysis of siTCR-T when blocked by anti-CD8 antibody TCR-expressing T cells (siTCR-T) prepared in Example 4 3 days after virus infection were incubated with 10-, 90-, or 270-fold diluted anti-CD8-APC (clone RPA-T8) (Biolegend) and anti-CD8-FITC (clone LT8) (Biorad) at 4°C for 20 minutes to block CD8 on the T cell surface. Then, HLA-A*02:01 Dextramer-PE (GPC3 １４４－１５２ The T cells were stained with dextramer (FVGEFFTDV; Imdex) at room temperature for 10 minutes. As a control without CD8 blocking, the T cells prepared in Example 4 3 days after virus infection were stained with the same dextramer for 10 minutes at room temperature, and then stained with anti-CD8-APC (clone RPA-T8) for 20 minutes at 4°C. Flow cytometry was performed using a FACSCant II flow cytometer (BD Biosciences). The results for CD8-positive cells are shown in Figure 6, and the results for CD8-negative cells are shown in Figure 7. As shown in Figure 6, 5B6 siTCR-T and 5B4 siTCR-T maintained a high dextramer positivity rate in two donors (HC814, HC021) compared to Pt16 siTCR-T, even under anti-CD8 antibody blocking. Furthermore, as shown in Figure 7, 5B6 siTCR-T and 5B4 siTCR-T confirmed dextramer-positive cells among CD8-negative cells, which were hardly observed with Pt16 siTCR-T. These results demonstrate that 5B6 siTCR-T and 5B4 siTCR-T have stronger binding ability with respect to dextramer reactivity than Pt16 siTCR-T.

[0095] Example 6 Peptide titration assay for anti-CD8 antibody blocking of siTCR-T TCR-expressing T cells 6 days after virus infection prepared in Example 4 were incubated with 10-fold diluted anti-CD8-APC (clone RPA-T8) and anti-CD8-FITC (clone LT8) at 4°C for 20 minutes, then treated with Brefeldin A and cocultured with T2 target cells at 1 x 10 ５The cells were mixed and co-cultured overnight. As a control, a condition in which no anti-CD8 antibody was used and CD8 was not blocked was also prepared. T2 cells (HLA-A*02:01 positive, TAP negative) were treated with GPC3 １４４－１５２ The cells were pulsed with peptide at final concentrations of 1E+02, 1E+03, 1E+04, 1E+05, 1E+06, and 1E+07 pg / mL. After co-culture, the cells were stained with anti-CD8-APC at room temperature for 15 minutes, permeabilized with IntraPrep (trademark, Beckman Coulter), and stained with anti-IFNγ-PE (Beckman Coulter) at room temperature for 15 minutes. Flow cytometry was performed using a FACSCant II flow cytometer. For each clone, the IFNγ positive rate was plotted against the peptide concentration, and the peptide concentration at which the positive rate reached 50 (EC50) was calculated, with the IFNγ positive rate at a peptide concentration of 1E+07 pg / mL set at 100. The results are shown in Table 2. As can be seen from Table 2, 5B6 siTCR-T and 5B4 siTCR-T had lower EC50 values ​​than Pt16 siTCR-T for both CD8-positive and CD8-negative cells. Furthermore, in CD8-positive cells, 5B6 siTCR-T and 5B4 siTCR-T had lower EC50 values ​​than Pt16 siTCR-T, even under CD8 blockade. These results confirmed that 5B6 siTCR-T and 5B4 siTCR-T exhibit high functional avidity.

[0096]

[0097] Example 7: Evaluation of cytotoxic activity of siTCR-T The cytotoxic activity of siTCR-T was measured using an xCelligence real-time cell analysis system (Agilent Technologies). Specifically, the human liver cancer cell line HepG2 and the HepG2-GPC3-KO cell line, in which the GPC3 gene of HepG2 was knocked out, were seeded onto a biosensor plate (E-plate) as target cells, and then cultured for 24 hours on a device installed in an incubator. As effector cells, TCR-expressing T cells prepared in Example 4 7 days after virus infection and non-transfected cells as a control were added at an E / T (effector / target) ratio of 10 and co-cultured for 4 days on the device. The results of the cell number (cell index) of target cells over time are shown in Figure 8.

[0098] As shown in Figure 8, for the HepG2-GPC3-KO cell line, both 5B6 siTCR-T and 5B4 siTCR-T showed a cell index similar to that of non-transfected cells, whereas for the HepG2 cell line, 5B6 siTCR-T and 5B4 siTCR-T, unlike non-transfected cells, reduced the cell index, demonstrating that 5B6 siTCR-T and 5B4 siTCR-T have GPC3-specific cytotoxic activity.

[0099] Example 8: Evaluation of in vivo cytotoxic activity of siTCR-T The cytotoxic activity of siTCR-T was measured by simultaneously transplanting it into immunodeficient mice with a target cancer cell line. Specifically, human liver cancer cell line HepG2 strain was used as the target cell, and 5B6 siTCR-expressing T cells (5B6 siTCR-T) prepared in Example 4 7 days after virus infection or control non-transfected cells (NGMC) were mixed at an E / T (effector / target) ratio of 1.0. NSG mice (NOD.Cg-Prkdc) that had been irradiated with 1.5 Gy of X-rays immediately before the experiment were used to obtain 20 million cells, i.e., 10 million cells of each type. ｓｃｉｄ Il2rg ｔｍ１ＷｊｌThe tumors were subcutaneously transplanted into mice (SzJ) and changes in tumor formation were observed for 21 days. The changes in tumor were calculated by measuring the long and short diameters with a vernier caliper and dividing the product of the square of the short diameter and the long diameter by 2. The results of the tumor volume over time are shown in Figure 9.

[0100] As shown in Figure 9, in the HepG2-5B6 siTCR-T mixed administration group, some individuals showed no visible tumor masses around 7 days after administration, and no tumor masses were visible in any individuals when measured 14 days later. On the other hand, in the group administered with non-transfected cells, tumor masses were visible in all individuals. These results demonstrate that 5B6 siTCR-T has cytotoxic activity against GPC3-expressing cancer cell lines in vivo.

[0101] Example 9: Evaluation of in vivo antitumor activity of siTCR-T The antitumor activity of siTCR-T was evaluated in immunodeficient mice bearing subcutaneous tumors. Specifically, 10 million HepG2 cells used in Example 8 were implanted subcutaneously into immunodeficient NSG mice. After confirming tumor mass formation 7 days after implantation, the mice were subjected to 1.5 Gy of whole-body X-ray irradiation to eliminate any remaining immune cells in the body. The following day (8 days after implantation), 10 million 5B6 siTCR-T or control cells (NGMC) were administered via the tail vein, and changes in tumor mass were observed for 7 days (Figure 10a). The change in tumor volume over time was calculated by measuring the long and short diameters with calipers and dividing the square of the short diameter by the product of the long diameter and the long diameter by 2. The 5B6 siTCR-T-treated group showed a significant decrease compared to the control group 7 days after implantation (15 days after cell line implantation) (Figure 10b). This result was also evident from the state of the excised tumors (Figure 11). Furthermore, in the 5B6 siTCR-T administration group, where tumor shrinkage was observed, flow cytometry analysis revealed that human CD3 and human CD8 were positive and GPC3 were positive in the tumor tissue, spleen, and blood. １４４－１５２The presence of / HLA-A*02:01 dextramer-positive cells was confirmed (Figure 12). This indicates that in the 5B6 siTCR-T administration group, the administered 5B6 siTCR-T infiltrated into tumor tissue and induced an antitumor effect, and that even 7 days after administration, the administered 5B6 siTCR-T remained in the tumor tissue, spleen, and blood. These results demonstrate that 5B6 siTCR-T has a therapeutic effect on tumor tissue expressing GPC3.

[0102] According to the present invention, GPC3 peptide (GPC3 １４４－１５２ The present invention provides a T cell receptor or a fragment thereof capable of binding to a peptide containing the GPC3 peptide (GPC3 peptide) or a complex of the GPC3 peptide and HLA-A02, as well as nucleic acids encoding the same. The nucleic acid encoding the T cell receptor or a fragment thereof can confer cytotoxic activity to T cells against cells presenting HLA-A02 and the GPC3 peptide, and is therefore useful for the prevention or treatment of diseases expressing GPC3.

Claims

1. A T cell receptor (TCR) or a fragment thereof comprising, as the complementarity determining region of the α chain, the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and as the complementarity determining region of the β chain, the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, or comprising, as the complementarity determining region of the α chain, the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and as the complementarity determining region of the β chain, the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, wherein the TCR or the fragment thereof is capable of binding to a complex of a peptide comprising the amino acid sequence shown in SEQ ID NO:20 and HLA-A02.

2. A nucleic acid comprising, as an α-chain variable region, the amino acid sequence shown in SEQ ID NO: 13, an amino acid sequence shown in SEQ ID NO: 13 in which one or several amino acids have been deleted, substituted or added, or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 13; and, as a β-chain variable region, the amino acid sequence shown in SEQ ID NO: 15, an amino acid sequence shown in SEQ ID NO: 15 in which one or several amino acids have been deleted, substituted or added, or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15; or, as an α-chain variable region, the amino acid sequence shown in SEQ ID NO: 14, an amino acid sequence shown in SEQ ID NO: 14 in which one or several amino acids have been deleted, substituted or added, or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 14; and, as a β-chain variable region, the amino acid sequence shown in SEQ ID NO: 16, an amino acid sequence shown in SEQ ID NO: 16 in which one or several amino acids have been deleted, substituted or added, or The TCR or a fragment thereof according to claim 1, comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:

16.

3. comprising, as the full-length amino acid sequence of the α chain, the amino acid sequence shown in SEQ ID NO:21, an amino acid sequence shown in SEQ ID NO:21 in which one or several amino acids have been deleted, substituted or added, or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:21, and, as the full-length amino acid sequence of the β chain, the amino acid sequence shown in SEQ ID NO:23, an amino acid sequence shown in SEQ ID NO:23 in which one or several amino acids have been deleted, substituted or added, or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:23; or, as the full-length amino acid sequence of the α chain, the amino acid sequence shown in SEQ ID NO:22, an amino acid sequence shown in SEQ ID NO:22 in which one or several amino acids have been deleted, substituted or added, or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:22, and, as the full-length amino acid sequence of the β chain, the amino acid sequence shown in SEQ ID NO:24, 3. The TCR or a fragment thereof according to claim 1 or 2, comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO:24, or an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:

24.

4. A nucleic acid encoding the TCR or a fragment thereof according to any one of claims 1 to 3.

5. A vector comprising the nucleic acid of claim 4.

6. A cell comprising the nucleic acid of claim 4 or the vector of claim 5.

7. The cell of claim 6, wherein the cell is a T cell.

8. The cell according to claim 7, which has CD8-independent cytotoxic activity.

9. A method for producing a cell according to any one of claims 6 to 8, comprising the step of introducing the nucleic acid according to claim 4 or the vector according to claim 5 into a cell.

10. A medicine comprising the cells according to any one of claims 6 to 8.

11. The pharmaceutical composition according to claim 10 for use in the prevention or treatment of cancer.

12. An agent for killing a cell expressing glypican 3, comprising the cell according to any one of claims 6 to 8.

13. The cell according to any one of claims 6 to 8 for use in the prevention or treatment of cancer.

14. Use of the cell according to any one of claims 6 to 8 for producing a preventive or therapeutic agent for cancer.

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