Immune cells that specifically recognize human mesothelin, express IL-7, and CCL19

Abstract

To provide an immunocompetent cell that targets mesothelin.SOLUTION: An immunocompetent cell that expresses a cell surface molecule specifically recognizing human mesothelin, interleukin-7 (IL-7), and chemokine (C-C motif) ligand 19 (CCL19) is produced. It is preferable that the cell surface molecule that specifically recognizes human mesothelin is a chimera antigen receptor (CAR) provided with a single-chain antibody, a membrane-spanning domain and a signal transmission domain which induces the activation of the immunocompetent cell, and that the heavy chain variable domain and the light chain variable domain are linked via a peptide linker comprising a sequence of 2-30 amino acids.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a cell surface molecule that specifically recognizes human mesothelin, an immune cell that expresses interleukin-7 (IL-7) and chemokine (C-C motif) ligand 19 (CCL19), a pharmaceutical composition containing such immune cells, a nucleic acid encoding a cell surface molecule that specifically recognizes mesothelin, a nucleic acid encoding IL-7, an expression vector containing a nucleic acid encoding CCL19, and a method for producing an immune cell that expresses a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19, which comprises introducing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19 into immune cells.

Background Art

[0002] Malignant tumors are diseases that affect many patients worldwide, and generally chemotherapy, radiotherapy, or surgical therapy is widely performed. However, there are various problems such as side effects, loss of some functions, and inability to treat recurrence or metastasis. Therefore, in recent years, the development of immunocytotherapy has been promoted in order to better maintain the quality of life (QOL) of patients. This immunocytotherapy is a therapy in which immune cells are collected from a patient, treated and amplified so as to enhance the immune function of such immune cells, and then re-introduced into the patient. Specifically, a therapy is known in which T cells are collected from a patient, a nucleic acid encoding a chimeric antigen receptor (hereinafter also referred to as "CAR") is introduced into such T cells and amplified, and then re-introduced into the patient (see Non-Patent Document 1). Such a therapy is currently undergoing clinical trials worldwide, and results showing effectiveness have been obtained in hematological malignancies such as leukemia and lymphoma.

[0003] On the one hand, the inventors of the present invention have proposed an immunocytotherapy (see Patent Documents 1 and 2) that significantly suppresses solid cancer by simultaneously expressing IL-7 and CCL19. By such a method, it is possible to enhance the activation of endogenous immune cells and the ability to accumulate in tumor cells.

[0004] Incidentally, mesothelin is known to be expressed in cancer cells such as mesothelioma, colorectal cancer (rectal cancer, colon cancer), pancreatic cancer, ovarian cancer, lung cancer, breast cancer, and head and neck cancer. CAR-T cells targeting such mesothelin have been disclosed (see Patent Documents 3 and 4).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] As described above, immunotherapy using CAR-expressing T cells, TCR-expressing T cells, etc. that simultaneously express IL-7 and CCL19 has been developed, significantly improving the proliferative ability, survival ability of immune cells, or the ability of host immune cells to accumulate, and the development of a technology applicable to solid cancers for which sufficient therapeutic effects have not been recognized in conventional immunotherapy is progressing. On the other hand, in the development of CAR-expressing immune cells targeting mesothelin, which is highly expressed in cancer cells such as mesothelioma and pancreatic cancer, the current situation is that the accumulation of immune cells at the cancer site is insufficient, and the anti-tumor effect is short-lived and there is a risk of tumor recurrence, and satisfactory clinical results have not been obtained. Therefore, an object of the present invention is to provide new immune cells targeting mesothelin.

Means for Solving the Problems

[0008] The present inventors examined further possibilities of T cells expressing CAR, IL-7, and CCL19 that they had developed so far. As a result, by selecting and using a CAR containing a specific amino acid sequence as a cell surface molecule that specifically recognizes human mesothelin and containing a single-chain antibody that specifically binds to human mesothelin, it has cytotoxic activity against cancer cells expressing mesothelin and can suppress the decrease in survival rate caused by tumors formed by cancer cells expressing mesothelin, and the present invention was completed.

[0009] That is, the present invention is as follows. 〔1〕Immune cells expressing a cell surface molecule that specifically recognizes human mesothelin, interleukin 7 (IL-7), and chemokine (C-C motif) ligand 19 (CCL19). 〔2〕The immune cells according to the above 〔1〕, which are immune cells isolated from a living body. 〔3〕The immune cells according to the above 〔1〕 or 〔2〕, characterized by containing a nucleic acid encoding a cell surface molecule that specifically recognizes exogenous human mesothelin, a nucleic acid encoding exogenous IL-7, and a nucleic acid encoding exogenous CCL19. 〔4〕The immune cells described in 〔3〕 above, wherein the nucleic acid encoding exogenous IL-7 and the nucleic acid encoding exogenous CCL19 are a nucleic acid encoding exogenous human IL-7 and a nucleic acid encoding exogenous human CCL19. 〔5〕The immune cells described in 〔3〕 or 〔4〕 above, wherein the nucleic acid encoding a cell surface molecule that specifically recognizes exogenous human mesothelin, the nucleic acid encoding exogenous IL-7, and the nucleic acid encoding exogenous CCL19 are integrated into the genome. 〔6〕The immune cells according to any one of 〔1〕 to 〔5〕 above, wherein the cell surface molecule that specifically recognizes human mesothelin is a chimeric antigen receptor (CAR) comprising a single-chain antibody, a transmembrane region, and a signal transduction region that induces activation of immune cells. 〔7〕The immune cells described in 〔6〕 above, wherein the single-chain antibody in the CAR is any of the following. (1-1) A single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (2-1) A single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; A single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 21, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 23, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 24, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 25; [8] The immune cell according to the above [6] or [7], wherein the single-chain antibody in the CAR is any one of the following. A single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and a light-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; A single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and a light-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; A single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5 and a light-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6; A single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and a light-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; A single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and a light-chain variable region consisting of an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; [9] The immune cell according to any one of the above [6] to [8], wherein the single-chain antibody in the CAR is any one of the following. A single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (2-3) A single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (3-3) A single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; (4-3) A single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (5-3) A single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2;

[10] The immune cell according to any one of [6] to [9] above, wherein the transmembrane region in the CAR comprises an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7.

[11] The immune cell according to any one of [6] to

[10] above, wherein the signal transduction region that induces activation of the immune cell in the CAR comprises the amino acid sequences shown in SEQ ID NOs: 8, 9, and 10.

[12] The immune cell according to any one of [7] to

[11] above, wherein the heavy-chain variable region and the light-chain variable region are linked via a peptide linker consisting of 2 to 30 amino acid sequences.

[13] The immune cell according to

[12] above, wherein the peptide linker consists of the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 27.

[14] The immune cell according to any one of [6] to

[13] above, wherein the single-chain antibody in the CAR is any of the following. (1-4) A single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; A single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (3-4) A single-chain antibody sequentially comprising a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1; (4-4) A single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (5-4) A single-chain antibody sequentially comprising a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1; (6-4) A single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; (7-4) A single-chain antibody sequentially comprising a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5; (8-4) A single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; (9-4) A single-chain antibody sequentially comprising a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5; 〔15〕The immune cell according to any one of 〔6〕 to 〔14〕 above, wherein the signal transduction region that induces activation of immune cells in CAR contains a polypeptide of the intracellular region of CD28, a polypeptide of the intracellular region of 4-1BB, and a polypeptide of the intracellular region of CD3ζ. 〔16〕The immune cell according to any one of 〔1〕 to 〔15〕 above, wherein the immune cell is a T cell. 〔17〕The immune cell according to any one of 〔1〕 to 〔16〕 above, wherein the immune cell is a T cell derived from or isolated from a human. 〔18〕A pharmaceutical composition containing the immune cell according to any one of 〔1〕 to 〔17〕 above and a pharmaceutically acceptable additive. 〔19〕The pharmaceutical composition according to 〔18〕 above, for use in the treatment of cancer. 〔20〕An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19. 〔21〕A method for producing immune cells that express a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19, characterized by introducing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19 into immune cells.

Advantages of the Invention

[0010] The immune cells of the present invention have cancer cell cytotoxic activity that expresses human mesothelin, and can suppress the formation of tumors that express human mesothelin. In addition, the immune cells of the present invention also have an effect of suppressing cancer cell recurrence.

Brief Description of the Drawings

[0011]

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Modes for Carrying Out the Invention

[0012] The immune cells of the present invention may be immune cells that express a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19, but are preferably immune cells that contain a nucleic acid encoding a foreign cell surface molecule, a nucleic acid encoding a foreign IL-7, and a nucleic acid encoding a foreign CCL19. Such immune cells can suppress tumor formation by cancer cells expressing human mesothelin.

[0013] (Human mesothelin) Human mesothelin is a 40 kDa protein that is hardly expressed in normal cells and is highly expressed in cancer cells such as mesothelioma and pancreatic cancer. The sequence information of human mesothelin can be appropriately obtained by searching known literature and databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). For example, as the amino acid sequence information of human mesothelin, Genbank accession numbers NP_037536.2, AAV87530.1, and their isoforms can be mentioned.

[0014] (Cell surface molecule) Examples of the cell surface molecule that specifically recognizes human mesothelin include a chimeric antigen receptor (CAR) that specifically recognizes human mesothelin, a T cell receptor (TCR) that specifically recognizes a peptide derived from human mesothelin, and a molecule or factor that confers specific discrimination ability against human mesothelin by being expressed on the cell surface, such as a protein or nucleic acid that specifically binds to human mesothelin. Note that CAR is an artificial chimeric protein in which a single-chain antibody (scFv) that recognizes a cell surface antigen of cancer cells is fused with a signal transduction region that induces activation of T cells.

[0015] The above cell surface molecule is preferably localized on the cell surface of immune cells by a signal peptide (leader sequence). Examples of the signal peptide include polypeptides of signal peptides (leader sequences) derived from immunoglobulin heavy chain, immunoglobulin light chain, CD8, α and β chains of T cell receptor, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR. Specifically, it consists of an amino acid sequence having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more in the amino acid sequence shown in SEQ ID NO: 11 or 12, and having an action equivalent to that of SEQ ID NO: 11 or 12; or an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 11 or 12, and having an action equivalent to that of the amino acid sequence shown in SEQ ID NO: 11 or 12. Note that the signal peptide is removed in the mature protein after the localization is completed.

[0016] In the present specification, the "amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted, and / or added" includes, for example, an amino acid sequence in which the number of amino acid residues deleted, substituted, inserted, and / or added is in the range of 1 to 30, preferably in the range of 1 to 20, more preferably in the range of 1 to 15, still more preferably in the range of 1 to 10, still more preferably in the range of 1 to 5, still more preferably in the range of 1 to 3, and still more preferably in the range of 1 to 2. These amino acid residue mutation treatments can be performed by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, and mutagenesis.

[0017] As used herein, the term "identity" refers to the degree of polypeptide or polynucleotide sequence similarity, which is determined by matching a query sequence with other preferably identical type sequences (nucleic acid or protein sequences). Preferred computer program methods for calculating and determining "identity" include, for example, GCG BLAST (Basic Local Alignment Search Tool) (Altschul et al., J. Mol. Biol. 1990, 215:403-410; Altschul et al., Nucleic Acids Res. 1997, 25:3389-3402; Devereux et al., Nucleic Acid Res. 1984, 12:387), as well as BLASTN 2.0 (Gish W., http: / / blast.Wustl.edu, 1996-2002), as well as FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 1988, 85:2444-2448), and GCG GelMerge (Wilbur and Lipman, SIAM J. Appl. Math. 1984, 44:557-567; Needleman and Wunsch, J. Mol. Biol. 1970, 48:443-453) that determines and aligns the longest pair of overlapping contigs.

[0018] (Single-chain antibody that specifically recognizes human mesothelin) When the cell surface molecule is a CAR, it preferably contains a single-chain antibody (scFv) that specifically recognizes human mesothelin as a molecule that specifically recognizes human mesothelin. In such a single-chain antibody that specifically recognizes human mesothelin, the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody that specifically recognizes human mesothelin may be linked by a peptide linker for linking the heavy chain variable region and the light chain variable region. Examples of the combination of the heavy chain variable region and the light chain variable region in such a single-chain antibody that specifically recognizes human mesothelin include, for example, the following combinations. Note that the light chain variable region may be located upstream (N-terminal side) or downstream (C-terminal side) of the heavy chain variable region.

[0019] (1-1) A combination of a heavy-chain variable region comprising a heavy-chain CDR (complementarity determining region) 1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light-chain variable region comprising a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (2-1) A combination of a heavy-chain variable region comprising a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light-chain variable region comprising a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (3-1) A combination of a heavy-chain variable region comprising a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 21, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22, and a light-chain variable region comprising a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 23, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 24, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 25;

[0020] In addition, based on the amino acid sequences of the heavy chain variable region and the light chain variable region of known antibodies that specifically recognize human mesothelin described in the following documents (U.S. Patent No. 8,357,783, Japanese Patent Application Laid-Open No. 2017-518053), etc., the CDRs of the heavy chain variable region and the light chain variable region that specifically recognize human mesothelin are identified based on numbering systems such as IMGT, Kabat, Chothia, North, or Contact, and combinations of the heavy chain variable region and the light chain variable region having such CDRs can also be mentioned. The CDRs can be identified by the following AbodyBuilder website (http: / / opig.stats.ox.ac.uk / webapps / sabdab-sabpred / Modelling.php).

[0021] In addition, as combinations of the heavy chain variable region and the light chain variable region in the single-chain antibody that specifically recognizes the above human mesothelin, the following combinations can also be mentioned. (1-2) A combination of a heavy chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and a light chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; (2-2) A combination of a heavy chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (3-2) A combination of a heavy chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and a light chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6; (4-2) A combination of a heavy chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and a light chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; (5-2) A combination of a heavy chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2;

[0022] In addition, for example, a combination of a heavy chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the heavy chain variable region of a known antibody that specifically recognizes human mesothelin described in the following documents (U.S. Patent No. 8,357,783, JP-T 2017-518053, etc.), and a light chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the light chain variable region of the known antibody that specifically recognizes human mesothelin may also be used.

[0023] Furthermore, as combinations of the heavy chain variable region and the light chain variable region in the single-chain antibody that specifically recognizes human mesothelin, the following combinations can also be mentioned. (1-3) The combination of a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (2-3) The combination of a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (3-3) The combination of a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; (4-3) The combination of a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (5-3) The combination of a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0024] In addition, combinations of heavy chain variable regions and light chain variable regions of known antibodies that specifically recognize human mesothelin described in the following documents (US Patent No. 8,357,783, JP-T 2017-518053), etc. may also be used.

[0025] (Peptide linker) The heavy chain variable region and the light chain variable region are linked via a peptide linker. The length of such a peptide linker is 2 to 30, preferably 15 to 25, more preferably 15 or 25. Specifically, in the amino acid sequence shown in SEQ ID NO: 26 or 27 containing a glycine-serine continuous sequence, it consists of an amino acid sequence having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more, and has the same function as SEQ ID NO: 26 or 27. Polypeptides, or amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 26 or 27, and having the same function as the amino acid sequence shown in SEQ ID NO: 26 or 27. Polypeptides can be preferably cited.

[0026] Examples of the combination of the heavy chain variable region, the light chain variable region, and the peptide linker in the single-chain antibody that specifically recognizes the above-mentioned human mesothelin include the following combinations. Here, "sequentially" means in order from the N-terminal side. (1-4) A combination sequentially including a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (2-4) A combination sequentially including a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; (3-4) A combination sequentially including a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1; (4-4) A combination sequentially including a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; (5-4) A combination sequentially including a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1; (6-4) A combination sequentially including a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; (7-4) A combination sequentially including a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5; A combination sequentially comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; A combination sequentially comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5;

[0027] (IL-7, CCL19) The above IL-7 is a cytokine essential for the survival of T cells and is produced by non-hematopoietic cells such as stromal cells in the bone marrow, thymus, and lymphoid organs / tissues. On the other hand, the production ability of T cells themselves is hardly recognized.

[0028] Also, the above CCL19 is mainly produced by dendritic cells and macrophages in lymph nodes and has a function of inducing the migration of T cells, B cells, and mature dendritic cells via its receptor CCR7.

[0029] The organisms from which IL-7 and CCL19 are derived are not particularly limited, but are preferably human. The amino acid sequences of these proteins are available from known sequence databases such as GenBank. For example, examples of the amino acid sequence of human IL-7 include the sequence registered as GenBank accession number: NM_000880.3 (SEQ ID NO: 28) and its isoforms. Also, examples of the amino acid sequence of human CCL19 include the sequence registered as GenBank accession number: NM_006274.2 (SEQ ID NO: 29) and its isoforms. Note that IL-7 and CCL19 may have a signal peptide, but in the mature protein, the signal peptide is removed. For example, in the amino acid sequence of human IL-7 described in SEQ ID NO: 28, the sequence at positions 1 to 25 corresponds to the signal peptide. Also, for example, in the amino acid sequence of human CCL19 described in SEQ ID NO: 29, the sequence at positions 1 to 21 corresponds to the signal peptide.

[0030] In addition, IL-7 and CCL19 may also be variants of the natural proteins as described above. Examples of variants of IL-7 include amino acid sequences having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more in the amino acid sequence of human IL-7 described in SEQ ID NO: 28, and polypeptides having an enhancing effect on the cell proliferation rate or cell survival rate in IL-7, or amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of human IL-7 described in SEQ ID NO: 28, and polypeptides having an enhancing effect on the cell proliferation rate or cell survival rate in IL-7. Examples of variants of human CCL19 include amino acid sequences having a sequence identity of 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more in the amino acid sequence of human CCL19 described in SEQ ID NO: 29, and polypeptides having a cell migration effect in CCL19, or amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of human CCL19 described in SEQ ID NO: 29, and polypeptides having a cell migration effect in CCL19.

[0031] (Other immune function regulators) The immune cells of the present invention may further express other immune function regulators such as IL-15, CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, Interferon-γ, MIP-1alpha, GM-CSF, M-CSF, TGF-beta, TNF-alpha, etc., but among the above other immune regulators, it is preferably an immune function regulator other than IL-12.

[0032] (Transmembrane region) Examples of the transmembrane region in the present invention include polypeptides of transmembrane regions derived from CD8, α and β chains of the T cell receptor, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR, and include an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more sequence identity with the amino acid sequence of the human CD8 transmembrane region shown in SEQ ID NO: 7, and having an action equivalent to that of the amino acid sequence shown in SEQ ID NO: 7. Polypeptides, and polypeptides consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 7 and having an action equivalent to that of the amino acid sequence shown in SEQ ID NO: 7 can be preferably cited. By such a transmembrane region, the CAR is fixed to the cell membrane of T cells.

[0033] The transmembrane region may consist of any oligopeptide or polypeptide and may include a hinge region having a length of 1 to 100 amino acids, preferably 10 to 70 amino acids. Examples of the hinge region include the hinge region of human CD8.

[0034] (Immune cell activation signal transduction region) The immune cell activation signal transduction region is a region capable of transmitting signals intracellularly when the cell surface molecule recognizes mesothelin, and preferably contains at least one or more polypeptides selected from the intracellular region polypeptides of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ, or Fc Receptor-associated γ chain. More preferably, it contains three polypeptides: the polypeptide of the intracellular region of CD28, the polypeptide of the intracellular region of 4-1BB, and the polypeptide of the intracellular region of CD3ζ. The amino acid sequence of the intracellular region of CD28 includes an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, and polypeptides having the same function as the amino acid sequence shown in SEQ ID NO: 8, or amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 8, and polypeptides having the same function as the amino acid sequence shown in SEQ ID NO: 8 can be mentioned. The amino acid sequence of the intracellular region of 4-1BB includes an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and polypeptides having the same function as the amino acid sequence shown in SEQ ID NO: 9, or amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 9, and polypeptides having the same function as the amino acid sequence shown in SEQ ID NO: 9 can be mentioned. The amino acid sequence of the intracellular region of CD3ζ includes an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 10, and polypeptides having the same function as the amino acid sequence shown in SEQ ID NO: 10, or amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 10, and polypeptides having the same function as the amino acid sequence shown in SEQ ID NO: 10 can be mentioned.When using T cells as immune cells, a polypeptide capable of signal transduction within the T cells can be selected. Similarly, when using other immune cells, a polypeptide capable of signal transduction to such immune cells can be selected. Examples of the immune cell activation signal transduction region when using T cells as immune cells include polypeptides containing the amino acid sequences shown in SEQ ID NOs: 8, 9, and 10, and preferably, polypeptides containing the amino acid sequences shown in SEQ ID NOs: 8, 9, and 10 in order from the N-terminal side can be preferably cited.

[0035] (Extracellular hinge region, spacer) In addition, an extracellular hinge region consisting of an arbitrary oligopeptide or polypeptide may be provided between the cell surface molecule that recognizes mesothelin and the transmembrane region. Examples of the length of the extracellular hinge region include 1 to 100 amino acid residues, preferably 10 to 70 amino acid residues. Examples of such extracellular hinge regions include hinge regions derived from CD8, CD28, CD4, etc., and hinge regions of immunoglobulins.

[0036] Furthermore, a spacer region consisting of an arbitrary oligopeptide or polypeptide may be provided between the transmembrane region and the immune cell activation signal transduction region. Examples of the length of the spacer region include 1 to 100 amino acid residues, preferably 10 to 50 amino acid residues. Examples of such spacer regions include glycine-serine continuous sequences.

[0037] (Arrangement of each region) In the above CAR, each of the above regions can be arranged in the order of single-chain antibody, transmembrane region, and immune cell activation signal transduction region from the N-terminus. Specifically, examples include a CAR arranged in the order of a single-chain antibody that specifically recognizes human mesothelin, the extracellular hinge region of human CD8, the transmembrane region of human CD8, the T cell activation signal transduction region of human CD28, the T cell activation signal transduction region of human 4-1BB, and the T cell activation signal transduction region of human CD3ζ from the N-terminal side.

[0038] (Protein expressed by a suicide gene) In addition, the immune cells of the present invention may express a protein having a function of causing its own cells to die, such as herpes simplex virus thymidine kinase (HSV-TK) or inducible caspase 9 (inducible caspase 9) (protein expressed by a suicide gene). By expressing these proteins based on suicide genes, it becomes possible to directly or secondarily induce a cytotoxic substance and have a function of causing its own cells to die. Therefore, for example, depending on the course of cancer treatment, when the tumor disappears, a drug that activates the above function can be administered to control the immune cells of the present invention in the living body. That is, it is possible to surely reduce the risk of cytokine release syndrome in the immune cells of the present invention as needed.

[0039] Examples of drugs that activate the functions of herpes simplex virus thymidine kinase (HSV-TK) or inducible caspase 9 (inducible caspase 9) include ganciclovir for the former and AP1903, a dimerization-inducing compound (chemical induction of dimerization: CID), for the latter (Cooper LJ., et. al. Cytotherapy. 2006;8(2):105-17., Jensen M. C.et.al. Biol Blood Marrow Transplant. 2010 Sep;16(9):1245-56., Jones BS.FrontPharmacol.2014 Nov 27;5:254., Minagawa K., Pharmaceuticals (Basel). 2015May8;8(2):230-49., Bole-Richard E., Front Pharmacol. 2015 Aug 25;6:174).

[0040] (Cell types in immune cells) As the types of cells in the immune cells, there are no particular restrictions as long as they are cells that are involved in the immune response and can express a cell surface molecule that specifically recognizes human mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19 by introducing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19. However, it is preferably immune cells isolated from a living body, and examples include lymphocyte cells such as T cells, natural killer cells (NK cells), and B cells, antigen-presenting cells such as monocytes, macrophages, and dendritic cells, and granulocytes such as neutrophils, eosinophils, basophils, and mast cells that are isolated from a living body. Specifically, T cells derived from mammals such as humans, dogs, cats, pigs, and mice or isolated from mammals can be preferably mentioned, and more preferably T cells derived from humans or isolated from humans. In addition, the T cells derived from mammals such as humans, dogs, cats, pigs, and mice include T cells artificially cultured in vitro from T cells isolated (collected) from mammals such as humans, dogs, cats, pigs, and mice, or T cells subcultured from the T cells. Further, the isolated T cells may be a cell population mainly containing T cells, and may contain other cells in addition to T cells, but it is preferably contained at a ratio of 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, and most preferably 90%. Also, T cells can be obtained by separating a cell population containing immune cells from body fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, or immune cells infiltrating cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites. In order to increase the ratio of T cells contained in the cell population, the separated cell population can also be further isolated or purified by a conventional method as necessary. Furthermore, cells prepared from ES cells or iPS cells may be used as the cells in the immune cells. Such T cells include alpha-beta T cells, gamma-delta T cells, CD8 + T cells, CD4 +Examples include T cells, tumor-infiltrating T cells, memory T cells, naive T cells, and NKT cells. Note that the origin of the immune cells and the administration target may be the same or different. Furthermore, in the case of human administration, as the immune cells, autologous cells collected from the patient himself / herself as the administration target or allogeneic cells collected from others may be used. That is, the donor and the recipient may or may not match, but it is preferable that they match.

[0041] (Method for producing immune cells) Examples of the method for producing the immune cells of the present invention include a method of producing by introducing a nucleic acid encoding a cell surface molecule, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19 into immune cells. For example, a method of producing by introducing the expression vector of the present invention described below into immune cells by the method described in Patent Document 1 or 2 above can be preferably mentioned. Alternatively, a method of purifying immune cells from a transgenic mammal produced by injecting a vector expressing a cell surface molecule that specifically recognizes human mesothelin, IL-7, and / or CCL19 into a fertilized egg, or a method of further introducing a vector expressing a cell surface molecule that specifically recognizes human mesothelin, IL-7, and / or CCL19 into the immune cells purified from such a transgenic mammal as needed can also be mentioned.

[0042] When introducing a nucleic acid encoding a cell surface molecule, a nucleic acid encoding IL-7, a nucleic acid encoding CCL19, or the vector of the present invention described below into immune cells, any method for introducing a nucleic acid or a vector into immune cells may be used. For example, the electroporation method (Cytotechnology, 3, 133 (1990)), the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987)), the virus infection method, etc. can be mentioned. As such a virus infection method, a method of transfecting a packaging cell such as GP2-293 cells (manufactured by Takara Bio Inc.), Plat-GP cells (manufactured by Cosmo Bio Co., Ltd.), PG13 cells (ATCC CRL-10686), PA317 cells (ATCC CRL-9078) with a vector to be introduced and a packaging plasmid to produce a recombinant virus, and infecting such a recombinant virus into immune cells (the above Patent Document 2) can be mentioned.

[0043] When preparing the above-mentioned "immune cells expressing a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19" using a vector, it can be prepared by any of the following methods.

[0044] (1) A method of introducing into immune cells a vector that contains a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, and expresses a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19; (2) A method of introducing into immune cells simultaneously or stepwise two types of vectors, namely, a vector that contains a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and expresses a cell surface molecule that specifically recognizes human mesothelin, and a vector that contains a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expresses IL-7 and CCL19; (3) A method of introducing two types of vectors, namely, a vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding IL-7 and expressing the cell surface molecule that specifically recognizes human mesothelin and IL-7, and a vector containing a nucleic acid encoding CCL19 and expressing CCL19, into immune cells simultaneously or stepwise; (4) A method of introducing two types of vectors, namely, a vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding CCL19 and expressing the cell surface molecule that specifically recognizes human mesothelin and CCL19, and a vector containing a nucleic acid encoding IL-7 and expressing IL-7, into immune cells simultaneously or stepwise; (5) A method of introducing two types of vectors, namely, a vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding IL-7 and expressing the cell surface molecule that specifically recognizes human mesothelin and IL-7, and a vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding CCL19 and expressing the cell surface molecule that specifically recognizes human mesothelin and CCL19, into immune cells simultaneously or stepwise; (6) A method of introducing two types of vectors, namely, a vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding IL-7 and expressing the cell surface molecule that specifically recognizes human mesothelin and IL-7, and a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19, into immune cells simultaneously or stepwise; (7) A method of introducing two types of vectors, namely, a vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding CCL19 and expressing the cell surface molecule that specifically recognizes human mesothelin and CCL19, and a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19, into immune cells simultaneously or stepwise; A method for introducing simultaneously or stepwise into immune cells three types of vectors: a vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and expressing the cell surface molecule that specifically recognizes human mesothelin, a vector containing a nucleic acid encoding IL-7 and expressing IL-7, and a vector containing a nucleic acid encoding CCL19 and expressing CCL19;

[0045] Furthermore, when preparing the above-mentioned "immune cells expressing a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19" using a vector, immune cells expressing a cell surface molecule that specifically recognizes human mesothelin are prepared in advance, and the immune cells expressing a cell surface molecule that specifically recognizes human mesothelin can also be prepared by any of the following methods using such immune cells. (1) A method for introducing a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19 into the immune cells expressing a cell surface molecule that specifically recognizes human mesothelin; (2) A method for introducing simultaneously or stepwise into the immune cells expressing a cell surface molecule that specifically recognizes human mesothelin two types of vectors: a vector containing a nucleic acid encoding IL-7 and expressing IL-7, and a vector containing a nucleic acid encoding CCL19 and expressing CCL19;

[0046] When using each of the above immune cells, a culture of the immune cells containing the immune cells may be used. Further, even when nucleic acids encoding cell surface molecules that specifically recognize human mesothelin, nucleic acids encoding IL-7, and nucleic acids encoding CCL19 are integrated into the genome of immune cells, they may be used in a state where they are not integrated into the genome (for example, an episomal state). Furthermore, when using each of the above immune cells, a mixture of immune cells in which nucleic acids encoding cell surface molecules that specifically recognize human mesothelin, nucleic acids encoding IL-7, and nucleic acids encoding CCL19 are integrated into the genome of the immune cells and immune cells in which the nucleic acids are not integrated into the genome may be used.

[0047] In addition, as described above, the "immune cells expressing a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19" can be produced by incorporating a cell surface molecule that specifically recognizes human mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19 into the genome of a cell so as to be expressible under the control of an appropriate promoter using a known gene editing technique. Examples of known gene editing techniques include techniques using endonucleases such as zinc finger nucleases, TALEN (transcription activator-like effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas systems. Also, for example, when expressing another foreign protein in CAR (anti-human mesothelin CAR)-expressing immune cells that specifically recognize human mesothelin, a polynucleotide containing a base sequence encoding the other foreign protein may be incorporated into the genome of the cell so as to be expressible under the control of an appropriate promoter using gene editing technology. Specifically, methods such as incorporating a polynucleotide containing a base sequence encoding an anti-human mesothelin CAR (or other protein) functionally linked to an appropriate promoter into a non-coding region of the cell genome; and incorporating a polynucleotide containing a base sequence encoding an anti-human mesothelin CAR (or other protein) downstream of an endogenous promoter of the cell genome can be mentioned. Examples of endogenous promoters include the promoters of TCRα and TCRβ.

[0048] (Administration subject) Examples of the administration subject preferably include mammals or mammalian cells. Among such mammals, humans, mice, dogs, rats, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, monkeys, and chimpanzees can be more preferably mentioned, and humans can be particularly preferably mentioned.

[0049] (Expression vector) The expression vector of the present invention may be any one that can be introduced into cells by contacting with immune cells or their precursor cells, and the predetermined protein (polypeptide) encoded therein is expressed in the immune cells, thereby producing the immune cells of the present invention. There is no particular limitation on the specific embodiment. A person skilled in the art can design and produce an expression vector capable of expressing a desired protein (polypeptide) in immune cells. For example, as an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, the following expression vectors (a) to (e) for producing the immune cells of the present invention (hereinafter, also referred to as "IL-7 / CCL19 expression - anti-human mesothelin vector") can be mentioned. Hereinafter, the "two expression vectors" means a set of two types of expression vectors, and the "three expression vectors" means a set of three types of expression vectors. (a) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19: (b) The following two expression vectors (b-1) and (b-2): (b-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin; (b-2) An expression vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19; (c) The following two expression vectors (c-1) and (c-2): (c-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding IL-7; (c-2) An expression vector containing a nucleic acid encoding CCL19; (d) The following two expression vectors (d-1) and (d-2): (d-1) An expression vector containing a nucleic acid encoding IL-7; An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding CCL19; (e) The following two expression vectors (e-1) and (e-2): (e-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding IL-7; (e-2) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding CCL19; (f) The following two expression vectors (f-1) and (f-2): (f-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding IL-7; (f-2) An expression vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19; (g) The following two expression vectors (g-1) and (g-2): (g-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin and a nucleic acid encoding CCL19; (g-2) An expression vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19; (h) The following three expression vectors (h-1), (h-2) and (h-3): (h-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes human mesothelin; (h-2) An expression vector containing a nucleic acid encoding IL-7; (h-3) An expression vector containing a nucleic acid encoding CCL19;

[0050] The above IL-7 / CCL19 expression - anti - human mesothelin vector may further contain nucleic acids encoding other immune function regulatory factors such as IL-15, CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, Interferon - γ, MIP-1alpha, GM-CSF, M-CSF, TGF-β, TNF-α, checkpoint inhibitory antibodies or fragments thereof. However, as the nucleic acids encoding the above other immune regulatory factors, it is preferably a nucleic acid encoding an immune function regulatory factor other than IL-12.

[0051] (Nucleic acid) As used herein, the "nucleic acid" may be any molecule in which nucleotides and molecules having functions equivalent to the nucleotides are polymerized. For example, it includes RNA which is a polymer of ribonucleotides, DNA which is a polymer of deoxyribonucleotides, a polymer in which ribonucleotides and deoxyribonucleotides are mixed, and a nucleotide polymer containing nucleotide analogs. Further, it may be a nucleotide polymer containing nucleic acid derivatives. The nucleic acid may be a single - stranded nucleic acid or a double - stranded nucleic acid. The double - stranded nucleic acid also includes a double - stranded nucleic acid in which one strand hybridizes with the other strand under stringent conditions.

[0052] As the above nucleotide analogs, any molecule may be used as long as it is a molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA or DNA in order to improve nuclease resistance, stabilize, increase the affinity for complementary - strand nucleic acids, increase cell permeability, or enable visualization, as compared with RNA or DNA. The nucleotide analogs may be natural or non - natural molecules. For example, sugar - modified nucleotide analogs and phosphodiester - bond - modified nucleotide analogs are included.

[0053] As the sugar moiety-modified nucleotide analogs, any chemical substance can be added or substituted for part or all of the chemical structure of the sugar of the nucleotide. Specific examples thereof include nucleotide analogs substituted with 2'-O-methylribose, nucleotide analogs substituted with 2'-O-propylribose, nucleotide analogs substituted with 2'-methoxyethoxyribose, nucleotide analogs substituted with 2'-O-methoxyethylribose, nucleotide analogs substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogs substituted with 2'-fluororibose, bridged nucleic acid (BNA) having two cyclic structures by introducing a cross-linked structure into the sugar moiety. More specifically, locked nucleic acid (LNA) in which the oxygen atom at the 2' position and the carbon atom at the 4' position are cross-linked via a methylene group, and ethylenebridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)] can be mentioned. Furthermore, peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxy peptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)], etc. can be mentioned.

[0054] As the phosphodiester bond-modified nucleotide analogs, any chemical substance can be added or substituted for part or all of the chemical structure of the phosphodiester bond of the nucleotide. Specific examples thereof include nucleotide analogs substituted with phosphorothioate bonds, nucleotide analogs substituted with N3'-P5' phosphoramidate bonds, etc. [Cell Engineering, 16, 1463-1473 (1997)] [RNAi Method and Antisense Method, Kodansha (2005)].

[0055] As the above nucleic acid derivative, any molecule may be used as long as it is a molecule obtained by adding another chemical substance to the nucleic acid in order to improve nuclease resistance, stabilize, increase the affinity for complementary strand nucleic acid, increase cell permeability, or enable visualization compared to the nucleic acid. Specific examples thereof include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0056] (Nucleic acids encoding cell surface molecules that specifically recognize human mesothelin, IL-7, CCL19, etc.) The nucleic acids encoding the cell surface molecule that specifically recognizes human mesothelin, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 can each be a nucleic acid derived from a mammal, and preferably a nucleic acid derived from a human. Each of the above nucleic acids can be appropriately selected according to the type of cell into which the expression vector of the present invention is introduced, and the sequence information of each such nucleic acid can be appropriately obtained by searching known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0057] Examples of the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin include a nucleic acid encoding a CAR that specifically recognizes human mesothelin.

[0058] Specific examples of the nucleic acid encoding the single-chain antibody contained in the above CAR that specifically recognizes human mesothelin include the following (1-1D) to (3-1D). A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (2-1D) A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (3-1D) A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 21, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 23, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 24, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 25;

[0059] As another embodiment 1 of the nucleic acid encoding the single-chain antibody contained in the CAR that specifically recognizes the above-mentioned human mesothelin, specifically, the following (1-2D) to (5-2D) can be mentioned. (1-2D) A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and a light-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2; A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and a light-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and a light-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 6; A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and a light-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4; A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, and a light-chain variable region consisting of an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2;

[0060] As another embodiment 2 of the nucleic acid encoding the single-chain antibody contained in the CAR that specifically recognizes the above-mentioned human mesothelin, specifically, the following (1-3D) to (5-3D) can be mentioned. A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; A nucleic acid encoding a single-chain antibody comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3 and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0061] As another aspect 3 of the nucleic acid encoding a single-chain antibody contained in the CAR that specifically recognizes the above-mentioned human mesothelin, specifically, the following (1-4D) to (9-4D) can be mentioned. A nucleic acid encoding a single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; A nucleic acid encoding a single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 3, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 4; A nucleic acid encoding a single-chain antibody sequentially comprising a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1; A nucleic acid encoding a single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2; A nucleic acid encoding a single-chain antibody sequentially comprising a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1; A nucleic acid encoding a single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; A nucleic acid encoding a single-chain antibody sequentially comprising a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5; A nucleic acid encoding a single-chain antibody sequentially comprising a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6; A nucleic acid encoding a single-chain antibody sequentially comprising a light-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 27, and a heavy-chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 5;

[0062] Examples of the nucleic acid encoding the transmembrane region polypeptide contained in the above CAR include a nucleic acid encoding a human CD8 transmembrane region polypeptide containing an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8. Further, as the nucleic acid encoding the polypeptide of the intracellular regions of CD28, 4-1BB, and CD3ζ in the immune cell activation signal transduction region contained in the above CAR, it includes an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more sequence identity with the amino acid sequence of the intracellular region of human CD28 shown in SEQ ID NO: 8, and encodes a polypeptide having an action equivalent to that of the amino acid sequence shown in SEQ ID NO: 8; an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more sequence identity with the amino acid sequence of the intracellular region of human 4-1BB shown in SEQ ID NO: 9, and encodes a polypeptide having an action equivalent to that of the amino acid sequence shown in SEQ ID NO: 9; an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more sequence identity with the amino acid sequence of the intracellular region of human CD3ζ shown in SEQ ID NO: 10, and encodes a polypeptide having an action equivalent to that of the amino acid sequence shown in SEQ ID NO: 10, or a combination thereof. Preferably, in order from the upstream (5'-terminal side), it includes a nucleic acid encoding the polypeptide of the intracellular region of the intracellular region of the polypeptide of the intracellular region of human CD28 shown in SEQ ID NO: 8, a nucleic acid encoding the polypeptide of the intracellular region of human 4-1BB shown in SEQ ID NO: 9, and a nucleic acid encoding the polypeptide of the intracellular region of human CD3ζ shown in SEQ ID NO: 10.

[0063] As the nucleic acid encoding IL-7, it includes an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28, and encodes a polypeptide having an action equivalent to that of the amino acid sequence shown in SEQ ID NO: 28. Specifically, a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 30 can be mentioned. As long as it has the action of enhancing the cell proliferation rate or cell survival rate in IL-7, a nucleic acid having 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, most preferably 98% or more sequence identity with the nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 30 may also be used. As the nucleic acid encoding CCL19, it includes an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 29, and encodes a polypeptide having an action equivalent to that of the amino acid sequence shown in SEQ ID NO: 29. Specifically, a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 31 can be mentioned. As long as it has the action of cell migration in CCL19, a nucleic acid having 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, most preferably 98% or more sequence identity with the nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 31 may be used.

[0064] (suicide gene) In addition, the expression vector of the present invention may contain a nucleic acid encoding a suicide gene. The suicide gene refers to a gene that has a function of directly or secondarily inducing a cytotoxic substance by expression and causing its own cells to die. By including a nucleic acid encoding a suicide gene in the expression vector of the present invention, depending on the course of cancer treatment, for example, when the tumor disappears, a drug that activates the function of the suicide gene can be administered to control immune cells in the living body. In addition, unlike other cytokines, IL-7 or CCL19 has a low possibility of causing cytokine release syndrome or tumorigenesis of gene-introduced cells as side effects. However, due to the enhanced function of immune cells into which the expression vector of the present invention is introduced, cytokines and the like released when attacking the target cancer tissue may unexpectedly affect surrounding tissues. In such a case, by including a nucleic acid encoding a suicide gene in the expression vector of the present invention, it is possible to surely reduce the risk of developing cytokine release syndrome.

[0065] Examples of the suicide gene include genes encoding thymidine kinase of herpes simplex virus (HSV-TK) and inducible caspase 9 described in the following literature. Examples of drugs that activate the functions of such genes include ganciclovir for the former and AP1903, a dimerization-inducing compound (chemical induction of dimerization: CID), for the latter.

[0066] (Obtaining nucleic acid sequence information and preparing nucleic acids) Each of the above nucleic acids contained in the expression vector of the present invention may be a naturally derived nucleic acid or a synthetic nucleic acid, and can be appropriately selected according to the type of cells into which the expression vector of the present invention is introduced. The sequence information can be appropriately obtained by searching known literature and databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0067] Each of the above nucleic acids can be prepared by known techniques such as chemical synthesis or amplification by PCR based on the information of the base sequences of the nucleic acids encoding them. The codons selected for encoding amino acids may be modified to optimize the expression of the nucleic acids in the target host cell.

[0068] (Arrangement of each nucleic acid) In the expression vector of the present invention, the expression vector containing the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 may have any nucleic acid arranged upstream or downstream of any other. Specifically, taking the case of containing the nucleic acid encoding anti-human mesothelin CAR as the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin as an example, the nucleic acid encoding anti-human mesothelin CAR, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 in order from the upstream (5'-end side), or the nucleic acid encoding anti-human mesothelin CAR, the nucleic acid encoding CCL19, and the nucleic acid encoding IL-7, or the nucleic acid encoding IL-7, the nucleic acid encoding CCL19, and the nucleic acid encoding anti-human mesothelin CAR, or the nucleic acid encoding IL-7, the nucleic acid encoding anti-mesothelin CAR, and the nucleic acid encoding CCL19, or the nucleic acid encoding CCL19, the nucleic acid encoding anti-mesothelin CAR, and the nucleic acid encoding IL-7, or the nucleic acid encoding CCL19, the nucleic acid encoding IL-7, and the nucleic acid encoding anti-human mesothelin CAR may all be acceptable.

[0069] In the expression vector of the vector of the present invention containing the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 in (b-2), (f-2), and (g-2), the arrangement of the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 is not particularly limited, and the nucleic acid encoding CCL19 may be arranged upstream or downstream of the nucleic acid encoding IL-7.

[0070] In the vector of the present invention, in the expression vector containing the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin in (c-1), (e-1) and (f-1), and the nucleic acid encoding IL-7, the arrangement of the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin and the nucleic acid encoding IL-7 is not particularly limited, and the nucleic acid encoding IL-7 may be arranged upstream or downstream of the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin.

[0071] In the vector of the present invention, in the expression vector containing the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin in (d-2), (e-2) and (g-1), and the nucleic acid encoding CCL19, the arrangement of the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin and the nucleic acid encoding CCL19 is not particularly limited, and the nucleic acid encoding CCL19 may be arranged upstream or downstream of the nucleic acid encoding the cell surface molecule that specifically recognizes human mesothelin.

[0072] (Transcription) Note that the nucleic acid encoding the cell surface molecule that specifically recognizes mesothelin, the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19, and the nucleic acid encoding the suicide gene may each be transcribed by a different promoter, or may be transcribed by one promoter using an internal ribosome entry site (IRES) or a self-cleaving 2A peptide.

[0073] When transcribing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 using an internal ribosome entry site (IRES) or a self-cleaving 2A peptide under one promoter, between each of the above nucleic acids, or when including a nucleic acid encoding a cell surface molecule that specifically recognizes the above mesothelin, between the said nucleic acid and the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19, any nucleic acid may be included as long as each nucleic acid can be expressed, but it is preferably linked via a self-cleaving peptide (2A peptide) or a sequence encoding an IRES, preferably a sequence encoding a 2A peptide. By linking using such a sequence, it becomes possible to efficiently express each nucleic acid.

[0074] The 2A peptide is a virus-derived self-cleaving peptide and has the characteristic that the position between G and P (the position of 1 residue from the C-terminus) in the amino acid sequence shown in SEQ ID NO: 32 is cleaved in the endoplasmic reticulum (Szymczak et al., Expert Opin. Biol. Ther. 5(5):627-638(2005)). Therefore, The nucleic acids incorporated before and after via the 2A peptide will be expressed independently of each other in the cell.

[0075] The said 2A peptide is preferably a 2A peptide derived from picornavirus, rotavirus, insect virus, aftovirus or trypanosoma virus, and more preferably the 2A peptide (F2A) derived from picornavirus shown in SEQ ID NO: 33.

[0076] (Type of vector) As for the type of vector in the expression vector of the present invention, it may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector, or a vector by a transposon. Further, such a vector may contain a control sequence such as a promoter or a terminator, and a selection marker sequence such as a drug resistance gene or a reporter gene. By arranging a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 operably downstream of the promoter sequence, each nucleic acid can be efficiently transcribed.

[0077] Examples of the promoter include viral-derived promoters such as the LTR promoter of retrovirus, the SV40 early promoter, the cytomegalovirus promoter, and the thymidine kinase promoter of herpes simplex virus, and mammalian-derived promoters such as the phosphoglycerate kinase (PGK) promoter, the Xist promoter, the β-actin promoter, and the RNA polymerase II promoter. Further, a tetracycline-responsive promoter induced by tetracycline, an Mx1 promoter induced by interferon, etc. may be used. By using a promoter induced by the above specific substance in the expression vector of the present invention, for example, when used as a pharmaceutical composition for using immune cells containing the vector of the present invention for cancer treatment, it is possible to control the induction of the expression of IL-7 and CCL19 according to the course of cancer treatment.

[0078] Examples of the viral vector include a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector, and a retroviral vector can be preferably mentioned, and the pMSGV vector (Tamada k et al., Clin Cancer Res 18:6436-6445(2002)) and the pMSCV vector (manufactured by Takara Bio Inc.) can be more preferably mentioned. If a retroviral vector is used, the introduced gene is incorporated into the genome of the host cell, so that it can be expressed stably for a long period of time.

[0079] Confirmation of the presence of the expression vector of the present invention in immune cells can be carried out, for example, when it contains a nucleic acid encoding a CAR, by examining the expression of the CAR by PCR such as flow cytometry, Northern blotting, Southern blotting, RT-PCR, ELISA, Western blotting. When the expression vector of the present invention contains a marker gene, it can be confirmed by examining the expression of the marker gene inserted into the expression vector.

[0080] (Pharmaceutical composition) The pharmaceutical composition of the present invention only needs to contain the immune cells of the present invention and pharmaceutically acceptable additives. Examples of the additives include physiological saline, buffered physiological saline, cell culture medium, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonic agents, fillers, and lubricants. In addition, since the immune cells in the pharmaceutical composition of the present invention are provided with a signal transduction region that induces activation of the immune cells, the pharmaceutical composition of the present invention may also be a pharmaceutical composition for use in the treatment of cancer. Such a pharmaceutical composition for use in the treatment of cancer may include an attached document, label, package, etc. that describe a method of use for treating cancer. Furthermore, since the immune cells in the pharmaceutical composition of the present invention have an effect of suppressing tumor recurrence, the pharmaceutical composition of the present invention may also be a pharmaceutical composition for use in suppressing tumor recurrence. Such a pharmaceutical composition for use in suppressing tumor recurrence may include an attached document, label, package, etc. that describe a method of use for suppressing tumor recurrence.

[0081] The pharmaceutical composition of the present invention can be administered to a subject in need thereof using methods known to those skilled in the art. Examples of the administration method include injection into the vein, tumor, dermis, subcutaneous tissue, muscle, abdominal cavity, artery, bone marrow, heart, joint, bursa synovialis, intracranial cavity, intrathecal cavity, and subarachnoid (cerebrospinal fluid).

[0082] The pharmaceutical composition of the present invention can be administered once or in several divided doses independently 4 times a day, 3 times a day, 2 times a day or once a day, once every other day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once a week, once every 7 days, once every 8 days, once every 9 days, twice a week, once a month or twice a month.

[0083] Although the cancer in the pharmaceutical composition of the present invention is not particularly limited, it is preferably a cancer type that expresses mesothelin in cancer tissue or a cancer type derived from cancer cells that express mesothelin, such as mesothelioma, colorectal cancer (colon cancer or rectal cancer), pancreatic cancer, thymic cancer, bile duct cancer, lung cancer (adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma), skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, etc., as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue and hematopoietic tissue. In addition, sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant angioendothelioma, malignant Schwannoma, osteosarcoma, soft tissue sarcoma, etc., and blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, etc., and germ cell tumors can be mentioned. As the dosage, a therapeutically effective amount can be administered. For example, in a single administration, the number of administered cells can be 1×10 4 ~1×10 10 cells, preferably 1×10 5 ~1×10 9 cells, more preferably 5×10 6 ~5×10 8 cells.

[0084] The pharmaceutical composition of the present invention can be used in combination with other anticancer agents. Examples of other anticancer agents include alkylating agents such as cyclophosphamide, bendamustine, ifosfamide, and dacarbazine; antimetabolites such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enocitabine; molecular target drugs such as rituximab, cetuximab, and trastuzumab; kinase inhibitors such as imatinib, gefitinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib; proteasome inhibitors such as bortezomib; calcineurin inhibitors such as cyclosporine and tacrolimus; anticancer antibiotics such as idarubicin, doxorubicin, and mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum preparations such as cisplatin, oxaliplatin, and carboplatin; hormonal therapy drugs such as tamoxifen and bicalutamide; and immunomodulatory drugs such as interferon, nivolumab, and pembrolizumab.

[0085] Examples of the method of "using in combination with the pharmaceutical composition of the present invention and other anticancer agents" include a method of treating with other anticancer agents and then using the pharmaceutical composition of the present invention, a method of using the pharmaceutical composition of the present invention and other anticancer agents simultaneously, and a method of treating with the pharmaceutical composition of the present invention and then using other anticancer agents. When the pharmaceutical composition for treating cancer of the present invention is used in combination with other anticancer agents, the therapeutic effect of cancer is further improved, and by reducing the number of administrations or the dose of each anticancer agent, it is possible to reduce the side effects caused by each anticancer agent. Further, the pharmaceutical composition of the present invention may contain the above other anticancer agents.

[0086] (Another aspect of the present invention) As another aspect 1 of the present invention, there can be mentioned 1) a method for treating cancer, characterized by administering the immune cells of the present invention to a patient in need of cancer treatment; 2) the immune cells of the present invention for use as a pharmaceutical composition; and 3) the use of the immune cells of the present invention in the preparation of a pharmaceutical composition.

[0087] In addition, as another aspect 2 of the present invention, a chimeric antigen receptor (CAR) comprising any of the following single-chain antibodies, transmembrane regions, and signal transduction regions that induce activation of immune cells can be mentioned. By expressing such a CAR in immune cells, it becomes possible to activate immune cells by stimulation with human mesothelin. . (1-1) A single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (2-1) A single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 19, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18; (3-1) A single-chain antibody comprising a heavy-chain variable region containing a heavy-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 20, a heavy-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 21, and a heavy-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 22, and a light-chain variable region containing a light-chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 23, a light-chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 24, and a light-chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 25;

[0088] Furthermore, as another aspect 3 of the present invention, there can be mentioned a kit for producing immune cells, which comprises the expression vector of the present invention. Such a kit is not particularly limited as long as it comprises the expression vector of the present invention, and may include instructions for producing the immune cells of the present invention and reagents used for introducing the expression vector of the present invention into immune cells.

[0089] Furthermore, as another aspect 4 of the present invention, there can be mentioned a method for suppressing cancer recurrence, which comprises administering to immune cells that simultaneously express a cell surface molecule (preferably, a CAR equipped with a single-chain antibody that specifically recognizes human mesothelin), IL-7, and CCL19.

[0090] Hereinafter, the present invention will be described more specifically by way of examples, but the technical scope of the present invention is not limited to these exemplifications.

[0091] [Example 1] Preparation of anti-human mesothelin CAR (Synthesis of scFv sequence and DNA fragment of anti-human mesothelin CAR) In order to compare the sequences, orders, and types of appropriate signal peptides of VL and VH, the sequences of nine types of anti-human mesothelin scFv shown in FIG. 1 were designed. VH07(15)VL07 consists of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1, the amino acid sequence of the peptide linker shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2. VH36(15)VL36 consists of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 3, the amino acid sequence of the peptide linker shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 4. VL07(15)VH07 consists of the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2, the amino acid sequence of the peptide linker shown in SEQ ID NO: 26, and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1. VH07(25)VL07 consists of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1, the amino acid sequence of the peptide linker shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2. VL07(25)VH07 consists of the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2, the amino acid sequence of the peptide linker shown in SEQ ID NO: 27, and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1. VHMO(15)VLMO consists of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 5, the amino acid sequence of the peptide linker shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 6. VLMO(15)VHMO consists of the amino acid sequence of the light chain variable region shown in SEQ ID NO: 6, the amino acid sequence of the peptide linker shown in SEQ ID NO: 26, and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 5. VHMO(25)VLMO consists of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 5, the amino acid sequence of the peptide linker shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 6. VLMO(25)VHMO consists of the amino acid sequence of the light chain variable region shown in SEQ ID NO: 6, the amino acid sequence of the peptide linker shown in SEQ ID NO: 27, and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 5. The amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 differ in that the 127th position in SEQ ID NO: 1 is glycine (G), while it is leucine (L) in the amino acid sequence shown in SEQ ID NO: 3. Also, the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 differ in that tyrosine (Y) at the 33rd position in SEQ ID NO: 2 is deleted in the amino acid sequence shown in SEQ ID NO: 4.

[0092] Next, DNA fragments encoding the amino acid sequences of the above respective anti-human mesothelin scFvs were synthesized.

[0093] (Preparation of anti-human mesothelin IL-7 / CCL19 CAR expression vector expressing IL-7 / CCL19 and HSV-TK, and conventional (Conv.) anti-human mesothelin CAR expression vector not expressing IL-7 / CCL19) In CAR-T cell therapy, systemic side effects such as cytokine release syndrome may occur due to a strong immune response against the target antigen. To address this problem, the herpes simplex virus-derived thymidine kinase gene HSV-TK was introduced as a suicide gene into a CAR construct. A lact construct was prepared. When this construct is transfected into T cells to express HSV-TK in CAR-expressing T cells, the addition of ganciclovir, a cytomegalovirus therapeutic agent, induces apoptosis and kills the CAR-T cells. Therefore, it becomes possible to control the CAR-T cells in the body by administering ganciclovir.

[0094] According to the method described in Patent Document 2 above, first, a third-generation CAR construct having an anti-human mesothelin scFv, a human CD8 transmembrane region, and a human CD28-4-1BB-CD3ζ intracellular signaling region was prepared in sequence from the N-terminal side. F2A, a 2A peptide, was added to the C-terminus of the construct, and further, human IL-7-F2A-human CCL19-F2A-HSV-TK was added downstream thereof. The obtained construct having scFv, a human CD8 transmembrane region, a human CD28-4-1BB-CD3ζ intracellular signaling region, human IL-7, human CCL19, and HSV-TK was inserted into the pMSGV1 retroviral expression vector (Tamada k et al., Clin Cancer Res 18:6436-6445(2012)) to obtain an anti-human A pMSGV vector expressing mesothelin scFv, human CD8 transmembrane region, human CD28-4-1BB-CD3ζ intracellular signaling region, human IL-7, human CCL19, and HSV-TK was constructed. Next, the anti-human mesothelin scFv region in the pMSGV vector was replaced with each anti-human mesothelin scFv DNA fragment synthesized by the method described in the "Synthesis of scFv Sequences and DNA Fragments of Anti-Human Mesothelin CAR" section by restriction enzyme (NcoI and NotI) treatment and ligation to prepare each "IL-7 / CCL19-expressing - anti-human mesothelin CAR vector". Note that the pMSGV1 vector has a signal peptide T (SEQ ID NO: 11) derived from immunoglobulin G on the N-terminal side of scFv. For the one in which the scFv region was replaced with the VH07(15)VL07 DNA fragment, a construct was also prepared in which the signal peptide T shown in SEQ ID NO: 11 was replaced with the signal peptide P shown in SEQ ID NO: 12 as the signal peptide. Furthermore, as a control without IL-7 and CCL19, a "conventional anti-human mesothelin CAR vector" was prepared in the same manner as above except that HSV-TK was used instead of the above human IL-7-F2A-human CCL19-F2A-HSV-TK.

[0095] (Preparation of Retrovirus Introduced with IL-7 / CCL19-Expressing - Anti-Human Mesothelin CAR Vector or Conventional Anti-Human Mesothelin CAR Vector) Retrovirus was prepared for gene transfer into T cells. Using Lipofectamine 3000 (manufactured by Life Technologies), each of the above IL-7 / CCL19-expressing - anti-human mesothelin CAR vector or conventional anti-human mesothelin CAR vector and the p-Ampho plasmid (manufactured by Takara Bio Inc.) were transfected into the GP2-293 packaging cell line (manufactured by Takara Bio Inc.) to prepare a retrovirus introduced with the IL-7 / CCL19-expressing - anti-human mesothelin CAR vector or conventional anti-human mesothelin CAR vector. The supernatant containing the retrovirus was collected 48 hours after transfection.

[0096] As the culture medium for the GP2-293 cells, DMEM supplemented with 10% FCS and 1% Penicillin-Streptmycin (manufactured by Wako Pure Chemical Industries, Ltd.) was used. Also, as the culture medium for the T cells used in the examples described later, GT-T551 containing 2.0% human AB serum (manufactured by Sigma-Aldrich), 1% Penicillin-Streptmycin (manufactured by Wako Pure Chemical Industries, Ltd.), and 2.5 μg / m l of amphotericin B (manufactured by Bristol-Myers Squibb) was used. was used.

[0097] (Transduction of T cells) 2 × 10 6 peripheral blood mononuclear cells collected from the blood of healthy donors were cultured for 3 days at 37°C and 5% CO2 in an incubator together with IL-2 (manufactured by Peprotech) on a plate coated with anti-CD3 monoclonal antibody (5 μg / ml) and retronectin (registered trademark: manufactured by Takara Bio Inc., 25 μg / ml) for activation of T cells. On the second day after the start of the culture, the supernatant containing the retrovirus introduced with the IL-7 / CCL19 expression - anti-human mesothelin CAR vector or the conventional anti-human mesothelin CAR vector prepared above was added at 500 μl per well to a surface-untreated 24-well plate pre-coated with 25 μg / ml of retronectin (manufactured by Takara Bio Inc.), and a retrovirus pre-loaded plate was prepared by centrifugation at 2000 g for 2 hours. A total of 2 plates were prepared, washed with 1.5% BSA / PBS after centrifugation, and stored at 4°C until use. On the third day of the culture, the activated cells were collected from the above plate, and the cell suspension (1 × 10 cells / ml) was added to the retrovirus pre-loaded plate, and centrifuged at 2000 g for 2 hours to allow the cells to take up the retrovirus. After centrifugation, the cells were resuspended in RPMI 1640 medium supplemented with 10% FCS, 1% Penicillin-Streptmycin, 2.5 μg / ml of amphotericin B, and cultured at 37°C and 5% CO2 in an incubator. 5Adjusted to (cells / ml). 1 ml of this cell suspension was added to each well of a retrovirus preloading plate, and cultured in a 37°C, 5% CO2 incubator for 24 hours in the presence of IL-2 to perform the first retroviral infection. The next day (the 4th day of culture), the cell solution in each well was transferred to the second stored virus preloading plate, centrifuged at 500 g for 1 minute, and then cultured at 37°C for 4 hours to perform the second infection. After culturing at 37°C for 4 hours, 1 ml of the cell suspension in each well was transferred to a new 12-well cell culture plate, diluted 4-fold with a new culture medium (GT-T551) containing IL-2, and cultured in a 37°C, 5% CO2 incubator. Cultured until the 7th day counted from the start date of culturing peripheral blood mononuclear cells, and obtained "anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells", which are T cells transfected with an IL-7 / CCL19 expression-anti-human mesothelin CAR vector, or "anti-human mesothelin CAR-expressing T cells", which are T cells transfected with a conventional anti-human mesothelin CAR vector. Anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells contain a nucleic acid encoding a foreign anti-human mesothelin CAR, a nucleic acid encoding a foreign IL-7, and a nucleic acid encoding a foreign CCL19. Anti-human mesothelin CAR-expressing T cells contain a nucleic acid encoding an anti-human mesothelin CAR and do not contain a nucleic acid encoding a foreign IL-7 and a nucleic acid encoding a foreign CCL19. At the same time, as a CAR-negative cell control, peripheral blood mononuclear cells obtained from the same healthy donor were activated by the same method but not infected with retrovirus to prepare "CAR, IL-7, and CCL19 non-expressing-T cells" (non-transfected cells: Non-infection).

[0098] Here, as described above, a retroviral vector is used to introduce nucleic acids encoding anti-human mesothelin CAR, nucleic acids encoding IL-7, and nucleic acids encoding CCL19 into T cells. Therefore, when T cells into which each of the above nucleic acids has been introduced are cultured and proliferated, some of the T cells contain a retroviral vector in their cytoplasm, but in many cases, in T cells, the nucleic acids encoding anti-human mesothelin CAR, the nucleic acids encoding IL-7, and the nucleic acids encoding CCL19 are integrated into the genome. When the nucleic acids encoding anti-human mesothelin CAR, the nucleic acids encoding IL-7, and the nucleic acids encoding CCL19 are integrated into the genome in the T cells, anti-human mesothelin CAR, IL-7, and CCL19 will be expressed from the introduced foreign recombinant construct.

[0099] [Example 2] Measurement of CAR Expression by Flow Cytometry (Flow Cytometry Analysis) Analysis of the expression level of CAR that recognizes mesothelin as an antigen was performed by flow cytometry analysis. The prepared anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells were reacted with recombinant human mesothelin (containing 6-His at the C-terminus) (manufactured by BioLegend), phycoerythrin (PE)-labeled anti-6-His monoclonal antibody (manufactured by abcam), and allophycocyanin (APC)-labeled anti-CD8 monoclonal antibody (manufactured by Affymetrix) for staining. The flow cytometer used was EC800 (manufactured by Sony), and data analysis was performed using FlowJo software (manufactured by Tree Star).

[0100] (Results) First, the flow cytometry analysis results of anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells having VH07(15)VL07 (expressed by signal peptide T), VH07(15)VL07 (expressed by signal peptide P), or VH36(15)VL36 as the scFv region are shown in Figure 2. In Figure 2, in each graph, the horizontal axis represents the expression of CAR, and the vertical axis represents the expression of CD8. As shown in Figure 2, compared with CAR, IL-7, and CCL19 non-expressing T cells (Non-infection), it was confirmed that all three types of anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells had high expression of CAR.

[0101] Next, the flow cytometry analysis results of anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells having VH07(15)VL07, VL07(15)VH07, VH07(25)VL07, or VL07(25)VH07 as the scFv region are shown in Figure 3. In Figure 3, in each graph, the horizontal axis represents the expression of CAR, and the vertical axis represents the expression of CD8. (a) represents CAR, IL-7, and CCL19 non-expressing T cells (Non-infection), and (b) to (e) are the results of anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells having each scFv region. Also, the numerical values in the figure represent the percentages of each population. As shown in Figures 3(b) to (e), the expression of CAR was confirmed in anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells.

[0102] [Example 3] Mesothelin Expression in Each Tumor Cell (Flow Cytometry Analysis) To confirm tumor cell lines expressing mesothelin, the expression levels of mesothelin in each tumor cell line were confirmed. Cell lines of malignant mesothelioma, ACC-MESO-1, Y-MESO8A, NCI-H2052, NCI-H226, MSTO211H, and cell line of renal cancer, A498, were stained with a commercially available mesothelin antibody labeled with PE (Catalog Number FAB32652P: manufactured by R&D systems), and the expression of mesothelin in each tumor cell was measured by flow cytometry analysis. Note that the PE-labeled anti-human mesothelin antibody was used for staining at 3 μg / sample. The flow cytometer used was EC800 (manufactured by Sony), and the data analysis was performed using FlowJo software (manufactured by Tree Star).

[0103] (Results) The results are shown in Figure 4. The expression of mesothelin was confirmed in the cell lines of malignant mesothelioma, ACC-MESO-1, Y-MESO8A, NCI-H2052, NCI-H226, MSTO211H. On the other hand, the expression of mesothelin was not confirmed in the cell line of renal cancer, A498.

[0104] [Example 4] Cytotoxicity Test - 1 (Co-culture Test) Anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells (scFv region is VH07(15)VL07 or VH07(25)VL07) were used as effectors and co-cultured on a culture plate with mesothelin-positive tumor cell lines (ACC-MESO-1, NCI-H2052) or mesothelin-negative tumor cell line (A498) after adjusting the effector:tumor cell ratio to 1:1, 1:3, 1:5 (1:5 was only for the measurement and analysis of IFN-γ) at 37°C in an incubator. The explanation of such co-culture is shown in Fig. 5. The culture medium used was RPMI containing 10% fetal calf serum (FCS), 1% Penicillin-Streptmycin (manufactured by Wako Pure Chemical Industries, Ltd.), 50 μM 2-ME (manufactured by Gibco), and 25 mM HEPES (manufactured by Sigma-Aldrich). Two days after the start of co-culture, the remaining tumor cell lines were measured by flow cytometry, and IFN-γ produced in the culture supernatant was measured using a commercially available IFN-γ ELISA kit (manufactured by BioLegend). The measurement results of the remaining tumor cell lines by flow cytometry two days after the start of co-culture are shown in Figs. 6A to 6C, and the measurement results of IFN-γ produced after co-culture are shown in Figs. 7A to 7C. As a control for anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells, CAR, IL-7, and CCL19 non-expressing T cells (Non infection) were used. During flow cytometry, dead cells were stained with Zombie Yellow (registered trademark: manufactured by BioLegend) for discrimination, and T cells were stained with a PE-labeled anti-CD45 monoclonal antibody (manufactured by BioLegend). The flow cytometer used was BD LSRFortessa X-20 (manufactured by BD Biosciences), and data analysis was performed using FlowJo software (manufactured by Tree Star).

[0105] (Results) As shown in FIGS. 6A to 6C, in the co-culture of each of the control CAR, IL-7, and CCL19 non-expressing T cells (Non-infection) with target tumor cells, it was shown that all target tumor cells grew to the same extent as in the wells with tumor only. On the other hand, in anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells, in the co-culture with mesothelin-negative target cells (A498: FIG. 6C), the target tumor cells grew in the same manner as in the wells with tumor only. However, in the co-culture with mesothelin-positive tumor cells (ACC-MESO-1: FIG. 6A, NCI-H2052: FIG. 6B), it was observed that the number of tumor cells was clearly decreased compared to the wells with mesothelin-positive tumor cells only and the wells of co-culture with control cells. From this, it was confirmed that anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells specifically damaged tumor cells antigenically.

[0106] Furthermore, as shown in FIGS. 7A to 7C, in the ELISA analysis of IFN-γ using the supernatant after co-culture, significant production of IFN-γ was confirmed only in the supernatant of the co-culture of anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells with mesothelin-positive tumor cells (ACC-MESO-1: FIG. 7A, NCI-H2052: FIG. 7B).

[0107] [Example 5] Cytotoxicity Test - 2 (Co-culture Test) In the same manner as in Example 4, anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells (scFv regions are VHMO(15)VLMO, VLMO(15)VHMO, VHMO(25)VLMO, or VLMO(25)VHMO), or "CAR, IL-7, and CCL19 non-expressing - T cells" (non-transduced cells: Non-infection) were adjusted to an effector:tumor cell ratio of 1:1 or 1:3 on a culture plate together with the PAN02 tumor cell line, which is a mesothelin-positive tumor cell line, or a mesothelin-negative tumor cell line, and then co-cultured in a 37°C incubator. The measurement results of leukocytes or the PAN02 tumor cell line remaining 3 days or 5 days after the start of co-culture by flow cytometry are shown in Fig. 8, and the measurement results of IFN-γ produced after co-culture are shown in Fig. 9. In addition, the anti-human mesothelin CAR, IL-7, and CCL19-expressing T cells used in the "treatment effect in the tumor model" of this Example and Example 5 described later were prepared using a pMSGV1 retroviral expression vector in which mouse IL-7-F2A-mouse CCL19-F2A-HSV-TK was used instead of human IL-7-F2A-human CCL19-F2A-HSV-TK, and the mouse CD8 transmembrane region and the mouse CD28-4-1BB-CD3ζ intracellular signaling region were inserted instead of the human CD8 transmembrane region and the human CD28-4-1BB-CD3ζ intracellular signaling region. As T cells, mouse T cells derived from the spleen and lymphocytes were used and prepared according to the method of Example 1 (hereinafter referred to as "anti-human mesothelin CAR-mouse IL-7 / mouse CCL19-expressing mouse T cells"). Also, the "CAR, IL-7, and CCL19 non-expressing - T cells" (non-transduced cells: Non-infection) used in this Example used mouse T cells derived from the spleen and lymphocytes as T cells.

[0108] (Results) As shown in Fig. 8, it was confirmed that anti-human mesothelin CAR-mouse IL-7 / mouse CCL19-expressing mouse T cells damaged tumor cells. Also, as shown in Fig. 9, in the ELISA of IFN-γ using the supernatant after co-culture, significant production of IFN-γ was confirmed only in the supernatant of the co-culture of anti-human mesothelin CAR-mouse IL-7 / mouse CCL19-expressing mouse T cells and PAN02 tumor cell line.

[0109] [Example 6] Therapeutic effect in tumor model (Administration of anti-mouse mesothelin CAR-IL-7 / CCL19-expressing T cells to tumor model mice) 5×10 5 PAN02 pancreatic cancer cell lines were subcutaneously inoculated into 7- to 10-week-old C57BL / 6 mice (purchased from SLC). On the 7th day after inoculation, the anticancer agent cyclophosphamide (CPA, 100 mg / kg) was intraperitoneally administered, and on the 10th day, 1×10 6 The anti-human mesothelin CAR-mouse IL- 7 / mouse CCL19-expressing mouse T cells (scFv region is VHMO(15)VLMO, VLMO(15)VHMO, VHMO(25)VLMO, or VLMO(25)VHMO)) or "anti-human mesothelin CAR-expressing mouse T cells" were intravenously administered. The results of the survival rate of mice in VHMO(15)VLMO and VHMO(25)VLMO are shown in Fig. 10, and the results of the tumor volume in VHMO(15)VLMO, VLMO(15)VHMO, VHMO(25)VLMO, or VLMO(25)VHMO) are shown in Fig. 11. In Fig. 10, the horizontal axis represents the number of days after subcutaneous inoculation of PAN02 (the day when PAN02 was subcutaneously inoculated into the mouse was set as day 0), and the vertical axis represents the survival rate. Also, in Fig. 11, the horizontal axis represents the number of days after subcutaneous inoculation of PAN02, and the vertical axis represents the tumor volume (long axis of the tumor × (short axis of the tumor) 2 / 2 (mm 3)(It is as follows). "No treatment" indicates the group administered with only CPA, "Conv." indicates the group administered with anti-human mesothelin CAR-expressing mouse T cells after CPA administration, and "7×19" indicates the group administered with anti-human mesothelin CAR-mouse IL-7 / mouse CCL19-expressing mouse T cells after CPA administration. The above-mentioned "anti-human mesothelin CAR-expressing mouse T cells" were prepared in the same manner as the "anti-human mesothelin CAR-expressing T cells" described in Example 1, except that in the method for preparing the pMSGV1 retroviral expression vector, instead of the human IL-7-F2A-human CCL19-F2A-HSV-TK, HSV-TK was used, and instead of the human CD8 transmembrane region and the human CD28-4-1BB-CD3ζ intracellular signaling region, a pMSGV1 retroviral expression vector was prepared by inserting the mouse CD8 transmembrane region and the mouse CD28-4-1BB-CD3ζ intracellular signaling region, and mouse T cells derived from the spleen and lymphocytes were used as T cells.

[0110] (Results) As shown in Figure 10, it was revealed that the survival rate was significantly increased by administering the anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells of the present invention. Also, as shown in Figure 11, it was revealed that the growth of tumors was clearly suppressed by administering the anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells of the present invention. From this, it became clear that in tumor model mice, the anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells exhibit excellent antitumor activity.

[0111] [Example 7] Therapeutic effect in tumor model - 2 Although it was revealed that anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells exhibit excellent antitumor activity, in order to further confirm the long-term antitumor effect and the antitumor effect other than pancreatic cancer, a human malignant pleural mesothelioma cell line was administered to immunodeficient mice to form tumors, and then the presence or absence of tumor recurrence was examined for 143 days depending on the presence or absence of administration of anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells. The "method for preparing the ACC-MESO-1-GFP-Luc strain" and the "method for activating T cells" used in this example are as follows.

[0112] (Preparation of ACC-MESO-1-GFP-Luc Strain) The human malignant mesothelioma cell line ACC-MESO-1, a mesothelin-positive tumor cell line provided by Dr. Yoshitaka Sekido of the Aichi Cancer Center Research Institute, was transfected with the gene of green fluorescent protein-luciferase (GFP-Luc) using a lentivirus.

[0113] On day 0, ACC-MESO-1 was seeded in a 96-well plate at 1×10 3 cells / well. As the medium, RPMI1640 supplemented with 10% FBS (manufactured by Gibco) was used. On day 1, RediFect Red-FLuc-GFP (manufactured by PerkinElmer), a lentiviral particle for producing luminescent cells, was added at an MOI of 100 to initiate transduction. At that time, in order to enhance the gene transduction efficiency, Hexadimethrine Bromide (manufactured by Sigma-aldrich) was added to the medium to a final concentration of 4 μg / mL. Twenty-four hours after the virus addition (day 2), the medium containing the virus was removed and the medium was changed. After continuous culture, only the cells expressing GFP were sorted with SH800 (manufactured by SONY) to obtain ACC-MESO-1 expressing GFP, that is, "ACC-MESO-1-GFP-Luc".

[0114] (Preparation of Anti-Human Mesothelin CAR-IL-7-CCL19-Expressing T Cells and Anti-Human Mesothelin CAR-Expressing T Cells) In Example 7, the IL-7 / CCL19 expression - anti - human mesothelin CAR vector obtained by Example 1 (the scFv region was replaced with the VH07(15)VL07 DNA fragment; the signal peptide was the signal peptide T shown in SEQ ID NO: 11) or the conventional anti - human mesothelin CAR vector (the scFv region was replaced with the VH07(15)VL07 DNA fragment; the signal peptide was the signal peptide T shown in SEQ ID NO: 11) was used.

[0115] (Activation of T cells) On day 0, 2×10 6 peripheral blood mononuclear cells collected from healthy donors were cultured in a 6 - well cell culture plate coated with 25 μL / mL of RetroNectin (manufactured by Takara Bio Inc.) and 5 μg / mL of anti - human CD3 monoclonal antibody (manufactured by Invitrogen, 5 μg / mL) together with IL - 2 (manufactured by Peprotech) in a 37°C, 5% CO2 incubator. The culture medium used was OpTmizer CTS (manufactured by Gibco) supplemented with 2 mM of L - glutamine (manufactured by Gibco), 1% penicillin - streptomycin (manufactured by Wako Pure Chemical Industries, Ltd.) and 2.5 μg / mL of Fungizone (manufactured by Bristol - Myers Squibb). After culturing for 3 days, it was confirmed under a microscope that the T cells were activated and morphological changes had occurred on day 3 to obtain activated T cells.

[0116] (Observation of tumor recurrence) First, on day 0, 2×10 6The above ACC-MESO-1-GFP-Luc was administered intrathoracically at cells / mouse. On day 1, in vivo tumor engraftment was confirmed using an in vivo imaging system (IVIS). On day 1, the anti-human mesothelin CAR-expressing T cells, anti-human mesothelin CAR-IL-7-CCL19-expressing T cells (scFv region: VH07(15)VL07), and the T cells activated by the above method that had been frozen after preparation in Example 1 were thawed. Since the CAR expression rates of the above anti-human mesothelin CAR-expressing T cells and the above anti-human mesothelin CAR-IL-7-CCL19-expressing T cells were 49.6% and 32.5% respectively, after adding the above activated T cells to the anti-human mesothelin CAR-expressing T cells and combining their CAR expression rates, 1×10 5 cells of the above anti-human mesothelin CAR-expressing T cells were administered to a group (N = 5), and 1×10 5 cells of the above anti-human mesothelin CAR-IL-7-CCL19-expressing T cells were administered to a group (N = 5). The administration of the anti-human mesothelin CAR-expressing T cells and the anti-human mesothelin CAR-IL-7-CCL19-expressing T cells was performed by intravenous injection via the tail vein. Furthermore, from day 3 onwards, the fluorescence intensity of the tumor was measured using IVIS (luminescence amount: Total Flux (photons / sec)). The results are shown in FIGS. 12A and 12B. Also, FIG. 13 shows a graph of the relationship between the number of days from the administration in the above results and the survival rate of the mice, and FIG. 14 shows a graph of the relationship between the number of days from the administration and the total fluorescence amount (photons / second). In FIGS. 12A, 12B, 13, and 14, those administered with anti-human mesothelin CAR-IL-7-CCL19-expressing T cells are indicated as "7×19 CAR-T", and those administered with anti-human mesothelin CAR-expressing T cells are indicated as "Conventional CAR-T". In addition, in this Example 7, since NSG immunodeficient mice lacking endogenous T cells were used as recipients, the influence of the recipients' endogenous T cells was excluded, and the effect of the administered anti-human mesothelin CAR-IL-7-CCL19-expressing T cells themselves was evaluated.

[0117] As shown in FIGS. 12A, 12B, 13, and 14, on day 21, almost no tumor fluorescence intensity was observed in either 7×19 CAR-T or Conventional CAR-T. In 7×19 CAR-T, no tumor fluorescence was observed until day 143, and it was confirmed that recurrence was completely suppressed. On the other hand, in Conventional CAR-T, tumor fluorescence began to be observed around day 45, the tumor fluorescence intensity increased on day 115, one mouse died on day 129, and the remaining four mice also died on day 143. Therefore, it was revealed that administration of CAR-IL-7-CCL19-expressing T cells has cytotoxic activity against cancer cells expressing human mesothelin such as pancreatic cancer and human malignant pleural mesothelioma, and has a long-term antitumor effect.

[0118] This application is based on Japanese Patent Application No. 2017-247109 filed on December 24, 2017, the content of which is incorporated herein in its entirety.

Claims

1. The present invention provides an immunocompetent cell that expresses a single chain antibody that specifically recognizes human mesothelin, a chimeric antigen receptor (CAR) having an extracellular hinge region, a transmembrane region, and a signal transduction region that induces activation of an immunocompetent cell, interleukin 7 (IL-7), and chemokine (C-C motif) ligand 19 (CCL19), the single chain antibody in the CAR is (1-1) a single chain antibody comprising a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18, the signal transduction region in the CAR that induces activation of an immunocompetent cell comprises a polypeptide of the intracellular domain of 4-1BB and a polypeptide of the intracellular domain of CD3ζ; The transmembrane domain of the CAR comprises a polypeptide of the transmembrane domain of CD8; and The immunocompetent cell, wherein the extracellular hinge region in the CAR comprises a polypeptide of the extracellular hinge region of CD8.

2. The immunocompetent cell according to claim 1, which is an immunocompetent cell isolated from a living body.

3. The immunocompetent cell according to claim 1 or 2, characterized in that it contains a nucleic acid encoding a CAR that specifically recognizes exogenous human mesothelin, a nucleic acid encoding exogenous IL-7, and a nucleic acid encoding exogenous CCL19.

4. The immunocompetent cell according to claim 3, characterized in that the nucleic acid encoding foreign IL-7 and the nucleic acid encoding foreign CCL19 are a nucleic acid encoding foreign human IL-7 and a nucleic acid encoding foreign human CCL19.

5. The immunocompetent cell according to claim 3 or 4, characterized in that a nucleic acid encoding a CAR that specifically recognizes exogenous human mesothelin, a nucleic acid encoding exogenous IL-7, and a nucleic acid encoding exogenous CCL19 are integrated into the genome.

6. The immunocompetent cell according to any of claims 1 to 5, wherein the single chain antibody in the CAR is a single chain antibody comprising: (1-2) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and including a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15; and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and including a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

18.

7. The immunocompetent cell according to any of claims 1 to 6, wherein the single chain antibody in the CAR is a single chain antibody comprising (1-3) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

2.

8. The immunocompetent cell according to any one of claims 1 to 7, characterized in that the cell membrane-spanning domain in the CAR comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

7.

9. The immunocompetent cell according to any one of claims 1 to 8, characterized in that the signal transduction region in the CAR that induces activation of an immunocompetent cell comprises an amino acid sequence having 90% or more sequence identity to the combined amino acid sequences of SEQ ID NOs: 9 and 10.

10. The immunocompetent cell according to any one of claims 1 to 9, characterized in that the heavy chain variable region and the light chain variable region are linked via a peptide linker consisting of a sequence of 2 to 30 amino acids.

11. The immunocompetent cell according to claim 10, characterized in that the peptide linker consists of the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO:

27.

12. The immunocompetent cell according to any one of claims 1 to 11, wherein the light chain variable region is located on the C-terminal side relative to the heavy chain variable region.

13. The immunocompetent cell according to any of claims 1 to 12, wherein the single chain antibody in the CAR is a single chain antibody sequentially comprising: (1-4) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1, a peptide linker consisting of the amino acid sequence shown in SEQ ID NO: 26, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:

2.

14. The immunocompetent cell according to any one of claims 1 to 13, characterized in that the immunocompetent cell is a T cell.

15. The immunocompetent cell according to any one of claims 1 to 14, characterized in that the immunocompetent cell is a T cell derived from a human or isolated from a human.

16. A pharmaceutical composition comprising the immunocompetent cell according to any one of claims 1 to 15 and a pharma- ceutical acceptable additive.

17. 17. The pharmaceutical composition of claim 16 for use in the treatment of cancer.

18. An expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) having a single chain antibody that specifically recognizes human mesothelin, an extracellular hinge region, a cell membrane-spanning region, and a signal transduction region that induces activation of an immunocompetent cell, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, the single chain antibody in the CAR is (1-1) a single chain antibody comprising a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18, the signal transduction region in the CAR that induces activation of an immunocompetent cell comprises a polypeptide of the intracellular domain of 4-1BB and a polypeptide of the intracellular domain of CD3ζ; The transmembrane domain of the CAR comprises a polypeptide of the transmembrane domain of CD8; and The expression vector, wherein the extracellular hinge region in the CAR comprises a polypeptide of the extracellular hinge region of CD8.

19. A method for producing immunocompetent cells expressing a CAR that specifically recognizes human mesothelin, IL-7, and CCL19, the method comprising introducing a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain antibody that specifically recognizes human mesothelin, an extracellular hinge region, a cell membrane-spanning region, and a signaling region that induces activation of an immunocompetent cell, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19 into an immunocompetent cell isolated from a living body, the method comprising the steps of: the single chain antibody in the CAR is (1-1) a single chain antibody comprising a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 13, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 14, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 16, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 17, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 18, the signal transduction region in the CAR that induces activation of an immunocompetent cell comprises a polypeptide of the intracellular domain of 4-1BB and a polypeptide of the intracellular domain of CD3ζ; The transmembrane domain of the CAR comprises a polypeptide of the transmembrane domain of CD8; and The method for producing an immunocompetent cell, wherein the extracellular hinge region in the CAR comprises a polypeptide of the extracellular hinge region of CD8.

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