Immunologically active cells and expression vectors that express immune function control factors

Abstract

The objective of this invention is to provide an immune function control factor that expresses immune-active cells in immune-active cells, an immune-active cell that has proliferative capacity, survival capacity, and T cell aggregation capacity, and a carrier for producing the immune function control factor that expresses the immune-active cells. Immunoactive cells are created that specifically recognize cell surface molecules of cancer antigens, interleukin-7 (IL-7), and CCL19. Preferably, the cell surface molecules that specifically recognize cancer antigens are T cell receptors that specifically recognize cancer antigens, or the immunoactive cells are T cells.

Description

Immune active cells expressing immune function control factors and expression vectors The present invention relates to immune active cells expressing cell surface molecules that specifically recognize cancer antigens, interleukin-7 (IL-7), and CCL19, an anticancer agent containing the immune active cells, and a vector for producing the immune active cells expressing the same. Cancer is a disease that affects a large number of people worldwide. Generally speaking, chemotherapy, radiotherapy, or surgical treatment are widely used in the industry. However, there are various problems such as side effects, loss of some functions, or difficulty in treating metastases. Therefore, in order to further maintain a relatively high QOL (Quality Of Life) of patients, immunotherapy has been developed in recent years. In this immunotherapy, immunocyte therapy is a therapy in which immunocompetent cells are collected from a patient, treated and expanded in a way that enhances their immune function, and then re-introduced into the patient's body. Specifically, there is a known therapy in which T cells are collected from a patient, a gene encoding CAR (Constitutive Androstane Receptor) is introduced into the T cells for expansion, and then re-introduced into the patient's body (see Non-Patent Document 1). This therapy is currently undergoing clinical trials worldwide and has obtained results showing effectiveness in hematopoietic organ malignancies such as leukemia and lymphoma. Also, as immune function regulators of immunocompetent cells such as T cells, at least several hundred factors such as cytokines, chemokines, and signaling control proteins are known. Among them, interleukin 7 (IL-7) is known to be a cytokine essential for the survival of T cells, and it is produced by non-hematopoietic cells such as stromal cells in the bone marrow, thymus, and lymphoid organs / tissues. As T cells that utilize the function of IL-7, T cells expressing a chimeric cytokine receptor formed by fusing IL-7 and IL-7Rα have been disclosed (see Patent Document 1). However, the chimeric cytokine receptor in these T cells is merely a fusion protein, which is expressed limited to the cell membrane surface of the introduced T cells, and only transmits cytokine signals such as IL-7R to its own cells without depending on ligands, and cannot enhance the function of T cells that do not introduce the above receptor. Also, it has been disclosed that a decrease in the expression of CCL19 or CCL21 and IL-7 leads to a defect in the maintenance of the T cell area in the spleen of SIRP (Signal Regulatory Protein) α mutant mice (see Non-Patent Document 2), and that CCL19 or CCL21 and IL-7 have the effect of maintaining the homeostasis of T cells in secondary lymphoid tissues (spleen or lymph nodes) (see Non-Patent Document 3). However, the above Non-Patent Documents 2 and 3 show the effects on non-activated T cells that are constantly present in the T cell area of secondary lymphoid tissues, and do not show a direct association with anti-tumor immune responses. Furthermore, the CCL19 or CCL21 and IL-7-expressing cells in the above Non-Patent Documents 2 and 3 are cells of the reticuloendothelial system present in secondary lymphoid tissues rather than T cells. On the other hand, the T cell receptor (hereinafter, also referred to as "TCR") is an antigen receptor molecule expressed on the cell membrane of T cells.It is known to exist in the form of heterodimers containing an α chain and a β chain, or a γ chain and a Δ chain, and activates T cells by recognizing antigen molecules bound to major histocompatibility complex (MHC) molecules. The development of immunotherapy has been continuously carried out in the industry, that is, applying the function of this TCR, introducing the TCR gene that can recognize tumor antigens expressed in cancer cells into T cells obtained from cancer patients, and re-introducing them into the patient's body after expansion. Specifically, a pharmaceutical composition for treating meningioma is disclosed, which contains cells expressing a TCR that specifically recognizes WT1-expressing cells (see Patent Document 2). Although anti-tumor effects against hematopoietic organ malignancies have been confirmed in a part of the above technologies, there are no examples showing significant effects on solid cancers. Considering the problems of low survival efficiency of the transferred immunocompetent cells in vivo, or activation of endogenous immunocompetent cells induced by the transferred immunocompetent cells, or insufficient accumulation in the tumor local area, technologies to solve these problems are sought. [Prior Art Documents] [Patent Documents] Patent Document 1: International Publication No. 2013 / 123061 Specification Patent Document 2: Japanese Patent Application Laid-Open No. 2013-116891 Non-Patent Document 1: Yozo Nakazawa, Shinshu Igaku, 61(4): 197-203 (2013) Non-Patent Document 2: SATO-HASHIMOTO M. et al., J. Immunol., 2011, vol. 187, no. 1, 291-7 Non-Patent Document 3: SIEGERT S. et al., Front. Immunol., 2012, vol. 3, article 285. [Problems to be Solved by the Invention] Among the immunocompetent cells used in previous immunotherapies, the immune induction effect of endogenous immunocompetent cells, or the proliferation ability, viability, or T cell aggregation ability of immunocompetent cells has not been sufficiently enhanced. Therefore, an object of the present invention is to provide an immunocompetent cell that expresses an immunofunctional regulator of immunocompetent cells and has proliferation ability, viability, and T cell aggregation ability, and a vector for expressing the immunofunctional regulator for producing the immunocompetent cell. [Technical Means for Solving the Problems] The inventors attempted to improve cells expressing immunofunctional regulators in order to achieve more excellent immune induction effects or antitumor activities in cancer immunotherapy using immunocompetent cells. In this process, cytokines, chemokines, and signaling control proteins, which are factors that control the immune functions of immunocompetent cells, were focused on, and a vector for expressing the factors that control the immune functions of immunocompetent cells was constructed. When this expression vector was introduced into immunocompetent cells, it was found that immunocompetent cells with more excellent immune induction effects, proliferation ability, viability, and T cell aggregation ability than previous immunocompetent cells could be produced, thus completing the present invention. That is, the present invention is disclosed as follows in (1) to (9) below. (1) An immunocompetent cell that expresses a cell surface molecule that specifically recognizes a cancer antigen, interleukin-7 (IL-7), and CCL19. (2) The immunocompetent cell according to (1) above, characterized in that the cell surface molecule that specifically recognizes a cancer antigen is a T cell receptor that specifically recognizes a cancer antigen. (3) The immunocompetent cell according to (1) or (2) above, characterized in that the immunocompetent cell is a T cell. (4) The immunocompetent cell according to any one of (1) to (3) above, characterized in that the cancer antigen is WT1, MART-1, NY-ESO-1, MAGE (Melanoma antigen)-A1, MAGE-A3, MAGE-A4, Glypican-3, KIF20A, Survivin, AFP (Alpha Fetal Protein)-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR (Vascular Endothelial Growth Facotr Receptor) 1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, Mesothelin, NKG2D, P1A, GD2, or GM2.(5) A expression vector, which is used to produce any one of the following (a) to (e) of the immunocompetent cells described in any one of the above (1) to (4): (a) A expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19; (b) Two expression vectors of the following (b-1) and (b-2): (b-1) A expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen; (b-2) A expression vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19; (c) Two expression vectors of the following (c-1) and (c-2): (c-1) A expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding IL-7; (c-2) A expression vector containing a nucleic acid encoding CCL19; (d) Two expression vectors of the following (d-1) and (d-2): (d-1) A expression vector containing a nucleic acid encoding IL-7; (d-2) A expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding CCL19; (e) Three expression vectors of the following (e-1), (e-2) and (e-3): (e-1) A expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen; (e-2) A expression vector containing a nucleic acid encoding IL-7; (e-3) A expression vector containing a nucleic acid encoding CCL19. (6) The expression vector described in the above (5), characterized in that: the cell surface molecule that specifically recognizes a cancer antigen is a T cell receptor that specifically recognizes a cancer antigen. (7) The expression vector described in the above (5) or (6), characterized in that: the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 in the expression vector of (a), the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 in the expression vector of (b-2), the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and the nucleic acid encoding IL-7 in the expression vector of (c-1), or the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and the nucleic acid encoding CCL19 in the expression vector of (d-2) are linked via a self-cleaving peptide. (8) The expression vector described in any one of the above (5) to (7), characterized in that: it contains a nucleic acid encoding a suicide gene. (9) An anticancer agent, which contains the immunocompetent cells described in any one of the above (1) to (4) and a pharmaceutically acceptable additive.[Effects of the Invention] When immunocompetent cells that specifically recognize cancer antigens, IL-7, and CCL19 (hereinafter, also referred to as "IL-7×CCL19-expressing immunocompetent cells") using the expression of the present invention are used, they have antitumor activity and can inhibit the reduction in survival rate caused by tumors formed by cancer cells having antigens specifically recognized by cell surface molecules. Further, when the expression vector of the present invention is used, immunocompetent cells having proliferation ability, survival ability, and T cell aggregation ability can be produced. The IL-7×CCL19-expressing immunocompetent cells of the present invention are not particularly limited as long as they express cell surface molecules that specifically recognize cancer antigens, interleukin-7 (IL-7), and CCL19. Furthermore, they may also express other immunoregulatory factors such as IL-15, CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, CCL4, Flt3L, interferon-γ, MIP (Macrophage Inflammatory Protein)-1α, GM-CSF (Granulocyte Macrophage-Colony Stimulating Factor), M-CSF (Macrophage Colony Stimulating Factor), TGF (Transforming Growth Factor)-β, TNF (Tumor Necrosis Factor)-α, etc. The so-called cancer antigen refers to substances such as proteins and glycolipids that are expressed at a higher level in cancer cells than in normal cells or are specifically expressed in cancer cells. Examples of such cancer antigens include tumor-associated antigens or cancer-testis antigens, angiogenesis-related antigens, antigenic determinant peptides of cancer neoantigens (neoantigens) generated by gene mutations. Specifically, examples include WT1, MART-1, NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, glypican-3, KIF20A, survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, mesothelin, NKG2D, P1A and other proteins, or GD2, GM2 and other glycolipids, but are not limited thereto. Examples of cell surface molecules that specifically recognize cancer antigens include cell surface receptors, artificial receptors, and adhesion factors that specifically recognize cancer antigens. Specifically, examples include T cell receptors that specifically recognize cancer antigens or chimeric antigen receptors (CARs) that specifically recognize cancer antigens, etc., which are molecules that confer cancer-specific recognition ability by being expressed on the cell surface. More specifically, TCRs are examples. As TCRs, as long as they specifically recognize cancer antigens, they can be heterodimers containing α and β chains (α / β-TCR) or heterodimers containing γ and δ chains (γ / δ-TCR).Furthermore, regarding cell surface molecules that specifically recognize cancer antigens, as long as the recognition of cancer antigens is specific, those that indirectly recognize them are acceptable. For example, molecules such as antibodies that specifically recognize cancer antigens are administered to a subject simultaneously or continuously with the immunocompetent cells of the present invention, and the antibodies or the labels labeled on the antibodies are recognized. Thereby, the immunocompetent cells of the present invention can indirectly and specifically recognize cancer antigens. As an example of the case of recognizing antibodies, CD16 etc. can be cited as cell surface molecules, and as an example of the labels labeled on the antibodies etc., FITC (fluorescein isothiocyanate) etc. can be cited. As the type of immunocompetent cells in the IL-7×CCL19-expressing immunocompetent cells of the present invention, any cells related to immune responses are acceptable, and examples include: lymphocyte cells such as T cells, natural killer cells (NK cells), and B cells, or antigen-presenting cells such as monocytes, macrophages, and dendritic cells, or granulocytes such as neutrophils, eosinophils, basophils, and mast cells. T cells derived from mammals such as humans, dogs, cats, pigs, and mice can be appropriately cited, and T cells derived from humans are preferred. Also, T cells can be isolated and purified from immune cells infiltrating body fluids such as blood and bone marrow fluid, or tissues such as the spleen, thymus, and lymph nodes, or cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites. Also, those produced from ES cells (embryonic stem cells) or iPS cells (induced pluripotent stem cells) can also be used. As such T cells, α / β-T cells, γ / Δ-T cells, CD8 can be cited. + T cells, CD4 +T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells. As a method for producing the IL-7×CCL19-expressing immunocompetent cells of the present invention, the following methods of introducing the expression vector of the present invention into immunocompetent cells can be mentioned. Alternatively, methods of inducing fertilized eggs, ES cells, or iPS cells by introducing a vector expressing a cell surface molecule that specifically recognizes a cancer antigen, interleukin 7 (IL-7), and / or CCL19 can also be mentioned; or immunocompetent cells isolated from a transgenic mammal expressing a cell surface molecule that specifically recognizes a cancer antigen by gene transfer, and further introducing a vector expressing a cell surface molecule that specifically recognizes a cancer antigen, interleukin 7 (IL-7), and / or CCL19 into the immunocompetent cells as needed. As a method for introducing the following expression vector of the present invention into the above immunocompetent cells, there is no particular limitation, and methods of introducing by known methods such as virus infection method, calcium phosphate method, liposome transfection method, microinjection method, electroporation method, etc. can be mentioned, and a method of introducing by virus infection method can be appropriately mentioned. As the virus infection method, a method of producing a recombinant virus by transfecting the expression vector of the present invention and a packaging plasmid into packaging cells such as GP2-293 cells (manufactured by TAKARA BIO INC.), Plat-GP cells (manufactured by Cosmobio Co., Ltd.), PG13 cells (ATCC CRL-10686), PA317 cells (ATCC CRL-9078), etc., and infecting the immunocompetent cells with the recombinant virus can be used, and commercially available kits such as Retrovirus packagin Kit Eco (retrovirus packaging kit Eco) (manufactured by TAKARA BIO INC.) can be used. In addition, the immunocompetent cells of the present invention can be produced by the following method: using a known gene editing technique, a polynucleotide containing a base sequence encoding a cell surface molecule that specifically recognizes a cancer antigen, IL-7, and CCL19 is incorporated into the genome of the cell so as to be able to be expressed under the control of an appropriate promoter. As a known gene editing technique, techniques using endonucleases such as zinc finger nucleases, TALEN (Transcription Activator-like Effector Nuclease), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system can be mentioned. The same gene editing technique can also be used when expressing other foreign proteins in the immunocompetent cells of the present invention, and a polynucleotide containing a base sequence encoding other foreign proteins is incorporated into the genome of the cell so as to be able to be expressed under the control of an appropriate promoter.As a method of incorporating a polynucleotide into the cell genome in such a way that it can be expressed under the control of an appropriate promoter, examples include: functionally linking the base sequences encoding cell surface molecules that specifically recognize cancer antigens, IL-7, and CCL19 (or other proteins) downstream of an appropriate promoter, and incorporating the obtained polynucleotide (i.e., a polynucleotide in which the coding sequence is linked in such a way that it can be expressed under the control of the promoter) into the non-coding region of the cell genome; a method of incorporating a polynucleotide containing the base sequences encoding cell surface molecules that specifically recognize cancer antigens, IL-7, and CCL19 (or other proteins) downstream of an endogenous promoter in the cell genome; etc. As endogenous promoters, for example, the promoters of TCRα and TCRβ can be mentioned. Also, it is possible to attempt to express the following herpes simplex virus thymidine kinase (HSV-TK) or inducible caspase 9 in the IL-7×CCL19-expressing immunocompetent cells of the present invention. Since the IL-7×CCL19-expressing immunocompetent cells of the present invention express cell surface molecules that specifically recognize cancer antigens, IL-7, and CCL19, they have high proliferative ability, viability, and the ability to aggregate endogenous T cells, and can be applied to adoptive immunotherapy using various immunocompetent cells. Examples of adoptive immunotherapy include dendritic cell therapy, NK cell therapy, γ / Δ-T cell therapy, α / β-T cell therapy, CTL (Cytotoxic T Lymphocyte) therapy, TIL (Tumor Infiltrating Lymphocyte) therapy, etc., but are not limited thereto. A method of amplifying by introducing the following expression vector of the present invention into immunocompetent cells collected from a patient and administering it to the patient can be mentioned. Hereinafter, specific examples are listed, but are not limited thereto. Dendritic cell therapy includes steps of introducing surgically removed cancer tissue or its lysate into dendritic cells differentiated from monocytes collected from a patient and administering it into the patient's body, but may also include steps of introducing the expression vector of the present invention into dendritic cells. Here, it is also possible to artificially synthesize and use antigenic determinant peptides of cancer antigen molecules instead of the above-mentioned cancer tissue or lysate. NK cell therapy includes steps of activating and proliferating NK cells using a plurality of stimulatory substances such as IL-2 on lymphocytes collected from a patient and then administering it to the patient, but may also include steps of introducing the vector of the present invention into NK cells. Furthermore, by using an antibody medicine against cancer in combination with activated NK cells, an effect of efficiently attacking cancer cells can be expected. γ / Δ-T cell therapy includes steps of culturing and stimulating lymphocytes collected from a patient using IL-2 or zoledronic acid to proliferate γ / Δ-T cells and then administering it to the patient, but may also include steps of introducing the expression vector of the present invention into γ / Δ-T cells.The α / β-T cell therapy includes steps of culturing lymphocytes collected from a patient using an anti-CD3 antibody or IL-2, activating them, and administering the obtained α / β-T cells to the patient, but may also include a step of introducing the expression vector of the present invention into the α / β-T cells. The CTL therapy includes steps of stimulating lymphocytes collected from a patient with cancer cells collected from the patient, culturing them by adding an anti-CD3 antibody or IL-2, proliferating specific CTLs in the cancer cells, and then administering them to the patient, but may also include a step of introducing the expression vector of the present invention into the CTLs. Further, antigen-presenting cells presenting cancer antigen epitope peptides can be used to replace the above-mentioned cancer cells. The TIL therapy includes steps of collecting lymphocytes from cancer tissues collected from a patient, stimulating and culturing them using IL-2, etc., and then administering them to the patient, but may also include a step of introducing the expression vector of the present invention into the lymphocytes. The expression vector of the present invention is any one of the following (a) to (e) for producing the IL-7×CCL19-expressing immunocompetent cells of the present invention. (a) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19; (b) Two expression vectors of the following (b-1) and (b-2): (b-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen; (b-2) An expression vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19; (c) Two expression vectors of the following (c-1) and (c-2): (c-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding IL-7; (c-2) An expression vector containing a nucleic acid encoding CCL19; (d) Two expression vectors of the following (d-1) and (d-2): (d-1) An expression vector containing a nucleic acid encoding IL-7; (d-2) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding CCL19; (e) Three expression vectors of the following (e-1), (e-2), and (e-3): (e-1) An expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen; (e-2) An expression vector containing a nucleic acid encoding IL-7; (e-3) An expression vector containing a nucleic acid encoding CCL19. The expression vector of the present invention may further contain nucleic acids encoding other immunomodulatory factors such as IL-15, CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, CCL4, Flt3L, Interferon-γ, MIP-1α, GM-CSF, M-CSF, TGF-β, TNF-α, etc.The nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the nucleic acid encoding interleukin 7 (IL-7), and the nucleic acid encoding CCL19 may each be exemplified by a nucleic acid derived from a mammal, and preferably a nucleic acid derived from a human. Each of the above nucleic acids may 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 of the nucleic acids may be appropriately obtained by retrieving publicly known literature or databases such as NCBI (National Center of Biotechnology Information) (http: / / www.ncbi.nlm.nih.gov / guide / ). As the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, a nucleic acid derived from a human is preferably exemplified. The nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen may be exemplified by a nucleic acid encoding a T cell receptor (TCR) or a nucleic acid encoding a chimeric antigen receptor (CAR), and may be a nucleic acid derived from nature or a synthetic nucleic acid, and may be appropriately selected according to the type of cell into which the expression vector of the present invention is introduced, and the sequence information may be appropriately obtained by retrieving publicly known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). The nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 can be produced by known techniques such as a method of chemical synthesis based on the information of the base sequence of the nucleic acid encoding each of them, or a method of amplification by PCR (Polymerase Chain Reaction). Furthermore, the selected codons for encoding amino acids may be modified to optimize the expression of the nucleic acid in the target host cell. As the TCR in the nucleic acid encoding TCR, it may be a heterodimer (α / β-TCR) containing an α chain and a β chain, or a heterodimer (γ / Δ-TCR) containing a γ chain and a δ chain. Furthermore, the nucleic acid encoding α / β-TCR includes both a nucleic acid encoding the α chain of TCR and a nucleic acid encoding the β chain, and the nucleic acid encoding γ / Δ-TCR includes both a nucleic acid encoding the γ chain of TCR and a nucleic acid encoding the δ chain. The sequence information of the nucleic acid encoding TCR can be identified by using a publicly known method in the art based on the nucleic acids of the α chain and the β chain, which are TCR subunits of CTL induced by using a specific antigen peptide (International Publication No. 2007 / 032255, and Morgan et al., J Immunol, 171, 3288 (2003)). For example, for analyzing TCR, the PCR method is preferably used.PCR primers for analysis can be, for example, a 5'-R primer (5'-gtctaccaggcattcgcttcat-3': SEQ ID NO: 3) as a 5'-side primer, and a 3-TRa-C primer (5'-tcagctggaccacagccgcagcgt-3': SEQ ID NO: 4) specific for the C region of the TCRα chain, a 3-TRb-C1 primer (5'-tcagaaatcctttctcttgac-3': SEQ ID NO: 5) specific for the C1 region of the TCRβ chain, or a 3-TRβ-C2 primer (5'-ctagcctctggaatcctttctctt-3': SEQ ID NO: 6) specific for the C2 region of the TCRβ chain, but are not limited to these. The TCR derivative can bind to target cells presenting antigen peptides with a relatively high binding force, and can optionally mediate the efficient killing of target cells presenting antigen peptides in vivo and in vitro. As the nucleic acid encoding the above TCR, for example, as long as it can recognize an MHC molecule-bound antigen molecule that encodes a TCR such as MART1-specific TCR (Cancer Res. 54, 5265-5268 (1994)), MAGE-A3-specific TCR (Anticancer Res., 20, 1793-1799 (2000)), gp100-specific TCR (J. Immunol. 170, 2186-2194 (2003)), NY-ESO-1-specific TCR (J. Immunol., 174, 4415-4423 (2005)), WT1-specific TCR (Blood, 106, 470-476 (2005)), MAGE-A1-specific TCR (Int. Immunol., 8, 1463-1466 (1996)), P1A-specific TCR (Sarma, S., Y. Guo, Y. Guilloux, C. Lee, X.-F. Bai, Y. Liu. 1999. Cytotoxic T lymphocytes to an unmutated tumor antigen P1A: normal development but restrained effector function. J. Exp. Med. 189: 811.), etc., and activates T cells, it can be a nucleotide sequence having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more with the nucleotide sequence encoding the TCR described in the above literature.Furthermore, it is also possible to specify the sequence encoding the CDR in the base sequence encoding the TCR described in the above-mentioned document, maintain the sequence encoding the CDR, and have a base sequence having an identity of 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more with the base sequence encoding the TCR described in the above-mentioned document in the sequence other than the sequence encoding the CDR. As the nucleic acid encoding IL-7, a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1 can be exemplified, and as long as it has the effect of enhancing the cell proliferation rate or cell survival rate of IL-7, it can be a base sequence having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more with the base sequence encoding the amino acid sequence shown in SEQ ID NO: 1. As the nucleic acid encoding CCL19, a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 can be exemplified, and as long as it has the cell migration effect of CCL19, a base sequence having an identity of 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more with the base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 can also be used. In addition, the expression vector of the present invention may also contain a nucleic acid encoding a suicide gene. The so-called suicide gene refers to a gene having the following function, that is, by expressing, it directly or secondarily induces a cytotoxic substance to cause the death of its own cells. By including a nucleic acid encoding a suicide gene in the expression vector of the present invention, according to the cancer treatment process, for example, when the tumor disappears, a drug that activates the function of the suicide gene is administered, and the immune active cells in the living body can be controlled. In addition, IL-7 or CCL19 is different from other cytokines and has a lower possibility of causing cytokine release syndrome or tumorigenesis of gene-introduced cells as a side effect. However, by enhancing the function of the immune active 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 the surrounding tissues. In this case, by including a nucleic acid encoding a suicide gene in the expression vector of the present invention, the risk of becoming cytokine release syndrome can be surely reduced.As suicide genes, genes encoding thymidine kinase (HSV-TK) of herpes simplex virus or inducible caspase 9 described in the following documents can be cited. As an agent for activating the function of the gene, for the former, ganciclovir can be cited, and for the latter, AP1903 as a dimerization-inducing compound (CID, chemical induction of dimerization) can be cited (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. Front Pharmacol. 2014 Nov 27; 5: 254., Minagawa K., Pharmaceuticals (Basel). 2015 May 8; 8 (2): 230-49., Bole-Richard E., Front Pharmacol. 2015 Aug 25; 6: 174). In the vector of the present invention, in the expression vector (a) containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, any of the nucleic acids can be arranged upstream or downstream of any other. Specifically, for example, in the case where a nucleic acid encoding a TCR is used as the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the nucleic acids can be, in order from upstream, a nucleic acid encoding a TCR, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19; or a nucleic acid encoding a TCR, a nucleic acid encoding CCL19, and a nucleic acid encoding IL-7; or a nucleic acid encoding IL-7, a nucleic acid encoding CCL19, and a nucleic acid encoding a TCR; or a nucleic acid encoding IL-7, a nucleic acid encoding a TCR, and a nucleic acid encoding CCL19; or a nucleic acid encoding CCL19, a nucleic acid encoding a TCR, and a nucleic acid encoding IL-7; or a nucleic acid encoding CCL19, a nucleic acid encoding IL-7, and a nucleic acid encoding a TCR. In the expression vector (b-2) in the vector of the present invention, which contains a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19, the arrangement of the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 is not particularly limited. The nucleic acid encoding CCL19 can be arranged upstream or downstream of the nucleic acid encoding IL-7.In the expression vector of (c-1) in the vector of the present invention, which contains a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding IL-7, the arrangement of the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and the nucleic acid encoding IL-7 is not particularly limited. Relative to the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the nucleic acid encoding IL-7 can be arranged upstream or downstream. In the expression vector of (d-2) in the vector of the present invention, which contains a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding CCL19, the arrangement of the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and the nucleic acid encoding CCL19 is not particularly limited. Relative to the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the nucleic acid encoding CCL19 can be arranged upstream or downstream. Furthermore, the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 can be transcribed using other promoters respectively, or an internal ribosome entry site (IRES, internal ribozyme entry site) or a self-cleaving 2A peptide can be used and transcribed using one promoter. When using an internal ribosome entry site (IRES) or a self-cleaving 2A peptide to transcribe the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 using one promoter, between the above-mentioned respective nucleic acids, or when including the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, between the nucleic acid and the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19, or when including a nucleic acid encoding α / β-TCR, between the nucleic acid encoding the α chain and the nucleic acid encoding the β chain, or when encoding γ / Δ-TCR, between the nucleic acid encoding the γ chain and the nucleic acid encoding the Δ chain, as long as each nucleic acid can be expressed, any nucleic acid can be contained, but it is preferably linked via a self-cleaving peptide (2A peptide) or a sequence encoding IRES, preferably a sequence encoding 2A peptide. By using this sequence for linking, each nucleic acid can be expressed efficiently. Also, when containing a nucleic acid encoding a suicide gene, the position of the suicide gene is not particularly limited. For example, it can be arranged upstream or downstream of the above-mentioned respective nucleic acids via a sequence encoding 2A peptide or IRES under the downstream of the promoter used to express the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the nucleic acid encoding IL-7, or the nucleic acid encoding CCL19, or it can also be arranged under the downstream of other promoters. The so-called 2A peptide is a self-cleaving peptide derived from a virus and has the characteristic that the cleavage occurs between G and P (at a position 1 residue from the C-terminus) in the amino acid sequence shown in SEQ ID NO: 7 (Szymczak et al., Expert Opin. Biol. Ther. 5 (5): 627-638 (2005)).Therefore, the nucleic acids incorporated before and after the 2A peptide are independently expressed in cells. As the above 2A peptide, a 2A peptide derived from picornavirus, rotavirus, insect virus, Candida virus or trypanosome virus is preferred, and a 2A peptide derived from picornavirus (F2A) shown in SEQ ID NO: 8 is more preferred. As the vector in the expression vector of the present invention, it can be linear or circular, can be a non-viral vector such as a plasmid, can be a viral vector, or can also be a transposon vector. In addition, control sequences such as promoters or terminators, or selection marker sequences such as drug resistance genes or reporter genes can be contained in the vector. By efficiently arranging the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 downstream of the promoter sequence, each nucleic acid can be transcribed with high efficiency. Examples of the above promoters include viral-derived promoters such as the LTR (Long Terminal Repeat) promoter of retrovirus, the SV40 early promoter, the cytomegalovirus promoter, and the thymidine kinase promoter of herpes simplex virus; mammalian-derived promoters such as the phosphoglycerate kinase (PGK) promoter, the Xist (X-inactive specific transcript) promoter, the β-actin promoter, and the RNA (Ribonucleic Acid) polymerase II promoter. In addition, a tetracycline-responsive promoter induced by tetracycline, an Mx1 promoter induced by interferon, etc. can also be used. By using a promoter induced by the above specific substances in the expression vector of the present invention, the induction of the expression of IL-7 and CCL19 can be controlled according to the treatment process of cancer. Examples of the above viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. A retroviral vector can be preferably exemplified, and a pMSGV vector (Tamada k et al., Clin Cancer Res 18: 6436-6445 (2002)) or a pMSCV vector (manufactured by TAKARA BIO Inc.) can be more preferably exemplified. 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 time.It is confirmed that the expression vector of the present invention is contained in immunocompetent cells. For example, when the nucleic acid encoding TCR is contained, the expression of TCR can be investigated by flow cytometry, Northern Blotting, Southern Blotting, PCR such as RT-PCR (Reverse Transcription-Polymerase Chain Reaction), ELISA (Enzyme Linked Immunosorbent Assay), and Western blotting. When the expression vector of the present invention contains a marker gene, it can be confirmed by investigating the expression of the marker gene inserted into the expression vector. When the nucleic acid encoding TCR is contained in the expression vector contained in the IL-7×CCL19-expressing immunocompetent cells of the present invention, the variable region of the expressed TCR is extracellular. By having the variable region of the TCR, the TCR-expressing immunocompetent cells can recognize and bind to antigen molecules bound to MHC molecules. If the anticancer agent of the present invention contains the IL-7×CCL19-expressing immunocompetent cells of the present invention and pharmaceutically acceptable additives, there is no particular limitation. Examples of the above additives include physiological saline, buffered physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers, and surfactants, buffers and preservatives, isotonic agents, fillers, and lubricants. The anticancer agent of the present invention can be administered to a recipient in need of cancer treatment by methods known to those skilled in the art. Examples of the administration method include injection into the vein, tumor, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intraspinal, and subarachnoid (cerebrospinal fluid). The amount of the IL-7×CCL19-expressing immunocompetent cells of the present invention contained in the administered anticancer agent can be appropriately adjusted according to the type, location, severity of cancer, age, weight, and condition of the recipient undergoing treatment, etc. Preferably, it can be cited as 1×10 4 ~1×10 10 cells, preferably 1×10 5 ~1×10 9 cells, more preferably 5×10 6 ~5×10 8The number of administrations can be, for example, 4 times a day, 3 times a day, 2 times a day or 1 time 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, 2 times a week, once a month or 2 times a month, and can be independently administered. As the anticancer agent of the present invention, or the cancer in the cancer treatment method described below, it can be a solid cancer or a blood cancer, and examples include adenocarcinoma, squamous cell carcinoma, adenosquamous cell 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, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, gastric cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, etc., or cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue and hematopoietic tissue. In addition, examples also include sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant angioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, etc., or embryonal cell tumors such as hepatoblastoma, neuroblastoma, nephroblastoma, neuroectodermal tumor, pancreaticoblastoma, pleuropulmonary blastoma, retinoblastoma, etc., or germ cell tumors, or lymphomas, or leukemias. The anticancer agent of the present invention can be used in combination with other anticancer agents. As other anticancer agents, examples include: alkylating agents such as cyclophosphamide, bendamustine, ifosfamide, dacarbazine, etc., antimetabolites such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, enocitabine, etc., molecularly targeted drugs such as rituximab, cetuximab, trastuzumab, etc., kinase inhibitors such as imatinib, gefitinib, erlotinib, afatinib, dasatinib, sunitinib, trametinib, etc., proteasome inhibitors such as bortezomib, etc., calcineurin inhibitors such as cyclosporine, tacrolimus, etc., anticancer antibiotics such as idarubicin, doxorubicin, mitomycin C, etc., plant alkaloids such as irinotecan, etoposide, etc., platinum preparations such as cisplatin, oxaliplatin, carboplatin, etc., hormone therapy drugs such as tamoxifen, bicalutamide, etc., immunomodulatory drugs such as interferon, nivolumab, pembrolizumab, etc., and alkylating agents or antimetabolites can be appropriately listed. As the method of "using the anticancer agent of the present invention in combination with other anticancer agents", examples include: a method of treating with other anticancer agents and then using the anticancer agent of the present invention; or a method of simultaneously using the anticancer agent of the present invention and other anticancer agents; or a method of treating with the anticancer agent of the present invention and then using other anticancer agents; and a method of treating with other anticancer agents and then using the anticancer agent of the present invention can be appropriately listed. Further, when the anticancer agent of the present invention is used in combination with other anticancer agents, the treatment effect of cancer can be further improved, and the number of administrations or the dosage of each anticancer agent can be reduced, thereby reducing the side effects caused by each anticancer agent. Further, the above other anticancer agents can also be included in the anticancer agent of the present invention.As another aspect 1 of the present invention, the following can be cited: 1) A method for treating cancer, characterized by administering to a patient in need of cancer treatment immunocompetent cells that express cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19; or 2) An immunocompetent cell that is used as an anticancer agent and expresses cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19; or 3) The use of an immunocompetent cell for the preparation of an anticancer agent, and the immunocompetent cell expresses cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19. Further, as another aspect 2 of the present invention, a kit can be cited, which is used to produce the above-mentioned immunocompetent cells that express cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19 and have the expression vector of the present invention. As this kit, as long as it has the expression vector of the present invention, there is no particular limitation, and it may also include an instruction manual for producing the IL-7×CCL19-expressing immunocompetent cells of the present invention, or a reagent for introducing the expression vector of the present invention into immunocompetent cells. Example 1 (Selection of immunomodulatory factors) There are at least several hundred molecules in the living body that can control the functions of T cells. Based on the insights and experiences to date, the inventors first selected IL-7 and CCL19 from a large number of combinations as control molecules for further enhancing the immunomodulatory effect of immunocompetent cells, and selected the combination of the two, that is, the combination of IL-7 and CCL19 rather than each alone, to produce a vector for the immunomodulatory factor of the immunocompetent cells. (Production of a vector expressing IL-7 and CCL19 - 1) Artificially synthesize an anti-FITC CAR DNA (Deoxyribonucleic Acid) fragment (SEQ ID NO: 9) encoding an anti-FITC CAR containing an anti-FITC scFv (Single Chain Fragment Variable), a mouse CD8 transmembrane region, and a mouse CD28-4-1BB-CD3ζ intracellular signaling motif, an F2A-MCS DNA fragment (SEQ ID NO: 10) encoding the 2A peptide (F2A) shown in SEQ ID NO: 8 and a restriction enzyme site (MCS (Multiple Cloning Site)) following this peptide, and an IL-7-F2A-CCL19 DNA fragment (SEQ ID NO: 11) encoding mouse IL-7 (without a stop codon), the F2A following it, and mouse CCL19 (manufactured by Life Technology). In order to produce a vector expressing IL-7 and CCL19, the above anti-FITC CAR DNA fragment and the above F2A-MCS DNA fragment were ligated to produce an anti-FITC CAR-F2A-MCS construct.Next, the constructed product was cloned into the pMSGV retroviral expression vector (Tamada k et al., Clin Cancer Res 18: 6436-6445 (2002)) to produce the pMSGV vector containing anti-FITC CAR-F2A-MCS. On the MCS of the pMSGV vector, the above IL-7-F2A-CCL19 DNA fragment was inserted by treatment with restriction enzymes (NsiI and SalI) and ligation, thereby obtaining the pMSGV vector containing anti-FITC CAR-F2A-IL-7-F2A-CCL19 (IL-7×CCL19 expression vector (1)). The gene map of the obtained vector is shown in Figure 1. Also, as a control, the above anti-FITC CAR DNA fragment was cloned into the above pMSGV retroviral expression vector to produce the pMSGV vector without IL-7 and CCL19 (control vector (1)). (Production of retrovirus introduced with IL-7×CCL19 expression vector) To transduce mouse T cells, retrovirus was produced. Using Lipofectamine 2000 or 3000 (manufactured by Life Technology), the above IL-7×CCL19 expression vector (1) or control vector (1) and the pCL-Eco plasmid (manufactured by Imgenex) were transfected into the GP2-293 packaging cell line (manufactured by TAKARA BIO) to produce the retrovirus introduced with the IL-7×CCL19 expression vector (1) or control vector (1). DMEM (Dulbecco Modified Eagle Medium) supplemented with 10% FCS (Fetal Calf Serum), 100 U / ml penicillin, and 100 mg / ml streptomycin was used as the culture medium for the above GP2-293 cells. Also, RPMI-1640 supplemented with 10% FCS, 100 U / ml penicillin, 100 mg / ml streptomycin, 50 mM 2-mercaptoethanol, and 2 mM L-glutamine was used as the culture medium for T cells used in the following examples. (Transduction of mouse T cells) To transduce mouse T cells, 3×10 derived from the spleen and lymph nodes were used with immobilized anti-CD3 mAb (3 μg / ml) and IL-2 (100 IU / ml). 6Purified mouse T cells were activated for 48 hours. Next, the supernatant of the retrovirus containing the IL-7×CCL19 expression vector (1) or the control vector (1) prepared as described above was mixed with the above-mentioned mouse T cells (1×10 6 cells / ml) activated in a culture dish coated with 25 μg / ml of RetroNectin (manufactured by TAKARA BIO Inc.). After centrifugation at 1500 rpm for 2 hours, the cells were cultured in the presence of IL-2 (100 IU / ml) for 6 hours. To remove the retrovirus from the culture solution, the mouse T cells were recovered and transferred to a new proliferation culture medium (RPMI) containing IL-2 (100 IU / ml), and then cultured for 42 hours to obtain mouse T cells transfected with the IL-7×CCL19 expression vector (1) (IL-7 / CCL19-expressing T cells (1)) or mouse T cells transfected with the control vector (1) (control T cells (1)). (Production of the expression vector expressing IL-7 and CCL19 - 2) In the production of the IL-7×CCL19 expression vector (1) described above, the sequence of the anti-FITC scFv region contained in the sequence shown in SEQ ID NO: 9 was replaced with the sequence of the anti-human CD20 scFv (SEQ ID NO: 12) synthesized by Life Technology based on the sequence of rituximab. Except for this, a pMSGV vector containing anti-human CD20 CAR-F2A-IL-7-F2A-CCL19 (IL-7×CCL19 expression vector (2)) was produced by the same method as in the above-mentioned "Production of the expression vector expressing IL-7 and CCL19 - 1". Similarly, in the production of the control vector (1) described above, the sequence of the anti-FITC scFv region contained in the sequence shown in SEQ ID NO: 9 was replaced with the sequence of the above-mentioned anti-human CD20 scFv (SEQ ID NO: 12). Except for this, a pMSGV vector without IL-7 and CCL19 (control vector (2)) was produced by the same method as in the above-mentioned "Production of the expression vector expressing IL-7 and CCL19 - 1". By the same method as above, the IL-7×CCL19 expression vector (2) or the control vector (2) was introduced into mouse T cells using retrovirus to produce IL-7 / CCL19-expressing T cells (2) or control T cells (2). Example 2 (Cell number and survival rate of IL-7 / CCL19-expressing T cells) The study was conducted on whether IL-7 or CCL19 produced by IL-7 / CCL19-expressing T cells exerted a biological function and showed an immune induction effect. The prepared IL-7 / CCL19-expressing T cells (2) (4×10 5Samples of IL-7 / CCL19-expressing T cells (2) or control T cells (2) were cultured for 5 days. The above culture was carried out without antigen stimulation with CD20 in order to exclude the influence of human CD20 CAR on the expression of IL-7 and CCL19. Next, trypan blue was used to investigate the cell count and survival rate. The results are shown in FIGS. 2A and 2B. FIG. 2A shows the cell count, and FIG. 2B shows the survival rate. The black bars represent IL-7 / CCL19-expressing T cells, and the white bars represent control T cells. (Results) As shown in FIGS. 2A and 2B, in IL-7 / CCL19-expressing T cells (2), the cell count increased by about 5 times and the survival rate increased by about 2 times compared to control T cells (2). Therefore, it was clarified that by using IL-7 / CCL19-expressing T cells obtained by introducing the expression vector of the present invention into T cells, the biological functions of IL-7 or CCL19 were exerted, and an immune induction effect was shown. Example 3 [T cell migration test] (T cell migration test using IL-7 / CCL19-expressing T cells) The effect of inducing migration of CCL19 was studied by using a cell migration test with Transwell. The migratory ability of the responder T cells was measured by allowing them to migrate through a polycarbonate filter with a pore size of 5 μm using a 96-well Transwell (registered trademark) chamber (manufactured by Corning Costar). Specifically, IL-7 / CCL19-expressing T cells (1) or control T cells (1) were cultured in the lower layer of the chamber. The above culture was carried out without antibody stimulation with FITC in order to exclude the influence of FITC CAR on the expression of IL-7 and CCL19. The responder T cells were prepared from the spleen or lymph nodes by negative selection using MACS (Magnetic Activated Cell Sorter) (manufactured by Miltenyi Biotec). The responder T cells were labeled with CytoTell blue (manufactured by AAT Bioquest) and cultured in the upper layer for 3 hours. The migration from the upper layer to the lower layer of the chamber was investigated using a flow cytometer (EC800: manufactured by Sony), and the data analysis was performed using FlowJo software (manufactured by Tree Star). The results are shown in FIG. 3. In FIG. 3, the black bars represent IL-7 / CCL19-expressing T cells (1), the white bars represent control T cells (1), and the vertical axis represents the absolute number of responder T cells that migrated to the lower layer of the chamber. Also, the statistically significant difference was studied by Student's t-test. (Results) As shown in FIG. 3, IL-7 / CCL19-expressing T cells (1) caused about 1.8 times more T cells to migrate to the lower layer compared to control T cells (1).In lymphocyte transfer therapy such as T cell therapy, the cancer cell damage caused by the administered T cells is of course important. However, in addition, it is also important to activate the endogenous T cells (host-side immune cells) originally present in cancer patients and mobilize them as cells to attack cancer cells. Therefore, in terms of the immunotherapy effect, it is preferable not only to transfer lymphocytes with antitumor activity from the outside, but also to use certain methods to trigger the active interaction between the transferred T cells and the endogenous T cells, so that the endogenous T cells accumulate at the cancer site. The results in Figure 3 show that the IL-7 / CCL19-expressing T cells (1) can induce the active interaction between the transferred T cells and the endogenous T cells because they have the ability to accumulate the endogenous T cells. In addition, the results in Figures 2A, 2B, and 3 show that the T cells expressing IL-7 and CCL19 proliferate effectively through IL-7, have a higher survival rate, and have an important effect essential for immune induction of accumulating T cells through CCL19, and have excellent immune induction effects. That is, it is shown that in immune active cells, the two control molecules expressing "IL-7" and "CCL19" can improve the proliferation ability, survival rate, and immune induction effect of the immune active cells. Furthermore, as described above, the T cells expressing IL-7 and CCL19 have the ability of proliferation, survival, and T cell accumulation, thus suggesting the possibility of having the infiltration effect of T cells or dendritic cells in cancer tissues or the tumor growth inhibition effect. Example 4 [Production of IL-7×CCL19×HSV-TK expression vector] By cloning the following base sequences at the multiple cloning sites of the pMSGV1 vector, a vector expressing IL-7, CCL19, and HSV-TK can be produced. The base sequences are arranged in series with the base sequences encoding IL-7, CCL19, and the base sequence of HSV-TK as a suicide gene sandwiching the base sequence encoding the 2A peptide as a self-cleaving peptide. The gene of this vector is shown in Figure 4. The immune active cells transfected with the IL-7×CCL19×HSV-TK expression vector produced by the above method can be controlled in the recipient by administering ganciclovir to the recipient administered with the above immune active cells. Example 5 [Production of TCR×IL-7×CCL19 expression vector] By cloning the following base sequences at the multiple cloning sites of the pMSGV1 vector, a vector expressing TCR, IL-7, and CCL19 can be produced. The base sequences are arranged in series with the base sequences encoding TCR, IL-7, and CCL19 sandwiching the base sequence encoding the 2A peptide as a self-cleaving peptide. The gene of this vector is shown in Figure 5.The introduced immune-activated cells with the TCR×IL-7×CCL19 expression vector produced by the above method can not only specifically bind to the cancer antigens existing on the surface of cancer cells, but also specifically bind to the complex of peptides derived from cancer antigens within cancer cells presented on MHC, and become capable of inducing specific T cells against a wider range of tumor-related target molecules. Example 6 [Production of an expression vector expressing IL-7, CCL19, and eGFP] An IL-7-F2A-CCL19 DNA fragment (manufactured by Life Technology) encoding mouse IL-7 (without a stop codon), followed by F2A, and mouse CCL19 was artificially synthesized. To produce a vector expressing IL-7, CCL19, and eGFP, the above-synthesized IL-7-F2A-CCL19 DNA fragment was inserted into the MCS of the pMSGV retroviral expression vector (Tamada k et al., Clin Cancer Res 18: 6436-6445 (2002)) having an F2A-eGFP sequence by restriction enzyme (NCOI and ECORI) treatment and ligation, to obtain a pMSGV vector (IL-7×CCL19 expression vector (3)) containing an IL-7-F2A-CCL19-F2A-eGFP DNA fragment (SEQ ID NO: 13). The gene of the obtained vector is shown in FIG. 6. Also, as a control, a pMSGV vector (control vector (3)) containing eGFP and not containing IL-7 and CCL19 was produced. Furthermore, in SEQ ID NO: 13, bases 1 to 462 are IL-7 (bases 1 to 75 are the message sequence of IL-7), bases 463 to 537 are F2A, bases 538 to 861 are CCL19 (bases 538 to 612 are the message sequence of CCL19), 868 to 942 are F2A, bases 946 to 1662 are the nucleic acid encoding eGFP, and bases 1663 to 1665 are stop codons. Also, the amino acid sequence corresponding to the above base sequence 13 is shown in SEQ ID NO: 14. Furthermore, in order to use the restriction enzyme NcoI, thymine (t) at the 4th base in SEQ ID NO: 13 was replaced with guanine (g) (phenylalanine (F) at the 2nd amino acid in SEQ ID NO: 14 was replaced with valine (V)). [Production of T cells expressing P815 tumor antigen P1A-specific TCR, IL-7, CCL19, and eGFP] From Y. Liu obtained expression of H-2L. dTransgenic mice expressing a restricted P815 tumor antigen P1A-specific TCR (Sarma, S., Y. Guo, Y. Guilloux, C. Lee, X.-F. Bai, Y. Liu. 1999. J. Exp. Med. 189: 811.) were used to collect spleen cells, and murine T cells expressing a P815 tumor antigen P1A-specific TCR derived from spleen cells (P1A-specific TCR-T cells) were obtained. Next, retroviruses carrying the IL-7×CCL19 expression vector (3) and the control vector (3) were produced by the same method as in Example 1 and transduced into cells obtained by activating spleen cells containing the above P1A-specific TCR-T cells (3×10 6 cells / well) for 48 hours with P1A peptide, to obtain P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells or P1A-specific TCR / eGFP-expressing T cells. The transduction of each expression vector was confirmed by flow cytometry analysis detecting eGFP as a surrogate marker. The expression level of eGFP in each of the obtained T cells was 70 - 80% in any experiment. On day 0, 5×10 5 P815 mastocytoma cells suspended in 0.1 ml of HBSS (Hank's Balanced Salt Solution) were subcutaneously inoculated into the flanks of male DBA / 2 mice (n = 30) aged 6 - 10 weeks. On day 6, the mice were pretreated with a sub-lethal dose (3 - 5 Gy) of irradiation. On day 7, the mice (n = 10) were divided into 3 groups and intravenously administered 1×10 6Individual P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells, or P1A-specific TCR / eGFP-expressing T cells (either type of cell is 70-80% eGFP positive). Subsequently, the survival rate of each mouse was analyzed, and the tumor volume of the dead mice was measured. The analysis results of the survival rate of each mouse are shown in Figure 7, and the results of measuring the tumor volume of the dead mice are shown in Figure 8. In Figure 7, ▲ represents the results of untreated mice, ■ represents the results of mice administered with P1A-specific TCR / eGFP-expressing T cells, ● represents the results of mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells. The horizontal axis is the number of days (day) after subcutaneous inoculation of P815 mastocytoma, and the vertical axis is the survival rate (%). 80% of the mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells were still alive on the 60th day, and 50% were still alive even after more than 100 days. Therefore, it is shown that by using immunocompetent cells expressing P1A-specific TCR, IL-7, and CCL19, an antitumor effect is exerted, and the reduction in the survival rate caused by the tumor is suppressed. Also, in Figure 8, the horizontal axis is the number of days (day) after subcutaneous inoculation of P815 mastocytoma, and the vertical axis is the tumor volume (mm 3 ). It is shown from Figure 8 that for the mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells, the increase in tumor volume was significantly suppressed, and P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells have excellent antitumor activity and exert a therapeutic effect against solid cancer. [Industrial Applicability] The IL-7×CCL19-expressing immunocompetent cells of the present invention can be used in the field of immunotherapy because they have proliferation ability, survival ability, and lymphocyte aggregation ability. Figure 1 is a diagram showing the genetic map of the IL-7×CCL19 expression vector. Figure 2A is a diagram showing the results obtained by investigating the cell number of IL-7 / CCL19-expressing T cells. Figure 2B is a diagram showing the results obtained by investigating the survival rate of IL-7 / CCL19-expressing T cells. Figure 3 is a diagram showing the results of a T cell migration assay performed using IL-7 / CCL19-expressing T cells. Figure 4 is a diagram showing the genetic map of the IL-7×CCL19×HSV-TK expression vector. Figure 5 is a diagram showing the genetic map of the TCR×IL-7×CCL19 expression vector. Figure 6 is a diagram showing the genetic map of the IL-7×CCL19×eGFP (enhanced green fluorescent protein) expression vector. Figure 7 is a diagram showing the survival rates of untreated mice, mice administered with P1A-specific TCR / eGFP-expressing T cells, and mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells. Figure 8 is a diagram showing the results obtained by investigating the tumor volumes of untreated mice, mice administered with P1A-specific TCR / eGFP-expressing T cells, and mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells.

Claims

1. An immune-active cell that specifically recognizes cell surface molecules of cancer antigens, interleukin-7 (IL-7), and a cell-closing cell-cell receptor (CCL) capable of aggregating T cells, wherein the cell surface molecules are T cell receptors or chimeric antigen receptors, and the CCL capable of aggregating T cells is CCL21.

2. The immune-active cells as claimed in claim 1, wherein the immune-active cell line contains nucleic acid encoding extracellularly introduced IL-7 and nucleic acid encoding extracellularly introduced CCL21.

3. The immune-active cells as claimed in claim 1 or 2, wherein the cell surface molecules that specifically recognize cancer antigens are T-cell receptors that specifically recognize cancer antigens.

4. Immune cells as requested in item 1 or 2, wherein the immune cells are T cells, natural killer cells (NK cells), B cells, antigen-presenting cells, or granulocytes.

5. Immune cells as requested in item 1 or 2, wherein the cancer antigens are WT1, MART-1, NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, phosphatidylinositol proteoglycan-3, KIF20A, survivability protein, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, mesothelin, NKG2D, P1A, GD2, or GM2.

6. An expression vector for producing immune-active cells as claimed in any one of claims 1 to 5, comprising any one of the following (a) to (e): (a) an expression vector containing nucleic acids encoding cell surface molecules that specifically recognize cancer antigens, nucleic acids encoding IL-7, and nucleic acids encoding CCLs that can aggregate T cells; (b) two expression vectors of the following (b-1) and (b-2): (b-1) an expression vector containing nucleic acids encoding cell surface molecules that specifically recognize cancer antigens; (b-2) an expression vector containing nucleic acids encoding IL-7 and nucleic acids encoding CCLs that can aggregate T cells; (c) two expression vectors of the following (c-1) and (c-2): (c-1) an expression vector containing nucleic acids encoding cell surface molecules that specifically recognize cancer antigens and nucleic acids encoding IL-7; (c-2) an expression vector containing nucleic acids encoding CCLs that can aggregate T cells; (d) Two types of expression vectors (d-1) and (d-2) below: (d-1) An expression vector containing nucleic acid encoding IL-7; (d-2) An expression vector containing nucleic acid encoding a cell surface molecule that specifically recognizes cancer antigens and nucleic acid encoding a CCL that can aggregate T cells; (e) Three types of expression vectors (e-1), (e-2) and (e-3) below: (e-1) An expression vector containing nucleic acid encoding a cell surface molecule that specifically recognizes cancer antigens; (e-2) An expression vector containing nucleic acid encoding IL-7; (e-3) An expression vector containing nucleic acid encoding a CCL that can aggregate T cells; and the above-mentioned cell surface molecule is a T cell receptor or a chimeric antigen receptor, and the above-mentioned CCL that can aggregate T cells is CCL21.

7. The expression vector as claimed in claim 6, wherein the cell surface molecule that specifically recognizes cancer antigens is a T cell receptor that specifically recognizes cancer antigens.

8. The expression vectors of claim 6 or 7, wherein the nucleic acid encoding cell surface molecules that specifically recognize cancer antigens, the nucleic acid encoding IL-7, and the nucleic acid encoding CCLs that can aggregate T cells in the expression vector of (a), the nucleic acid encoding IL-7 and the nucleic acid encoding CCLs that can aggregate T cells in the expression vector of (b-2), the nucleic acid encoding IL-7 and the nucleic acid encoding CCLs that can aggregate T cells in the expression vector of (c-1), or the nucleic acid encoding cell surface molecules that specifically recognize cancer antigens and the nucleic acid encoding IL-7 in the expression vector of (d-2), are linked via self-cleaving peptides.

9. The vector for expression, as requested in item 6 or 7, contains nucleic acid encoding a suicide gene.

10. An anticancer agent comprising immune-active cells as claimed in any one of claims 1 to 5 and pharmaceutically permissible additives.

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