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

[Technical Field]

[0001] This invention relates to an immune-active cell that specifically recognizes cancer antigens, interleukin-7 (IL-7), and CCL19, an anticancer agent containing the immune-active cell, and a carrier for producing the immune-active cell. [Previous Technology]

[0002] Cancer is a disease affecting a large number of people worldwide. Generally, chemotherapy, radiation therapy, or surgery are widely used in the industry. However, these methods have various problems, such as side effects, loss of bodily functions, and difficulty in treating metastases. Therefore, in order to further maintain a higher quality of life for patients, immunotherapy has been developed in recent years. In immunotherapy, immune cell therapy involves collecting immune-active cells from the patient, processing and expanding these cells to enhance their immune function, and then reintroducing them into the patient's body. Specifically, a known method involves collecting T cells from the patient, introducing the gene encoding CAR (Constitutive Androstane Receptor) into these T cells for expansion, and then reintroducing them into the patient's body (see Non-Patent Literature 1). This therapy is currently undergoing clinical trials worldwide and has shown effectiveness in hematopoietic malignancies such as leukemia and lymphoma. Furthermore, at least hundreds of factors, including cytokines, chemokines, and signaling control proteins, are known to be immune function control factors for immune-active cells such as T cells. Among them, interleukin-7 (IL-7) is known to be an essential cytokine for T cell survival, produced by non-hematopoietic cells such as stromal cells of bone marrow, thymus, and lymphoid organs / tissues. As T cells utilizing IL-7, T cells exhibiting a chimeric cytokine receptor formed by the fusion of IL-7 and IL-7Rα have been revealed (see Patent Document 1). However, the chimeric cytokine receptor in this T cell is merely a fusion protein, limited to the membrane surface of the introduced T cell, and only transmits cytokine signals such as IL-7R to its own cells without ligand dependence, failing to enhance the function of T cells without the aforementioned receptor. Furthermore, it was revealed that reduced expression of CCL19 or CCL21, and IL-7 leads to a deficiency in the maintenance of the T cell region in the spleen of SIRP (Signal Regulatory Protein) α-mutant mice (see Non-Patent Literature 2), and that CCL19 or CCL21, and IL-7 play a role in maintaining T cell homeostasis in secondary lymphoid tissues (spleen or lymph nodes) (see Non-Patent Literature 3). However, Non-Patent Literature 2 and 3 show an effect on inactive T cells that are homeostatically present in the T cell region of secondary lymphoid tissues, and do not show a direct correlation with anti-tumor immune responses. Furthermore, the CCL19 or CCL21, IL-7 expressing cells in Non-Patent Literature 2 and 3 are cells of the reticuloendothelial system present in secondary lymphoid tissues, not 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 that T cells exist in the form of heterodimers containing α and β chains, or γ and Δ chains, and activate T cells by recognizing antigen molecules that bind to major histocompatibility complex (MHC) molecules. The industry is continuously developing immunotherapies that utilize the function of this TCR, introducing a TCR gene that recognizes tumor antigens expressed in cancer cells into T cells obtained from cancer patients, and then amplifying and reintroducing it into the patient. Specifically, a pharmaceutical composition for the treatment of meningiomas is disclosed, which contains cells that express TCRs that specifically recognize WT1-expressing cells (see Patent Document 2). While some of the above-mentioned technologies have been shown to have anti-tumor effects against hematopoietic organ malignancies, there are no examples showing significant effects against solid tumors. Considering the problems of low survival efficiency of the transferred immune-active cells in vivo, activation of endogenous immune-active cells induced by the transferred immune-active cells, or insufficient aggregation at the tumor site, a technology is sought to solve these problems. [Previous Art Documents] [Patent Documents] Patent Document 1: International Publication No. 2013 / 123061 Patent Document 2: Japanese Patent Application Publication No. 2013-116891 Non-Patent Document 1: Nakazawa Yozo Shinshu Medical Journal 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. [Summary of the Invention]

[0003] [Problem to be Solved by the Invention] In the immunotherapies used in previous immunotherapies, the immune induction effect, proliferative capacity, survival capacity, or T cell aggregation capacity of endogenous immunotherapeutic cells have not been sufficiently enhanced. Therefore, the object of the present invention is to provide an immunotherapeutic cell that expresses immune function control factors of immunotherapeutic cells, possesses proliferative capacity, survival capacity, and T cell aggregation capacity, and an immunotherapeutic cell expression vector for producing the immunotherapeutic cell's immunotherapeutic control factors. [Technical Means for Solving the Problem] In order to achieve better immune induction effects or antitumor activity in cancer immunotherapy using immunotherapeutic cells, the inventors have attempted to improve cells that express immune function control factors. In this process, focusing on cytokines, chemokines, and signal control proteins as factors controlling the immune function of immunotherapeutic cells, a vector expressing the aforementioned factors controlling the immune function of immunotherapeutic cells is constructed. The expression vector was introduced into immune-active cells, and it was found that immune-active cells with superior immune induction effects, proliferative capacity, survival capacity, and T cell aggregation capacity compared to previous immune-active cells could be produced, thus completing the present invention. That is, the present invention is disclosed as follows (1) to (9). (1) An immune-active cell that specifically recognizes cell surface molecules of cancer antigens, interleukin-7 (IL-7), and CCL19. (2) The immune-active cell described in (1) above, characterized in that: the cell surface molecules that specifically recognize cancer antigens are T cell receptors that specifically recognize cancer antigens. (3) The immune-active cell described in (1) or (2) above, characterized in that: the immune-active cell is a T cell. (4) The immune-active cells described in any one of (1) to (3) above are characterized in that: the cancer antigen is WT1, MART-1, NY-ESO-1, MAGE (Melanoma antigen)-A1, MAGE-A3, MAGE-A4, phosphatidylinositol proteoglycan-3, KIF20A, Survivin, AFP (Alpha Fetal Protein)-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR (Vascular Endothelial Growth Factor) Receptor (vascular endothelial growth factor receptor) 1, VEGFR 2, NEIL 3, MPHOSPH 1, DEPDC 1, FOXM 1, CDH 3, TTK, TOMM 34, URLC 10, KOC 1, UBE 2T, TOPK, ECT 2, mesothelin, NKG 2D, P1A, GD 2, or GM 2.(5) An expression vector for preparing an immune-active cell as described in any one of (1) to (4) above, which is one of the following (a) to (e): (a) an expression vector containing nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, nucleic acid encoding IL-7, and nucleic acid encoding CCL19; (b) two expression vectors of the following (b-1) and (b-2): (b-1) an expression vector containing nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen; (b-2) an expression vector containing nucleic acid encoding IL-7 and nucleic acid encoding CCL19; (c) two expression vectors of the following (c-1) and (c-2): (c-1) an expression vector containing nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and nucleic acid encoding IL-7; (c-2) an expression vector containing nucleic acid encoding CCL19; (d) two expression vectors of the following (d-1) and (d-2): (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 CCL19; (e) The following three expression vectors (e-1), (e-2) and (e-3): (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 CCL19. (6) The expression vector described in (5) above is characterized in that: the cell surface molecule that specifically recognizes cancer antigens is a T cell receptor that specifically recognizes cancer antigens. (7) The expression vector as described in (5) or (6) above is characterized in that: (a) the expression vector contains nucleic acids encoding cell surface molecules that specifically recognize cancer antigens, nucleic acids encoding IL-7, and nucleic acids encoding CCL19; (b-2) the expression vector contains nucleic acids encoding IL-7 and nucleic acids encoding CCL19; (c-1) the expression vector contains nucleic acids encoding cell surface molecules that specifically recognize cancer antigens and nucleic acids encoding IL-7; or (d-2) the expression vector contains nucleic acids encoding cell surface molecules that specifically recognize cancer antigens and nucleic acids encoding CCL19 linked via self-cleaving peptides. (8) The expression vector as described in any one of (5) to (7) above is characterized in that: it contains nucleic acids encoding suicide genes. (9) An anticancer agent containing immune-active cells as described in any one of (1) to (4) above and pharmaceutically permissible additives.[Effects of the Invention] When using the expression vector of the present invention, which specifically recognizes cell surface molecules, IL-7, and CCL19 (hereinafter also referred to as "IL-7×CCL19 expression-activated immune cells"), they exhibit anti-tumor activity and can inhibit the decrease in survival rate caused by tumors formed by cancer cells that specifically recognize antigens on their cell surface molecules. Furthermore, when using the expression vector of the present invention, it is possible to produce immune-activated cells that possess proliferative capacity, viability, and T-cell aggregation capacity.

Implementation Method

[0005] The IL-7×CCL19 immune-active 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 can also express 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, and other immune function control factors. Cancer antigens refer to proteins, glycolipids, and other substances that are expressed at higher levels in cancer cells than in normal cells, or that are specifically expressed in cancer cells. Examples of cancer antigens include: tumor-associated antigens (TAAs) or cancer-testis antigens (CTIs), angiogenesis-associated antigens (AGIs), and antigenic determinants of neoantigens (cancer antigens) resulting from gene mutations. Specifically, examples include: WT1, MART-1, NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, phosphatidylinositol proteoglycan-3, and KI. Proteins such as F20A, 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, and P1A, or glycolipids such as GD2 and GM2, are included, but are not limited to these. Cell surface molecules that specifically recognize cancer antigens can include cell surface receptors, artificial receptors, and adhesion factors that specifically recognize cancer antigens. Suitable examples include T-cell receptors that specifically recognize cancer antigens, or chimeric antigen receptors (CARs) that specifically recognize cancer antigens, which are molecules that, through expression on the cell surface, possess the ability to specifically recognize cancer. TCRs are even more suitable examples.As a TCR, as long as it specifically recognizes cancer antigens, it can be a heterodimer containing α and β chains (α / β-TCR), or a heterodimer 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, the recognition can be indirect. For example, by simultaneously or continuously delivering antibodies or other molecules that specifically recognize cancer antigens to a target along with the immune-active cells of the present invention, the immune-active cells of the present invention can indirectly and specifically recognize cancer antigens by recognizing the antibodies or other molecules, or by recognizing the tags labeled on the antibodies or other molecules. Examples of recognition antibodies include cell surface molecules such as CD16, and examples of tags labeled on antibody or other molecules include FITC (fluorescein isothiocyanate). The types of immune-active cells in the IL-7×CCL19-expressing immune-active cells of this invention can be any cells related to the immune response, including: T cells, natural killer cells (NK cells), B cells and other lymphocytes; antigen-presenting cells such as monocytes, macrophages, dendritic cells and other antigen-presenting cells; and granulocytes such as neutrophils, eosinophils, basophils, and basophils. Suitable examples include T cells derived from mammals such as humans, dogs, cats, pigs, and mice, with human-derived T cells being preferred. Furthermore, T cells can be obtained by isolating and purifying immune cells infiltrating bodily fluids such as blood and bone marrow fluid, or tissues such as the spleen, thymus, and lymph nodes, or cancerous tissues such as primary tumors, metastatic tumors, and cancerous ascites. Alternatively, they can be made from ES cells (embryonic stem cells) or iPS cells (induced pluripotent stem cells). Examples of such T cells include: α / β-T cells, γ / Δ-T cells, CD8+ T cells, CD4+ T cells, tumor-infiltrating T cells, memory T cells, naive T cells, and NKT cells. As for the method of producing the IL-7×CCL19-expressing immune-active cells of the present invention, the following methods can be used: introducing the expression vector of the present invention into immune-active cells. Alternatively, methods can be used to induce the expression of cell surface molecules that specifically recognize cancer antigens, interleukin-7 (IL-7), and / or CCL19 in fertilized eggs, ES cells, or iPS cells; or methods can be used to produce immune-active cells isolated from gene-transfected mammals that express expression of cell surface molecules that specifically recognize cancer antigens through gene introduction, and then, as needed, introducing a vector that specifically recognizes cancer antigens, interleukin-7 (IL-7), and / or CCL19 into those immune-active cells.There are no particular limitations on the method for introducing the expression vector of the present invention into the aforementioned immune-active cells. Methods such as viral infection, calcium phosphate method, liposome transfection, microinjection, and electroporation can be used, and viral infection can be appropriately cited. As for viral infection, methods for transfecting the expression vector and packaging plasmid of the present invention into packaging cells such as GP2-293 cells (manufactured by TAKARA BIO), Plat-GP cells (manufactured by Cosmobio), PG13 cells (ATCC CRL-10686), and PA317 cells (ATCC CRL-9078) to create a recombinant virus, and then infecting the immune-active cells with the recombinant virus, can be performed using commercially available kits such as the Retrovirus Packagin Kit Eco (manufactured by TAKARA BIO). Furthermore, the immune-active cells of the present invention can be produced by means of a known gene-editing technique, in a manner that allows expression under the control of a suitable promoter, by introducing a polynucleotide sequence containing base sequences encoding cell surface molecules that specifically recognize cancer antigens, IL-7, and CCL19 into the cell's genome. Examples of known gene-editing techniques include zinc finger nucleases, TALEN (Transcription Activator-like Effector Nuclease), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas systems. Similarly, the expression of other foreign proteins in the immune-active cells of the present invention can also be achieved by using gene-editing techniques, in a manner that allows expression under the control of a suitable promoter, by introducing a polynucleotide sequence containing base sequences encoding other foreign proteins into the cell's genome. Methods for incorporating polynucleotides into the cellular genome in a manner that allows them to be expressed under the control of an appropriate promoter 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 resulting polynucleotide (i.e., a polynucleotide with the coding sequence linked in a manner that allows it to be expressed under the control of that promoter) into the non-coding region of the cellular genome; and methods for incorporating polynucleotides containing 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 cellular genome. Examples of endogenous promoters include the promoters of TCRα and TCRβ.Furthermore, the IL-7×CCL19-expressing immune-active cells of this invention can also express thymidine kinase (HSV-TK) or inducible caspase 9 of herpes simplex virus. Because the IL-7×CCL19-expressing immune-active cells of this invention specifically recognize cell surface molecules of cancer antigens, IL-7, and CCL19, they exhibit high proliferative capacity, survival capacity, and endogenous T cell aggregation capacity, and can be applied to adoptive immunotherapy using various immune-active 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, and TIL (Tumor Infiltrating Lymphocyte) therapy, but are not limited to these. A method of amplifying immune-active cells collected from a patient by introducing the expression vector of this invention and administering it to the patient can also be described. The following are specific examples, but are not limited to these. Dendritic cell therapy includes the steps of introducing surgically removed cancer tissue or its lysate into dendritic cells differentiated from mononuclear globules collected from a patient, and administering them to the patient, but may also include the step of introducing the expression vector of the present invention into the dendritic cells. Here, antigen-determining peptides of cancer antigen molecules can also be artificially synthesized and used instead of the aforementioned cancer tissue or lysate. NK cell therapy includes the steps of activating and proliferating NK cells from lymphocytes collected from a patient using multiple stimulating substances such as IL-2, and then administering them to the patient, but may also include the step of introducing the vector of the present invention into the NK cells. Furthermore, by using anti-cancer antibody drugs in combination with activated NK cells, a highly efficient attack on cancer cells can be expected. γ / Δ-T cell therapy includes culturing and stimulating lymphocytes collected from a patient using IL-2 or zoledronic acid to induce γ / Δ-T cell proliferation, followed by administration to the patient. However, it may also include the step of introducing the expression vector of the present invention into γ / Δ-T cells. α / β-T cell therapy includes culturing and activating lymphocytes collected from a patient using anti-CD3 antibodies or IL-2, followed by administration of the obtained α / β-T cells to the patient. However, it may also include the step of introducing the expression vector of the present invention into α / β-T cells. CTL therapy includes stimulating lymphocytes collected from a patient using cancer cells, adding anti-CD3 antibodies or IL-2 for culturing, inducing specific CTL proliferation in the cancer cells, followed by administration to the patient. However, it may also include the step of introducing the expression vector of the present invention into CTLs. Furthermore, antigen-presenting cells displaying cancer antigen-determining peptides may be used instead of the aforementioned cancer cells.TIL therapy includes the steps of collecting lymphocytes from cancer tissue collected from a patient, stimulating and culturing them with IL-2, and then administering them to the patient, but may also include the 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) used to prepare the IL-7×CCL19 immune-expressing cells of the present invention described above. (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 CCL19; (b) Two expression vectors of (b-1) and (b-2) below: (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 CCL19; (c) Two expression vectors of (c-1) and (c-2) below: (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 CCL19; (d) Two expression vectors of (d-1) and (d-2) below: (d-1) An expression vector containing nucleic acids encoding IL-7; (d-2) An expression vector containing nucleic acids encoding cell surface molecules that specifically recognize cancer antigens and nucleic acids encoding CCL19; (e) The following three expression vectors (e-1), (e-2), and (e-3) are: (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; and (e-3) an expression vector containing nucleic acid encoding CCL19. The expression vectors of this invention may further contain nucleic acids encoding other immune function control 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, and TNF-α. The aforementioned nucleic acids encoding cell surface molecules that specifically recognize cancer antigens, the nucleic acid encoding interleukin-7 (IL-7), and the nucleic acid encoding CCL19 can be examples derived from mammalian nucleic acids, and can appropriately include nucleic acids derived from humans. The aforementioned nucleic acids can be appropriately selected according to the type of cells into which the expression vector of the present invention is introduced. The sequence information of each nucleic acid can be appropriately obtained by searching well-known literature or databases such as NCBI (National Center of Biotechnology Information) (http: / / www.ncbi.nlm.nih.gov / guide / ).Nucleic acids encoding cell surface molecules that specifically recognize cancer antigens can be appropriately listed as human-derived nucleic acids. These nucleic acids encoding cell surface molecules that specifically recognize cancer antigens can include nucleic acids encoding T-cell receptors (TCRs) or chimeric antigen receptors (CARs). They can be naturally derived or artificially synthesized nucleic acids, and can be appropriately selected according to the type of cell into which the expression vector of this invention is introduced. Sequence information can be appropriately obtained by searching well-known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ). Nucleic acids encoding cell surface molecules that specifically recognize cancer antigens, nucleic acids encoding IL-7, and nucleic acids encoding CCL19 can be produced using known techniques such as chemical synthesis based on the base sequence information of the nucleic acids encoding each, or amplification using PCR (Polymerase Chain Reaction). Furthermore, the selected codons used to encode amino acids can be modified to optimize the expression of nucleic acids in the target host cells. The TCR in the aforementioned nucleic acid encoding the TCR can be a heterodimer comprising both α and β chains (α / β-TCR) or a heterodimer comprising both γ and Δ chains (γ / Δ-TCR). Furthermore, the nucleic acid encoding α / β-TCR includes both the nucleic acid encoding the α chain of the TCR and the nucleic acid encoding the β chain, and the nucleic acid encoding γ / Δ-TCR includes both the nucleic acid encoding the γ chain of the TCR and the nucleic acid encoding the Δ chain. The sequence information of the aforementioned nucleic acid encoding the TCR can be identified using methods known in the art, based on the α and β chains of the nucleic acid, which are the TCR subunits of CTLs induced by a specific antigenic peptide (International Publication No. 2007 / 032255, and Morgan et al., J Immunol, 171, 3288 (2003)). For example, PCR is preferred for analyzing the TCR. PCR primers used for analysis may be, for example, the 5'-R primer (5'-gtctaccaggcattcgcttcat-3': sequence number 3) as the 5' side primer, and the 3' side primers that are specific to the C region of the TCRα chain (5'-tcagctggaccacagccgcagcgt-3': sequence number 4), the 3' side primers that are specific to the C1 region of the TCRβ chain (5'-tcagaaatcctttctcttgac-3': sequence number 5), or the 3' side primers that are specific to the C2 region of the TCRβ chain (5'-ctagcctctggaatcctttctctt-3': sequence number 6), but are not limited to these.TCR derivatives can bind to target cells presenting antigenic peptides with high binding affinity and can mediate the efficient killing of target cells presenting antigenic peptides in vivo and in vitro. Nucleic acids encoding the aforementioned TCRs can be, for example, those that can recognize MART1-specific TCRs (Cancer Res. 54, 5265-5268 (1994)), MAGE-A3-specific TCRs (Anticancer Res., 20, 1793-1799 (2000)), gp100-specific TCRs (J. Immunol. 170, 2186-2194 (2003)), NY-ESO-1-specific TCRs (J. Immunol., 174, 4415-4423 (2005)), WT1-specific TCRs (Blood, 106, 470-476 (2005)), MAGE-A1-specific TCRs (Int. Immunol., 8, 1463-1466 (1996)), and P1A-specific TCRs (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.) Nucleic acids encoding TCR, or antigen molecules bound to MHC molecules, that activate T cells can be base sequences with at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%, and most preferably at least 98% identity with the base sequences encoding TCR described in the aforementioned literature. Furthermore, a sequence encoding a CDR can be specifically identified from the base sequence encoding TCR described in the aforementioned literature. This CDR-encoding sequence, in addition to the CDR-encoding sequence, possesses at least 60%, preferably at least 70%, more preferably at least 80%, further preferably at least 90%, and most preferably at least 95% identity with the base sequence encoding TCR described in the aforementioned literature. As for the nucleic acid encoding IL-7, the base sequence encoding the amino acid sequence shown in Sequence Number 1 can be used. As long as it has an enhancing effect on IL-7's cell proliferation rate or cell survival rate, it can be a base sequence possessing at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%, and most preferably at least 98% identity with the base sequence encoding the amino acid sequence shown in Sequence Number 1.As the nucleic acid encoding CCL19, the base sequence encoding the amino acid sequence shown in Sequence Number 2 can be used. As long as it has the cell migration function of CCL19, a base sequence with at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%, and most preferably at least 98% identity with the base sequence encoding the amino acid sequence shown in Sequence Number 2 can also be used. Furthermore, the expression vector of the present invention may also contain nucleic acid encoding a suicide gene. A suicide gene refers to a gene that, through expression, directly or indirectly induces cytotoxic substances, causing the cell to die. By including 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 has disappeared, an agent that activates the function of the suicide gene can be administered to control the immune-active cells in vivo. Furthermore, IL-7 or CCL19, unlike other cytokines, has a lower likelihood of causing cytokine release syndrome as a side effect or tumorigenesis of the gene-introduced cells. However, by enhancing the function of immune-active cells introduced with the expression vector of the present invention, the cytokines released during their attack on the target cancerous tissue may unexpectedly affect surrounding tissues. In such cases, by including nucleic acids encoding suicide genes in the expression vector of the present invention, the risk of developing cytokine release syndrome can be reliably reduced. As suicide genes, the following genes encoding thymidine kinase (HSV-TK) or inducible caspase 9, as described in the literature, can be listed. For the former, ganciclovir can be cited; for the latter, AP1903, a chemical induction of dimerization compound (CID), can be cited (Cooper LJ., et al. Cytotherapy. 2006; 8 (2): 105-17., Jensen MC 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, (a) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes cancer antigens, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, any one of the nucleic acids can be configured in either upstream or downstream. Specifically, taking the case where a nucleic acid encoding TCR is used as the nucleic acid encoding a cell surface molecule that specifically recognizes cancer antigens as an example, then from upstream, the nucleic acid can be sequentially encoded as TCR, IL-7, and CCL19; or it can be encoded as TCR, CCL19, and IL-7; or it can be encoded as IL-7, CCL19, and TCR; or it can be encoded as IL-7, TCR, and CCL19; or it can be encoded as CCL19, TCR, and IL-7; or it can be encoded as CCL19, IL-7, and TCR. In the expression vector (b-2) of the present invention, which contains nucleic acids encoding IL-7 and CCL19, the configuration of the nucleic acids encoding IL-7 and CCL19 is not particularly limited. The nucleic acid encoding CCL19 can be configured upstream or downstream of the nucleic acid encoding IL-7. In the expression vector (c-1) of the present invention, which contains nucleic acids encoding cell surface molecules that specifically recognize cancer antigens and IL-7, the configuration of the nucleic acids encoding cell surface molecules that specifically recognize cancer antigens and IL-7 is not particularly limited. The nucleic acid encoding IL-7 can be configured upstream or downstream of the nucleic acid encoding cell surface molecules that specifically recognize cancer antigens. In the expression vector (d-2) of the present invention, which contains a nucleic acid encoding a cell surface molecule that specifically recognizes cancer antigens and a nucleic acid encoding CCL19, there are no particular restrictions on the configuration of the nucleic acid encoding the cell surface molecule that specifically recognizes cancer antigens and the nucleic acid encoding CCL19. The nucleic acid encoding CCL19 can be configured upstream or downstream of the nucleic acid encoding the cell surface molecule that specifically recognizes cancer antigens. Furthermore, the nucleic acid encoding the cell surface molecule that specifically recognizes cancer antigens, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 can be transcribed using other promoters, or they can be transcribed using an internal ribozyme entry site (IRES) or a self-cleaving 2A peptide and a single promoter.In cases where an internal ribosome entry site (IRES) or a self-cleaving 2A peptide is used to transcribe nucleic acids encoding IL-7 and CCL19 using a single promoter, the following nucleic acids may be linked: between the aforementioned nucleic acids; between the nucleic acids encoding cell surface molecules that specifically recognize cancer antigens and the nucleic acids encoding IL-7 and CCL19; between the nucleic acids encoding α / β-TCR and the nucleic acids encoding β; or between the nucleic acids encoding γ / Δ-TCR and the nucleic acids encoding Δ. Any nucleic acid may be present, provided it can express each nucleic acid. However, it is preferred to link via a self-cleaving peptide (2A peptide), a sequence encoding IRES, or, more preferably, a sequence encoding a 2A peptide. Using this sequence for linking allows for efficient expression of each nucleic acid. Furthermore, in cases where nucleic acids encoding suicide genes are present, the location of the suicide gene is not particularly restricted. For example, it can be positioned upstream or downstream of the promoters of nucleic acids encoding cell surface molecules that specifically recognize cancer antigens, nucleic acids encoding IL-7, or nucleic acids encoding CCL19, via sequences encoding 2A peptides or IRES. It can also be positioned downstream of other promoters. The so-called 2A peptide is a self-cleaving peptide derived from viruses, characterized by the endoplasmic reticulum cleavage of the GP (one residue from the C-terminus) in the amino acid sequence represented by sequence number 7 (Szymczak et al., Expert Opin. Biol. Ther. 5 (5): 627-638 (2005)). Therefore, nucleic acids incorporated before and after the 2A peptide can express themselves independently within the cell. The aforementioned 2A peptide is preferably derived from piconelasmosis, rotavirus, insect virus, thrush virus, or trypanosome virus, and more preferably from piconelasmosis as indicated in sequence number 8 (F2A). The vector used in the expression vector of this invention can be linear or circular, can be a non-viral vector such as a plastid, can be a viral vector, or can be a translocase vector. Furthermore, the vector may contain control sequences such as promoters or terminators, or selection marker sequences such as drug resistance genes or reporter genes. By efficiently configuring nucleic acids encoding IL-7 and CCL19 downstream of the promoter sequence, transcription of each nucleic acid can be performed efficiently.Examples of promoters mentioned above include: viral promoters such as the LTR (Long Terminal Repeat) promoter of retroviruses, the SV40 initial promoter, the cytomegalovirus promoter, and the thymidine kinase promoter of herpes simplex virus; and mammalian promoters such as the PGK (Phosphoglycerate kinase) promoter, the Xist (X-inactive specific transcript) promoter, the β-actin promoter, and the RNA (ribonucleic acid) polymerase II promoter. Additionally, tetracycline-responsive promoters induced by tetracycline and the Mx1 promoter induced by interferon can also be used. By using promoters induced by the aforementioned specific substances in the expression vector of this invention, the induction of IL-7 and CCL19 expression can be controlled according to the cancer treatment process. Examples of viral vectors mentioned above include: retroviral vectors, lentiviral vectors, adenovirus vectors, and adeno-associated virus vectors. Retroviral vectors are a suitable example, with pMSGV vectors (Tamada k et al., Clin Cancer Res 18: 6436-6445 (2002)) or pMSCV vectors (manufactured by TAKARA BIO) being more appropriate. When a retroviral vector is used, the introduced gene is inserted into the host cell's genome, thus allowing for long-term and stable expression. When the expression vector of the present invention is confirmed to be present in immune-active cells, for example, when it contains nucleic acid encoding TCR, the expression of TCR can be investigated by flow cytometry, Northern Blotting, Southern Blotting, PCR (Reverse Transcription-Polymerase Chain Reaction), ELISA (Enzyme-Linked Immunosorbent Assay), or 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 expression vector contained in the IL-7×CCL19 immune-active cells of the present invention contains nucleic acid encoding TCR, the variable region of the expressed TCR is located extracellularly. By possessing this variable region of the TCR, TCR-expressing immune-active cells can recognize antigen molecules bound to MHC molecules.The anticancer agent of this invention is not particularly limited in that it contains the IL-7×CCL19 immune-active cells of this invention and pharmaceutically permissible additives. Examples of such 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 this invention can be administered to subjects requiring cancer treatment using methods known to those in the art. Administration methods include intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intra-articular, intra-articular, intra-articular, intramuscular, intra-articular, intra-articular, intra-articular, intracranial, intraspinal, and subarachnoid (medullary) injections. The amount of IL-7×CCL19 immune-active cells contained in the administered anticancer agent can be appropriately adjusted according to the type, location, severity of the cancer, and the age, weight, and condition of the patient receiving treatment. Preferably, 1×10⁴ to 1×10¹⁰ cells, more preferably 1×10⁵ to 1×10⁹ cells, and even more preferably 5×10⁶ to 5×10⁸ cells can be administered in a single administration. The administered anticancer agent can be administered independently in the following ways: 4 times, 3 times, 2 times, or once a day; every 1 day; every 2 days; every 3 days; every 4 days; every 5 days; once a week; every 7 days; every 8 days; every 9 days; twice a week; once a month; or twice a month. The cancer mentioned in this invention as an anticancer agent or a treatment method for the following cancers can be solid cancer or hematologic cancer, including 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, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, and ovarian cancer. This invention relates to cancers of bone, cartilage, adipose, muscle, blood vessels, and hematopoietic tissues. Examples of other cancers include chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma, hepatoblastoma, neuroblastoma, renal blastoma, neuroblastoma, pancreatic blastoma, pleural pulmonary blastoma, retinal blastoma, blastoma, and other germ cell tumors, as well as lymphoma and leukemia. The anticancer agent of this invention can be used in combination with other anticancer agents.Other anticancer agents include: alkylating agents such as cyclophosphamide, bendamustine, ifosfamide, and dacarbazine; metabolic antagonists such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enoxabin; molecularly targeted drugs such as rituximab, cetuximab, and trastuzumab; and agonists such as imatinib, gefitinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib. Enzyme inhibitors, proteasome inhibitors such as bortezomib, calcineurin inhibitors such as cyclosporine and tacrolimus, anticancer antibiotics such as adamycin, doxorubicin, and mitomycin C, plant alkaloids such as irinotecan and etoposide, platinum preparations such as cisplatin, oxaliplatin, and carboplatin, hormone therapy drugs such as tamoxifen and bicalutamide, immunosuppressants such as interferon, nivolumab, and pembrolizumab, and alkylating agents or metabolic antagonists may be appropriately listed. As a method of "using the anticancer agent of the present invention in combination with other anticancer agents" as described above, examples include: treatment with other anticancer agents followed by the use of the anticancer agent of the present invention; or simultaneous use of the anticancer agent of the present invention with other anticancer agents; or treatment with the anticancer agent of the present invention followed by the use of other anticancer agents; and methods of treatment with other anticancer agents followed by the use of the anticancer agent of the present invention may be appropriately listed. Furthermore, when the anticancer agent of the present invention is used in combination with other anticancer agents, the therapeutic effect of cancer is further improved, and the frequency or dosage of each anticancer agent is reduced, thereby reducing the side effects caused by each anticancer agent. Also, the anticancer agent of the present invention may include the aforementioned other anticancer agents. Another embodiment 1 of the present invention can be listed as follows: 1) a method of treating cancer, characterized by administering immune-active cells that specifically recognize cell surface molecules, interleukin-7 (IL-7), and CCL19 to a patient requiring cancer treatment; or 2) an immune-active cell used as an anticancer agent, and specifically recognizing cell surface molecules, interleukin-7 (IL-7), and CCL19 of cancer antigens; or 3) the use of an immune-active cell in the preparation of an anticancer agent, wherein the immune-active cell specifically recognizes cell surface molecules, interleukin-7 (IL-7), and CCL19 of cancer antigens. Furthermore, as another embodiment 2 of the present invention, a kit can be provided, which is used to prepare immune-active cells that specifically recognize cancer antigens, interleukin-7 (IL-7), and CCL19 by the expression vector of the present invention. This kit is not particularly limited as long as it contains the expression vector of the present invention, and may also include instructions for preparing the IL-7×CCL19 immune-active cells of the present invention, or reagents for introducing the expression vector of the present invention into immune-active cells. Example 1 (Selection of Immune Function Control Factors) At least hundreds of molecules that can control the function of T cells exist in organisms.Based on their insights and experience to date, the inventors first selected IL-7 and CCL19 from a large number of combinations as control molecules to further enhance the immune function control effect of immune-active cells, and chose the combination of the two, i.e., the combination of IL-7 and CCL19, rather than each alone, to create a carrier of the immune function control factor that expresses immune-active cells. (Preparation of vectors for expressing IL-7 and CCL19 - 1) A synthetically produced anti-FITC CAR DNA (Deoxyribonucleic acid) fragment (Sequence No. 9) encoding anti-FITC CAR containing anti-FITC scFv (Single Chain Fragment Variable), mouse CD8 transmembrane region, mouse CD28-4-1BB-CD3ζ intracellular signaling motifs, the F2A-MCS DNA fragment (Sequence No. 10) encoding the 2A peptide (F2A) shown in Sequence No. 8 and the restriction enzyme site (MCS (Multiple Cloning Site)) following the peptide, and the IL-7-F2A-CCL19 DNA fragment (Sequence No. 11) encoding mouse IL-7 (without a stop codon), and the subsequent F2A and mouse CCL19 (manufactured by Life Technology). To create vectors expressing IL-7 and CCL19, the anti-FITC CAR DNA fragment was ligated to the F2A-MCS DNA fragment to create an anti-FITC CAR-F2A-MCS construct. Next, the constructed construct was colonized into a pMSGV retroviral expression vector (Tamada k et al., Clin Cancer Res 18: 6436-6445 (2002)) to create a pMSGV vector containing anti-FITC CAR-F2A-MCS. The IL-7-F2A-CCL19 DNA fragment was inserted into the MCS of this pMSGV vector by restriction enzyme treatment (NsiI and SalI) and ligation, thereby obtaining a pMSGV vector containing anti-FITC CAR-F2A-IL-7-F2A-CCL19 (IL-7×CCL19 expression vector (1)). The gene diagram of the obtained vector is shown in Figure 1. Furthermore, as a control, the above-mentioned anti-FITC CAR DNA fragment was selected and inserted into the above-mentioned pMSGV retroviral expression vector to prepare a pMSGV vector without IL-7 and CCL19 (control vector (1)). (Preparation of retrovirus with IL-7×CCL19 expression vector) Retrovirus was prepared for transduction of mouse T cells.Using Lipofectamine 2000 or 3000 (manufactured by Life Technology), the above-mentioned IL-7×CCL19 expression vector (1) or control vector (1) and pCL-Eco plasmids (manufactured by Imgenex) were transfected into the GP2-293 packaging cell line (manufactured by TAKARA BIO) to produce retroviruses infused 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-mentioned GP2-293 cells. Furthermore, 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 the T cells used in the following examples. (Transduction of mouse T cells) To perform transduction of mouse T cells, 3 × 10⁶ purified mouse T cells derived from the spleen and lymph nodes were activated for 48 hours using immobilized anti-CD3 mAb (3 μg / ml) and IL-2 (100 IU / ml). Next, the supernatant containing the retrovirus prepared above and infused with the IL-7×CCL19 expression vector (1) or control vector (1) was mixed with the mouse T cells (1×10⁶ cells / ml) activated in a culture dish coated with 25 μg / ml RetroNectin (registered trademark: TAKARA BIO). After centrifugation at 1500 rpm for 2 hours, the mixture was cultured for 6 hours in the presence of IL-2 (100 IU / ml). To remove the retrovirus from the culture medium, the mouse T cells were recovered and transferred to a new proliferative culture medium (RPMI) containing IL-2 (100 IU / ml), and cultured for 42 hours to obtain mouse T cells infused with the IL-7×CCL19 expression vector (1) (IL-7 / CCL19 expression T cells (1)) or mouse T cells infused with the control vector (1) (control T cells (1)).(Preparation of IL-7 and CCL19 expression vectors-2) In the preparation of the IL-7×CCL19 expression vector (1) above, the sequence of the anti-FITC scFv region contained in the sequence shown in sequence number 9 was replaced with the sequence of anti-human CD20 scFv (sequence number 12) synthesized by Life Technology based on the sequence of rituximab. Otherwise, a pMSGV vector (IL-7×CCL19 expression vector (2)) containing anti-human CD20 CAR-F2A-IL-7-F2A-CCL19 was prepared by means of the same method as in the above "Preparation of IL-7 and CCL19 expression vectors-1". Similarly, in the preparation of the control vector (1) mentioned above, the sequence containing the anti-FITC scFv region in the sequence shown in sequence number 9 was replaced with the sequence of the anti-human CD20 scFv (sequence number 12). Otherwise, a pMSGV vector (control vector (2)) without IL-7 and CCL19 was prepared by means of the same method as described in "Preparation of expression vectors for IL-7 and CCL19 - 1". Using the same method as described above, the IL-7×CCL19 expression vector (2) or control vector (2) was introduced into mouse T cells using a retrovirus to prepare IL-7 / CCL19 expression T cells (2) or control T cells (2). Example 2 (Number and survival rate of IL-7 / CCL19 expression T cells) The study investigated whether IL-7 or CCL19 produced by IL-7 / CCL19 expression T cells could exert biological functions and show an immune-inducing effect. Samples containing IL-7 / CCL19-expressing T cells (2) (4 × 10⁵ cells) or control T cells (2) were cultured for 5 days. This culture was performed without CD20 antigen stimulation to exclude the influence of human CD20 CAR on IL-7 and CCL19 expression. Next, cell number and survival rate were investigated using trypan blue. The results are shown in Figures 2A and 2B. Figure 2A shows cell number, and Figure 2B shows survival rate. Black bars represent IL-7 / CCL19-expressing T cells, and white bars represent control T cells. (Results) As shown in Figures 2A and 2B, the number of IL-7 / CCL19-expressing T cells (2) was approximately 5-fold higher than that of control T cells (2), and the survival rate was approximately 2-fold higher. Therefore, it is clear that IL-7 / CCL19-expressing T cells, prepared by introducing the expression vector of the present invention into T cells, can exert the biological functions of IL-7 or CCL19 and exhibit an immune-inducing effect. Example 3 [T Cell Migration Assay] (T cell migration assay using IL-7 / CCL19-expressed T cells) The migration-inducing effect of CCL19 was studied using a cell migration assay with Transwell.The migration of responder T cells was measured using 96-well Transwell chambers (manufactured by Cornig Costar) through a 5 μm polycarbonate filter. Specifically, IL-7 / CCL19-expressing T cells (1) or control T cells (1) were cultured in the lower layer of the chamber. This culture was performed without FITC antibody stimulation to exclude the influence of FITC CAR on IL-7 and CCL19 expression. Responder T cells were prepared from the spleen or lymph nodes using negative selection with a MACS (Magnetic Activated Cell Sorter) (manufactured by Miltenyi Biotec). 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 to the lower chambers was investigated using a flow cytometer (EC800: Sony Corporation), and data analysis was performed using FlowJo software (Tree Star Corporation). The results are shown in Figure 3. In Figure 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 responding T cells that migrated to the lower chambers. Furthermore, statistically significant differences were investigated using the Student's t-test. (Results) As shown in Figure 3, IL-7 / CCL19-expressing T cells (1) resulted in approximately 1.8 times more T cells migrating to the lower chambers compared to control T cells (1). In T cell and other lymphocyte transplantation therapy, the damage to cancer cells caused by the injected T cells is certainly important, but it is also important to activate and mobilize endogenous T cells (=host-side immune cells) that are originally present in the cancer patient to attack cancer cells. Therefore, in terms of the efficacy of immunotherapy, it is preferable not only to introduce lymphocytes with anti-tumor activity from the outside, but also to use certain methods to induce the active interaction between the introduced T cells and endogenous T cells, so that the endogenous T cells accumulate in the cancer site. The results of Figure 3 show that IL-7 / CCL19 expressed T cells (1) have the ability to induce the accumulation of endogenous T cells, thus inducing the active interaction between the introduced T cells and endogenous T cells. Furthermore, the results of Figures 2A, 2B, and 3 show that T cells expressing IL-7 and CCL19 proliferate effectively through IL-7, have a higher survival rate, and possess the important effect of accumulating T cells through CCL19, which is essential for immune induction, and have excellent immune induction effect. That is, it shows that the expression of the two control molecules "IL-7" and "CCL19" in immune active cells can improve the proliferative capacity, survival rate, and immune induction effect of the immune active cells.Furthermore, as described above, T cells expressing IL-7 and CCL19 possess proliferative, surviving, and T cell aggregation capabilities, suggesting the potential for T cell or dendritic cell penetration into cancerous tissues or tumor proliferation inhibition. Example 4 [Preparation of IL-7×CCL19×HSV-TK Expression Vector] By selecting the following base sequence at the multiple selection site of the pMSGV1 vector, a vector expressing IL-7, CCL19, and HSV-TK can be prepared. This base sequence is formed by tandemly arranging the base sequences encoding IL-7, CCL19, and the suicide gene HSV-TK with the base sequence encoding a 2A peptide, which is a self-cleaving peptide. The gene representation of this vector is shown in Figure 4. Immunoactive cells containing the IL-7×CCL19×HSV-TK expression vector prepared by the above method are introduced into the body. Ganciclovir is then administered to subjects containing these immunoactive cells to control the immunoactive cells in the subjects. Example 5 [Preparation of TCR×IL-7×CCL19 Expression Vector] By selecting the following base sequence at the multiple selection site of the pMSGV1 vector, a vector expressing TCR, IL-7, and CCL19 can be prepared. This base sequence is formed by tandemly arranging the base sequences encoding the genes for TCR, IL-7, and CCL19 with the base sequence encoding the 2A peptide, which is a self-cleaving peptide. The gene illustration of this vector is shown in Figure 5. Immunoactive cells infused with the TCR×IL-7×CCL19 expression vector prepared using the above method can not only specifically bind to cancer antigens present on the surface of cancer cells, but also specifically bind to the MHC complex of peptides of cancer antigens derived from cancer cells, thus becoming capable of inducing specific T cells targeting a wider range of tumor-related molecules. Example 6 [Preparation of expression vectors for IL-7, CCL19 and eGFP] IL-7-F2A-CCL19 DNA fragments encoding mouse IL-7 (without a stop codon), and subsequently F2A and mouse CCL19 were artificially synthesized (manufactured by Life Technology). To prepare vectors expressing IL-7, CCL19, and eGFP, the synthesized IL-7-F2A-CCL19 DNA fragment was inserted into the MCS of a pMSGV retroviral expression vector (Tamada k et al., Clin Cancer Res 18: 6436-6445 (2002)) containing the F2A-eGFP sequence by restriction enzyme treatment and ligation, thus obtaining a pMSGV vector (IL-7×CCL19 expression vector (3)) containing the IL-7-F2A-CCL19-F2A-eGFP DNA fragment (sequence number 13). The gene diagram of the obtained vector is shown in Figure 6.Furthermore, as a control, a pMSGV vector containing eGFP but lacking IL-7 and CCL19 was prepared (control vector (3)). In sequence number 13, bases 1 to 462 are IL-7 (bases 1 to 75 are the IL-7 message sequence), bases 463 to 537 are F2A, bases 538 to 861 are CCL19 (bases 538 to 612 are the CCL19 message sequence), bases 868 to 942 are F2A, bases 946 to 1662 are the nucleic acid encoding eGFP, and bases 1663 to 1665 are the stop codons. The amino acid sequences corresponding to the above-mentioned base sequence 13 are shown in sequence number 14. Furthermore, in order to use the restriction enzyme NcoI, the fourth base in sequence number 13, thymine (t), was replaced with guanine (g) (and the second amino acid in sequence number 14, phenylalanine (F), was replaced with valine (V)). [Production of T cells expressing P815 tumor antigen P1A-specific TCR, IL-7, CCL19, and eGFP] Splenic cells were collected from genetically modified mice (Sarma, S., Y. Guo, Y. Guilloux, C. Lee, X.-F. Bai, Y. Liu. 1999. J. Exp. Med. 189: 811.) expressing H-2Ld-restricted P815 tumor antigen P1A-specific TCR derived from Y. Liu to obtain mouse T cells (P1A-specific TCR-T cells) expressing P815 tumor antigen P1A-specific TCR derived from spleen cells. Next, using the same method as in Example 1, retroviruses containing IL-7×CCL19 expression vector (3) and control vector (3) were prepared and transduced into cells obtained by activating spleen cells (3×10⁶ cells / well) containing the above-mentioned P1A-specific TCR-T cells with P1A peptide for 48 hours, thereby obtaining P1A-specific TCR / IL-7 / CCL19 / eGFP expression T cells or P1A-specific TCR / eGFP expression T cells. The transduction of each expression vector was confirmed by flow cytometry analysis using eGFP as a substitute marker. The eGFP expression level of each T cell obtained was 70-80% in any experiment. On day 0, 5×10⁵ P815 obesity cell tumors suspended in 0.1 ml of HBSS (Hank's Balanced Salt Solution) were subcutaneously inoculated into the flank of 6-10 week old male DBA / 2 mice (n=30). On day 6, mice were irradiated with a sublethal dose (3-5 Gy) as a pretreatment.On day 7, mice (n=10) were divided into three groups and intravenously injected with either 1×10⁶ P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells or P1A-specific TCR / eGFP-expressing T cells (both types of cells were 70-80% eGFP positive). Subsequently, the survival rate of each mouse was analyzed, and the tumor volume of the deceased mice was measured. The results of the survival rate analysis are shown in Figure 7, and the results of the tumor volume measurement of the deceased mice are shown in Figure 8. In Figure 7, ▲ represents the results of untreated mice, ■ represents the results of mice injected with P1A-specific TCR / eGFP-expressing T cells, and ● represents the results of mice injected with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells. The horizontal axis represents the number of days after subcutaneous inoculation with P815 obese cell tumors, and the vertical axis represents the survival rate (%). Mice injected with P1A-specific TCR / IL-7 / CCL19 / eGFP expressing T cells maintained an 80% survival rate at day 60 and a 50% survival rate even after 100 days. This indicates that the use of immune-active cells expressing P1A-specific TCR, IL-7, and CCL19 can exert an anti-tumor effect and inhibit the tumor-induced decrease in survival. Furthermore, in Figure 8, the horizontal axis represents the number of days after subcutaneous inoculation with P815 obese cell tumors, and the vertical axis represents tumor volume (mm3). Figure 8 shows that the increase in tumor volume was significantly inhibited in mice injected with P1A-specific TCR / IL-7 / CCL19 / eGFP expressing T cells, demonstrating that P1A-specific TCR / IL-7 / CCL19 / eGFP expressing T cells exhibit excellent anti-tumor activity and therapeutic effects against solid tumors. [Industrial Applicability] The IL-7×CCL19 immune-active cells of the present invention have the ability to proliferate, survive and aggregate lymphocytes, and therefore can be used in the field of immunotherapy. [Simplified Explanation of the Diagram]

[0004] Figure 1 is a genetic map of the IL-7×CCL19 expression vector. Figure 2A is a graph showing the results of investigating the number of IL-7 / CCL19-expressing T cells. Figure 2B is a graph showing the results of investigating the survival rate of IL-7 / CCL19-expressing T cells. Figure 3 is a graph showing the results of a T cell migration assay using IL-7 / CCL19-expressing T cells. Figure 4 is a genetic map of the IL-7×CCL19×HSV-TK expression vector. Figure 5 is a genetic map of the TCR×IL-7×CCL19 expression vector. Figure 6 is a genetic map of the IL-7×CCL19×eGFP (enhanced green fluorescent protein) expression vector. Figure 7 shows the survival rates of untreated mice, mice injected with P1A-specific TCR / eGFP-expressing T cells, and mice injected with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells. Figure 8 shows the results of investigating tumor volume in untreated mice, mice injected with P1A-specific TCR / eGFP-expressing T cells, and mice injected with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells. [Sequence List]

Claims

1. A natural killer cell (NK cell) whose exogenous expression specifically recognizes chimeric antigen receptor (CAR), interleukin-7 (IL-7), and CCL19 of cancer antigens.

2. Natural killer cells (NK cells) as described in claim 1, containing extracellularly introduced nucleic acids encoding CAR, extracellularly introduced nucleic acids encoding IL-7, and extracellularly introduced nucleic acids encoding CCL19.

3. Natural killer cells (NK 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, 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, GD2, or GM2.

4. An anticancer agent containing natural killer cells (NK cells) as claimed in claim 1 or 2, and pharmaceutically permissible additives.

5. A method for preparing natural killer cells (NK cells) that specifically recognize cell surface molecules of cancer antigens, interleukin-7 (IL-7), and CCL19, comprising the use of nucleic acids encoding IL-7 and CCL19, wherein the nucleic acids encoding IL-7 and CCL19 are introduced into natural killer cells (NK cells) that specifically recognize cell surface molecules of cancer antigens.

6. As requested in item 5, wherein an expression vector is used to introduce nucleic acids encoding IL-7 and CCL19 into natural killer cells (NK cells).

7. As requested in item 6, wherein an expression vector containing nucleic acids encoding IL-7 and CCL19 is introduced into natural killer cells (NK cells).

8. If any of the claims 5 to 7 are used together, wherein the cell surface molecule that specifically recognizes the cancer antigen is a chimeric antigen receptor (CAR) that specifically recognizes the cancer antigen, a T-cell receptor that specifically recognizes the cancer antigen, or an antibody that specifically recognizes the cancer antigen.

9. If any one of the requests 5 to 7 is used together, it contains nucleic acid encoding a suicide gene.

10. If any of the requests in items 5 to 7 are used together, wherein the cancer antigen is WT1, MART-1, NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, phosphatidylinositol proteoglycan-3, KIF20A, survival 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.

Images