Enhancer of t-cells or b-cells having memory function, inhibitor of malignant tumor recurrence, and inducer inducing memory function in t-cells or b-cells

Abstract

To provide an enhancer of endogenous T-cells or B cells having a memory function and an inhibitor of malignant tumor recurrence that continue rejecting malignant tumors for a long period of time.SOLUTION: An enhancer of T-cells and B-cells having a memory function in an administration target comprising a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7) and a nucleic acid encoding chemokine (C-C motif) ligand 19 (CCL19) or an inducer that induces memory function in T-cells and B-cells in an administration target is prepared. Also, an inhibitor of malignant tumor recurrence containing a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7) and a nucleic acid encoding CCL19 is prepared.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] The present invention relates to an agent for enhancing T cells or B cells having memory function in a subject, an agent for suppressing the recurrence of malignant tumor, and an inducer for inducing memory function in T cells or B cells in a subject, each of which comprises a nucleic acid delivery vehicle, a nucleic acid encoding interleukin-7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19). [Background technology]

[0002] Malignant tumors are diseases that affect many people around the world, and chemotherapy, radiation therapy, or surgery are generally widely used. However, there are various problems, such as side effects, loss of some functions, and inability to treat recurrence or metastasis. In order to maintain a high quality of life (QOL) for patients, the development of immune cell therapy has been promoted in recent years. This immune cell therapy is a therapy in which immunocompetent cells are collected from a patient, treated to enhance the immune function of the immunocompetent cells, amplified, and then transferred back to the patient. Specifically, a therapy is known in which T cells are collected from a patient, a nucleic acid encoding a CAR is introduced into the T cells, the T cells are amplified, and then transferred back to the patient (see Non-Patent Document 1). Clinical trials of this therapy are currently underway around the world, and results have been obtained that show its effectiveness in hematopoietic malignant tumors such as leukemia and lymphoma.

[0003] In addition, at least several hundred types of factors, such as cytokines, chemokines, and signal control proteins, are known as immune function regulators of immune cells such as T cells. Among them, interleukin 7 (IL-7) is a cytokine essential for the survival of T cells, and is known to be produced by non-hematopoietic cells such as stromal cells in bone marrow, thymus, and lymphoid organs and tissues. As a T cell utilizing the function of IL-7, a T cell expressing a chimeric cytokine receptor in which IL-7 and IL-7R alpha are fused (see Patent Document 1) has been disclosed. However, the chimeric cytokine receptor in such a T cell is expressed as a single fusion protein limited to the membrane surface of the T cell into which it is introduced, and merely transmits cytokine signals such as IL-7R in a ligand-independent manner only to self cells, and it was not possible to enhance the function of a T cell into which the above receptor has not been introduced.

[0004] It has also been disclosed that reduced expression of CCL19, CCL21, and IL-7 causes defective maintenance of the T cell region in the spleen in SIRP alpha mutant mice (see Non-Patent Document 2), and that CCL19, CCL21, and IL-7 have the function of maintaining T cell homeostasis in secondary lymphatic tissues (spleen and lymph nodes) (see Non-Patent Document 3). However, the above Non-Patent Documents 2 and 3 show the effect on non-activated T cells that are constantly present in the T cell region of secondary lymphatic tissues, and do not show a direct relationship with anti-tumor immune responses. Furthermore, the CCL19, CCL21, and IL-7 expressing cells in the above Non-Patent Documents 2 and 3 were not T cells but reticuloendothelial cells present in secondary lymphatic tissues.

[0005] Meanwhile, the present inventors have proposed an immune cell therapy that significantly suppresses solid cancer by simultaneously expressing IL-7 and CCL19 (see Patent Documents 2 and 3). This method can increase the activation of endogenous immune cells in the host (recipient) and their ability to accumulate in tumor cells, but it was unclear whether this immune cell therapy can prevent recurrence and continue to reject malignant tumors over the long term. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2013 / 123061 Brochure [Patent Document 2] International Publication No. 2016 / 056228 Brochure [Patent Document 3] International Publication No. 2017 / 159736 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Hirozo Nakazawa 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 [Problem to be solved by the invention]

[0008] As described above, immune cell therapy using CAR-expressing T cells and TCR-expressing T cells that simultaneously express IL-7 and CCL19 has been developed, and the proliferation and survival ability of immune cells or the accumulation ability of immune cells in the host have been significantly improved, and the development of technology applicable to solid cancers for which sufficient therapeutic effects have not been observed with conventional immune cell therapy is progressing. However, malignant tumors often recur, and even if the above-mentioned immune cell therapy can temporarily treat malignant tumors, it is unclear whether malignant tumors will continue to be rejected for a long period of time, and no preventive measures against recurrence have been considered. Therefore, the object of the present invention is to provide an enhancer of endogenous T cells or B cells with memory function, an inhibitor of malignant tumor recurrence, and an inducer that induces memory function in endogenous T cells or B cells in order to continue rejecting malignant tumors for a long period of time. [Means for solving the problem]

[0009] The present inventors have been investigating further possibilities of the T cells expressing CAR, IL-7 and CCL19 that they have developed, and have found that when such T cells are administered to a subject, memory function is induced not only in the administered T cells but also in endogenous T cells in the subject (host) to increase the absolute number of cells having memory function, and that in a malignant tumor recurrence model experiment, malignant tumor formation is suppressed in cells that do not have an antigen recognized by CAR. Furthermore, they have found that the same effect is obtained when TCR is used instead of CAR or when a virus is used instead of T cells, and have completed the present invention.

[0010] That is, the present invention is as follows. (1) An agent for enhancing T cells or B cells having memory function in a subject, the agent comprising a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19). (2) An agent for enhancing T cells or B cells with memory function in a subject as described in (1) above, characterized in that the nucleic acid delivery vehicle is at least one selected from the group consisting of immunocompetent cells, viruses, anaerobic bacteria, liposomes, mesenchymal stem cells (MSCs), and nanoparticles. (3) An agent for enhancing T cells or B cells with memory function in a subject to be administered according to (1) or (2) above, characterized in that the nucleic acid delivery vehicle has the ability to accumulate in malignant tumor cells or the ability to specifically proliferate in malignant tumor cells. (4) The agent for enhancing T cells or B cells with memory function in a subject according to any one of (1) to (3) above, wherein the nucleic acid delivery vehicle has the ability to cytotoxicity against malignant tumor cells. (5) An agent for enhancing T cells or B cells with memory function in a subject to be administered according to any one of (1) to (4) above, characterized in that the nucleic acid delivery vehicle is an immunocompetent cell, and the immunocompetent cell has a cell surface molecule that recognizes a malignant tumor antigen. (6) An agent for enhancing T cells or B cells with memory function in a subject to be administered according to (5) above, characterized in that the cell surface molecule that recognizes a malignant tumor antigen is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). (7) An agent for enhancing T cells or B cells having memory function in a subject to be administered according to (5) or (6) above, characterized in that the immunocompetent cells are T cells. (8) An agent for enhancing T cells or B cells with memory function in a subject of administration according to any one of (1) to (7) above, characterized in that the T cells or B cells with memory function are central memory T cells. (9) A pharmaceutical composition comprising an enhancer for T cells or B cells having memory function in a subject according to any one of (1) to (8) above and a pharma- ceutical acceptable additive. (10) An agent for suppressing the recurrence of a malignant tumor, comprising a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19). (11) The agent for suppressing recurrence of malignant tumors according to (10) above, characterized in that the nucleic acid delivery vehicle is at least one selected from the group consisting of immunocompetent cells, viruses, anaerobic bacteria, liposomes, mesenchymal stem cells (MSCs), and nanoparticles. (12) The agent for suppressing the recurrence of malignant tumor according to (10) or (11) above, wherein the nucleic acid delivery vehicle has an ability to accumulate in malignant tumor cells or an ability to specifically proliferate in malignant tumor cells. (13) The agent for suppressing the recurrence of malignant tumor according to any one of (10) to (12) above, wherein the nucleic acid delivery vehicle has an ability to cytotoxicity against malignant tumor cells. (14) The agent for suppressing the recurrence of a malignant tumor according to any one of (10) to (13) above, wherein the nucleic acid delivery vehicle is an immunocompetent cell, and the immunocompetent cell has a cell surface molecule that recognizes a malignant tumor antigen. (15) The agent for suppressing malignant tumor recurrence described in (14) above, characterized in that the nucleic acid delivery vehicle is an immunocompetent cell having a cell surface molecule that recognizes a malignant tumor cell antigen, and the malignant tumor recurrence is caused by a malignant tumor cell that does not have a malignant tumor antigen specifically recognized by the cell surface molecule. (16) The agent for suppressing recurrence of malignant tumors according to (14) or (15) above, characterized in that the cell surface molecule that recognizes a malignant tumor cell antigen is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). (17) The agent for suppressing the recurrence of malignant tumor according to any one of (14) to (16) above, wherein the immunocompetent cells are T cells. (18) The agent for suppressing the recurrence of malignant tumors according to (11) above, wherein the nucleic acid delivery vehicle is an oncolytic virus. (19) A pharmaceutical composition comprising the malignant tumor recurrence inhibitor according to any one of (10) to (18) above and a pharma- ceutical acceptable additive. (20) An inducer for inducing a memory function in T cells or B cells in a subject, the inducer comprising a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19).

[0011] Further, other aspects of the present invention are as follows. 1) Use of a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19) for the preparation of an enhancer of T cells or B cells having memory function in a subject; 2) A method for enhancing T cells or B cells with memory function, comprising administering to a subject a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19); 3) Use of a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19) for the preparation of a malignant tumor recurrence inhibitor; 4) a method for suppressing recurrence of a malignant tumor, comprising administering to a subject a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19); 5) Use of a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19) for the preparation of an inducer for inducing memory function in T cells or B cells in a subject; 6) A method for inducing memory function in T cells or B cells, comprising administering to a subject a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19); 7) The inducer for inducing a memory function in T cells or B cells in a subject of administration described in (20) above, characterized in that the nucleic acid delivery vehicle is at least one selected from the group consisting of immunocompetent cells, viruses, anaerobic bacteria, liposomes, mesenchymal stem cells (MSCs), and nanoparticles. 8) An inducer for inducing a memory function in T cells or B cells in a subject of administration as described in (20) or (7) above, characterized in that the nucleic acid delivery vehicle has the ability to accumulate in malignant tumor cells or the ability to specifically proliferate in malignant tumor cells. 9) An inducer for inducing a memory function in T cells or B cells in a subject of administration according to any one of (20), (7) and (8) above, characterized in that the nucleic acid delivery vehicle has the ability to cytotoxicity against malignant tumor cells. 10) An inducer for inducing a memory function in T cells or B cells in a subject of administration described in any one of (20), 7) to 9) above, characterized in that the nucleic acid delivery vehicle is an immunocompetent cell, and the immunocompetent cell has a cell surface molecule that recognizes a malignant tumor antigen. 11) An inducer for inducing memory function in T cells or B cells in a subject of administration as described in 10) above, characterized in that the cell surface molecule that recognizes a malignant tumor antigen is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). 12) An inducer for inducing memory function in T cells or B cells in a subject of administration as described in 10) or 11) above, characterized in that the immunocompetent cells are T cells. Effect of the Invention

[0012] By using the enhancer of T cells or B cells having memory function of the present invention, it is possible to enhance T cells or B cells having memory function in the administration subject. Furthermore, by using the malignant tumor recurrence suppressor of the present invention, it is possible to suppress recurrence after malignant tumor treatment. Furthermore, by using the inducer of the present invention that induces memory function in T cells or B cells, it is possible to induce memory function in T cells or B cells in the administration subject. [Brief description of the drawings]

[0013] [Figure 1] (a) is a diagram showing the results of microscopic observation of the localization of T cells in malignant tumor tissue stained with a labeled antibody in Example 1. (b) is a graph showing the areas (μm2) of administered donor T cells and endogenous T cells of the recipient, obtained by image analysis processing of the results observed in (a) above. [Diagram 2]13 is a graph showing the malignant tumor volume (mm3) 14 days after administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells (7x19) or such T cells together with anti-CD90.2 antibody (anti-CD90.2) to mice in Example 2. [Diagram 3] 1 shows graphs illustrating the results of examining memory conversion of administered donor T cells and endogenous T cells of the recipient in Example 3. [Figure 4] 1 is a graph showing the results of examining the memory function of administered donor T cells and endogenous T cells of the recipient by IFN-γ production in Example 3. (a) shows the ratio of IFN-γ positive cells in CD90.1-positive donor T cells, and (b) shows the results of detecting IFN-γ positive cells in CD90.2-positive endogenous CD8-positive T cells by flow cytometry. [Figure 5A] 13 is a graph showing the results of investigating changes in T cell receptor (TCR) repertoire before treatment of CAR-IL-7 / CCL19-expressing T cells (α chain: before treatment, CAR-positive) in Example 4. [Figure 5B] 13 is a graph showing the results of investigating changes in T cell receptor (TCR) repertoire before treatment of CAR-IL-7 / CCL19-expressing T cells (α chain: before treatment, CAR-negative) in Example 4. [Figure 5C] 13 is a graph showing the results of investigating changes in T cell receptor (TCR) repertoire after treatment with CAR-IL-7 / CCL19-expressing T cells in Example 4 (α chain: CAR-positive after treatment). [Figure 5D] 13 is a graph showing the results of investigating changes in T cell receptor (TCR) repertoire after treatment with CAR-IL-7 / CCL19-expressing T cells (α chain: after treatment, CAR-negative) in Example 4. [Figure 6A] This is a graph showing the results of investigating changes in T cell receptor (TCR) repertoire before treatment of CAR-IL-7 / CCL19-expressing T cells (β chain: before treatment, CAR-positive) in Example 4. [Figure 6B] 13 is a graph showing the results of investigating changes in T cell receptor (TCR) repertoire before treatment of CAR-IL-7 / CCL19-expressing T cells (β chain: before treatment, CAR-negative) in Example 4. [Figure 6C] 13 is a graph showing the results of investigating changes in T cell receptor (TCR) repertoire after treatment with CAR-IL-7 / CCL19-expressing T cells in Example 4 (β chain: CAR-positive after treatment). [Figure 6D] 13 is a graph showing the results of investigating changes in T cell receptor (TCR) repertoire after treatment with CAR-IL-7 / CCL19-expressing T cells (β chain: after treatment, CAR-negative) in Example 4. [Figure 7A] In Example 5, on the 140th day after inoculation with P815-hCD20, tumor-rejected mice or control naive mice were inoculated with P815-hCD20 or the parent strain P815 not expressing hCD20 into the left and right flanks, respectively, and this figure shows the results of measuring the tumor volume. [Figure 7B] In Example 5, 140 days after inoculation with 3LL-hCD20, tumor-rejected mice or control naive mice were inoculated with 3LL-hCD20 or the parent strain 3LL not expressing hCD20 into the left and right flanks, respectively, and this figure shows the results of measuring the tumor volume. [Figure 8] FIG. 13 is a diagram showing the experimental protocol for confirming the inhibitory effect on tumor recurrence in Example 6. [Figure 9A]FIG. 13 shows the results of photographing mice on days 1, 3, 7, 10, 14, 21, 31, and 38 with an exposure time of 30 seconds in a group administered ACC-MESO1-GFP-Luc described below and anti-human mesothelin CAR-IL-7-CCL19-expressing T cells (7×19 CAR-T) on day 1 and a group administered conventional anti-human mesothelin CAR-expressing T cells (conventional CAR-T) in Example 6. [Figure 9B] This figure shows the results of photographing mice on days 45, 59, 73, 87, 101, 115, 129, and 143 with an exposure time of 30 seconds in a group administered with ACC-MESO1-GFP-Luc described below and anti-human mesothelin CAR-IL-7-CCL19 expressing T cells (7×19 CAR-T) on day 1 and a group administered with conventional anti-human mesothelin CAR expressing T cells (conventional CAR-T) in Example 6. The second individual from the left on day 129 has died, so a photograph is omitted. [Figure 10] 1 is a graph showing the relationship between the number of days after administration of ACC-MESO1-GFP-Luc described below and the survival rate of mice in Example 6. [Figure 11] 13 is a graph showing the relationship between the number of days after administration of ACC-MESO1-GFP-Luc described below and the total amount of fluorescence in Example 6. [Figure 12] FIG. 13A shows the results of flow cytometric analysis of the proportion of CD8+GFP+ cells in spleen cells, and FIG. 13B shows the results of flow cytometric analysis of the absolute number of CD8+GFP+ cells in Example 7. [Figure 13] In Example 7, (a) is a diagram showing the number of CD44+ cells in CD8+ spleen cells from naive BDA / 2 mice and in CD8+GFP- or CD8+GFP+ spleen cells from mice treated with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells, and (b) is a diagram showing the proportion of CD44+ cells in (a). [Figure 14]FIG. 13 shows the results of Example 7, in which cells were co-cultured with mucosal mast cells (MMC) treated with P815 for about 5 days, and the concentration of IFN-γ in the culture medium supernatant was detected by ELISA (Enzyme-Linked Immunosorbent Assay). [Figure 15] In Example 8, (a) shows the structure of the recombinant vaccinia virus LC16mO TK-SP-mouse IL-7-F2A-mouse CCL19-F2A-eGFP (TK-ICE), and (b) shows the structure of the recombinant vaccinia virus LC16mO TK-SP-Luc-F2A-eGFP (TK-LE). [Figure 16] FIG. 13 is a diagram showing, by images, the relationship between the cytopathic effect of vaccinia virus and eGFP fluorescence expression in A549 cells and CT26 cells infected with recombinant vaccinia virus TK-ICE or TK-LE in Example 8. [Figure 17] FIG. 13 shows the results of investigating the amounts of IL-7 and CCL19 secreted in A549 cells or CT26 cells infected with recombinant vaccinia virus TK-ICE or TK-LE in Example 8. [Figure 18] FIG. 13 shows the concept of subcutaneously transplanting mouse colon cancer CT26 cells into both abdominal regions of a mouse in Example 8. [Figure 19] FIG. 13 is a graph showing the change in tumor size (mm3) after intratumoral administration of recombinant vaccinia virus TK-ICE or TK-LE to a BALB / c mouse model in which tumors using mouse colon cancer cell CT26 were subcutaneously transplanted on both sides in Example 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The "enhancer for T cells or B cells having memory function" in this specification is not particularly limited as long as it contains a nucleic acid delivery vehicle, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, and such an enhancer can enhance T cells or B cells having memory function. Furthermore, the "inducer for inducing memory function in T cells or B cells" in this specification is not particularly limited as long as it contains a nucleic acid delivery vehicle, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, and such an inducer can induce memory function in T cells or B cells. Hereinafter, in this specification, the above-mentioned "enhancer for T cells or B cells having memory function" or the above-mentioned "inducer for inducing memory function in T cells or B cells" are also collectively referred to as "the enhancer or inducer of the present invention."

[0015] The "malignant tumor recurrence suppressor" in this specification is not particularly limited as long as it contains a nucleic acid delivery vehicle, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, and such a malignant tumor recurrence suppressor makes it possible to suppress the recurrence of a malignant tumor. Hereinafter, in this specification, the above-mentioned "malignant tumor recurrence suppressor" is also referred to as the "malignant tumor recurrence suppressor of the present invention."

[0016] Examples of the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 include human-derived nucleic acids. Each of the above nucleic acids can be appropriately selected depending on the type of cells to be introduced, and sequence information of each of the nucleic acids can be appropriately obtained by searching known literature or databases such as NCBI (https: / / www.ncbi.nlm.nih.gov / guide / ). Examples of the nucleic acid encoding IL-7 include a base sequence encoding the amino acid sequence shown in SEQ ID NO: 1. As long as it has an effect of enhancing the cell proliferation rate or cell survival rate in IL-7, it may be a base sequence encoding amino acids having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more with the amino acid sequence shown in SEQ ID NO: 1. Examples of the nucleic acid encoding CCL19 include a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2. As long as it has a cell migration effect in CCL19, it may be a base sequence encoding amino acids having a sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more with the amino acid sequence shown in SEQ ID NO: 2. The enhancing effect of IL-7 on cell proliferation rate or cell survival rate and the cell migration effect of CCL19 can be confirmed by the method described in Patent Document 2 above.

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

[0018] The nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 may be incorporated into a vector containing a control sequence such as a promoter or a terminator, or a selection marker sequence such as a drug resistance gene or a reporter gene. Furthermore, the vector may contain a nucleic acid encoding a suicide gene, or a nucleic acid encoding a 2A peptide or an IRES. The vector, the nucleic acid encoding a suicide gene, and the nucleic acid encoding a 2A peptide or an IRES can be obtained or prepared by referring to Patent Documents 2 and 3. Examples of the promoter include a promoter of the IE (immediate early) gene of cytomegalovirus (CMV), an early promoter of SV40, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, an SRα promoter, an NFAT promoter, and an HIF promoter. Examples of the vector include a retrovirus vector such as a pMSGV vector (Tamada k et al., Clin Cancer Res 18:6436-6445 (2002)) or a pMSCV vector (manufactured by Takara Bio Inc.), or a vector derived from such a vector.

[0019] In addition to the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19, the nucleic acid may contain nucleic acids encoding other immune function regulators 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, checkpoint inhibitor antibodies or fragments thereof, etc. However, as long as the enhancer or inducer and the malignant tumor recurrence suppressor contain at least a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 as nucleic acids encoding immune function regulators, they can fully exert their effects as the enhancer or inducer and the malignant tumor recurrence suppressor even if they do not contain nucleic acids encoding the other immune function regulators.

[0020] The above-mentioned "memory function" means a function of T cells that have been stimulated by a tumor antigen in the past, and when they come into contact with malignant tumor cells again, they are activated more quickly and strongly than naive T cells that have not been stimulated by a tumor antigen in the past, and exert a high immune function against malignant tumor cells. Examples of T cells or B cells with memory function include central memory T cells and memory B cells. Such T cells or B cells with memory function can be confirmed by comprehensively evaluating the positivity / negative (+ / -) and expression intensity of CD44, CD62L, CD127 (IL-7R), etc., and it is possible to select which CD should be measured appropriately depending on the subject, such as human or mouse. In the present application, positive may be described as "+" and negative as "-".

[0021] The above-mentioned "enhancement of T cells or B cells having memory function" means an increase in the proportion of T cells or B cells having memory function in a cell population containing T cells or B cells having memory function, an increase in the absolute number of T cells or B cells having memory function, or an enhancement of the memory function per cell of T cells or B cells having memory function.

[0022] The above-mentioned "induction of memory function in T cells or B cells" means inducing naive T cells or naive B cells in a cell population containing naive T cells or naive B cells to have memory function by acquiring immune memory upon stimulation with a tumor antigen, inducing T cells or B cells that already have memory function to have a higher memory function, or increasing the number of T cells or B cells that already have memory function in vivo. Specific examples include inducing naive T cells to become central memory T cells.

[0023] The subjects for administration can preferably include mammals or mammalian cells, and among such mammals, more preferably include humans, mice, dogs, rats, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, monkeys, and chimpanzees, with humans being particularly preferably included.

[0024] The above-mentioned "nucleic acid delivery vehicle" may be at least one selected from immunocompetent cells, viruses, anaerobic bacteria, liposomes, mesenchymal stem cells (MSCs), and nanoparticles, and a mixture of multiple types may be used.

[0025] Here, the above-mentioned immunocompetent cells are not particularly limited as long as they are involved in immune responses and can express IL-7 and CCL19 by introducing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19, but are preferably immunocompetent cells isolated (collected) from a living body, and examples of such isolated cells include lymphocyte cells such as T cells, natural killer cells (NK cells), and B cells, antigen-presenting cells such as monocytes, macrophages, and dendritic cells, and granulocytes such as neutrophils, eosinophils, basophils, and mast cells, and are preferably T cells isolated from mammals such as humans, dogs, cats, pigs, and mice, and preferably T cells isolated from humans. The above-mentioned isolated T cells may contain other cells in addition to T cells, but may contain T cells at a ratio of 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and most preferably 90%. Furthermore, T cells can be obtained by isolating a cell population containing immunocompetent cells from immune cells infiltrating body fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, or cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites. In order to increase the proportion of T cells contained in the cell population, the separated cell population can be further isolated or purified by a standard method as necessary. T cells produced from ES cells or iPS cells may also be used. Examples of such T cells include alpha-beta T cells, gamma-delta T cells, CD8 T cells, and CD8 T cells. + T cells, CD4 +Examples of the immunocompetent cells include T cells, tumor-infiltrating T cells, memory T cells, naive T cells, and NKT cells. The origin of the immunocompetent cells and the recipient may be the same or different, but it is preferable that they are the same. Furthermore, when the recipient is a human, the immunocompetent cells may be autologous cells collected from the patient himself / herself who is the recipient, or allogeneic cells collected from another person. In other words, the donor and the recipient may be matched or mismatched, but it is preferable that they are matched.

[0026] The above-mentioned virus is not particularly limited as long as it can encapsulate a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and can infect malignant tumor cells, but is preferably an oncolytic virus. The above-mentioned oncolytic virus means a virus that hardly grows when it infects normal cells, but grows when it infects malignant tumor cells and has the ability to kill malignant tumor cells (malignant tumor cell damage), and is reviewed, for example, in Molecular Therapy, Vol. 18, No. 2, February 2010, pp. 233-234. Such oncolytic viruses are not particularly limited as long as they have the ability to infect and kill malignant tumor cells, but include oncolytic vaccinia virus, oncolytic adenovirus, oncolytic herpes simplex virus, oncolytic reovirus, oncolytic measles virus, oncolytic Newcastle disease virus, oncolytic cowpox virus, oncolytic mumps virus, oncolytic coxsackie virus, etc. Furthermore, the oncolytic vaccinia virus may be, but is not limited to, those described in Kim MK et al. Science Translational Medicine. 2013 May 15;5(185):185ra63, Heo J, et al. Nature Medicine, 2013 (3):329-36. doi: 10.1038 / nm.3089. Epub 2013 Feb 10, and WO 2012 / 094386.In addition, the oncolytic adenovirus may be, but is not limited to, those described in Tedcastle A et al. Mol Ther. 2016;24:796-804, Marino N, Illingworth S, Kodialbail P, Patel A, Calderon H, Lear R, Fisher KD, Champion BR, Brown ACN. PLoS One 2017;12(5):e0177810, Freedman JD, et al. EMBO Mol Med 9:1067-1087 (2017), Lang FF et al., Journal of Clinical Oncology (2018), James M. et al. The Journal of Oncology, 188(6):2391-7, 2012, Japanese Patent No. 3867968 and Japanese Patent No. 5574284. In addition, the oncolytic herpes simplex virus may be, but is not limited to, those described in Mazzacurati et al., Mol Ther, 2015 Jan;23(1):99-107, Hirooka Y, et al. BMC Cancer 2018, 18, 596, Nakatake R, et al. Cancer Sci. 2018 Mar, 109(3);600-610. and AndtbackaRHI, et al. J Clin Oncol. 2015;33:2780-2788. In addition, the oncolytic reovirus may be, but is not limited to, those described in Mahalingam, et al., Cancers 2018, 10, 160. In addition, the oncolytic Newcastle disease virus may be, but is not limited to, those described in Journal of Virology. 2016 Jun;90(11):5343-5352. Furthermore, the oncolytic vesicular stomatitis virus may be, but is not limited to, the one described in Muik A. et al. Cancer Res; 74(13); 3567-78.Some oncolytic viruses have been genetically modified to have the ability to express proteins, and instead of or in addition to such proteins, the above-mentioned IL-7 and CCL19 may be expressed.

[0027] The anaerobic bacteria are not particularly limited as long as they are anaerobic bacteria that can express IL-7 and CCL19 by introducing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 into the cells, but are preferably anaerobic gram-positive bacteria that have the ability to accumulate in malignant tumor cells, and examples of such bacteria include Bifidobacterium genus bacteria such as Bifidobacterium, Lactobacillus genus bacteria, and Listeria genus bacteria. It is known that anaerobic bacteria tend to grow in an environment with little oxygen and therefore tend to accumulate in malignant tumor cells.

[0028] The above-mentioned liposome is not particularly limited as long as it is a lipid nanocapsule composed of a phospholipid bilayer membrane that can encapsulate a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 in tumor cells, and such liposomes can be obtained using commercially available products or by synthesis using standard methods.

[0029] The above-mentioned MSCs are not particularly limited, so long as they are capable of expressing IL-7 and CCL19 by introducing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 into the cells and have the ability to accumulate in malignant tumor cells.

[0030] The above-mentioned nanoparticles are not particularly limited as long as they have the ability to deliver a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 to tumor cells and are particulate bodies on the order of nanometers, preferably 5 to 800 nm in diameter, and examples of such nanoparticles include metal nanoparticles such as gold nanoparticles and silica nanoparticles. Such nanoparticles can be obtained using commercially available products or by synthesis using a conventional method.

[0031] The above "nucleic acid delivery vehicle has the ability to accumulate in malignant tumor cells" means the ability of the nucleic acid delivery vehicle to specifically accumulate in malignant tumor cells. Specifically, examples of the nucleic acid delivery vehicle include a) a nucleic acid delivery vehicle having a substance that recognizes cell surface molecules of malignant tumor cells, and the ability of the nucleic acid delivery vehicle to accumulate in malignant tumor cells by the action of such a substance, and b) an ability of the nucleic acid delivery vehicle to accumulate in malignant tumor cells by the EPR (enhanced permeability and retention) effect that utilizes the fact that the blood vessel walls of tumor tissues have gaps of several hundred nanometers, which are one order of magnitude wider than the blood vessels of normal tissues. Examples of the substance that the nucleic acid delivery vehicle recognizes on the cell surface molecules of malignant tumor cells include chimeric antigen receptors (CARs) and T cell receptors (TCRs). As such CARs and TCRs, those described in Patent Documents 2 and 3 can be used. Therefore, examples of the nucleic acid delivery vehicle that has the ability to accumulate in malignant tumor cells include CAR-expressing immunocompetent cells and TCR-expressing immunocompetent cells. A chimeric antigen receptor (CAR) is an artificial chimeric protein that combines a single-chain antibody (single chain Fv:scFv) that recognizes cell surface antigens on cancer cells with a signal transduction domain that induces T cell activation.

[0032] The above-mentioned "having specific proliferation ability in malignant tumor cells" means the ability to hardly proliferate when infecting normal cells, but to proliferate when infecting malignant tumor cells, and for example, the ability of oncolytic viruses to proliferate specifically in malignant tumor cells can be mentioned. Therefore, an example of a nucleic acid delivery vehicle having specific proliferation ability in malignant tumor cells can be an oncolytic virus.

[0033] The above-mentioned "having a malignant tumor cell cytotoxicity" means an ability to damage malignant tumor cells and lyse or kill the malignant tumor cells. By lysing or killing malignant tumor cells, malignant tumor antigens in the malignant tumor cells are released around the lysed or killed cells.

[0034] The above-mentioned "cell surface molecule that recognizes a malignant tumor antigen" may be any cell surface molecule that recognizes a malignant tumor antigen present on the cell surface of a malignant tumor, and examples of such a molecule include chimeric antigen receptors (CARs) and T cell receptors (TCRs). Such CARs and TCRs can be those described in the above-mentioned Patent Documents 2 and 3. Thus, examples of immunocompetent cells having a cell surface molecule that recognizes a malignant tumor antigen include CAR-expressing immunocompetent cells and TCR-expressing immunocompetent cells.

[0035] The malignant tumor antigen refers to a substance such as a protein or glycolipid that is expressed more highly in malignant tumor cells than in normal cells or is specifically expressed in malignant tumor cells. Examples of such malignant tumor antigens include tumor-associated antigens, cancer-testis antigens, angiogenesis-associated antigens, and epitope peptides of malignant tumor neoantigens (neoantigens) caused by genetic mutations. Specific examples of such malignant tumor antigens 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, Examples of such proteins include TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, MESOTHELIN, NKG2D, P1A, 5T4, B7-H6, BCMA, CD123, CD133, CD138, CD171, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CEA, cMet, CS1, EGFR, EGFRvIII, EphA2, ErbB2, FAP, FR-α, HER2, IL13Ra2, MUC1, MUC16, NKG2D, PSCA, PSMA, ROR1, TARP, DLL3, PRSS21, Claudin18.2, Claudin18, CAIX, L1-CAM, FAP-α, CTAG1B, and FR-α, as well as glycolipids such as GD2 and GM2.

[0036] The above "comprising a nucleic acid delivery vehicle, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19" also includes an embodiment in which the nucleic acid delivery vehicle contains a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19. For example, when the nucleic acid delivery vehicle is an immunocompetent cell, it is sufficient that the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 are contained in the immunocompetent cell. In addition, the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 may be incorporated into the genome of the immunocompetent cell, or may not be incorporated into the genome (e.g., in an episomal state).

[0037] When the nucleic acid delivery vehicle is an immunocompetent cell, the present enhancer or inducer or the present malignant tumor recurrence suppressor can be produced by introducing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 into the immunocompetent cell, i.e., by introducing an exogenous nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 (preferably an exogenous nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 operably linked downstream of a promoter) into the immunocompetent cell. The introduction method may be any method for introducing DNA into an immunocompetent cell, and examples of the introduction method include electroporation (Cytotechnology, 3, 133 (1990)), calcium phosphate method (JP Patent Publication 2-227075), lipofection (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), and virus infection. An example of such a viral infection method is a method in which a vector containing the nucleic acid to be introduced and a packaging plasmid are transfected into packaging cells such as GP2-293 cells (Takara Bio), Plat-GP cells (Cosmo Bio), PG13 cells (ATCC CRL-10686), PA317 cells (ATCC CRL-9078), etc. to produce a recombinant virus, and then the recombinant virus is infected into T cells (Patent Document 2 mentioned above).

[0038] Furthermore, when the nucleic acid delivery vehicle is a virus, a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 can be introduced into the virus, i.e., an exogenous nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 (preferably an exogenous nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 operably linked downstream of a promoter) can be introduced into the virus to prepare a "virus having a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19." Such a prepared virus can be used as the present enhancer or inducer or the present malignant tumor recurrence suppressant.

[0039] Similarly, when the nucleic acid delivery vehicle is an anaerobic bacterium, liposome, or mesenchymal stem cell (MSC), a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 can be introduced into the anaerobic bacterium, liposome, or mesenchymal stem cell (MSC), i.e., an exogenous nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 (preferably, an exogenous nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 operably linked downstream of a promoter) can be introduced into the anaerobic bacterium, liposome, or mesenchymal stem cell (MSC) to produce "anaerobic bacterium, liposome, or mesenchymal stem cell (MSC) having a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19." The thus produced anaerobic bacterium, liposome, or mesenchymal stem cell (MSC) can be used as the enhancer or inducer of the present invention or the malignant tumor recurrence inhibitor of the present invention. Furthermore, the above-mentioned "virus having a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19", "anaerobic bacteria having a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19", "liposome having a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19", or "mesenchymal stem cells (MSCs) having a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19" may be used as an inhibitor of malignant tumor proliferation.

[0040] Another method for introducing the above-mentioned IL-7-encoding nucleic acid and CCL19-encoding nucleic acid into immunocompetent cells may be to incorporate the IL-7-encoding nucleic acid and CCL19-encoding nucleic acid into the genome of the immunocompetent cells using a known gene editing technique so that they can be expressed under the control of an appropriate promoter. Examples of known gene editing techniques include techniques using endonucleases such as zinc finger nucleases, TALENs (transcription activation-like effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas systems.

[0041] When preparing "immunocompetent cells that contain a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and have a CAR as a cell surface molecule that recognizes a malignant tumor antigen, i.e., immunocompetent cells that contain nucleic acids encoding CAR, IL-7, and CCL19 and express CAR, IL-7, and CCL19" as the enhancer or inducer or the malignant tumor recurrence inhibitor, the immunocompetent cells can be prepared by any of the following methods. (1) A method of simultaneously or stepwise introducing two types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19, and a vector containing a nucleic acid encoding CAR and expressing CAR; (2) A method of simultaneously or stepwise introducing two types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding a CAR and a nucleic acid encoding IL-7 and expressing CAR and IL-7, and a vector containing a nucleic acid encoding a CAR and a nucleic acid encoding CCL19 and expressing CAR and CCL19; (3) A method of simultaneously or stepwise introducing two types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding CAR and a nucleic acid encoding IL-7 and expressing CAR and IL-7, and a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19; (4) A method of simultaneously or stepwise introducing two types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding CAR and a nucleic acid encoding CCL19 and expressing CAR and CCL19, and a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19; (5) A method of simultaneously or stepwise introducing two types of vectors, a vector containing a nucleic acid encoding CAR and a nucleic acid encoding IL-7 and expressing CAR and IL-7, and a vector containing a nucleic acid encoding CCL19 and expressing CCL19, into immunocompetent cells; (6) A method of simultaneously or stepwise introducing two types of vectors, a vector containing a nucleic acid encoding CAR and a nucleic acid encoding CCL19 and expressing CAR and CCL19, and a vector containing a nucleic acid encoding IL-7 and expressing IL-7, into immunocompetent cells; (7) A method for simultaneously or stepwise introducing three types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding CAR and expressing CAR, a vector containing a nucleic acid encoding IL-7 and expressing IL-7, and a vector containing a nucleic acid encoding CCL19 and expressing CCL19;

[0042] Similarly, when preparing "immunocompetent cells that contain a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and have a TCR as a cell surface molecule that recognizes a malignant tumor antigen, i.e., immunocompetent cells that contain nucleic acids encoding TCR, IL-7, and CCL19 and express TCR, IL-7, and CCL19" as the enhancer or inducer of the present invention or the malignant tumor recurrence inhibitor of the present invention, the cells can be prepared by any of the following methods. (1) A method of simultaneously or stepwise introducing two types of vectors, i.e., a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19, and a vector containing a nucleic acid encoding TCR and expressing TCR, into immunocompetent cells; (2) A method of simultaneously or stepwise introducing two types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding a TCR and a nucleic acid encoding an IL-7 and expressing TCR and IL-7, and a vector containing a nucleic acid encoding a TCR and a nucleic acid encoding CCL19 and expressing TCR and CCL19; (3) A method of simultaneously or stepwise introducing two types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding TCR and a nucleic acid encoding IL-7 and expressing TCR and IL-7, and a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19; (4) A method of simultaneously or stepwise introducing two types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding TCR and a nucleic acid encoding CCL19 and expressing TCR and CCL19, and a vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expressing IL-7 and CCL19; (5) A method of simultaneously or stepwise introducing two types of vectors, a vector containing a nucleic acid encoding TCR and a nucleic acid encoding IL-7 and expressing TCR and IL-7, and a vector containing a nucleic acid encoding CCL19 and expressing CCL19, into immunocompetent cells; (6) A method of simultaneously or stepwise introducing two types of vectors, a vector containing a nucleic acid encoding TCR and a nucleic acid encoding CCL19 and expressing TCR and CCL19, and a vector containing a nucleic acid encoding IL-7 and expressing IL-7, into immunocompetent cells; (7) A method of simultaneously or stepwise introducing three types of vectors into immunocompetent cells: a vector containing a nucleic acid encoding TCR and expressing TCR, a vector containing a nucleic acid encoding IL-7 and expressing IL-7, and a vector containing a nucleic acid encoding CCL19 and expressing CCL19;

[0043] Furthermore, when producing the above-mentioned "immunocompetent cells expressing TCR, IL-7, and CCL19," immunocompetent cells expressing a TCR specific to a desired tumor antigen are prepared in advance, and the immunocompetent cells can be used to produce the cells by any of the following methods. (1) A method for introducing a vector that contains a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 and expresses IL-7 and CCL19 into the TCR-expressing immunocompetent cells; (2) A method of simultaneously or stepwise introducing two vectors, a vector containing a nucleic acid encoding IL-7 and expressing IL-7, and a vector containing a nucleic acid encoding CCL19 and expressing CCL19, into the TCR-expressing immunocompetent cells;

[0044] In addition, "immunocompetent cells expressing CAR, TCR, IL-7, and CCL19" may be prepared as the enhancer or inducer of the present invention or the malignant tumor recurrence suppressor of the present invention. Specifically, the following methods can be mentioned: a method in which all or any of the vectors described above in "When preparing immunocompetent cells expressing CAR, IL-7, and CCL19" are further prepared by including a nucleic acid encoding a TCR so as to express TCR; a method in which all or any of the vectors described above in "When preparing immunocompetent cells expressing TCR, IL-7, and CCL19" are further prepared by including a nucleic acid encoding a CAR so as to express CAR; or a method in which each of the vectors described above in "When preparing immunocompetent cells expressing CAR, IL-7, and CCL19" are introduced into the "TCR-expressing immunocompetent cells" to prepare the cells.

[0045] Furthermore, in the case of the present enhancer or inducer or the present malignant tumor recurrence suppressor, when the immunocompetent cells have a CAR, "immunocompetent cells expressing multiple, preferably two types of CARs that recognize different malignant tumor antigens" may be prepared. Specifically, in each vector described above in "When preparing immunocompetent cells expressing CAR, IL-7, and CCL19", a method of preparing multiple, preferably two types of CARs including nucleic acids encoding multiple, preferably two types of CARs that recognize different malignant tumor antigens can be mentioned. In particular, for example, a vector containing a nucleic acid encoding a CAR that recognizes malignant tumor antigen X and a nucleic acid encoding IL-7 and expressing a CAR that recognizes malignant tumor antigen X and IL-7, and a vector containing a nucleic acid encoding a CAR that recognizes malignant tumor antigen Y and a nucleic acid encoding CCL19 and expressing a CAR that recognizes malignant tumor antigen Y and CCL19 are prepared and introduced into immunocompetent cells, IL-7 and CCL19 are secreted around the cells that have X and Y as malignant tumor antigens, thereby making it possible to further increase tumor specificity.

[0046] In addition, when the nucleic acid delivery vehicle is an immunocompetent cell, a virus, an anaerobic bacterium, or a mesenchymal stem cell, a culture obtained by culturing the immunocompetent cell, a virus, an anaerobic bacterium, or a mesenchymal stem cell and containing the immune cell may be used.

[0047] The "malignant tumor recurrence" in the malignant tumor recurrence inhibitor means the occurrence of a malignant tumor again after malignant tumor treatment by general chemotherapy, radiotherapy, surgical therapy, etc. Such malignant tumor recurrence is preferably a recurrence caused by malignant tumor cells having resistance to the accumulation ability of a nucleic acid delivery vehicle in malignant tumor cells or to the specific proliferation ability of malignant tumor cells.

[0048] Here, "recurrence caused by malignant tumor cells having resistance to the ability to accumulate in malignant tumor cells" can be, for example, a case where the nucleic acid delivery vehicle is an immunocompetent cell having a cell surface molecule that recognizes a malignant tumor antigen, and the malignant tumor recurrence is caused by malignant tumor cells that do not have a malignant tumor antigen specifically recognized by the cell surface molecule, or that have lost the malignant tumor antigen specifically recognized by the cell surface molecule. In addition, "recurrence caused by malignant tumor cells having resistance to the specific proliferation ability of the malignant tumor cells of the nucleic acid delivery vehicle" can be, for example, a case where the malignant tumor cells are not susceptible to infection by a malignant tumor lytic virus, or that have lost susceptibility to infection by a malignant tumor lytic virus. Such "recurrence caused by malignant tumor cells having resistance to the ability to accumulate in malignant tumor cells" can be confirmed, for example, by examining the malignant tumor antigen in the malignant tumor cells of the recurrent tissue.

[0049] The malignant tumor recurrence suppressor can be administered to a subject with a malignant tumor that has been immunotherapeutic. For the purpose of long-term suppression of recurrence, it can also be administered to a subject with a malignant tumor that has been immunotherapeutic 100 days or more after the day of immunotherapeutic treatment.

[0050] The enhancer or inducer and the malignant tumor recurrence suppressor may contain a pharma- ceutical acceptable additive. Furthermore, the enhancer or inducer and the malignant tumor recurrence suppressor may include instructions for use. The enhancer or inducer and the malignant tumor recurrence suppressor may contain a pharma- ceutical acceptable additive to form a pharmaceutical composition. Hereinafter, "a pharmaceutical composition containing the enhancer and a pharma- ceutical acceptable additive", "a pharmaceutical composition containing the inducer and a pharma- ceutical acceptable additive", and "a pharmaceutical composition containing the malignant tumor recurrence suppressor and a pharma- ceutical acceptable additive" are collectively referred to as "the pharmaceutical composition". Examples of the additives include physiological saline, buffered physiological saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, fillers, and lubricants.

[0051] The present enhancer or inducer, the present malignant tumor recurrence inhibitor, or the present pharmaceutical composition can be administered to a subject in need of treatment or suppression of recurrence of a malignant tumor using methods known to those skilled in the art, and examples of the administration methods include intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intrathecal, and subarachnoid (cerebrospinal fluid) injection.

[0052] The enhancer or inducer, malignant tumor recurrence inhibitor, or pharmaceutical composition may be independently administered four times, three times, twice or once a day, every other day, every second day, every third day, every fourth day, every fifth day, once a week, every seventh day, every eighth day, or every ninth day, twice a week, once a month, or twice a month.

[0053] The dosage of the enhancer or inducer, the malignant tumor recurrence suppressor, or the pharmaceutical composition can be appropriately determined depending on the age, sex, health, weight, etc. of the subject. For example, when the nucleic acid delivery vehicle is an immunocompetent cell, the dosage is 1×10 per kg of body weight for a human adult. 3 ~1×10 9 pcs, preferably 1 x 10 4 ~1×10 8 pcs, more preferably 1×10 5 ~1×10 7 In addition, when the nucleic acid delivery vehicle is an oncolytic virus, the number of doses per administration for an adult human is about 10 2 ~10 10 Plaque forming units (PFU), preferably 10 5 ~10 6 Plaque forming units (PFU) may be mentioned.

[0054] The malignant tumor in this specification may be a solid malignant tumor or a hematological malignant tumor, and may be, for example, glioma, melanoma, malignant mesothelioma, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, thyroid cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, head and neck cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, Examples of cancers include stomach cancer, esophageal cancer, biliary tract cancer, gallbladder cancer, testicular cancer, ovarian cancer, and brain tumors; cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue; sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatic blastoma, pleuropulmonary blastoma, and retinoblastoma; germ cell tumors, lymphomas, leukemia, and myeloma.

[0055] The present enhancer or inducer, the present malignant tumor recurrence suppressor, or the present pharmaceutical composition can be used in combination with other antitumor agents. In addition, the method using the present enhancer or inducer, the present malignant tumor recurrence suppressor, or the present pharmaceutical composition may be combined with cancer therapy using radiation. Examples of the other antitumor agents include alkylating agents such as cyclophosphamide, bendamustine, iosphamide, and dacarbazine; metabolic antagonists such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enocitabine; molecular targeted drugs such as rituximab, cetuximab, and trastuzumab; and kinase inhibitors such as imatinib, gefetinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib. Examples of such anticancer drugs include anticancer agents, proteasome inhibitors such as bortezomib, calcineurin inhibitors such as cyclosporine and tacrolimus, anticancer antibiotics such as idarubicin, doxorubicin, and mitomycin C, plant alkaloids such as irinotecan and etoposide, platinum preparations such as cisplatin, oxaliplatin, and carboplatin, hormone therapy drugs such as tamoxifen and bicaldamide, and immunosuppressants such as interferon, nivolumab, and pembrolizumab.

[0056] Examples of the method of "using the present enhancer or inducer, the present malignant tumor recurrence suppressor, or the present pharmaceutical composition in combination with another anticancer drug" include a method of treating with another anticancer drug and then using the present enhancer or inducer, the present malignant tumor recurrence suppressor, or the present pharmaceutical composition, a method of simultaneously using the present enhancer or inducer, the present malignant tumor recurrence suppressor, or the present pharmaceutical composition with another anticancer drug, and a method of treating with the present enhancer or inducer, the present malignant tumor recurrence suppressor, or the present pharmaceutical composition and then using another anticancer drug. Furthermore, when the present enhancer or inducer, the present malignant tumor recurrence suppressor, or the present pharmaceutical composition is used in combination with another anticancer drug, the therapeutic effect of cancer is further improved, and by reducing the number of times or the dosage of each, it is possible to reduce the side effects caused by each.

[0057] All references cited herein, including journal articles, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth herein. EXAMPLES

[0058] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0059] (Generation of anti-human CD20 CAR-IL-7 / CCL19 expressing T cells and anti-human CD20 CAR expressing T cells) The "T cells expressing anti-human CD20 CAR, mouse IL-7, and mouse CCL19 (anti-human CD20 CAR-IL-7 / CCL19-expressing T cells: also referred to as "7x19" in the following examples or drawings)" and "anti-human CD20 CAR-expressing T cells: also referred to as "Conv." in the following examples or drawings)" used in the following examples were prepared according to the methods described in the above Patent Document 3 and the document by Tamada et al. (Nature Biotechnology doi:10.1038 / nbt.4086). The preparation method is briefly described below. The above "T cells expressing anti-human CD20 CAR, mouse IL-7, and mouse CCL19" are T cells having anti-human CD20 CAR as a cell surface molecule that recognizes a malignant tumor cell antigen, and include a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19.

[0060] The anti-human CD20 CAR-IL-7 / CCL19 expressing T cells were prepared by first preparing a pMSGV vector that contains a nucleic acid encoding an anti-human CD20 CAR, a nucleic acid encoding mouse IL-7, and a nucleic acid encoding mouse CCL19, and expresses anti-human CD20 CAR, IL-7, and CCL19. Next, the vector was transfected into CD90.1 positive (CD90.1 + ), CD90.2 negative (CD90.2 -The T cells were prepared by introducing a retrovirus into mouse T cells isolated from the spleen and lymph nodes of congenic mice (manufactured by Bar Harbor) using the Pan T Cell Isolation Kit II (manufactured by Miltenyi Biotec). On the other hand, T cells expressing anti-human CD20 CAR (anti-human CD20 CAR-expressing T cells) were prepared by first constructing a pMSGV vector expressing anti-human 20 CAR and introducing such a vector into the above-isolated mouse T cells using a retrovirus. Note that the above-isolated mouse T cells without gene transduction (non-transducted T cells) are also referred to as "non-transducted" in the following examples or drawings. Here, the pMSGV vector, which is a retroviral vector, is used to introduce a nucleic acid encoding anti-human CD20 CAR, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19 into the mouse T cells isolated as described above. Therefore, when the mouse T cells into which each of the above nucleic acids has been introduced are cultured and proliferated, some of the mouse T cells contain the retroviral vector in the cytoplasm, but in many cases, in the mouse T cells, the nucleic acid encoding anti-human CD20 CAR, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 are integrated into the genome. When the nucleic acid encoding anti-human CD20 CAR, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 are integrated into the genome in mouse T cells, anti-human CD20 CAR, IL-7, and CCL19 will be expressed from the introduced foreign recombinant construct.

[0061] For culturing T cells, RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μM 2-mercaptoethanol, 25 mM HEPES, and 2 mM L-glutamine was used.

[0062] [Example 1] <Localization of T cells> To investigate the antitumor effect of CAR-IL-7 / CCL19-expressing T cells, we investigated whether recipient-derived endogenous T cells infiltrated tumor tissues in the same way as donor T cells. First, 7- to 10-week-old C57BL / 6 mice (SLC) were injected with 2.5 × 10 6 3LL-hCD20 (3LL cells derived from mouse lung cancer genetically modified to express human CD20) were subcutaneously inoculated (day 0). On day 7, the anticancer drug cyclophosphamide (CPA: 100 mg / kg) was administered intraperitoneally. On day 10, CD90.1 positive (CD90.1 + ), CD90.2 negative (CD90.2 - ) 1 × 10 6 The anti-human CD20 CAR-expressing T cells, the anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, or the isolated mouse T cells without gene transfer were intravenously administered. On the 19th day, tumor tissues were excised from the mice. For primary staining, a combination of biotin-labeled anti-CD90.1 antibody (clone OX-7 BioLegend: binds to donor T cells) and anti-CD3 antibody (clone 17A2: Tonbo biosciences: binds to both donor T cells and endogenous T cells of the recipient) was used, and for secondary staining, Alexa Fluor488-conjugated streptavidin (Thermo Fisher Scientific: green) and Alexa Fluor647-conjugated anti-rat IgG2b (Abcam: red) were used. Nuclei were stained with DAPI (Thermo Fisher Scientific: blue). Microscopic observation was performed at 400x magnification. The results are shown in Figure 1(a). In the second staining, cells bound by biotin-labeled anti-CD90.1 antibody are shown in green, and cells bound by anti-CD3 antibody are shown in red. Therefore, donor T cells, anti-human CD20 CAR-IL-7 / CCL19 expressing T cells, are shown in yellow (green + red: CD90.1 + +CD3 + ), whereas the recipient's endogenous T cells are red (CD90.1 - +CD3 +), but is shown in grayscale in Figure 1(a).

[0063] Moreover, the results of quantifying each positive region labeled in FIG. 1(a) using the Hybrid Cell Count program (manufactured by KEYENCE) are shown in FIG. 1(b). In Figure 1(b), the black columns indicate the area of ​​the recipient's (host's) endogenous T cells (red in the right panel of Figure 1(a)), and the white columns indicate the area of ​​the administered donor T cells (yellow in the right panel of Figure 1(a)). Figures 1(a) and (b) reveal that when anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were administered, the accumulation of not only the administered T cells but also the recipient's (host's) endogenous T cells in the tumor local area was enhanced; in other words, by secreting IL-7 and CCL19 in the tumor local area, the accumulation of endogenous T cells in the tumor local area was also enhanced.

[0064] [Example 2] <Involvement of T cells in antitumor effects> 2.5 × 10 6 3LL-hCD20 was subcutaneously inoculated (day 0). + ), CD90.2 negative (CD90.2 - ) 1 × 10 6 Anti-human CD20 CAR-expressing T cells (Conventional: Conv.) or anti-human CD20 CAR-IL-7 / CCL19-expressing T cells (7×19) were administered intravenously. Anti-CD90.2 antibody (anti-CD90.2: produced by the inventors using a hybridoma purchased from ATCC) against CD90.2 (Thy1.2) expressed in endogenous T cells was administered intraperitoneally about twice a week from day 1. The dosage was 1 mg / mouse for the first two doses, and 0.5 mg / mouse thereafter. The mean ± SD of the tumor volume on day 14 for each group (n=5) is shown in FIG. 2. ○ indicates the value for each mouse.

[0065] As shown in Figure 2, when anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were administered, the growth of malignant tumors was suppressed, the anti-tumor effect was extremely high, and the tumor disappeared, but the anti-tumor effect was reduced by about 50% by administering anti-CD90.2 antibody. Therefore, it was revealed that the anti-tumor effect of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells is also involved in the endogenous T cells of the recipient, in other words, the anti-tumor effect of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells is largely involved not only in the anti-human CD20 CAR-IL-7 / CCL19-expressing T cells themselves but also in the endogenous T cells of the recipient.

[0066] [Example 3] <Memory of recipient's endogenous T cells> The acquisition of memory function of donor CAR-T cells and endogenous T cells and the increase in cells with memory function by anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were evaluated using memory cell markers CD44 and CD62L.

[0067] 2.5 × 10 6 3LL-hCD20 was subcutaneously inoculated (day 0). + ), CD90.2 negative (CD90.2 - ) 1 × 10 6 The above anti-human CD20 CAR-expressing T cells (Conv.) or the above anti-human CD20 CAR-IL-7 / CCL19-expressing T cells (7 × 19) were administered intravenously. On day 28, spleen cells were harvested and used for the following analysis.

[0068] Donor T cells were CD90.1 positive cells (CD90.1 + ), and recipient T cells were CD90.2 positive cells (CD90.2 +) were identified. Expression of memory T cell markers (CD44 and CD62L) and CAR in CD4-positive and CD8-positive T cells was performed by flow cytometry. The numbers in the dot plots or histograms indicate the percentage of cells in each gate. The results are shown in Figure 3.

[0069] In addition, the memory function, i.e., acquisition of responsiveness to stimuli, was examined by the production of IFN-γ. First, spleen cells collected on the 28th day were stimulated by co-culture with 3LL-hCD20 or its parental line 3LL that does not express hCD20, which had been treated with mitomycin C (Kyowa Hakko Kirin Co., Ltd.) for 90 minutes at 37°C. The production of IFN-γ was examined by intracellular cytokine staining. The results are shown in Figure 4. In Figure 4, the numbers in the histogram (a) indicate the proportion of IFN-γ positive cells in CD90.1 positive donor T cells. In Figure 4, (b) shows the results of detecting IFN-γ positive cells in CD90.2 positive endogenous CD8 positive T cells by flow cytometry. The numbers in the dot plots indicate the proportion of cells in each of the four quadrants.

[0070] As shown in Figure 3, when anti-human CD20 CAR-expressing T cells (Conv.) were administered, central memory T cells (memory T cell markers CD44-positive and CD62L-positive cells) in the CD90.2 gated cells (endogenous T cells of the recipient) were 5.5% in the CD4-positive cells and 24.8% in the CD8-positive cells. On the other hand, when anti-human CD20 CAR-IL-7 / CCL19-expressing T cells (7x19) were administered, central memory T cells in the CD90.2 gated cells (endogenous T cells of the recipient) were 6.76% in the CD4-positive cells and 49.2% in the CD8-positive cells, and the proportion of central memory T cells was increased in both cases. Therefore, it was revealed that administering anti-human CD20 CAR-IL-7 / CCL19-expressing T cells (7x19), i.e., secreting IL-7 and CCL19 from a single cell, activates the recipient's endogenous T cells and induces their differentiation into central memory T cells, thereby increasing the number of central memory T cells and increasing the proportion of central memory T cells in the spleen cell group. In other words, it was revealed that anti-human CD20 CAR-IL-7 / CCL19-expressing T cells can be used as an inducer of T cells with memory function in the recipient or as an enhancer of T cells with memory function in the recipient.

[0071] On the other hand, in the CD90.1 gated cells (donor T cells), central memory T cells accounted for 75.8% of CD4 positive cells and 90.7% of CD8 positive cells, confirming that the donor T cells themselves were also induced to become central memory T cells. Note that the CD90.1 gated cells were 0% in Conv., and CD90.1 positive cells were not detected (nd), because Conv. did not express IL-7 and CCL19, and the survival rate of CAR-expressing T cells was low.

[0072] Furthermore, as shown in FIG. 4, upon stimulation with 3LL, 90.5% of CD4-positive cells and 93.6% of CD8-positive cells in donor cells were producing IFN-γ. Also, in the recipient's endogenous T cells (without CAR-T expression), while 0.957% of CD4-positive cells produced IFN-γ in the control (Conv.), 14.9% of cells produced IFN-γ in the 7×19 group. Therefore, by secreting IL-7 and CCL19 with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, it became clear that both donor T cells and recipient cells were producing IFN-γ upon stimulation and had acquired memory function.

[0073] [Example 4] <Change in gene expression pattern of T cell receptor> To examine the epitope diversity of T cells, changes in the T cell receptor (TCR) repertoire before and after treatment with CAR-IL-7 / CCL19-expressing T cells were investigated. Five DBA / 2 mice (n = 5) were subcutaneously inoculated with 5×10 5 individuals of P815-hCD20 (mouse mastocytoma P815 genetically engineered to express human CD20) on day 0. Then, on day 10, cyclophosphamide (CPA: 100 mg / kg) and 1×10 6 individuals of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were intravenously administered to treat the tumor. On day 140 after P815 tumor cell administration, spleen cells were collected from the mice (tumor-rejected mice) that had been treated and cured of the tumor as described above, and CAR-positive T cells or CAR-negative T cells sorted by a cell sorter (SH800: manufactured by Sony Corporation) were cultured with P815-hCD20 or the parental strain that did not express hCD20 (hCD20-negative: hCD20 - ) P815 for 4 days to proliferate. Subsequently, for TCR repertoire analysis, CD8-positive and CAR-positive (CD8 + CAR + ) or CD8-positive and CAR-negative (CD8 + CAR -) population was sorted using a flow cytometer. As a control, CD8 positive and CAR positive or CD8 positive and CAR negative populations before injection into mice were sorted. The TCR repertoire was analyzed using a next-generation sequencer, and the frequency of V and J region usage in the α and β chains was shown in a 3-D graph. The results are shown in Figures 5A-D and 6A-D. Figures 5A-D are the α chain, Figures 6A-D are the β chain, and Figures 5A, B, and 6A, B are before injection of each cell, and Figures 5C, D, and 6C, D are after injection of each cell. The numbers in the upper left corner of the figure indicate diversity indexes calculated using the 1-Pielou evenness index (a higher number indicates a less diversity), and the lower the number, the higher the diversity.

[0074] The T cell receptor is an antigen receptor molecule expressed on the cell membrane of T cells. It exists as a heterodimer consisting of an α chain and a β chain, or a γ chain and a δ chain, and is known to activate T cells by recognizing antigen molecules bound to major histocompatibility complex (MHC) molecules.

[0075] As is clear from the diversity indexes in Figures 5A-D and 6A-D, the diversity index values ​​of both the α and β chains increased by administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. Therefore, it was revealed that the TCR diversity of not only the administered anti-human CD20 CAR-IL-7 / CCL19-expressing T cells but also the recipient's endogenous T cells was reduced, and T cells that secrete IL-7 and CCL19 at the tumor site and destroy tumor cells, which are thought to have memory function against tumor antigens, were selectively increased.

[0076] [Example 5] <Inhibition of tumor recurrence-1> A cancer recurrence model using animals was performed as follows. First, the tumor-specific memory response of recipient T cells was examined in mice treated with anti-human CD20 CAR-IL-7 / CCL19 expressing T cells. 5 × 10 5 Each mouse was subcutaneously inoculated with 1 × 10 P815-hCD20. On the 10th day, the anticancer drug cyclophosphamide (CPA: 100 mg / kg) was administered intraperitoneally. On the 14th day, 1 × 10 6 Anti-human CD20 CAR-IL-7 / CCL19 expressing T cells were inoculated intravenously. 140 days after inoculation with P815-hCD20, tumor-rejected mice or control naive mice were inoculated with P815-hCD20 or the parent strain P815 not expressing hCD20 into the left and right flanks. The tumor volume was measured twice a week. The results are shown in Figure 7A. In addition, a similar analysis was performed using C57BL / 6 mice inoculated with 3LL-hCD20 or the parent strain 3LL not expressing hCD20 instead of the above P815-hCD20. The results are shown in Figure 7B. Note that the parent strains P815 and 3LL do not express human CD20, and therefore the malignant tumor cells do not have the malignant tumor antigen that is specifically recognized by the cell surface molecules of anti-human CD20 CAR-IL-7 / CCL19 expressing T cells.

[0077] The horizontal axis in Figure 7A indicates the number of days from the day (day 0) when P815-hCD20 or P815 was first administered to naive mice, and the number of days from the day (day 0) when P815-hCD20 was again administered 140 days after the first P815-hCD20 inoculation in mice whose tumors were cured by treatment. The horizontal axis in Figure 7B indicates the number of days from the day (day 0) when 3LL-hCD20 or 3LL was first administered to naive mice, and the number of days from the day (day 0) when 3LL-hCD20 was again administered to mice whose tumors were cured by treatment. The vertical axis in Figures 7A and 7B indicates the tumor volume (mm 3 ).

[0078] As shown in the lower part of Figures 7A and 7B, when tumor-rejected mice were inoculated with P815-hCD20 or 3LL-hCD20, no tumors were formed. In other words, it was revealed that tumor recurrence caused by tumor cells having an antigen recognized by CAR can be suppressed by treating tumors with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. Furthermore, surprisingly, as shown in the upper part of Figures 7A and 7B, when tumor-rejected mice were inoculated with parental strains P815 or 3LL that do not have CD20 on the cell surface, tumor formation was significantly suppressed compared to naive mice. In other words, it was revealed that treatment with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells can also suppress tumor formation caused by tumor cells that do not have an antigen recognized by CAR, in other words, tumor recurrence caused by tumor cells that do not have an antigen recognized by CAR. Thus, the administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells affects the immune function of the recipient, and the recipient continues to reject the parent tumor that does not express the target antigen of CAR for a long time, suppressing recurrence. This was unexpected from the perspective of the target molecule-specific reactivity of CAR-T cells. Combined with the results of the change in gene expression pattern of the T cell receptor in Example 4 above, it is considered that the administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells causes the destruction of tumor cells, and as a result, the recipient's endogenous T cells react against the tumor antigen originally possessed by the tumor cells, inducing a long-term memory function. In other words, it is considered that the administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells destroys tumor cells and secretes IL-7 and CCL19 locally in the tumor, thereby extremely efficiently inducing the epitope spreading phenomenon, and suppressing the recurrence of malignant tumors by inducing and enhancing the memory function. In the above cancer recurrence model, P815-hCD20, P815, 3LL-hCD20, and 3LL were first inoculated subcutaneously, and on day 140, each of the above cells was inoculated into the left and right flanks, respectively.Therefore, administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells is thought to suppress not only the recurrence of malignant tumors but also the metastasis of malignant tumors. Therefore, it is thought that anti-human CD20 CAR-IL-7 / CCL19-expressing T cells can also be used as a "malignant tumor metastasis inhibitor."

[0079] [Example 6] <Inhibition of tumor recurrence-2> To confirm the effect of suppressing tumor recurrence, a human malignant pleural mesothelioma cell line was administered to mice to form tumors, and then the presence or absence of tumor recurrence over 143 days was examined with or without the administration of anti-human mesothelin CAR-IL-7 / CCL19-expressing T cells. The specific experimental protocol is shown in Figure 8. The preparation methods for "ACC-MESO1-GFP-Luc", "anti-mesothelin CAR-expressing T cells", and "anti-mesothelin CAR-expressing T cells" in Figure 8, and the activation method for T cells are as follows.

[0080] (Generation of ACC-MESO1-GFP-Luc strain) We used lentivirus to introduce the green fluorescent protein-luciferase (GFP-Luc) gene into the human malignant mesothelioma cell line ACC-MESO1, a mesothelin-positive tumor cell line provided by Dr. Yoshitaka Sekido of the Aichi Cancer Center Research Institute.

[0081] On day 0, 1 × 10 3ACC-MESO1 was plated at 100 cells / well. RPMI1640 (Gibco) supplemented with 10% FBS was used as the medium. On day 1, RediFect Red-FLuc-GFP (PerkinElmer), a lentiviral particle for producing luminescent cells, was added at MOI 100 to start transduction. To increase the efficiency of gene transduction, Hexadimethrine Bromide (Sigma-Aldrich) was added to the medium at a final concentration of 4 μg / mL. 24 hours after the addition of the virus (day 2), the medium containing the virus was removed and replaced. After continued cultivation, only cells expressing GFP were sorted using SH800 (SONY) and designated as the ACC-MESO1-GFP-Luc strain.

[0082] (Generation of anti-human mesothelin CAR-IL-7-CCL19 expressing T cells and anti-human mesothelin CAR expressing T cells) Anti-human mesothelin CAR-IL-7-CCL19 expressing T cells and anti-human mesothelin CAR expressing T cells (conventional CAR expressing T cells) were prepared based on the methods described in Japanese Patent Application No. 2017-247109 and International Publication No. 2016 / 056228. Briefly, a third-generation CAR construct (SEQ ID NO: 6) was prepared, which is an anti-human mesothelin single-chain antibody consisting of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 3, the amino acid sequence of the linker shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 5, a human CD8 transmembrane domain, and a human CD28-4-1BB-CD3ζ intracellular signal motif. A construct was prepared in which human IL-7 shown in SEQ ID NO: 1, followed by picornavirus-derived 2A peptide (F2A) shown in SEQ ID NO: 7, human CCL19 shown in SEQ ID NO: 2, and herpes virus-derived thymidine kinase gene (HSV-tk) were sequentially provided at the C-terminus of the construct, and inserted into a pMSGV1 retroviral expression vector (Tamada k et al., Clin Cancer Res 18:6436-6445(2002)) to prepare a pMSGV1 retroviral expression vector expressing human IL-7 / CCL19 and HSV-tk. The obtained pMSGV1 retroviral expression vector was introduced into mouse T cells by retrovirus to prepare anti-human mesothelin CAR-IL-7-CCL19-expressing T cells. Similarly, instead of the pMSGV1 retroviral expression vector expressing the above-mentioned human IL-7 / CCL19 and HSV-tk, a pMSGV1 retroviral expression vector expressing a third-generation CAR construct sequentially comprising the above-mentioned anti-human mesothelin single-chain antibody, human CD8 transmembrane domain, and human CD28-4-1BB-CD3ζ intracellular signal motif was introduced into mouse T cells by retrovirus to produce anti-human mesothelin CAR-expressing T cells (conventional anti-human mesothelin CAR-expressing T cells). The signal peptide used was the signal peptide shown in SEQ ID NO:8.

[0083] (T cell activation) 2 × 10 collected from healthy donors on day 0 6Peripheral blood mononuclear cells were cultured in a 6-well cell culture plate immobilized with 25 μL / mL Retronectin (Takara Bio) and 5 μg / mL anti-human CD3 monoclonal antibody (Invitrogen, 5 μg / mL) together with 200 IU / mL IL-2 (Peprotech) at 37°C in a 5% CO2 incubator. The culture medium used was OpTmizer CTS (Gibco) supplemented with 2 mM L-glutamine (Gibco), 1% penicillin-streptomycin (Wako Pure Chemical Industries, Ltd.) and 2.5 μg / mL Fungizone (Bristol-Myers Squibb). The cells were cultured for 3 days, and on day 3, activation of T cells and morphological changes were confirmed under a microscope.

[0084] (Monitoring for tumor recurrence) First, 8-week-old female NSG immunodeficient mice were treated with 2 × 10 6 The above ACC-MESO1-GFP-Luc was administered intrathoracically to 1 × 10 cells / mouse. Tumor engraftment in the thoracic cavity was confirmed using an in vivo imaging system (IVIS) on day 1. On day 1, the above conventional CAR-expressing T cells, anti-human mesothelin CAR-IL-7-CCL19-expressing T cells, and T cells activated by the above method, which had been prepared from peripheral blood mononuclear cells (PBMCs) of healthy donors and cryopreserved, were thawed. The CAR expression rates of the above conventional anti-human mesothelin CAR-expressing T cells and the above anti-human mesothelin CAR-IL-7-CCL19-expressing T cells were 49.6% and 32.5%, respectively. The above activated T cells were added to the conventional anti-human mesothelin CAR-expressing T cells to combine the CAR expression rates of both, and then 1 × 10 5 A group receiving the conventional anti-human mesothelin CAR-expressing T cells (N=5), 1×10 5A group (N=5) was prepared to receive the above-mentioned anti-human mesothelin CAR-IL-7-CCL19 expressing T cells from conventional mice. The administration of conventional anti-human mesothelin CAR-expressing T cells and anti-human mesothelin CAR-IL-7-CCL19 expressing T cells was performed intravenously via the tail vein. Furthermore, from day 3 onwards, the tumor fluorescence intensity was measured using IVIS (luminescence amount: Total Flux (photons / sec)). The results are shown in Figures 9A and 9B. FIG. 10 is a graph showing the relationship between the number of days from administration and the survival rate of the mice in the above results, and FIG. 11 is a graph showing the relationship between the number of days from administration and the total fluorescence amount (photons / second). In Figures 9A and 9B, 10 and 11, the mice administered with anti-human mesothelin CAR-IL-7-CCL19-expressing T cells are shown as "7x19 CAR-T", and the mice administered with conventional anti-mesothelin CAR-expressing T cells are shown as "Conventional CAR-T". In this Example 6, NSG immunodeficient mice lacking endogenous T cells are used as recipients, so the influence of the endogenous T cells of the recipients is eliminated, and the effect of the administered anti-human mesothelin CAR-IL-7-CCL19-expressing T cells themselves is evaluated.

[0085] As shown in Figures 9A and 9B, 10, and 11, almost no tumor fluorescence intensity was observed in either 7x19 CAR-T or conventional CAR-T around day 21. In 7x19 CAR-T, no tumor fluorescence was observed until day 143, confirming that recurrence was completely suppressed. On the other hand, in conventional CAR-T, tumor fluorescence began to be observed around day 45, tumor fluorescence intensity increased on day 115, one mouse died on day 129, and the remaining four mice died on day 143. Therefore, it was revealed that the administration of CAR-IL-7-CCL19-expressing T cells, that is, the secretion of IL-7 and CCL19 locally in the tumor and the destruction of tumor cells, can suppress the recurrence of malignant tumors.

[0086] [Example 7] <Memory of recipient's endogenous T cells> In the above, we used T cells bearing CAR as a cell surface molecule that recognizes malignant tumor antigens, but we also investigated the memory formation of the recipient's endogenous T cells using T cells bearing T cell receptor (TCR) as a cell surface molecule that recognizes malignant tumor antigens instead of CAR.

[0087] (Generation of P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells) T cells expressing TCR specific to P1A, the tumor antigen of P815, mouse IL-7, mouse CCL19, and GFP were produced according to the method described in the above-mentioned Patent Document 3. Briefly, the procedure is as follows.

[0088] An IL-7-F2A-CCL19 DNA fragment encoding mouse IL-7 (without a stop codon), followed by a picornavirus-derived 2A peptide (F2A), and mouse CCL19 was artificially synthesized (Life Technologies). The IL-7-F2A-CCL19 DNA fragment synthesized above was inserted into the multicloning site of a pMSGV retrovirus expression vector (Patent Document 3) having an F2A-eGFP sequence by restriction enzyme (NCOI and ECORI) treatment and ligation to obtain a pMSGV vector (IL-7×CCL19-eGFP expression vector) containing an IL-7-F2A-CCL19-F2A-eGFP DNA fragment (SEQ ID NO: 9). In addition, a pMSGV vector (eGFP control vector) containing eGFP but not containing IL-7 or CCL19 was prepared as a control. In SEQ ID NO:9, bases 1 to 462 are IL-7 (bases 1 to 75 are the IL-7 signal sequence), bases 463 to 537 are F2A, bases 538 to 861 are CCL19 (bases 538 to 612 are the CCL19 signal sequence), bases 868 to 942 are F2A, bases 946 to 1662 are a nucleic acid encoding eGFP, and bases 1663 to 1665 are a stop codon.

[0089] Obtained from Y. Liu, H-2L dSpleen cells were collected from a transgenic mouse expressing a TCR specific to P1A, a tumor antigen of the restricted P815 (Sarma, S., Y. Guo, Y. Guilloux, C. Lee, X.-F. Bai, Y. Liu. 1999. J. Exp. Med. 189:811.), and mouse T cells expressing a TCR specific to P1A, a tumor antigen of the P815 derived from the spleen cells (P1A-specific TCR-T cells) were obtained. Next, retroviruses were prepared by introducing an IL-7×CCL19-eGFP expression vector and an eGFP control vector, and spleen cells (3×10 6 7 × 19 cells / well) were transduced into cells activated with P1A peptide for 48 hours to obtain P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells (conv. P1A-CTL) or P1A-specific TCR / eGFP-expressing T cells (conv. P1A-CTL). 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 all experiments.

[0090] CD8 in spleen cells when mice were treated with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells or P1A-specific TCR / eGFP-expressing T cells, respectively + GFP + Cell percentage and CD8 + GFP + The results of flow cytometry analysis of the absolute cell counts are shown in Figure 12 (a) and (b). When spleen cells were treated with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells, CD8 + GFP + Cell percentage and CD8 + GFP + An increase in the absolute cell number was confirmed.

[0091] Next, CD8 +CD8 of mice treated with splenocytes and P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells + GFP - or CD8 + GFP + The results of analyzing the expression of CD44 in splenocytes by flow cytometry are shown in FIGS. 13(a) and (b). FIG. 13(a) shows the cell number of representative CD44 + cells, and FIG. 13(b) shows the proportion of CD44 + cells. As shown in FIGS. 13(a) and (b), not only the administered T cells (GFP + gated), but also the recipient's endogenous T cells (GFP - gated) had a more than two-fold increase in the proportion of CD44 + compared to naive T cells. Therefore, it was confirmed that P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells have the effect of memoryizing the recipient's endogenous T cells, that is, inducing the memory function of the recipient's endogenous T cells and enhancing the memory function of the recipient's endogenous T cells.

[0092] Furthermore, to confirm the function of memoryization, that is, the acquisition of responsiveness to stimulation, it was examined by the production of IFN-γ. T cells were magnetically isolated from splenocytes and co-cultured with mucosal mast cells (MMC) treated with P815 for about 5 days. The results of detecting the concentration of IFN-γ in the culture supernatant by ELISA (Enzyme-Linked Immuno Sorbent Assay) are shown in FIG. 14. As shown in FIG. 14, it was confirmed that P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells produced a large amount of IFN-γ. Therefore, it was confirmed that P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells improved the antitumor activity of the recipient's endogenous T cells. From this, it was considered that a recurrence suppression effect could be induced in the recipient.

[0093] [Example 8] [Virus expressing IL-7 and CCL19] In the above, we used immunocompetent cells as a nucleic acid delivery vehicle, but if IL-7 and CCL19 are secreted locally in the tumor, it should be possible to enhance T cells or B cells with memory function in the subject and suppress the recurrence of malignant tumors without using immunocompetent cells. Therefore, we performed an analysis using viruses instead of immunocompetent cells as a nucleic acid delivery vehicle.

[0094] (Construction of recombinant vaccinia viruses expressing mouse IL-7 and mouse CCL19) The recombinant vaccinia virus expressing mouse IL-7 and mouse CCL19 was prepared according to the method described in the above-mentioned Patent Document 3 and WO 2011 / 125469 pamphlet as follows. The blue fluorescent protein (BFP) gene region was amplified using two primers (5'-ATG GCC GGA CCG GCC ACC GGT CGC CAC CAT GAG CGA G-3': SEQ ID NO: 10) and (5'-TCG AAT TCG CTA GCG GCC GCT TAA TTA AGC TTG TGCCCC AG-3': SEQ ID NO: 11) with the template. The PCR product was cleaved with restriction enzymes SfiI and EcoRI, and cloned into the same restriction enzyme site of the pTK-SP-LG vector (International Publication No. 2011 / 125469), and pTK-SP-BFP was constructed by linking BFP under a synthetic vaccinia virus promoter (Hammond JM. et al., Journal of Virological Methods. 1997; 66(1):135-138). Next, pAmCyan1-N1 vector (Takara Bio) was cleaved with restriction enzymes AgeI and NotI, and the fluorescent protein AmCyan1 fragment was cloned into the site of pTK-SP-BFP treated with the same restriction enzymes to construct pTK-SP-AmCyan1.

[0095] The pMSGV plasmid (Patent Document 3) containing a DNA fragment encoding mouse IL-7, followed by picornavirus-derived 2A peptide (F2A), mouse CCL19, F2A, and eGFP was cleaved with the restriction enzyme BamHI, followed by blunt treatment and then cleavage with NcoI to obtain a mouse IL-7-F2A-mouse CCL19-F2A-eGFP fragment. pTK-SP-AmCyan1 was cleaved with the restriction enzyme NheI, followed by blunt treatment, and cloned into the site cleaved with NcoI to construct the transfer vector plasmid pTK-SP-mouse IL-7-F2A-mouse CCL19-F2A-eGFP.

[0096] Using pGL4.20 (Promega) DNA as a template, the firefly luciferase gene region was amplified with two primers (5'-GCT CCG GAC GCC ACC ATG GAA GAT GCC AAA AAC-3' (SEQ ID NO: 12) and 5'-GCG AAT TCC ACG GCG ATC TTG CCG CCC TTC T-3' (SEQ ID NO: 13)). The PCR product was digested with the restriction enzymes BspEI and EcoRI to obtain a Luc fragment, and pTK-SP-mouse IL-7-F2A-mouse CCL19-F2A-eGFP was digested with the restriction enzymes EcoRI and BsrGI to obtain a fragment containing a part of eGFP. These fragments were then simultaneously cloned into the site of pTK-SP-mouse IL-7-F2A-mouse CCL19-F2A-eGFP digested with the restriction enzymes BspEI and BsrGI to construct the transfer vector plasmid pTK-SP-Luc-F2A-eGFP.

[0097] To recover recombinant vaccinia viruses having the viral genome shown in Figures 15(a) and (b), CV1 cells cultured to 80% confluence in a 6-well dish were infected with vaccinia virus (LC16mO) at MOI = 0.02 to 0.1, and after adsorption at room temperature for 1 hour, transfer vector plasmid DNA (pTK-SP-mouse IL-7-F2A-mouse CCL19-F2A-eGFP or pTK-SP-Luc-F2A-eGFP) mixed with FuGENE HD (Promega) was added to the cells according to the manual for uptake, and cultured at 37 ° C for 2 to 5 days. The cells were recovered, frozen and thawed, sonicated, appropriately diluted, and inoculated into nearly confluent BSC1 cells, and Eagle MEM containing 0.8% methylcellulose and 5% FBS medium were added and cultured at 37 ° C for 2 to 5 days. The medium was removed, and the BFP-expressing plaques were scraped off with the tip of a tip and suspended in Opti-MEM medium (Invitrogen). This procedure was repeated three more times with BSC1 cells to purify the plaques. After sonication of the plaque suspension collected after plaque purification, genomic DNA was extracted from 200 μL of the suspension using the High Pure Viral Nucleic Acid Kit (Roche) according to the manual and subjected to PCR screening. PCR was performed using two primers (5'-ATT TCT CCG TGA TAG GTA TCG ATG-3' (SEQ ID NO: 14) and 5'-AAC GGT TTA CGT TGA AAT GTC C-3' SEQ ID NO: 15), and the base sequence of the PCR product was confirmed by direct sequencing for clones in which a PCR product of a predetermined size was detected. A viral clone with no problem in the base sequence was selected and amplified in A549 cells, after which the viral titer was measured in RK13 cells, and the recombinant vaccinia virus LC16mO TK-SP-mouse IL-7-F2A-mouse CCL19-F2A-eGFP (TK-ICE: Fig. 15(a)) or LC16mO TK-SP-Luc-F2A-eGFP (TK-LE: Fig. 15(b)) was used in the following experiments.

[0098] A549 cells (1×10 5cells / well) or CT26 cells (5 × 10 4 After seeding in a 24-well plate, cells (cells / well) were cultured at 37°C, and infected with TK-ICE or TK-LE at MOI=0.1 for A549 cells or MOI=10 for CT26 cells (n=3). 24, 48, and 72 hours after infection, the cells were observed under a fluorescent microscope (Olympus) (Figure 16), and the supernatant was collected. The supernatants collected after 24, 48, and 72 hours were diluted 100-fold, and the secretion amounts of IL-7 and CCL19 in 0.5 mL of the supernatant were measured using DuoSet ELISA Mouse IL-7 (DY407, R&D Systems), DuoSet ELISA Mouse CCL-19 (DY440, R&D Systems), and DuoSet Ancillary Reagent Kit2 (DY008, R&D Systems). The measurement results are shown in Figure 17.

[0099] As shown in Figures 16 and 17, eGFP fluorescence expression in A549 cells and CT26 cells at 24, 48, and 72 hours after infection was comparable between TK-ICE and TK-LE. In A549 cells, eGFP fluorescence was detected in almost all cells at 48 hours after infection, and in CT26 cells, eGFP fluorescence expression increased over time. In A549 cells and CT26 cells infected with TK-ICE, IL-7 and CCL19 were detected from 24 hours after infection, reached a plateau at 48 hours after infection in A549 cells, and increased over time in CT26 cells. On the other hand, IL-7 and CCL19 were below the detection limit when infected with TK-LE. These results confirmed that TK-ICE destroys tumor cells and secretes IL-7 and CCL19.

[0100] [Example 9] <Anti-tumor effect> It was confirmed from Example 8 that the recombinant vaccinia virus TK-ICE prepared above infects tumor cells, destroys the cancer cells, and secretes IL-7 and CCL19. Therefore, the antitumor effect of the recombinant vaccinia virus TK-ICE was examined.

[0101] As shown in FIG. 18, mouse colon cancer CT26 cells (5×10 5 The recombinant vaccinia viruses (3-5 × 10 7 The larger of the tumors formed in both abdomens was selected and injected with 1000 plaque-forming units (PFU) into the tumor three times (days 0, 2, and 4). The tumor diameters of both abdomens were then measured to determine the antitumor effect of the virus. 3 The tumor on the non-treated side was 24-82 mm 3 The results are shown in Figure 19.

[0102] From the results of FIG. 19, the TK-LE-administered group (N=7) which does not express IL-7 and CCL19 suppressed tumor growth in the tumor on the administration side compared to the PBS-administered group (N=8), but no inhibitory effect on the tumor was observed in the non-administered group. In contrast, the TK-ICE-administered group (N=4) which expresses IL-7 and CCL19 suppressed tumor growth not only in the tumor on the administration side but also in the tumor on the non-administered side compared to the PBS-administered group (N=4). Although not shown in the figure, it was confirmed that the recombinant vaccinia virus administered to the tumor on one ventral side did not transfer to the tumor on the other ventral side by a method of non-invasively detecting the virus by administering a substrate (luciferin) to the luminescent enzyme (luciferase) expressed in tumor cells infected with TK-LE and confirming the presence or absence of luminescence. Therefore, it is believed that the destruction of tumor cells by TK-ICE, along with the secretion of IL-7 and CCL19 locally in the tumor, induces tumor immunity in the treated subject and enhances the memory function of endogenous T cells, thereby not only causing the disappearance of the tumor in the treated subject but also inhibiting the growth of the tumor in the non-treated subject, thereby suppressing the recurrence of the malignant tumor.

Claims

1. a nucleic acid delivery vehicle, a nucleic acid encoding interleukin 7 (IL-7), and a nucleic acid encoding chemokine (C-C motif) ligand 19 (CCL19); Induce memory of endogenous immune cells, Inducer.

2. The inducer according to claim 1 , wherein the nucleic acid delivery vehicle is at least one selected from the group consisting of immunocompetent cells, viruses, anaerobic bacteria, liposomes, mesenchymal stem cells (MSCs), and nanoparticles.

3. The inducer according to claim 1 or 2, wherein the nucleic acid delivery vehicle has an ability to accumulate in malignant tumor cells or an ability to specifically proliferate in malignant tumor cells.

4. The inducer according to any one of claims 1 to 3, wherein the nucleic acid delivery vehicle has an ability to cytotoxicity against malignant tumor cells.

5. The inducer according to any one of claims 1 to 4, wherein the nucleic acid delivery vehicle is an immunocompetent cell, and the immunocompetent cell has a cell surface molecule that recognizes a malignant tumor antigen.

6. the nucleic acid delivery vehicle is an immunocompetent cell having a cell surface molecule that recognizes a malignant tumor antigen; The inducer according to any one of claims 1 to 5, characterized in that the inducer is useful for malignant tumor recurrence or malignant tumor metastasis caused by malignant tumor cells that do not have a malignant tumor antigen specifically recognized by the cell surface molecule.

7. The inducer according to claim 5 or 6, wherein the cell surface molecule that recognizes the malignant tumor antigen is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

8. The inducer according to any one of claims 5 to 7, wherein the immunocompetent cells are T cells.

9. The inducing agent according to any one of claims 1 to 8, wherein the nucleic acid delivery vehicle comprises a nucleic acid encoding interleukin 7 (IL-7) and a nucleic acid encoding chemokine (CC motif) ligand 19 (CCL19).

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