Car expression vector and car expression t cells

Abstract

To provide CAR-expressing T cells that express the immune function-promoting factor of T cells together with chimeric antigen receptor (CAR) in T cells and have high immune-inducing effect and antitumor activity; and to provide CAR expression vectors for producing such CAR expression T cells.SOLUTION: Produced are a CAR expression vector containing a nucleic acid encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding an immune function promoting factor of T cells, the nucleic acid encoding the immune function promoting factor being a nucleic acid encoding interleukin 7, a nucleic acid encoding CCL19, and a nucleic acid encoding a dominant negative variant for SHP-1, or a nucleic acid encoding a dominant negative variant for SHP-2; and a CAR-expressing T cell into which the CAR expression vector has been introduced.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to a CAR expression vector, a CAR-expressing T cell introduced with a CAR expression vector, and an anticancer agent containing the CAR-expressing T cell. [Background technology]

[0002] A chimeric antigen receptor (hereinafter, also referred to as "CAR") is an artificial chimeric protein that combines a single-chain antibody that recognizes cell surface antigens of cancer cells with a signal transduction domain that induces T cell activation. As shown in Figure 1, by introducing a gene encoding a CAR into normal peripheral blood T cells (peripheral blood T lymphocytes) that do not have tumor reactivity, it is possible to produce a large number of CAR-expressing T cells (hereinafter, also referred to simply as "CAR-T cells") that can express a CAR. Such CAR-T cells have tumor reactivity and can induce damage to cancer cells without depending on interactions with major histocompatibility complex (MHC).

[0003] Cancer immunotherapy using CAR-T cells, more specifically, a therapy in which T cells are collected from a patient, a gene encoding CAR is introduced into the T cells, the cells are amplified, and the cells are then transferred back to the patient (see Non-Patent Document 1), is currently undergoing clinical trials around the world, and results have shown its effectiveness in treating hematopoietic malignancies such as leukemia and lymphoma.

[0004] In recent years, various researches on CAR-T cells have been conducted. For example, there are a pharmaceutical composition comprising modified autologous human T cells comprising a nucleic acid encoding a CAR consisting of a CD19 antigen-binding region, a transmembrane region, a 4-1BB costimulatory signal region, and a CD3ζ signal region (see Patent Document 1), a T cell population expressing one or more therapeutically effective anti-tag chimeric antigen receptors (AT-CARs) that bind to a tagged protein and induce cancer cell death, which is administered to a subject simultaneously or separately with a combination of one or more tagged proteins that bind to cancer cells (see Patent Document 2), a cell comprising a nucleic acid encoding a chimeric antigen receptor comprising an antigen-binding domain of human antibody 139, an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain (see Patent Document 3), and a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor, the chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a sequence sequence represented by SEQ ID NO: 1. Proposed are cells containing a CD3ζ signaling domain containing the amino acid sequence of NO:24 (see Patent Document 4); genetically engineered CD19-specific T cells that express and possess a CD19-specific chimeric receptor on the cell surface membrane, the chimeric receptor consisting of an intracellular signaling domain for the effector function of immune cells, at least one transmembrane domain and at least one extracellular domain, the extracellular domain containing a CD19-specific receptor (see Patent Document 5); and chimeric antigen receptor-expressing cells into which a nucleic acid encoding a chimeric antigen receptor containing the intracellular domain of glucocorticoid-induced tumor necrosis factor receptor (GITR) as the intracellular domain has been introduced (see Patent Document 6).

[0005] However, the problems of low survival efficiency of CAR-T cells in vivo, insufficient activation of endogenous T cells induced by CAR-T cells, and insufficient accumulation in tumor sites, as well as the inhibition of CAR-T cell activity by immunosuppressive signals via the PD-L1 / PD-1 pathway, which is a tumor immune evasion mechanism possessed by cancer cells, and immunosuppressive factors such as TGF-β and IL-10 secreted in the tumor microenvironment, have not been resolved by the techniques used up to now. Therefore, there are cancer types and cases in which sufficient therapeutic effects are not observed, and there is a demand for the creation of more effective CAR-T cells and expression vectors for producing such CAR-T cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US Patent Application Publication No. 2014 / 0106449 [Patent Document 2] Special Publication No. 2014-504294 [Patent Document 3] Special Publication No. 2014-516510 [Patent Document 4] Special Publication No. 2014-507118 [Patent Document 5] JP 2011-004749 A [Patent Document 6] International Publication No. 2013 / 051718 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Hirozo Nakazawa Shinshu Medical Journal 61(4):197~203(2013) Summary of the Invention [Problem to be solved by the invention]

[0008] In conventional CAR-T cells, the activation ability of T cells has been enhanced by incorporating CD28, 4-1BB, CD3ζ, etc. into the signal transduction region of the CAR, but the immune induction effect of endogenous T cells by CAR-T cells and resistance to the immune suppressive mechanism in the tumor microenvironment have not been sufficiently enhanced, and the therapeutic effect of CAR-T cells against solid cancers has not yet been achieved. Therefore, the object of the present invention is to provide CAR-T cells that express a T cell immune function promoting factor together with a CAR in T cells and have high immune induction effect and antitumor activity, and a CAR expression vector for producing such CAR-T cells. [Means for solving the problem]

[0009] The inventors attempted to improve CAR-T cells in order to achieve better immune induction effects and antitumor activity in cancer immunotherapy using CAR-T cells. In the process, they focused on cytokines, chemokines, and signal regulatory proteins, which are factors that promote the immune function of T cells, and constructed vectors that express CAR and factors that promote the immune function of T cells. When such expression vectors were introduced into T cells, they found that CAR-T cells with better immune induction effects and antitumor activity than conventional CAR-T cells could be produced, and thus completed the present invention.

[0010] That is, the present invention is as disclosed below. (1) A CAR expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding a T cell immune function-enhancing factor, wherein the nucleic acid encoding the immune function-enhancing factor is a nucleic acid encoding interleukin-7 and a nucleic acid encoding CCL19, a nucleic acid encoding a dominant-negative mutant of SHP-1, or a nucleic acid encoding a dominant-negative mutant of SHP-2. (2) The CAR expression vector according to (1) above, characterized in that the nucleic acid encoding the immune function-promoting factor is a nucleic acid encoding interleukin-7 and a nucleic acid encoding CCL19. (3) The CAR expression vector according to (2) above, characterized in that a nucleic acid encoding a CAR and a nucleic acid encoding a T cell immune function-enhancing factor are linked via a sequence encoding a self-cleaving peptide. (4) The CAR expression vector according to (2) or (3) above, characterized in that the nucleic acid encoding interleukin-7 and the nucleic acid encoding CCL19 are linked via a sequence encoding a self-cleaving peptide. (5) A CAR expression vector according to any one of (1) to (4) above, characterized in that the nucleic acid encoding the CAR contains a nucleic acid encoding a polypeptide of a single-chain antibody that recognizes FITC or CD20. (6) A CAR expression vector according to any one of (1) to (5) above, characterized in that the nucleic acid encoding the CAR contains a nucleic acid encoding a polypeptide of the CD8 transmembrane domain. (7) A CAR expression vector according to any one of (1) to (6) above, characterized in that the nucleic acid encoding the CAR contains a nucleic acid encoding a polypeptide of the intracellular region of CD28, the intracellular region of 4-1BB, and the intracellular region of CD3ζ. (8) CAR-expressing T cells transfected with the vector shown in (a) or (b) below. (a) a CAR expression vector according to any one of (1) to (7) above; (b) a CAR expression vector containing a nucleic acid encoding a CAR and a nucleic acid encoding interleukin 7, and a CAR expression vector containing a nucleic acid encoding a CAR and a nucleic acid encoding CCL19; (9) An anticancer agent comprising the CAR-expressing T cells described in (8) above and a pharma- ceutical acceptable additive. Effect of the Invention

[0011] By using the CAR expression vector of the present invention, it is possible to produce CAR-T cells that have survival ability, lymphocyte accumulation ability, and tumor cytotoxicity, as well as CAR-T cells that are resistant to immunosuppression in the cancer microenvironment. If such CAR-T cells are used in immunotherapy for cancer patients, a strong cancer treatment effect is expected, and effective cancer immunotherapy can be achieved even against refractory and progressive cancers. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the structure of CAR and the basic system of cancer immunotherapy using CAR-T cells. [Diagram 2] FIG. 1 depicts vectors expressing CAR, interleukin 7 (IL-7) and CCL19. [Diagram 3] 1 shows the results of confirming the expression level of CAR in anti-FITC CAR-IL-7 / CCL19-expressing T cells by flow cytometry-1. The left shows unstained CAR, and the right shows stained CAR. [Figure 4] FIG. 1 shows the results of confirming the expression level of CAR in anti-FITC CAR-IL-7 / CCL19-expressing T cells by flow cytometry-2. [Diagram 5] This figure shows the results of confirming the CAR expression level in anti-human CD20 CAR-IL-7 / CCL19-expressing T cells by flow cytometry. [Figure 6] FIG. 1 shows the results of ELISA measurement of the concentrations of IL-7 and CCL19 in the cell supernatant of anti-FITC CAR-IL-7 / CCL19-expressing T cells. [Figure 7] FIG. 2 shows the results of ELISA measurement of the concentrations of IL-7 and CCL19 in the cell supernatant of anti-FITC CAR-IL-7 / CCL19-expressing T cells. [Figure 8] FIG. 1 shows the results of measuring the concentrations of IL-7 and CCL19 in the cell supernatants of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells by ELISA. [Figure 9]This figure shows the number of cells when anti-FITC CAR-IL-7 / CCL19-expressing T cells were stimulated and cultured for 3 days, 5 days, and 7 days. [Figure 10] FIG. 1 shows the survival rate of anti-FITC CAR-IL-7 / CCL19 expressing T cells when stimulated and cultured for 3, 5, and 7 days. [Figure 11] FIG. 1 shows the number of cells when anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were stimulated and cultured for 5 days. [Figure 12] FIG. 1 shows the results of a T cell migration test using anti-FITC CAR-IL-7 / CCL19-expressing T cells-1. [Figure 13] FIG. 13 shows the results of T cell migration test-2 using anti-FITC CAR-IL-7 / CCL19-expressing T cells. [Figure 14] FIG. 1 shows the results of a dendritic cell migration assay using anti-FITC CAR-IL-7 / CCL19-expressing T cells. [Figure 15] FIG. 1 shows the results of a T cell migration assay using anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. [Figure 16] FIG. 1 shows the results of examining the proliferation ability of anti-FITC CAR-IL-7 / CCL19-expressing T cells (5 days after stimulation). [Figure 17] FIG. 1 shows the results of examining the proliferation ability of anti-FITC CAR-IL-7 / CCL19-expressing T cells (3 and 7 days after stimulation). [Figure 18] Fig. 1 shows the results of examining CD127 expression in anti-FITC CAR-IL-7 / CCL19-expressing T cells. [Figure 19] FIG. 1 shows the results of examining CCR7 expression in anti-FITC CAR-IL-7 / CCL19-expressing T cells. [Figure 20] FIG. 1 shows the results of investigating changes in tumor volume when anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were administered to cancer-bearing mice. [Figure 21]FIG. 1 shows the results of investigating the survival rate of tumor-bearing mice when anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were administered to the mice. [Figure 22] This figure shows the results of investigating the mouse survival rate when anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were administered to mice that had been subcutaneously inoculated with P815-hCD20 and then administered cyclophosphamide. [Diagram 23] This figure shows the results of examining the tumor volume in mice that were subcutaneously inoculated with P815-hCD20 and then administered cyclophosphamide, and then administered anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. [Figure 24] This is a diagram in which the values ​​on the vertical axis of the graph of CPA+7×19 in FIG. 23 are divided by 10. [Diagram 25] FIG. 13 shows the results of H&E staining of tumor tissues in mice subcutaneously inoculated with P815-hCD20 and then administered anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. [Figure 26] FIG. 1 shows the results of immunohistochemical analysis of tumor tissues in mice subcutaneously inoculated with P815-hCD20 and then administered anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. [Figure 27] FIG. 27 shows the results of quantification of the positive areas labeled by fluorescent staining in FIG. 26. [Figure 28] FIG. 1 shows the results of examining the tumor volume when mice were subcutaneously inoculated with P815-hCD20 and then administered anti-human CD20 CAR-IL-7-expressing T cells, anti-human CD20 CAR-CCL19-expressing T cells, or anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. [Figure 29](a) Diagram of vector expressing dominant-negative mutants of CAR and SHP1 (Src homology region 2 domain-containing phosphatase-1). (b) Diagram of vector expressing dominant-negative mutants of CAR and SHP2 (Src homology region 2 domain-containing phosphatase-2). [Diagram 30] 1A and 1B show the results of a cytotoxicity test using anti-human CD20 CAR-SHP1DN and anti-human CD20 CAR-SHP2DN-expressing T cells, respectively. [Diagram 31] FIG. 1 shows the results of examining tumor cytotoxicity by mixing P815-hCD20 in the presence of anti-FITC CAR-IL-7 / CCL19-expressing T cells and FITC-conjugated rituximab. [Diagram 32] FIG. 1 shows the results of examining tumor cytotoxicity by mixing P815-hCD20 with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. [Diagram 33] This figure shows the results of flow cytometry analysis of the leukocyte surface phenotypes, CD4, CD8, CD44, and CD62L, in mice subcutaneously inoculated with P815-hCD20 and then administered anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. [Diagram 34] FIG. 1 shows the results of splenic leukocytes being stimulated by culturing with mitomycin C-treated P815-hCD20 for 4 days, and examining T cell proliferation by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The CAR expression vector of the present invention is not particularly limited as long as it contains a nucleic acid encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding a T cell immune function-enhancing factor, and the nucleic acid encoding the immune function-enhancing factor is a nucleic acid encoding interleukin-7 and a nucleic acid encoding CCL19, a nucleic acid encoding a dominant negative mutant of SHP-1, or a nucleic acid encoding a dominant negative mutant of SHP-2. The chimeric antigen receptor refers to an artificial chimeric protein in which a single-chain antibody that recognizes a cell surface antigen of a cancer cell is fused with a signal transduction domain that induces T cell activation via a cell membrane-spanning domain.

[0014] In the present invention, the nucleic acid encoding a CAR is not particularly limited as long as it encodes a polypeptide constituting a CAR, and includes nucleic acids encoding polypeptides of a single chain antibody that recognizes a cell surface antigen of a cancer cell, a cell membrane-spanning domain, and a signal transduction domain that induces T cell activation.

[0015] The single-chain antibody in the CAR can be an oligo- or polypeptide consisting of a light chain variable region and a heavy chain variable region (scFv) derived from the antigen-binding site of a monoclonal antibody, with a linker peptide located between the light chain variable region and the heavy chain variable region.

[0016] The cell surface antigen of a cancer cell recognized by the single chain antibody may be a biomolecule that is specifically expressed in cancer cells and their precursor cells, a biomolecule whose expression has been newly confirmed due to the malignant transformation of a cell, or a biomolecule whose expression level is increased in cancer cells compared to normal cells, and examples of such a biomolecule include CD20, EGFR, FITC, CD19, CD22, CD33, PSMA, GD2, EGFR variant, ROR1, c-Met, HER2, CEA, mesothelin, GM2, CD7, CD10, CD30, CD34, CD38, CD41, CD44, CD74, CD123 CD133, CD171, MUC16, MUC1, CS1 (CD319), IL-13Ra2, BCMA, LewisY, IgG kappa chain, Folate receptor-alpha, PSCA, and EpCAM.

[0017] The T cell activation signaling region is a region capable of transmitting a signal intracellularly when the single chain antibody recognizes a cell surface antigen of a cancer cell, and preferably comprises at least one or more polypeptides selected from the group consisting of polypeptides of the intracellular regions of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ, and Fc Receptor-associated γchain, and more preferably comprises polypeptides of the intracellular regions of the three types of CD28, 4-1BB, and CD3ζ.

[0018] The polypeptides of the respective intracellular domains may be linked via an oligopeptide linker or polypeptide linker consisting of 2 to 10 amino acids, and an example of such a linker sequence is a consecutive glycine-serine sequence.

[0019] The transmembrane domain in the present invention may be a polypeptide derived from a transmembrane domain of CD8, α or β chain of T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or GITR, and preferably a polypeptide derived from a transmembrane domain of human CD8. The transmembrane domain fixes the CAR to the cell membrane of the T cell.

[0020] The transmembrane domain may include a hinge domain consisting of any oligopeptide or polypeptide and having a length of 1 to 100 amino acids, preferably 10 to 70 amino acids. An example of the hinge domain is the hinge domain of human CD8.

[0021] Furthermore, a spacer region consisting of any oligopeptide or polypeptide may be provided between the single chain antibody that recognizes a cell surface antigen of a cancer cell and the transmembrane domain, and between the transmembrane domain and the T cell activation signal transduction domain. The length of the spacer region can be 1 to 100 amino acids, preferably 10 to 50 amino acids, and an example of such a spacer region is a consecutive glycine-serine sequence.

[0022] In the present invention, the nucleic acid encoding a T cell function-promoting factor is not particularly limited as long as it is a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 (hereinafter also referred to as "the present nucleic acid 1"), a nucleic acid encoding a dominant negative mutant of SHP-1 (hereinafter also referred to as "the present nucleic acid 2"), or a nucleic acid encoding a dominant negative mutant of SHP-2 (hereinafter also referred to as "the present nucleic acid 3"), and may contain a plurality of each of the present nucleic acids 1 to 3, specifically, a nucleic acid containing the present nucleic acid 1 and the present nucleic acid 2, the present nucleic acid 1 and the present nucleic acid 3, the present nucleic acid 2 and the present nucleic acid 3, or the present nucleic acid 1, the present nucleic acid 2 and the present nucleic acid 3.

[0023] The nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 in the present nucleic acid 1 may include a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19, and the nucleic acid encoding CCL19 may be located upstream or downstream of the nucleic acid encoding IL-7.

[0024] Nucleic acids encoding dominant-negative mutants of SHP1 are not particularly limited as long as they are nucleic acids encoding mutants of SHP1 that act dominantly over SHP1 and can inhibit the action of SHP1, and examples thereof include nucleic acids encoding mutants consisting of an amino acid sequence in which at least one amino acid in the amino acid sequence of SHP1 is substituted with another amino acid and capable of inhibiting the action of SHP1. Nucleic acids encoding dominant-negative mutants of SHP2 are not particularly limited as long as they are nucleic acids encoding mutants of SHP2 that act dominantly over SHP2 and can inhibit the action of SHP2, and examples thereof include nucleic acids encoding mutants consisting of an amino acid sequence in which at least one amino acid in the amino acid sequence of SHP2 is substituted with another amino acid and capable of inhibiting the action of SHP2.

[0025] In the CAR expression vector of the present invention, any nucleic acid may be contained between the nucleic acid encoding the chimeric antigen receptor and the nucleic acid encoding the T cell immune function enhancing factor, between each of the nucleic acids in the case where the vector contains a plurality of the present nucleic acid 1, the present nucleic acid 2, and the present nucleic acid 3, and between the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 in the present nucleic acid 1, as long as each of the nucleic acids can be expressed, but it is preferable that the nucleic acids are linked via a sequence encoding a self-cleaving peptide (2A peptide) or an IRES (internal ribozyme entry site), preferably a sequence encoding the 2A peptide. Linking the nucleic acids using such a sequence enables efficient expression of each of the nucleic acids.

[0026] The 2A peptide is a self-cleaving peptide derived from a virus, and is characterized by being cleaved in the endoplasmic reticulum between the GPs (one residue from the C-terminus) in the amino acid sequence shown in SEQ ID NO: 1 (Szymczak et al., Expert Opin. Biol. Ther. 5(5):627-638(2005)). Therefore, the nucleic acids incorporated before and after the 2A peptide are expressed independently of each other in the cell.

[0027] The 2A peptide is preferably a 2A peptide derived from a picornavirus, a rotavirus, an insect virus, an aphthovirus, or a trypanosoma virus, and more preferably a 2A peptide derived from a picornavirus shown in SEQ ID NO:2 (F2A).

[0028] A nucleic acid encoding a chimeric antigen receptor can be prepared by known techniques such as chemical synthesis or PCR amplification based on a nucleotide sequence encoding a polypeptide of a single-chain antibody against a cell surface antigen of a cancer cell, a cell membrane-spanning domain, and a T cell activation signaling domain. The codons selected for encoding amino acids may be modified to optimize expression of the nucleic acid in a host cell of interest.

[0029] Information on the nucleotide sequences encoding the polypeptides of single-chain antibodies against cell surface antigens of cancer cells, transmembrane domains, and T cell activation signal transduction domains can be appropriately obtained by searching publicly known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0030] For example, information on the nucleotide sequences encoding the polypeptides of the transmembrane domains of CD28, 4-1BB, and CD3ζ in the T cell activation signal transduction domain can be appropriately obtained by searching databases such as NCBI. For human CD28, Genbank number: NM_006139.2 (updated May 10, 2014); for human 4-1BB, Genbank number: NM_001561.5 (updated March 16, 2014); and for human CD3ζ, Genbank number: NM_000734.3 (updated August 12, 2014) can be given as examples.

[0031] Furthermore, information on the nucleotide sequence encoding the polypeptide of the cell membrane-spanning domain of human CD8 can be appropriately obtained by searching databases such as NCBI, and an example of such information is the one registered under Genbank number: NM_001768.6 (last updated: May 10, 2014).

[0032] Furthermore, information on the base sequence encoding the polypeptide of a single-chain antibody can be obtained by preparing a monoclonal antibody that recognizes a target cell surface antigen, determining the amino acid sequence of the monoclonal antibody by a known method such as the Edman method, and then based on the amino acid sequence. Methods for producing a monoclonal antibody include a method using a hybridoma, a method in which a host is transformed with an expression vector containing an antibody gene by genetic engineering techniques, and a method in which a transgenic animal is immunized with a desired antigen.

[0033] The nucleic acids encoding the T cell immune function-enhancing factors, ie, the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19, the nucleic acid encoding a dominant negative mutant of SHP-1, and the nucleic acid encoding a dominant negative mutant of SHP-2, can be prepared based on their respective nucleotide sequences by known techniques such as chemical synthesis and PCR amplification. Note that the codons selected for encoding amino acids may be modified to optimize the expression of the nucleic acid in the host cell of interest.

[0034] Information on the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19, the nucleic acid encoding a dominant-negative mutant of SHP-1, or the nucleic acid encoding a dominant-negative mutant of SHP-2 can be appropriately obtained by searching publicly known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0035] The nucleic acid encoding IL-7 can be appropriately selected depending on the type of cell into which the CAR expression vector of the present invention is introduced, and examples thereof include a nucleic acid encoding the amino acid sequence of human IL-7 (SEQ ID NO: 3). As long as it has the effect of enhancing the cell proliferation rate in IL-7, a nucleic acid having an 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 nucleic acid sequence shown in SEQ ID NO: 3 may be used.

[0036] The nucleic acid encoding CCL19 can be appropriately selected depending on the type of cell into which the CAR expression vector of the present invention is introduced, and examples thereof include a nucleic acid encoding the amino acid sequence of human CCL19 (SEQ ID NO: 4). As long as it has a T cell migration effect in CCL19, a nucleic acid having an 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 to the nucleic acid sequence shown in SEQ ID NO: 4 may be used.

[0037] The nucleic acid encoding a dominant negative mutant against SHP-1 can be appropriately selected depending on the type of cell into which the CAR expression vector of the present invention is introduced, and can include, for example, a nucleic acid encoding the amino acid sequence (SEQ ID NO: 5) of a dominant negative mutant against human SHP-1. As long as it can inhibit the action of SHP-1 in a dominant negative mutant against SHP-1, a nucleic acid having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more identity with the nucleotide sequence shown in SEQ ID NO: 5 may be used. In addition, in SEQ ID NO: 5, serine at position 453 is the mutation site.

[0038] The nucleic acid encoding a dominant negative mutant against SHP-2 can be appropriately selected depending on the type of cell into which the CAR expression vector of the present invention is introduced, and can include, for example, a nucleic acid encoding the amino acid sequence (SEQ ID NO: 6) of a dominant negative mutant against human SHP-2. As long as it can inhibit the action of SHP-2 in a dominant negative mutant against SHP-2, a nucleic acid having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more identity with the nucleotide sequence shown in SEQ ID NO: 6 may be used. In addition, in SEQ ID NO: 6, serine at position 459 is the mutation site.

[0039] The CAR expression vector of the present invention may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector. Such vectors may also contain 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. By operably arranging a nucleic acid encoding a CAR or a nucleic acid encoding a T cell immune function promoter downstream of a promoter sequence, it becomes possible to efficiently transcribe each nucleic acid. In addition, by including a marker gene, it becomes possible to easily confirm the expression of a nucleic acid encoding a chimeric antigen receptor.

[0040] The CAR expression vector of the present invention may also contain a nucleic acid encoding a suicide gene, and the position of such a suicide gene is not particularly limited, and may be located upstream or downstream of a promoter for expressing a nucleic acid encoding IL-7, a nucleic acid encoding CCL19, a nucleic acid encoding a dominant negative mutant of SHP-1, or a nucleic acid encoding a dominant negative mutant of SHP-2, via a sequence encoding a 2A peptide or an IRES, or may be located downstream of another promoter. By including a nucleic acid encoding a suicide gene in the CAR expression vector of the present invention, it becomes possible to control the number of CAR-expressing T cells in a living body by administering a drug that activates the function of the suicide gene depending on the course of cancer treatment, for example, when a tumor disappears.

[0041] Examples of suicide genes include herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9, which are described in the following literature. Drugs that activate the functions of these genes include ganciclovir for the former and AP1903, a chemical induction of dimerization (CID), for the latter (Cooper LJ., et. al. Cytotherapy. 2006;8(2):105-17., Jensen MC et.al. Biol Blood Marrow Transplant. 2010 Sep;16(9):1245-56., Jones BS. FrontPharmacol. 2014 Nov 27;5:254., Minagawa K., Pharmaceuticals (Basel). 2015 May8;8(2):230-49., Bole-Richard E., Front Pharmacol. 2015 Aug 2015). 25;6:174).

[0042] Examples of the viral vector include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors, and preferably include retroviral vectors, and more preferably include pMSGV vectors (Tamada k et al., Clin Cancer Res 18:6436-6445(2002)) and pMSCV vectors (Takara Bio Inc.). When a retroviral vector is used, the introduced gene is incorporated into the genome of the host cell, allowing for long-term and stable expression.

[0043] The CAR-expressing T cells of the present invention are not particularly limited as long as they are (a) T cells obtained by introducing the CAR expression vector of the present invention, or (b) T cells obtained by introducing at least two vectors, namely, a CAR expression vector containing a nucleic acid encoding a CAR and a nucleic acid encoding interleukin 7 (CAR-IL-7 expression vector), and a CAR expression vector containing a nucleic acid encoding a CAR and a nucleic acid encoding CCL19 (CAR-CCL19 expression vector). The method for introducing the CAR expression vector of the present invention, or the CAR-IL-7 expression vector and the CAR-CCL19 expression vector into T cells is not particularly limited, and examples of the method include known methods such as a viral infection method, a calcium phosphate method, a lipofection method, a microinjection method, and an electroporation method, with the viral infection method being preferred. The above-mentioned CAR-IL-7 expression vector is sufficient as long as it contains a nucleic acid encoding a CAR and a nucleic acid encoding interleukin-7, and the above-mentioned CAR-CCL19 expression vector is sufficient as long as it contains a nucleic acid encoding a CAR and a nucleic acid encoding CCL19, and similarly to the CAR expression vector of the present invention, it may contain other nucleic acids such as a nucleic acid encoding a 2A peptide, an IRES, or a suicide gene, so long as it is capable of expressing each nucleic acid.

[0044] Examples of viral infection methods include transfecting the CAR expression vector and packaging plasmid of the present invention into packaging cells such as GP2-293 cells (Takara Bio), Plat-GP cells (Cosmo Bio), PG13 cells (ATCC CRL-10686), and PA317 cells (ATCC CRL-9078) to produce a recombinant virus, and then infecting T cells with the recombinant virus. This may be performed using a commercially available kit such as the Retrovirus packaging Kit Eco (Takara Bio).

[0045] Introduction of the CAR expression vector of the present invention into T cells can be confirmed by examining the expression of the CAR by flow cytometry, Northern blotting, Southern blotting, PCR such as RT-PCR, ELISA, or Western blotting, or by examining the expression of a marker gene inserted into the vector.

[0046] Examples of T cells include T cells derived from humans and T cells derived from non-human mammals such as dogs, cats, pigs, and mice. T cells can be obtained by isolating and purifying them from body fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, or immune cells infiltrating cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites. Examples of such T cells include αβT cells, γδT cells, CD8 + T cells, CD4 + Examples include T cells, tumor-infiltrating T cells, memory T cells, naive T cells, and NKT cells.

[0047] In the CAR-expressing T cells of the present invention, the expressed single-chain antibody is located outside the cell, and the presence of such a single-chain antibody enables the CAR-expressing T cells to recognize tumor-associated antigens (TAA) expressed on the surface of cancer cells.

[0048] Furthermore, a vector containing a nucleic acid encoding a suicide gene may be introduced into the CAR-expressing T cells of the present invention together with the CAR expression vector of the present invention.

[0049] The anticancer agent of the present invention is not particularly limited as long as it contains the CAR-expressing T cells of the present invention and a pharma- ceutically acceptable additive, and examples of the additive include saline, buffered 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.

[0050] The anticancer agents of the present invention can be administered to a subject in need of cancer treatment using methods known to those skilled in the art, including intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intrathecal, and subarachnoid (cerebrospinal fluid) injection.

[0051] The amount of the CAR-expressing T cells of the present invention contained in the anticancer agent to be administered can be appropriately adjusted depending on the type, location, and severity of the cancer, as well as the age, weight, and condition of the subject to be treated. Preferably, the amount of the CAR-expressing T cells in one administration is 1×10 4 ~1×10 10 pcs, preferably 1 x 10 5 ~1×10 9 pcs, more preferably 5×10 6 ~5×10 8 I can mention a few.

[0052] The anticancer drugs to be administered can be independently administered 4 times, 3 times, 2 times or once a day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, once a week, every 7 days, every 8 days, every 9 days, twice a week, once a month, or twice a month.

[0053] Examples of cancers that can be treated with the anticancer agent of the present invention or the cancer treatment method described below include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated cancer, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, and ovarian cancer; cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue; as well as 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; and leukemia.

[0054] The anticancer agent of the present invention can be used in combination with other anticancer agents. Examples of other anticancer agents include alkylating agents such as cyclophosphamide, bendamustine, 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, kinase inhibitors such as imatinib, gefetinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib, proteasome inhibitors such as bortezomib, and cyclophosphamide. Examples of such anticancer antibiotics include calcineurin inhibitors such as porin 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. Preferred examples of such anticancer antibiotics include alkylating agents or metabolic antagonists, and cyclophosphamide.

[0055] The above-mentioned method of "using the anticancer agent of the present invention in combination with another anticancer agent" includes a method of treating with another anticancer agent and then using the anticancer agent of the present invention, a method of simultaneously using the anticancer agent of the present invention and another anticancer agent, a method of treating with the anticancer agent of the present invention and then using another anticancer agent, and a method of treating with another anticancer agent and then using the anticancer agent of the present invention is preferably exemplified. In addition, when the anticancer agent of the present invention is used in combination with another anticancer agent, the therapeutic effect of cancer is further improved, and the side effects of each anticancer agent can be reduced by reducing the number of times or the dosage of each anticancer agent. In addition, the anticancer agent of the present invention may contain the above-mentioned other anticancer agent.

[0056] Another aspect of the present invention includes 1) a method for treating cancer, comprising administering the CAR-expressing T cells of the present invention to a patient in need of cancer treatment, 2) the CAR-expressing T cells of the present invention for use as an anticancer agent, and 3) use of the CAR-expressing T cells of the present invention in the preparation of an anticancer agent.

[0057] Furthermore, another aspect 2 of the present invention can include a kit for producing CAR-expressing T cells, which is equipped with the CAR expression vector of the present invention. Such a kit is not particularly limited as long as it is equipped with the CAR expression vector of the present invention, and may also include instructions for producing CAR-expressing T cells and reagents used to introduce the CAR expression vector of the present invention into T cells. EXAMPLES

[0058] [Generation of T cells expressing IL-7 and CCL19] (Selection of factors that promote immune function of T cells) There are at least several hundred types of molecules capable of controlling the function of T cells in the body. Based on past knowledge and experience, the inventors first selected IL-7 and CCL19 from a vast number of combinations as control molecules for further enhancing the antitumor effect of CAR-T cells, and selected a combination of two of them, i.e., IL-7 and CCL19, rather than using either alone, and created a vector that expresses both the T cell immune function promoter and CAR.

[0059] IL-7 is a cytokine essential for the survival of T cells, and is produced by non-hematopoietic cells such as stromal cells in bone marrow, thymus, and lymphoid organs and tissues. On the other hand, T cells themselves have little ability to produce IL-7.

[0060] Moreover, the CCL19 is mainly produced by dendritic cells and macrophages in lymph nodes, and has the function of inducing the migration of T cells, B cells, and mature dendritic cells via its receptor CCR7.

[0061] (Construction of anti-FITC CAR expression vector expressing IL-7 and CCL19) Anti-FITC scFv, mouse CD8 transmembrane domain, anti-FITC CAR DNA fragment (SEQ ID NO: 7) encoding anti-FITC CAR consisting of mouse CD28-4-1BB-CD3ζ intracellular signal motif, 2A peptide (F2A) shown in SEQ ID NO: 1 and F2A-MCS DNA fragment (SEQ ID NO: 8) encoding the restriction enzyme site (MCS) following the peptide, mouse IL-7 (no stop codon), followed by F2A and IL-7-F2A-CCL19 DNA fragment (SEQ ID NO: 9) encoding mouse CCL19 were artificially synthesized. In SEQ ID NO: 7, 1 to 819 are sequences encoding anti-FITC scFv, 829 to 1074 are sequences encoding mouse CD8 transmembrane domain, 1075 to 1197 are sequences encoding mouse CD28 intracellular domain, 1198 to 1332 are sequences encoding 4-1BB intracellular domain, and 1333 to 1674 are sequences encoding the polypeptides of CD3ζ intracellular domain. In addition, in SEQ ID NO:9, positions 1 to 462 are a sequence encoding IL-7, positions 463 to 537 are a sequence encoding F2A, and positions 538 to 864 are a sequence encoding CCL19.

[0062] To prepare a CAR vector expressing CAR, IL-7, and CCL19, the anti-FITC CAR DNA fragment and the F2A-MCS DNA fragment were ligated to prepare an anti-FITC CAR-F2A-MCS construct. The prepared construct was then cloned into a pMSGV retrovirus expression vector (Tamada k et al., Clin Cancer Res 18:6436-6445(2002)) to prepare a pMSGV vector containing anti-FITC CAR-F2A-MCS. The IL-7-F2A-CCL19 DNA fragment was inserted into the MCS of the pMSGV vector by restriction enzyme (NsiI and SalI) treatment and ligation to obtain a pMSGV vector containing anti-FITC CAR-F2A-IL-7-F2A-CCL19 (IL-7 / CCL19 expression-anti-FITC CAR vector). The layout of the obtained vector is shown in FIG. 2. As a control, the anti-FITC CAR DNA fragment was cloned into the pMSGV retroviral expression vector to generate a pMSGV vector containing the anti-FITC CAR (control anti-FITC CAR vector).

[0063] (Construction of retrovirus carrying IL-7 / CCL19-expressing anti-FITC CAR vector) Retroviruses were prepared for transduction of mouse T cells. The above-mentioned IL-7 / CCL19 expression-anti-FITC CAR vector or control anti-FITC CAR vector and pCL-Eco plasmid (Imgenex) were transfected into GP2-293 packaging cell line (Takara Bio) using Lipofectamine 2000 or 3000 (Life Technologies), to produce retroviruses carrying the IL-7 / CCL19 expression-anti-FITC CAR vector or control anti-FITC CAR vector. The supernatant containing the retrovirus was collected 48 hours after transfection.

[0064] The culture medium for the GP2-293 cells was DMEM supplemented with 10% FCS, 100 U / ml penicillin, and 100 mg / ml streptomycin.The culture medium for the T cells used in the Examples below was RPMI-1640 supplemented with 10% FCS, 100 U / ml penicillin, 100 mg / ml streptomycin, 50 mM 2-mercaptoethanol, and 2 mM L-glutamine.

[0065] (Transduction of mouse T cells) For transduction of mouse T cells, 3 x 10 cells from spleen and lymph nodes were 6 The purified mouse T cells were activated for 48 hours with immobilized anti-CD3 monoclonal antibody (3 μg / ml), anti-CD28 monoclonal antibody (1 μg / ml), and IL-2 (100 IU / ml). Next, the supernatant containing the retroviruses carrying the IL-7 / CCL19 expression-anti-FITC CAR vector or the control anti-FITC CAR vector prepared above was applied to a plate coated with 25 μg / ml RetroNectin (registered trademark: manufactured by Takara Bio Inc.) to activate the above-mentioned mouse T cells (1 × 10 6 The cells were mixed with IL-7 / CCL19 expression-anti-FITC CAR vector-transduced mouse T cells (anti-FITC CAR-IL-7 / CCL19-expressing T cells) and centrifuged at 1500 rpm for 2 hours, and then cultured in the presence of IL-2 (100 IU / ml) for 6 hours. To remove retrovirus from the culture medium, the mouse T cells were collected, transferred to a new growth medium (RPMI) containing IL-2 (100 IU / ml), and cultured for an additional 42 hours to obtain mouse T cells transduced with IL-7 / CCL19 expression-anti-FITC CAR vector (anti-FITC CAR-IL-7 / CCL19-expressing T cells) or mouse T cells transduced with a control anti-FITC CAR vector (anti-FITC CAR-expressing T cells).

[0066] (Construction of anti-CD20 CAR expression vector expressing IL-7 and CCL19) In the above-mentioned IL-7 / CCL19 expression-anti-FITC CAR vector, the sequence of the anti-FITC scFv region contained in the sequence shown in SEQ ID NO: 7 was replaced with the sequence of anti-human CD20 scFv (SEQ ID NO: 10) synthesized by Life Technologies based on the sequence of rituximab, except that a pMSGV vector containing anti-human CD20 CAR-F2A-IL-7-F2A-CCL19 (IL-7 / CCL19 expression-anti-human CD20 CAR vector) was prepared in the same manner. Similarly, in the above-mentioned control anti-FITC CAR vector, the sequence of the anti-FITC scFv region contained in the sequence shown in SEQ ID NO: 7 was replaced with the sequence of the above-mentioned anti-human CD20 scFv (SEQ ID NO: 10), except that a pMSGV vector containing anti-human CD20 CAR (control anti-human CD20 CAR vector) was prepared in the same manner. The IL-7 / CCL19-expressing anti-human CD20 CAR vector or the control anti-human CD20 CAR vector was introduced into mouse T cells in the same manner as described above to generate anti-human CD20 CAR-IL-7 / CCL19-expressing T cells or anti-human CD20 CAR-expressing T cells. EXAMPLES

[0067] [CAR expression measurement by flow cytometry] (Flow cytometry analysis) The expression level of CAR that recognizes FITC as a model antigen was analyzed by two-color flow cytometry. The anti-FITC CAR-IL-7 / CCL19-expressing T cells were cultured in the presence of FITC-conjugated dextran and allophycocyanin (APC)-conjugated anti-CD8 monoclonal antibody (53-6.7, Affymetrix). Flow cytometry was performed using EC800 (Sony), and data analysis was performed using FlowJo software (Tree Star).

[0068] The expression level of CAR that recognizes human CD20 was also analyzed by two-color flow cytometry. The anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were analyzed using biotin-labeled protein L and APC-conjugated streptavidin.

[0069] (result) The results are shown in Figures 3 to 5. In Figure 3, the left side shows the results of anti-FITC CAR-IL-7 / CCL19 expressing T cells without CAR staining (no FITC-bound dextran added), and the right side shows the results of anti-FITC CAR-IL-7 / CCL19 expressing T cells with CAR staining (FITC-bound dextran added). In Figure 4, "transduction (-)" shows untransduced T cells, "Cont." shows anti-FITC CAR expressing T cells, and "7x19" shows anti-FITC CAR-IL-7 / CCL19 expressing T cells. In Figure 5, "transduction (-)" shows the results of untransduced T cells, "Cont." shows anti-human CD20 CAR expressing T cells, and "7x19" shows anti-human CD20 CAR-IL-7 / CCL19 expressing T cells. The numbers in the figures represent the percentages of each population. As shown in Figures 3 to 5, CAR expression was confirmed in anti-FITC CAR-IL-7 / CCL19-expressing T cells and anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. EXAMPLES

[0070] [Secretion of IL-7 and CCL19] (Measurement of IL-7 and CCL19 concentrations in culture supernatants of anti-FITC CAR-IL-7 / CCL19 expressing T cells-1) The prepared anti-FITC CAR-IL-7 / CCL19 expressing T cells or anti-FITC CAR expressing T cells were stimulated with 1 μg / ml of immobilized FITC-conjugated trastuzumab and cultured for 3 days, and the supernatant was collected and the concentrations of IL-7 and CCL19 were measured using a commercially available ELISA kit (manufactured by R&D systems). The results are shown in Figure 6.

[0071] (result) As shown in Figure 6, IL-7 was detected at 300 pg / ml or more, and CCL19 was detected at 75 pg / ml or more in the culture supernatant. Therefore, it was confirmed that anti-FITC CAR-IL-7 / CCL19 expressing T cells express IL-7 and CCL19, and that the expressed IL-7 and CCL19 are secreted outside the cells. In the control anti-FITC CAR expressing T cells, both IL-7 and CCL19 were below the detection limit (Not detected).

[0072] (Measurement of IL-7 and CCL19 concentrations in culture supernatants of anti-FITC CAR-IL-7 / CCL19 expressing T cells - 2) The concentrations of IL-7 and CCL-19 after 3, 5, and 7 days of culture with or without stimulation with immobilized FITC-conjugated trastuzumab or anti-CD3 monoclonal antibody were measured using an ELISA kit. The results are shown in Figure 7. In Figure 7, the white columns indicate no stimulation, the gray columns indicate stimulation with FITC-conjugated trastuzumab, and the black columns indicate stimulation with anti-CD3 monoclonal antibody. In addition, "Cont." indicates anti-FITC CAR-expressing T cells, and "7x19" indicates anti-FITC CAR-IL-7 / CCL19-expressing T cells.

[0073] (result) As is clear from Figure 7, anti-FITC CAR-IL-7 / CCL19-expressing T cells were found to secrete IL-7 and CCL-19 extracellularly not only when cultured for 3 days but also when cultured for 5 or 7 days.

[0074] (Measurement of IL-7 and CCL19 concentrations in culture supernatants of anti-human CD20 CAR-IL-7 / CCL19 expressing T cells) Furthermore, the concentrations of IL-7 and CCL-19 after 3 or 5 days of culture of the prepared anti-human CD20 CAR-IL-7 / CCL19-expressing T cells or anti-human CD20 CAR-expressing T cells with or without stimulation with P815 mastocytoma treated with mitomycin C, P815 mastocytoma genetically modified to express human CD20 (P815-hCD20), or immobilized anti-CD3 monoclonal antibody were measured using an ELISA kit. The results are shown in Figure 8. In Figure 8, the white column indicates no stimulation, the shaded column indicates stimulation with P815 treated with mitomycin C, the black column indicates stimulation with P815-hCD20, and the gray column indicates stimulation with immobilized anti-CD3 monoclonal antibody. In addition, "Cont." indicates anti-human CD20 CAR-expressing T cells, and "7x19" indicates anti-human CD20 CAR-IL-7 / CCL19-expressing T cells.

[0075] (result) As is clear from Figure 8, it was revealed that IL-7 and CCL-19 were also secreted extracellularly from anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. EXAMPLES

[0076] [Number of CAR-expressing T cells and survival rate] (Cell number and survival rate of anti-FITC CAR-IL-7 / CCL19-expressing T cells) We investigated whether IL-7 and CCL19 produced by anti-FITC CAR-IL-7 / CCL19-expressing T cells exert biological functions and show immune induction effects. The prepared anti-FITC CAR-IL-7 / CCL19-expressing T cells or anti-FITC CAR-expressing T cells were stimulated with 1 μg / ml of immobilized FITC-conjugated trastuzumab and cultured for 3, 5, and 7 days, and the cells and supernatants were collected. The cell count and viability were analyzed by trypan blue staining. The results are shown in Figures 9 and 10. In Figures 9 and 10, the black columns indicate anti-FITC CAR-IL-7 / CCL19-expressing T cells, the white columns indicate anti-FITC CAR-expressing T cells, and the horizontal axis indicates the number of days of culture. Statistical significance was examined using Student's t-test (*p<0.05, **p<0.01, ***p<0.005, †p<0.001).

[0077] (result) As shown in Figures 9 and 10, both the cell proliferation and survival rate of anti-FITC CAR-IL-7 / CCL19-expressing T cells were enhanced, demonstrating that IL-7 and CCL19 produced by anti-FITC CAR-IL-7 / CCL19-expressing T cells exert biological functions.

[0078] (Number of anti-human CD20 CAR-IL-7 / CCL19 expressing T cells) Anti-human CD20 CAR-IL-7 / CCL19 expressing T cells (4 × 10 5A sample containing 1000 cells was stimulated with P815-hCD20 together with mitomycin C in the presence of a rat IgG2a isotype control, an anti-CD127 monoclonal neutralizing antibody, or an anti-CCR7 monoclonal neutralizing antibody. After 5 days of culture, the absolute number of live cells was examined using trypan blue. CD127 is a receptor for IL-7, and CCR7 is a receptor for CCL19. The results are shown in FIG. 11. In FIG. 11, "Iso.Cntrl." indicates the case where the sample was stimulated with P815-hCD20 in the presence of the rat IgG2a isotype control, "anti-CD127" is an anti-CD127 monoclonal neutralizing antibody, and "anti-CCR7" is an anti-CCR7 monoclonal neutralizing antibody. In FIG. 11, the black columns indicate anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, and the white columns indicate anti-human CD20 CAR-expressing T cells. Each data is shown as the mean ± standard deviation of three wells, with *: P < 0.05, †: P < 0.001.

[0079] (result) As shown in Figure 11, the cell number also increased in anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, and the cell proliferation rate was enhanced. In addition, cell proliferation was inhibited by anti-CD127, demonstrating that the enhancement of the cell proliferation rate acts via CD127, the IL-7 receptor. EXAMPLES

[0080] [T cell migration test] (T cell migration test using anti-FITC CAR-IL-7 / CCL19 expressing T cells) The migration-inducing effect of CCL19 was examined by a cell migration test using a transwell. The migration of responder T cells was measured by using a 96-well Transwell (registered trademark) chamber (manufactured by Corning Costar) to allow migration through a polycarbonate filter with a pore size of 5 μm. Specifically, anti-FITC CAR-IL-7 / CCL19-expressing T cells or anti-FITC CAR-expressing T cells were stimulated with 1 μg / ml of immobilized FITC-conjugated trastuzumab in the lower layer of the chamber for 3 days. Responder T cells were prepared from the spleen or lymph nodes by negative selection using MACS (registered trademark) (manufactured by Miltenyi Biotec). Responder T cells were labeled with CytoTell blue (manufactured by AAT Bioquest) and cultured in the upper layer for 3 hours. Migration from the upper layer to the lower layer of the chamber was examined by flow cytometry. The results are shown in FIG. 12. In Figure 12, the black columns indicate anti-FITC CAR-IL-7 / CCL19 expressing T cells, the white columns indicate anti-FITC CAR expressing T cells, and the vertical axis indicates the absolute number of responder T cells that migrated to the lower chamber (similar to Figures 13 and 14 below). Statistical significance was examined using Student's t-test (*p<0.05).

[0081] (result) As shown in FIG. 12, anti-FITC CAR-IL-7 / CCL19 expressing T cells migrated more T cells to the lower layer compared to anti-FITC CAR expressing T cells. In lymphocyte transfer therapy using CAR expressing T cells, it is important to activate endogenous T cells (= host immune cells) that are originally present in the cancer patient and mobilize them as cells that attack cancer cells. For this purpose, it is preferable to not only transfer lymphocytes with antitumor activity from outside, but also to induce active interaction between the transferred T cells and endogenous T cells by some method and accumulate the endogenous T cells at the cancer site, in order to enhance the immunotherapy effect. From the results of FIG. 12, it was revealed that anti-FITC CAR-IL-7 / CCL19 expressing T cells have the ability to accumulate endogenous T cells, and therefore it is possible to induce active interaction between the transferred T cells and endogenous T cells.

[0082] (Migration test of T cells or dendritic cells using anti-FITC CAR-IL-7 / CCL19 expressing T cells) In the lower chamber of the transwell, samples containing anti-FITC CAR-IL-7 / CCL19-expressing T cells or anti-FITC CAR-expressing T cells (5 × 10 5 4 × 10 cells were stimulated with immobilized FITC-conjugated trastuzumab or anti-CD3 monoclonal antibody. 5 T cells were overlaid and incubated for 3 or 5 hours. Similarly, each sample was stimulated with immobilized FITC-conjugated trastuzumab and stained with CytoTell Blue on day 3. 5 Dendritic cells were placed in the upper layer and incubated for 3 hours. The responder cells that migrated from the upper layer to the lower layer were analyzed by flow cytometry. The results are shown in Figures 13 and 14. In Figures 13 and 14, the black columns indicate anti-FITC CAR-IL-7 / CCL19 expressing T cells, and the white columns indicate anti-FITC CAR expressing T cells. In Figures 13 and 14 and Figure 15 described below, each data is shown as the mean ± standard deviation of three wells, and *: P < 0.05, **: P < 0.01, †: P < 0.001, ††: P < 0.00001, ‡: P < 5 × 10 -5 Shows.

[0083] (result) The results in Figures 13 and 14 demonstrate that anti-FITC CAR-IL-7 / CCL19-expressing T cells have a high ability to accumulate endogenous T cells and dendritic cells.

[0084] (T cell migration assay using anti-human CD20 CAR-IL-7 / CCL19 expressing T cells) In the lower chamber of the transwell, anti-human CD20 CAR-IL-7 / CCL19-expressing T cells (1 × 10 5 Samples containing 4 × 10 cells were co-cultured with mitomycin C-treated P815-hCD20 cells. 5T cells were placed in the upper layer and incubated for 3 hours in the presence of rat IgG2a isotype control, anti-CD127 monoclonal antibody, or anti-CCR7 monoclonal antibody. Each responder T cell that migrated from the upper layer to the lower layer was analyzed by flow cytometry. The results are shown in Figure 15. In Figure 15, "Iso.Cntrl." indicates the rat IgG2a isotype control, "anti-CD127" indicates the case where P815-hCD20 was stimulated in the presence of anti-CD127 monoclonal neutralizing antibody, and "anti-CCR7" indicates the case where P815-hCD20 was stimulated in the presence of anti-CCR7 monoclonal neutralizing antibody. In Figure 15, the black columns indicate anti-human CD20 CAR-IL-7 / CCL19 expressing T cells, and the white columns indicate anti-human CD20 CAR expressing T cells.

[0085] (result) The results in Figure 15 show that anti-human CD20 CAR-IL-7 / CCL19-expressing T cells also have a high ability to accumulate endogenous T cells, and that the accumulation of endogenous T cells is suppressed by anti-CCR7, demonstrating that the accumulation of endogenous T cells acts via CCR7, the receptor for CCL19.

[0086] In addition, from the results of Figures 9 to 15, it was revealed that anti-FITC CAR-IL-7 / CCL19-expressing T cells and anti-human CD20 CAR-IL-7 / CCL19-expressing T cells proliferate effectively with IL-7, have high survival rates, and accumulate T cells and dendritic cells at the cancer site with CCL19, which are important effects essential for immune induction, and have excellent immune induction effects. In other words, it was revealed that by expressing two regulatory molecules, "IL-7" and "CCL19", in CAR-expressing T cells, it is possible to improve the proliferation ability, survival rate, and immune induction effect of such T cells. EXAMPLES

[0087] [T cell proliferation ability] Samples containing anti-FITC CAR-IL-7 / CCL19 expressing T cells and anti-FITC CAR expressing T cells as a control (5 × 10 5) were stained with CytoTell Blue (AAT Bioquest) and analyzed by flow cytometry after stimulation with immobilized FITC-conjugated trastuzumab. The results from the 5th day after the start of stimulation are shown in Figure 16, and the results from the 3rd and 7th days are shown in Figure 17. In Figure 16, the numbers in the histogram indicate the number of cell divisions, and in Figures 16 and 17, the numbers in the pie charts indicate the proportion of each gated fraction (0, 1, 2, 3, 4> cell division numbers) in the leukocyte population.

[0088] (result) The results in Figures 16 and 17 demonstrated that anti-FITC CAR-IL-7 / CCL19-expressing T cells had increased proliferation ability compared to anti-FITC CAR-expressing T cells. EXAMPLES

[0089] [Expression of CD127 or CCR7 on T cells, dendritic cells, and CAR-expressing T cells] Unstimulated splenic T cells (naive T cells), splenic T cells stimulated by culturing with anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody and IL-2 for 2 days (activated T cells), unstimulated splenic dendritic cells, and anti-FITC CAR-expressing T cells (Cont.) and anti-FITC CAR-IL-7 / CCL19-expressing T cells (7x19) activated in the same manner as in "Transduction of mouse T cells" in Example 1 were analyzed by flow cytometry to examine the expression of CD127 or CDR7. + CD19 - population, anti-FITC CAR expressing T cells, anti-FITC CAR-IL-7 / CCL19 expressing T cells are positive populations for FITC-conjugated dextran beads, and dendritic cells are CD11c + The population was determined as follows. The results of examining CD127 expression are shown in FIG. 18, and the results of examining CCR7 expression are shown in FIG. 19. In the figures, the numbers indicate the positive percentage, "Cont." indicates anti-FITC CAR-expressing T cells, and "7x19" indicates anti-FITC CAR-IL-7 / CCL19-expressing T cells.

[0090] (result) As shown in FIG. 18, the expression of CD127 was clearly decreased in activated T cells compared to naive T cells, but in anti-FITC CAR-IL-7 / CCL19-expressing T cells, it was found to be greater than that in activated T cells and to have recovered to a level greater than that of naive T cells. In addition, as shown in FIG. 19, it was found that the expression of CCR7 in anti-FITC CAR-IL-7 / CCL19-expressing T cells was decreased by activation, but the expression was maintained at approximately 67% compared to naive T cells. It has been known that the expression of CD127 or CCR7 decreases to approximately 1 / 2 to 1 / 3 when T cells are activated. Therefore, even if CAR-expressing T cells expressing IL-7 or CCL19 are produced, it is considered that the action of IL-7 and CCL19 is reduced by the activation of the CAR-expressing T cells. Therefore, it is generally considered difficult to expect that the immune induction effect or antitumor activity of CAR-expressing T cells will be enhanced by expressing IL-7 and CCL19 in CAR-expressing T cells. In this study, it was also confirmed that the expression of CD127 or CCR7 temporarily decreased on the second day after activation of splenic T cells. However, it was revealed that the expression of CD127 or CCR7 was restored on the fourth day in anti-FITC CAR-IL-7 / CCL19-expressing T cells. This demonstrated that expressing IL-7 and CCL19 in CAR-expressing T cells is useful for enhancing immune induction effects and antitumor activity. EXAMPLES

[0091] [Therapeutic effect in mouse tumor models] (Administration of anti-human CD20 CAR-IL-7 / CCL19 expressing T cells to mice) Tumor-bearing mice (DBA / 2 mice) were inoculated with 5 × 10 blastocytoma cells expressing human CD20 (P815-hCD20). 5 Three days later, 3 × 10 6Anti-human CD20 CAR-IL-7 / CCL19 expressing T cells or anti-human CD20 CAR expressing T cells were administered intravenously to the mice. As a control, a non-treated group was set up in which the P815 mastocytoma was inoculated and then left untreated (no administration of CAR expressing T cells). The tumor volume and survival rate of the mice were measured twice a week. In the tumor volume analysis, the standard deviation was calculated for each experimental group. The statistical significance of the differences among the three groups was examined by Student's t-tests in the tumor volume analysis and by the log-rank test in the survival rate investigation (*P<0.05, **P<0.01).

[0092] The results of changes in mouse tumor volume are shown in Figure 20, and the results of mouse survival rates are shown in Figure 21. In Figures 20 and 21, ○ indicates the cases where anti-human CD20 CAR-expressing T cells were administered, ● indicates the cases where anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were administered, and ◇ indicates the case where CAR-expressing T cells were not administered as a non-treatment group. In addition, the horizontal axis of Figure 20 shows the number of days that have passed since the cells were administered intravenously to mice, and the vertical axis shows the tumor volume (mm 3 21), and the horizontal axis of FIG. 21 represents the number of weeks since the cells were intravenously administered to mice, and the vertical axis represents the survival rate (%).

[0093] (result) As shown in Figures 20 and 21, when anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were administered, the tumor volume was reduced and the survival rate was improved (survival period was extended) compared to when anti-human CD20 CAR-expressing T cells were administered or when CAR-expressing T cells were not administered. Therefore, it was revealed that anti-human CD20 CAR-IL-7 / CCL19-expressing T cells have excellent anti-tumor activity.

[0094] (Inoculation of mice with anti-cancer drugs and anti-human CD20 CAR-IL-7 / CCL19-expressing T cells) 5x10 on mouse 5 Each mouse was subcutaneously inoculated with 1 × 10 P815-hCD20. On the 10th day after inoculation, the anticancer drug cyclophosphamide (CPA, 100 mg / kg) was administered intraperitoneally, and on the 14th day, 1 × 10 6Anti-human CD20 CAR-IL-7 / CCL19-expressing T cells or anti-human CD20 CAR-expressing T cells were intravenously administered. The results of mouse survival rate are shown in FIG. 22, and the results of tumor volume are shown in FIGS. 23 and 24. In FIGS. 22 to 24, the horizontal axis indicates the number of days after subcutaneous inoculation of P815-hCD20 (the day when P815-hCD20 was subcutaneously inoculated into the mouse was set as day 0), and the vertical axis indicates survival rate (FIG. 22), tumor volume (long axis of tumor × short axis of tumor) 2 / 2(mm 3 )) (Figures 23 and 24), where "no treatment" is no treatment, "CPA" is CPA only, "CPA+Cont." is CPA administration followed by administration of anti-human CD20 CAR-expressing T cells, and "CPA+7x19" is CPA administration followed by administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, and † indicates the death of the mouse. Note that Figure 24 is a graph in which the values ​​on the vertical axis of the graph for CPA+7x19 in Figure 23 are divided by 10.

[0095] (result) As shown in Figure 22, it was revealed that the combined use of the anti-human CD20 CAR-IL-7 / CCL19-expressing T cells of the present invention and an anticancer drug significantly increased the survival rate. In addition, as shown in Figures 23 and 24, it was revealed that the combined use of the anti-human CD20 CAR-IL-7 / CCL19-expressing T cells of the present invention and an anticancer drug completely eliminated the tumor. As shown in Figure 24, the tumor volume reached its maximum on day 10 after subcutaneous inoculation of P815-hCD20, with the short diameter at this time being 4.86 mm to 7.25 mm and the long diameter being 5.92 mm to 8.39 mm, resulting in a tumor volume of 69.91 mm. 3 ~220.50mm 3 , the average is 140.02 mm 3The above results also show that once-grown tumors disappeared due to treatment with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. When used in combination with other anticancer drugs, it is preferable to first reduce the number of lymphocytes using the other anticancer drug, as in the above method, and then administer the anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, in order to further enhance the antitumor activity of the CAR-expressing T cells of the present invention. This method can enhance the homeostasis of the CAR-expressing T cells in the body. EXAMPLES

[0096] [Tumor tissue penetration effect] 5x10 on mouse 5 Three days after inoculation, 1 × 10 6Anti-human CD20 CAR-IL-7 / CCL19 expressing T cells were administered, and tumor tissues were sectioned 21 days after inoculation. Each tissue was divided into two pieces. One was stained with hematoxylin and eosin (H&E) and the other was used for immunohistochemical analysis. In the immunohistochemical analysis, a combination of anti-CD4 and anti-CD8 monoclonal antibodies or a combination of anti-CD3 and anti-DEC205 monoclonal antibodies was used as the primary antibody. In addition, Alexa Fluor™ 488-conjugated anti-rat IgG2a (green) and Alexa Fluor™ 647-conjugated anti-rat IgG2b (red) were used as the secondary antibody. Cell nuclei were stained with DAPI (blue). H&E stained samples and immunolabeled sections were observed under a microscope at a magnification of ×100 or ×200. CD4 and CD8 are markers of T cells, and DEC205 is a marker of dendritic cells. The results of H&E staining are shown in Figure 25, and the results of immunohistochemical analysis are shown in Figures 26(a) and (b). In addition, the positive areas labeled by each fluorescent staining (CD4 staining (red), CD8 staining (green), CD3 staining (red), DEC205 staining (green), coexistence of CD3 and DEC205 (yellow)) in the data of Figures 26(a) and (b) were quantified using the Hybrid Cell Count program (manufactured by KEYENCE), and the results are shown in Figures 27(a) and (b), respectively. In Figures 25 to 27, "no treatment" or "no treat." refers to a group that was not treated, "Cont." refers to a group that was treated with anti-human CD20 CAR-expressing T cells, and 7x19 refers to a group that was treated with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells.

[0097] (result) As shown in the results of Figure 25, when treated with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, necrosis progressed (areas indicated by arrows) and areas where nuclei had disappeared were observed. Furthermore, the results of Figures 26(a) and 27(a) demonstrated that treatment with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells allowed T cells to infiltrate into the cancer tissue, and the results of Figures 26(b) and 27(b) demonstrated that treatment with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells allowed dendritic cells to infiltrate into the cancer tissue together with T cells. EXAMPLES

[0098] [Tumor treatment effect of the combination of IL-7 and CCL19] 5 × 10 in DBA / 2 mice 5 Three days after inoculation, 1 × 10 anti-human CD20 CAR-expressing T cells, anti-human CD20 CAR-IL-7-expressing T cells expressing only IL-7 (not expressing CCL19) as an immune function promoter, anti-human CD20 CAR-CCL19-expressing T cells expressing only CCL19 (not expressing IL-7) as an immune function promoter, or anti-human CD20 CAR-IL-7 / CCL19-expressing T cells expressing IL-7 and CCL19 were subcutaneously inoculated into the mice. 6 As a control, a group of mice was provided that did not receive CAR-expressing T cells that did not express IL-7 or CCL19. The long and short axes of the tumors were measured 10 days after administration, and the tumor volume (mm 3 ) was calculated in the same manner as above. The results are shown in Figure 28. In Figure 28, "No treat" indicates the case where CAR-expressing T cells were not administered, "Control CAR" indicates the case where anti-human CD20 CAR-expressing T cells, "IL-7 CAR" indicates the case where anti-human CD20 CAR-IL-7-expressing T cells, "CCL19 CAR" indicates the case where anti-human CD20 CAR-CCL19-expressing T cells, and "IL-7 / CCL19 CAR" indicates the case where anti-human CD20 CAR-IL-7 / CCL19-expressing T cells were administered.

[0099] The anti-human CD20 CAR-IL-7 expressing T cells were obtained by preparing a pMSGV vector (IL-7 expression-anti-human CD20 CAR vector) containing anti-human CD20 CAR-F2A-IL-7 and introducing it into mouse T cells in the same manner as in "Transduction of mouse T cells" in Example 1. Similarly, the anti-human CD20 CAR-CCL19 expressing T cells were obtained by preparing a pMSGV vector (CCL19 expression-anti-human CD20 CAR vector) containing anti-human CD20 CAR-F2A-CCL19 and introducing it into mouse T cells in the same manner as in "Transduction of mouse T cells" in Example 1. The preparation of each vector was performed according to the method of "Preparation of anti-FITC CAR expression vector expressing IL-7 and CCL19" and "Preparation of anti-CD20 CAR expression vector expressing IL-7 and CCL19" in Example 1. The sequence encoding IL-7 was the sequence from positions 1 to 462 and the subsequent termination codon sequence in SEQ ID NO: 9, and the sequence encoding CCL19 was the sequence from positions 538 to 864 in SEQ ID NO: 9. The results are shown in FIG.

[0100] (result) As shown in Figure 28, when anti-human CD20 CAR-IL-7 expressing T cells or anti-human CD20 CAR-CCL19 expressing T cells were administered, the tumor growth suppression effect was almost the same as or slightly lower than that of Conotrol's anti-human CD20 CAR expressing T cells, but when anti-human CD20 CAR-IL-7 / CCL19 expressing T cells were administered, the tumor almost disappeared. Therefore, it was revealed that although IL-7 and CCL19 alone have almost no tumor growth suppression effect, the combination of IL-7 and CCL19 can provide an extremely high tumor growth suppression effect. EXAMPLES

[0101] [ 51 Tumor cytotoxicity by Cr release assay-1] (Selection of factors that promote immune function of T cells) In the microenvironment of cancer tissue, inhibitory signals are transmitted to immune cells, inhibiting antitumor immune responses and weakening the efficacy of immunotherapy. Inhibitory signals to immune cells are transmitted by SHP-1 and SHP-2. Therefore, in T cell therapy for cancer, it is possible to enhance the antitumor effect by producing dominant-negative mutants that inhibit the action of SHP-1 and SHP-2 in T cells themselves. Therefore, we created vectors that express both dominant-negative mutants that inhibit the action of SHP-1 and SHP-2 and CAR, and examined their tumor cytotoxicity.

[0102] (Construction of CAR expression vector expressing dominant-negative mutant of SHP1 or SHP2) A DNA fragment encoding a dominant-negative mutant (SHP1DN) of mouse SHP1 containing a mutation (C453S) of the catalytic cysteine ​​residue at position 453 to serine was prepared by site-directed mutagenesis using PCR, and a DNA fragment encoding a dominant-negative mutant (SHP2DN) of mouse SHP2 containing a mutation (C459S) of the catalytic cysteine ​​residue at position 459 to serine was synthesized by Life Technologies. The nucleotide sequence encoding mouse SHP1DN is shown in SEQ ID NO: 11, and the nucleotide sequence encoding mouse SHP2DN is shown in SEQ ID NO: 12. The mutation sites are the three bases at positions 1357 to 1359 in SEQ ID NO: 11 and 1375 to 1377 in SEQ ID NO: 12. A DNA fragment encoding SHP1DN or SHP2DN was inserted into the MCS of a pMSGV vector containing anti-human CD20 scFv CAR-F2A-MCS in the process of constructing an IL-7 / CCL19 expression-anti-human CD20 CAR vector in Example 2 to obtain an SHP1DN expression-anti-human CD20 CAR vector and an SHP2DN expression-anti-human CD20 CAR vector, respectively. The layout of the obtained vectors is shown in Figure 29.

[0103] (Transduction of mouse T cells) The SHP1DN expression-anti-human CD20 CAR vector and the SHP2DN expression-anti-human CD20 CAR vector were introduced into mouse T cells in the same manner as in Example 1 to obtain anti-human CD20 CAR-SHP1DN-expressing T cells and anti-human CD20 CAR-SHP2DN-expressing T cells, respectively. As a control, the anti-human CD20 CAR-expressing T cells prepared in Example 1 were used.

[0104] ( 51 Tumor cytotoxicity by Cr release assay) Cytotoxic activity of CAR-expressing T cells against tumors was measured in a standard 4-hour 51 The IgG antibody titer was measured by Cr release assay. P815 expressing human CD20 (P815-hCD20) was used as the target tumor cell. The tumor cells were harvested and injected with 100 μCi Na 2 51 CrO 4 The cells were cultured in the presence of 10% Triton-X (Sigma-Aldrich) at 37°C for 1 hour and then washed three times. Then, the cells were co-cultured with anti-human CD20 CAR-expressing T cells, anti-human CD20 CAR-SHP1DN-expressing T cells, or anti-human CD20 CAR-SHP2DN-expressing T cells as effector T cells. The effector / target ratios were 0.6, 1.25, 2.5, 5, and 10. The maximum release and spontaneous release of target cells were measured by culturing the cells in a culture medium containing 10% Triton-X (Sigma-Aldrich) or in culture medium alone. The supernatant 51 Cr release was measured using a TopCount scintillation counter (PerkinElmer). The percentage of specific damage was calculated using the formula: specific damage (%) = [(test release - spontaneous release) / (maximum release - spontaneous release)] × 100. The results are shown in Figure 30. In Figure 30(a), ○ indicates anti-human CD20 CAR-expressing T cells, ● indicates anti-human CD20 CAR-SHP1DN-expressing T cells, and in Figure 30(b), ○ indicates anti-human CD20 CAR-expressing T cells, ● indicates anti-human CD20 CAR-SHP2DN-expressing T cells. The horizontal axis represents the effector (T cells) to target (tumor cells) ratio as E / T ratio, and the vertical axis indicates specific damage (%). Statistical significance was examined using Student's t-test (*p<0.05).

[0105] As shown in Figure 30, it was revealed that anti-human CD20 CAR-SHP1DN-expressing T cells and anti-human CD20 CAR-SHP2DN-expressing T cells have significantly higher tumor cytotoxicity than anti-human CD20 CAR-expressing T cells. EXAMPLES

[0106] [ 51 Tumor cytotoxicity by Cr release assay-2] P815-hCD20(1×10 4 7 × 19 cells / well) were mixed with anti-FITC CAR-expressing T cells (Cont, circles) or anti-FITC CAR-IL-7 / CCL19-expressing T cells (7 × 19, squares) in the presence of unlabeled (Ab, open) or FITC-conjugated (FITC-Ab, filled) rituximab at effector / target (E / T) ratios of 0.15625, 0.3125, 0.625, 2.5, 5, and 10, and the supernatants were analyzed in the same manner as described above. 51 Cr release was measured and the percentage of cytotoxic activity was calculated. The results are shown in Figure 31. In Figure 31, the case where anti-FITC CAR-expressing T cells were mixed in the presence of FITC-conjugated rituximab is shown as "●", the case where anti-FITC CAR-expressing T cells were mixed in the presence of unlabeled rituximab is shown as "○", the case where anti-FITC CAR-expressing T cells were mixed in the presence of FITC-conjugated rituximab is shown as "■", and the case where anti-FITC CAR-IL-7 / CCL19-expressing T cells were mixed in the presence of unlabeled rituximab is shown as "□".

[0107] In addition, 815-hCD20 (1 × 10 4 The cells (cells / well) were mixed with anti-human CD20 CAR-expressing T cells or anti-human CD20 CAR-IL-7 / CCL19-expressing T cells at effector / target (E / T) ratios of 0.3125, 0.625, 2.5, 5, 10, and 20, and the supernatants were analyzed in the same manner as above. 51The Cr release was measured, and the percentage of cytotoxic activity was calculated. The results are shown in Figure 32. In Figure 32, the cases where the cells were mixed with anti-human CD20 CAR-IL-7 / CCL19 expressing T cells are indicated by "●", and the cases where the cells were mixed with anti-human CD20 CAR expressing T cells are indicated by "○".

[0108] (result) As shown in Figures 31 and 32, it was revealed that the anti-FITC CAR-IL-7 / CCL19-expressing T cells maintained the same level of tumor cytotoxicity per cell as the anti-FITC CAR-expressing T cells, and similarly, the anti-human CD20 CAR-IL-7 / CCL19-expressing T cells maintained the same level of tumor cytotoxicity per cell as the anti-human CD20 CAR-expressing T cells. EXAMPLES

[0109] [In vivo survival of CAR-expressing T cells and differentiation into memory T cells] (Flow cytometry analysis) 5 × 10 in DBA / 2 mice 5 Each mouse was subcutaneously inoculated with 1 × 10 P815-hCD20. On the 10th day after inoculation, the anticancer drug cyclophosphamide (CPA, 100 mg / kg) was administered intraperitoneally, and on the 14th day, 1 × 10 6 Anti-human CD20 CAR-IL-7 / CCL19 expressing T cells or anti-human CD20 CAR expressing T cells were administered intravenously. Twenty-one days after administration of CAR expressing T cells, leukocytes were isolated from the spleen or tumor-draining lymph nodes (axillary, brachial, and inguinal). The phenotypes of the leukocyte surface were analyzed by flow cytometry for CD4, CD8, CD44, and CD62L, as shown in Figure 33. In addition, splenic leukocytes were stimulated by culturing with P815-hCD20 treated with mitomycin C for 4 days, and the proliferation of T cells was examined by flow cytometry, as shown in Figure 34. CAR expression was confirmed using biotin-labeled protein L and APC-bound streptavidin. The numbers in Figure 33 indicate CD4 + T cells, CD8 + Each gate region of T cells (CD62L + CD44 - is naive T cells, CD62L +CD44 + is central memory T cells, CD62L - CD44 + indicates the percentage of effector memory T cells), and the numbers in Fig. 34 indicate the percentage of protein L-positive T cells. In Figs. 33 and 34, "Cont." indicates anti-human CD20 CAR-expressing T cells, and "7x19" indicates anti-human CD20 CAR-IL-7 / CCL19-expressing T cells.

[0110] (result) As shown in Figures 33 and 34, it was revealed that in mice administered with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, memory T cells were increased in the spleen and lymph nodes, and that anti-human CD20 CAR-IL-7 / CCL19-expressing T cells surviving in the mice proliferated strongly when co-cultured with tumor cells expressing human CD20. Combined with the results of survival rates in Figures 21 and 22, it is believed that the CAR-expressing T cells of the present invention survive efficiently in the living body to which they are administered, and have the ability to eliminate cancer cells and increase survival rates by becoming memory T cells, and are also shown to be effective in preventing cancer recurrence. [Industrial Applicability]

[0111] The CAR expression vector of the present invention can be used in the field of cancer immunotherapy, since it is possible to produce CAR-T cells that have both survival ability and lymphocyte accumulation ability, and CAR-T cells that are resistant to immunosuppression in the cancer microenvironment.

Claims

1. An AT-CAR expression vector comprising a nucleic acid encoding an anti-tag chimeric antigen receptor (AT-CAR) and a nucleic acid encoding a T cell immune function enhancer, The AT-CAR expression vector, wherein the nucleic acid encoding the immune function-promoting factor is a nucleic acid encoding interleukin-7 and a nucleic acid encoding CCL19.

2. The AT-CAR expression vector according to claim 1, wherein the AT-CAR specifically recognizes FITC, which is a cell surface antigen of a cancer cell.

3. An AT-CAR-expressing T cell into which the following vector (a) or (b) has been introduced: (a) the AT-CAR expression vector according to claim 1 or 2; (b) an AT-CAR expression vector containing a nucleic acid encoding AT-CAR and a nucleic acid encoding interleukin 7, and an AT-CAR expression vector containing a nucleic acid encoding AT-CAR and a nucleic acid encoding CCL19;

4. An anticancer agent comprising the AT-CAR-expressing T cell according to claim 3 and a pharma- ceutical acceptable additive.

5. T cells expressing AT-CAR, interleukin 7, and CCL19.

6. The T cell according to claim 5, wherein the AT-CAR specifically recognizes FITC, a cell surface antigen of a cancer cell.

7. An anticancer agent comprising the T cell according to claim 5 or 6 and a pharma- ceutically acceptable additive.

8. The anticancer agent of claim 7 , which is administered in combination with cyclophosphamide or fludarabine.

9. A method for producing a T cell expressing AT-CAR, interleukin 7, and CCL19, the method comprising a step of introducing a nucleic acid encoding AT-CAR, a nucleic acid encoding interleukin 7, and a nucleic acid encoding CCL19 into a T cell using an expression vector.

10. The method for producing T cells according to claim 9, wherein the AT-CAR specifically recognizes FITC, which is a cell surface antigen of a cancer cell.

11. (a) an AT-CAR expression vector containing a nucleic acid encoding AT-CAR, a nucleic acid encoding interleukin-7, and a nucleic acid encoding CCL19; (b) a CAR expression vector containing a nucleic acid encoding AT-CAR and a nucleic acid encoding interleukin-7, and an AT-CAR expression vector containing a nucleic acid encoding AT-CAR and a nucleic acid encoding CCL19; A kit for generating a T cell expressing AT-CAR, interleukin 7, and CCL19, comprising the AT-CAR expression vector shown in (a) or (b) above.

12. The kit according to claim 11, wherein the AT-CAR specifically recognizes FITC, which is a cell surface antigen of a cancer cell.

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