CAR expression vector and CAR-expressing 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 the CAR expression vector, and an anticancer agent containing the CAR-expressing T cell. [Background technology]

[0002] A chimeric antigen receptor (CAR) is an artificial chimeric protein that combines a single-chain antibody that recognizes a cell surface antigen on cancer cells with a signaling 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 are not tumor-reactive, it is possible to produce a large number of CAR-expressing T cells (hereinafter simply referred to as "CAR-T cells") that can express the CAR. Such CAR-T cells are tumor-reactive and can induce damage to cancer cells independently of interactions with the 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 a CAR is introduced into the T cells, the cells are amplified, and the cells are then reintroduced into 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 studies 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 one or more tagged proteins that bind to cancer cells and induce cancer cell death, which is administered to a subject simultaneously or separately with a formulation of the tagged proteins (see Patent Document 2), a cell comprising a nucleic acid encoding a chimeric antigen receptor comprising the 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, wherein the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a sequence encoding a target gene sequence represented by SEQ ID NO: 1. Proposed examples include 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, existing technologies have not yet resolved the problems of low in vivo survival efficiency of CAR-T cells, insufficient activation of endogenous T cells induced by CAR-T cells, and insufficient accumulation in tumor locales. Furthermore, the activity of CAR-T cells is inhibited by immunosuppressive signals via the PD-L1 / PD-1 pathway, a tumor immune evasion mechanism possessed by cancer cells, and by immunosuppressive factors such as TGF-β and IL-10 secreted in the tumor microenvironment. Therefore, there are cancer types and cases in which sufficient therapeutic effects are not observed, and there is a need for more effective CAR-T cells and the creation of expression vectors for generating 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] Japanese Patent Application Laid-Open No. 2011-004749 [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, efforts have been made to enhance the activation ability of T cells by incorporating CD28, 4-1BB, CD3ζ, etc. into the signal transduction domain of the CAR, but the immune-inducing effect of endogenous T cells by CAR-T cells and resistance to immunosuppressive mechanisms in the tumor microenvironment have not been sufficiently enhanced, and CAR-T cells have not yet achieved therapeutic effects against solid cancers. Therefore, an object of the present invention is to provide CAR-T cells that express a T cell immune function-enhancing factor together with a CAR in T cells and have high immune-inducing effects and antitumor activity, as well as 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 CARs 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, thereby completing the present invention.

[0010] That is, the present invention is as disclosed below. (1) A chimeric antigen receptor (CAR) expression vector containing a nucleic acid encoding a CAR and a nucleic acid encoding a T cell immune function enhancer, wherein the nucleic acid encoding the immune function enhancer 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-enhancing factor is a nucleic acid encoding interleukin-7 and a nucleic acid encoding CCL19. (3) A 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) A 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 nucleic acids encoding polypeptides 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 pharmaceutically acceptable additive. [Effects of the Invention]

[0011] The CAR expression vector of the present invention can be used to generate CAR-T cells that have viability, lymphocyte accumulation, and tumor cytotoxicity, as well as CAR-T cells that are resistant to immunosuppression in the cancer microenvironment. Immunotherapy using such CAR-T cells in cancer patients is expected to be highly effective in treating cancer, enabling effective cancer immunotherapy even for refractory and progressive cancers. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the structure of CAR and the basic system of cancer immunotherapy using CAR-T cells. [Figure 2] FIG. 1 depicts vectors expressing CAR, interleukin 7 (IL-7), and CCL19. [Figure 3] This figure shows the results of flow cytometry to confirm the expression level of CAR in anti-FITC CAR-IL-7 / CCL19-expressing T cells - 1. The left image shows unstained CAR, and the right image shows stained CAR. [Figure 4] FIG. 10 shows the results of flow cytometry confirming the expression level of CAR in anti-FITC CAR-IL-7 / CCL19-expressing T cells - 2. [Figure 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]FIG. 1 shows the number of cells when anti-FITC CAR-IL-7 / CCL19-expressing T cells were stimulated and cultured for 3, 5, 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 anti-human CD20 CAR-IL-7 / CCL19-expressing T cells 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. 10 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 test 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 (days 3 and 7 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] This figure shows the results of examining 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 examining the survival rate of mice 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. [Figure 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. [Figure 25] FIG. 11 shows the results of H&E staining of tumor tissues obtained when mice subcutaneously inoculated with P815-hCD20 were administered with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells. [Figure 26] This figure 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 quantifying the positive areas labeled by fluorescent staining in FIG. 26. [Figure 28] This figure shows the results of examining 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 showing vectors expressing dominant-negative mutants of CAR and SHP1 (Src homology region 2 domain-containing phosphatase-1). (b) Diagram showing vectors expressing dominant-negative mutants of CAR and SHP2 (Src homology region 2 domain-containing phosphatase-2). [Figure 30] (a) and (b) show the results of a cytotoxicity test using anti-human CD20 CAR-SHP1DN-expressing T cells. [Figure 31] FIG. 1 shows the results of examining tumor cytotoxicity by mixing P815-hCD20 with anti-FITC CAR-IL-7 / CCL19-expressing T cells in the presence of FITC-conjugated rituximab. [Figure 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. [Figure 33] This figure shows the results of flow cytometry analysis of the leukocyte surface phenotypes of 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. [Figure 34] This figure shows the results of examining T cell proliferation by flow cytometry after splenic leukocytes were stimulated by culturing them with mitomycin C-treated P815-hCD20 for 4 days. DETAILED DESCRIPTION OF THE INVENTION

[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. A chimeric antigen receptor refers to an artificial chimeric protein in which a single-chain antibody that recognizes a cell surface antigen on cancer cells is fused with a signal transduction domain that induces T cell activation via a transmembrane domain.

[0014] In the present invention, the nucleic acid encoding a CAR is not particularly limited as long as it encodes a polypeptide that constitutes 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 transmembrane domain, and a signal transduction domain that induces T cell activation.

[0015] The single-chain antibody in the CAR may 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 antigens of cancer cells recognized by the single-chain antibodies may be biomolecules that are specifically expressed in cancer cells and their precursor cells, biomolecules whose expression has been newly confirmed due to the malignant transformation of cells, or biomolecules whose expression levels are increased in cancer cells compared to normal cells. Examples of such biomolecules 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 domain is a domain capable of transmitting a signal intracellularly when the single-chain antibody recognizes a cell surface antigen on a cancer cell, and preferably comprises at least one or more polypeptides selected from the group consisting of polypeptides of the intracellular domains of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ, and Fc Receptor-associated γ chain, and more preferably polypeptides of the intracellular domains 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] Examples of the transmembrane domain in the present invention include polypeptides derived from CD8, the α and β chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and GITR, and preferably the polypeptide of the human CD8 transmembrane domain. Such a transmembrane domain anchors the CAR to the plasma 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 that of human CD8.

[0021] Furthermore, a spacer region consisting of any oligopeptide or polypeptide may be inserted between the single-chain antibody that recognizes a cell surface antigen on 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 glycine-serine contiguous sequence.

[0022] In the present invention, the nucleic acid encoding a T cell function-enhancing 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"). The nucleic acid may contain a plurality of each of the present nucleic acids 1 to 3, and specifically may be 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 encode a mutant of SHP1 that acts dominantly over SHP1 and can inhibit the action of SHP1, and examples thereof include nucleic acids encoding mutants that have an amino acid sequence in which at least one amino acid in the SHP1 amino acid sequence has been substituted with another amino acid and can inhibit the action of SHP1. Nucleic acids encoding dominant-negative mutants of SHP2 are not particularly limited as long as they encode a mutant of SHP2 that acts dominantly over SHP2 and can inhibit the action of SHP2, and examples thereof include nucleic acids encoding mutants that have an amino acid sequence in which at least one amino acid in the SHP2 amino acid sequence has been substituted with another amino acid and can inhibit 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 a chimeric antigen receptor and the nucleic acid encoding a T cell immune function-enhancing factor, between each nucleic acid when multiple nucleic acids, such as Nucleic Acid 1, Nucleic Acid 2, and Nucleic Acid 3, are contained, or between the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 in Nucleic Acid 1, as long as each nucleic acid can be expressed. However, it is preferred that the nucleic acids be linked via a sequence encoding a self-cleaving peptide (2A peptide) or an internal ribozyme entry site (IRES), preferably a sequence encoding a 2A peptide. Linking using such a sequence enables efficient expression of each nucleic acid.

[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, nucleic acids incorporated before and after the 2A peptide are expressed independently of each other within the cell.

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

[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 transmembrane domain, and a T cell activation signaling domain. Note that the codons selected to encode 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 signaling 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. Examples of such sequences include those registered as Genbank number NM_006139.2 (updated May 10, 2014) for human CD28, Genbank number NM_001561.5 (updated March 16, 2014) for human 4-1BB, and Genbank number NM_000734.3 (updated August 12, 2014) for human CD3ζ.

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

[0032] Furthermore, information on the nucleotide sequence encoding the polypeptide of a single-chain antibody can be obtained by producing 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 using the amino acid sequence to obtain the information. Methods for producing monoclonal antibodies 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] Nucleic acids encoding factors that enhance T cell immune function, such as nucleic acids encoding IL-7 and CCL19, nucleic acids encoding dominant-negative mutants of SHP-1, and nucleic acids encoding dominant-negative mutants of SHP-2, can be prepared based on their respective nucleotide sequences by known techniques such as chemical synthesis or PCR amplification. Note that the codons selected to encode amino acids may be modified to optimize expression of the nucleic acids in a host cell of interest.

[0034] Information on nucleic acids encoding IL-7 and CCL19, nucleic acids encoding dominant-negative mutants of SHP-1, and nucleic acids encoding dominant-negative mutants 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 selected appropriately depending on the type of cell into which the CAR expression vector of the present invention is introduced. For example, a nucleic acid encoding the amino acid sequence of human IL-7 (SEQ ID NO: 3) can be used. As long as it has the effect of enhancing the cell proliferation rate in IL-7, 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 to the base sequence shown in SEQ ID NO: 3 may be used.

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

[0037] The nucleic acid encoding a dominant-negative mutant of SHP-1 can be selected appropriately 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 a dominant-negative mutant of human SHP-1 (SEQ ID NO: 5). As long as it is capable of inhibiting the action of SHP-1 in a dominant-negative mutant of 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 to the nucleotide sequence shown in SEQ ID NO: 5 may be used. In SEQ ID NO: 5, the serine at position 453 is the mutation site.

[0038] The nucleic acid encoding a dominant-negative mutant of SHP-2 can be selected appropriately 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 a dominant-negative mutant of human SHP-2 (SEQ ID NO: 6). As long as the action of SHP-2 in a dominant-negative mutant of SHP-2 can be inhibited, 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 to the nucleotide sequence shown in SEQ ID NO: 6 may be used. In SEQ ID NO: 6, the 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. Furthermore, such vectors may contain regulatory sequences such as promoters and terminators, as well as selectable marker sequences such as drug resistance genes and reporter genes. Placing a nucleic acid encoding a CAR or a nucleic acid encoding a T cell immune function-enhancing factor operably downstream of a promoter sequence enables efficient transcription of each nucleic acid. Furthermore, the inclusion of a marker gene allows for easy confirmation of the expression of a nucleic acid encoding a chimeric antigen receptor.

[0040] Furthermore, the CAR expression vector of the present invention may contain a nucleic acid encoding a suicide gene, and the location 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 the body by administering a drug that activates the function of the suicide gene depending on the course of cancer treatment, for example, when the tumor disappears.

[0041] Suicide genes include herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9, as 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 May 8;8(2):230-49, Bole-Richard E., FrontPharmacol. 2015 Aug 2015). 25;6:174).

[0042] Examples of the viral vector include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors, with retroviral vectors being preferred, and pMSGV vectors (Tamada k et al., Clin Cancer Res 18:6436-6445(2002)) and pMSCV vectors (Takara Bio Inc.) being more preferred. When a retroviral vector is used, the introduced gene is incorporated into the genome of the host cell, enabling 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: 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 include known methods such as viral infection, calcium phosphate method, lipofection, microinjection, and electroporation, with the viral infection method being preferred. The CAR-IL-7 expression vector may contain a nucleic acid encoding a CAR and a nucleic acid encoding interleukin-7, and the CAR-CCL19 expression vector may contain a nucleic acid encoding a CAR and a nucleic acid encoding CCL19. As with the CAR expression vector of the present invention, the vector may contain other nucleic acids such as a nucleic acid encoding a 2A peptide, an IRES, or a suicide gene, as 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 also be done 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 CAR expression 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 also be isolated and purified 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 αβ T cells, γδ T cells, and CD8 T cells. + 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 extracellularly, 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 pharmaceutically 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, isotonic 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 (spinal 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 adjusted appropriately 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 of the present invention contained in the anticancer agent to be administered is 1 × 10 4 ~1×10 10 pieces, preferably 1 x 10 5 ~1×10 9 pieces, more preferably 5 x 10 6 ~5×10 8 I can list some examples.

[0052] The anticancer drugs to be administered can 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, every ninth day, 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 and the cancer treatment methods 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; sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, 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, iosfamide, 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 suitable anti-cancer drugs include calcineurin inhibitors such as porin and tacrolimus, anti-cancer 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 bicaludamide, and immunosuppressants such as interferon, nivolumab, and pembrolizumab. Preferred examples of suitable anti-cancer drugs include alkylating agents or antimetabolites, and cyclophosphamide.

[0055] Examples of the above-mentioned method of "using the anticancer agent of the present invention in combination with another anticancer agent" include 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 preferred method of treating with another anticancer agent and then using the anticancer agent of the present invention. Furthermore, when the anticancer agent of the present invention is used in combination with another anticancer agent, the cancer therapeutic effect is further improved, and by reducing the number of administrations or the dosage of each anticancer agent, it is possible to reduce the side effects caused by each anticancer agent. Furthermore, the anticancer agent of the present invention may contain the above-mentioned other anticancer agent.

[0056] Another aspect 1 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 includes 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 for introducing the CAR expression vector of the present invention into T cells. [Example]

[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 molecules in the body that can regulate T cell function. Based on their knowledge and experience to date, the inventors first selected IL-7 and CCL19 from a vast number of combinations as regulatory molecules to further enhance the anti-tumor effect of CAR-T cells. They then selected a combination of two molecules, namely IL-7 and CCL19, rather than using either molecule alone, and created a vector that co-expresses this T cell immune function-enhancing factor 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 the bone marrow, thymus, and lymphoid organs and tissues. However, T cells themselves have little ability to produce IL-7.

[0060] Furthermore, 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) We artificially synthesized an anti-FITC CAR DNA fragment (SEQ ID NO: 7) encoding an anti-FITC CAR consisting of an anti-FITC scFv, a mouse CD8 transmembrane domain, and a mouse CD28-4-1BB-CD3ζ intracellular signal motif; an F2A-MCS DNA fragment (SEQ ID NO: 8) encoding the 2A peptide (F2A) shown in SEQ ID NO: 1 and the subsequent restriction enzyme site (MCS); and an IL-7-F2A-CCL19 DNA fragment (SEQ ID NO: 9) encoding mouse IL-7 (without a stop codon) followed by F2A and mouse CCL19. In SEQ ID NO: 7, positions 1 to 819 encode the anti-FITC scFv, positions 829 to 1074 the mouse CD8 transmembrane domain, positions 1075 to 1197 the intracellular domain of mouse CD28, positions 1198 to 1332 the intracellular domain of 4-1BB, and positions 1333 to 1674 the intracellular domain of CD3ζ. In addition, in SEQ ID NO: 9, positions 1 to 462 are the sequence encoding IL-7, positions 463 to 537 are the sequence encoding F2A, and positions 538 to 864 are the sequence encoding CCL19.

[0062] To construct a CAR vector expressing CAR, IL-7, and CCL19, the anti-FITC CAR DNA fragment and the F2A-MCS DNA fragment were ligated to generate the anti-FITC CAR-F2A-MCS construct. The resulting construct was then cloned into the pMSGV retroviral expression vector (Tamada k et al., Clin Cancer Res 18:6436-6445(2002)) to generate a pMSGV vector containing the anti-FITC CAR-F2A-MCS. The IL-7-F2A-CCL19 DNA fragment was inserted into the MCS of the pMSGV vector by restriction enzyme digestion (NsiI and SalI) and ligation to obtain a pMSGV vector containing the anti-FITC CAR-F2A-IL-7-F2A-CCL19 (IL-7 / CCL19 expression-anti-FITC CAR vector). The layout of the resulting vector is shown in Figure 2. As a control, the anti-FITC CAR DNA fragment was cloned into the pMSGV retroviral expression vector to prepare 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 IL-7 / CCL19 expression-anti-FITC CAR vector or control anti-FITC CAR vector and the pCL-Eco plasmid (Imgenex) were transfected into the GP2-293 packaging cell line (Takara Bio) using Lipofectamine 2000 or 3000 (Life Technologies). Retroviruses carrying the IL-7 / CCL19 expression-anti-FITC CAR vector or control anti-FITC CAR vector were prepared by transfection. 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 6 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 transfected with the IL-7 / CCL19 expression-anti-FITC CAR vector or the control anti-FITC CAR vector prepared above was applied to the above-mentioned mouse T cells (1 × 10 cells) activated on a plate coated with 25 μg / ml RetroNectin (registered trademark: Takara Bio Inc.). 6 The cells were mixed with 1000 μg / ml of IL-7 / CCL19-expressing anti-FITC CAR vector and centrifuged at 1500 rpm for 2 hours, followed by 6 hours of culture in the presence of IL-2 (100 IU / ml). To remove the retrovirus from the culture medium, the mouse T cells were collected, transferred to fresh growth medium (RPMI) containing IL-2 (100 IU / ml), and cultured for an additional 42 hours to obtain mouse T cells transfected with the IL-7 / CCL19-expressing anti-FITC CAR vector (anti-FITC CAR-IL-7 / CCL19-expressing T cells) or mouse T cells transfected with the control anti-FITC CAR vector (anti-FITC CAR-expressing T cells).

[0066] (Construction of anti-CD20 CAR expression vector expressing IL-7 and CCL19) A pMSGV vector (IL-7 / CCL19 expression-anti-human CD20 CAR vector) containing anti-human CD20 CAR-F2A-IL-7-F2A-CCL19 was prepared in the same manner as in the preparation of the IL-7 / CCL19 expression-anti-FITC CAR vector described above, except that 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. Similarly, a pMSGV vector (control anti-human CD20 CAR vector) containing anti-human CD20 CAR was prepared in the same manner as in the preparation of the control anti-FITC CAR vector described above, except that 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 anti-human CD20 scFv (SEQ ID NO: 10). 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. [Example]

[0067] [CAR expression measurement by flow cytometry] (Flow cytometry analysis) The expression level of CARs that recognize 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 an EC800 (Sony) system, and data analysis was performed using FlowJo software (Tree Star).

[0068] The expression level of CAR, which 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 panel shows the results for anti-FITC CAR-IL-7 / CCL19-expressing T cells without CAR staining (no FITC-conjugated dextran added), and the right panel shows the results for anti-FITC CAR-IL-7 / CCL19-expressing T cells with CAR staining (with FITC-conjugated dextran added). In Figure 4, "transduction (-)" indicates untransduced T cells, "Cont." indicates anti-FITC CAR-expressing T cells, and "7x19" indicates anti-FITC CAR-IL-7 / CCL19-expressing T cells. In Figure 5, "transduction (-)" indicates untransduced T cells, "Cont." indicates anti-human CD20 CAR-expressing T cells, and "7x19" indicates 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. [Example]

[0070] [IL-7 and CCL19 secretion] (Measurement of IL-7 and CCL19 concentrations in the culture supernatant 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. The supernatants were collected and the concentrations of IL-7 and CCL19 were measured using commercially available ELISA kits (R&D Systems). The results are shown in Figure 6.

[0071] (result) As shown in Figure 6, IL-7 was detected at levels of 300 pg / ml or more and CCL19 was detected at levels of 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 extracellularly. 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 the culture supernatant 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 represent no stimulation, the gray columns represent stimulation with FITC-conjugated trastuzumab, and the black columns represent stimulation with anti-CD3 monoclonal antibody. "Cont." represents anti-FITC CAR-expressing T cells, and "7x19" represents 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 secreted IL-7 and CCL-19 extracellularly, even when cultured for not only 3 days but also 5 and 7 days.

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

[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 induce immune responses. The generated 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, or 7 days, and the cells and supernatants were collected. Cell counts and viability were analyzed by trypan blue staining. The results are shown in Figures 9 and 10. In Figures 9 and 10, the black and white columns represent anti-FITC CAR-IL-7 / CCL19-expressing T cells, respectively, and the horizontal axis represents the number of days in culture. Statistical significance was determined 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 cell proliferation and survival rates 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 5Samples containing IL-7 / CCL19 were stimulated with P815-hCD20 in the presence of a rat IgG2a isotype control, an anti-CD127 monoclonal neutralizing antibody, or an anti-CCR7 monoclonal neutralizing antibody, along with mitomycin C. After 5 days of culture, the absolute number of viable cells was determined using trypan blue. CD127 is the receptor for IL-7, and CCR7 is the receptor for CCL19. The results are shown in Figure 11. In Figure 11, "Iso. Control" represents the results obtained by stimulation with P815-hCD20 in the presence of the rat IgG2a isotype control, "anti-CD127" represents the anti-CD127 monoclonal neutralizing antibody, and "anti-CCR7" represents the anti-CCR7 monoclonal neutralizing antibody. In Figure 11, the black columns represent anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, and the white columns represent anti-human CD20 CAR-expressing T cells. Each data point is shown as the mean ± standard deviation of triplicate wells, with *: P < 0.05 and †: P < 0.001.

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

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

[0081] (result) As shown in Figure 12, anti-FITC CAR-IL-7 / CCL19-expressing T cells induced greater migration of T cells to the subcellular layer compared with anti-FITC CAR-expressing T cells. In lymphocyte transfer therapy using CAR-expressing T cells, while tumor cell cytotoxicity by the administered T cells is of course important, it is also important to activate endogenous T cells (i.e., host immune cells) that are naturally present in the cancer patient and recruit them to attack cancer cells. To achieve this, rather than simply transferring lymphocytes with antitumor activity from an exogenous source, it is preferable to induce active interactions between the transferred T cells and endogenous T cells by some method, thereby allowing endogenous T cells to accumulate at the tumor site, thereby enhancing the immunotherapeutic effect. The results in Figure 12 demonstrate that anti-FITC CAR-IL-7 / CCL19-expressing T cells have the ability to accumulate endogenous T cells, thereby inducing active interactions 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. On day 3, 4 × 10 cells were stained with CytoTell Blue. 5 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. 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 represent anti-FITC CAR-IL-7 / CCL19-expressing T cells, and the white columns represent anti-FITC CAR-expressing T cells. In Figures 13 and 14 and Figure 15 described below, each data point is shown as the mean ± standard deviation of triplicate wells, with *: 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. On day 3, 4 × 10 cells were stained with CytoTell Blue. 5T cells were placed in the upper layer and incubated for 3 hours in the presence of a rat IgG2a isotype control, an anti-CD127 monoclonal antibody, or an anti-CCR7 monoclonal antibody. Responder T cells that migrated from the upper layer to the lower layer were analyzed by flow cytometry. The results are shown in Figure 15. In Figure 15, "Iso. Control" represents the rat IgG2a isotype control, "anti-CD127" represents the anti-CD127 monoclonal neutralizing antibody, and "anti-CCR7" represents the P815-hCD20 stimulation in the presence of an anti-CCR7 monoclonal neutralizing antibody. In Figure 15, the black columns represent anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, and the white columns represent anti-human CD20 CAR-expressing T cells.

[0085] (result) The results in Figure 15 demonstrate 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] Furthermore, the results in Figures 9 to 15 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 expressing the two regulatory molecules "IL-7" and "CCL19" in CAR-expressing T cells can improve the proliferation ability, survival rate, and immune induction effect of such T cells. [Example]

[0087] [T cell proliferation ability] Anti-FITC CAR-IL-7 / CCL19-expressing T cells and a sample containing anti-FITC CAR-expressing T cells as a control (5 × 10 5The cells (number of cells) were stained with CytoTell Blue (AAT Bioquest) and analyzed by flow cytometry after stimulation with immobilized FITC-conjugated trastuzumab. The results from day 5 after the start of stimulation are shown in Figure 16, and the results from days 3 and 7 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 divisions) in the leukocyte population.

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

[0089] [CD127 or CCR7 expression on T cells, dendritic cells, and CAR-expressing T cells] Unstimulated splenic T cells (naive T cells), splenic T cells stimulated by culturing for 2 days with anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, and IL-2 (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 for FITC-conjugated dextran beads, and dendritic cells are CD11c + The results of examining CD127 expression are shown in Figure 18, and the results of examining CCR7 expression are shown in Figure 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 Figure 18, CD127 expression was clearly reduced in activated T cells compared to naive T cells. However, it was found that the expression level was higher in anti-FITC CAR-IL-7 / CCL19-expressing T cells than in activated T cells and even more restored than in naive T cells. Furthermore, as shown in Figure 19, CCR7 expression was reduced in anti-FITC CAR-IL-7 / CCL19-expressing T cells upon activation, but maintained approximately 67% of the expression level compared to naive T cells. It has previously been known that CD127 or CCR7 expression decreases to approximately one-half to one-third upon T cell activation. Therefore, even if CAR-expressing T cells expressing IL-7 or CCL19 are generated, the effects of IL-7 and CCL19 are thought to be reduced upon activation of the CAR-expressing T cells. Therefore, it is generally unlikely that expressing IL-7 and CCL19 in CAR-expressing T cells will enhance the immune-inducing effects or antitumor activity of CAR-expressing T cells. In this study, we also confirmed that the expression of CD127 or CCR7 temporarily decreased on day 2 after splenic T cell activation. However, in anti-FITC CAR-IL-7 / CCL19-expressing T cells, the expression of CD127 or CCR7 was found to recover on day 4. This indicates that the expression of IL-7 and CCL19 in CAR-expressing T cells is useful for enhancing immune induction effects and antitumor activity. [Example]

[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 x 10 cells of P815 mastocytoma, which was genetically engineered to express 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 intravenously administered to the mice. A control group was inoculated with the P815 mastocytoma tumor and then left untreated (no CAR-expressing T cells were administered). Tumor volume and survival rates of the mice were measured twice weekly. Standard deviations were calculated for each experimental group in tumor volume analysis. Statistical differences among the three groups were determined using Student's t-tests for tumor volume analysis and log-rank tests for survival rate analysis (*P<0.05, **P<0.01).

[0092] The results of changes in tumor volume in mice are shown in Figure 20, and the results of mouse survival rates are shown in Figure 21. In Figures 20 and 21, ○ indicates the case where anti-human CD20 CAR-expressing T cells were administered, ● indicates the case 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 Figure 20, the horizontal axis represents the number of days after intravenous administration of cells to mice, and the vertical axis represents tumor volume (mm 3 21, the horizontal axis represents the number of weeks since the cells were intravenously administered to the mouse, and the vertical axis represents the survival rate (%).

[0093] (result) 20 and 21, administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells reduced tumor volume and improved survival rate (prolonged survival period) compared with administration of anti-human CD20 CAR-expressing T cells or administration of no CAR-expressing T cells. Therefore, it was demonstrated 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 the mouse 5 P815-hCD20 cells were subcutaneously inoculated. On the 10th day after inoculation, cyclophosphamide (CPA, 100 mg / kg), an anticancer drug, was intraperitoneally administered. 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 Figure 22, and the results of tumor volume are shown in Figures 23 and 24. In Figures 22 to 24, the horizontal axis represents the number of days after subcutaneous inoculation of P815-hCD20 (the day when P815-hCD20 was subcutaneously inoculated into the mice was set as day 0), and the vertical axis represents survival rate (Figure 22) and tumor volume (long axis of tumor × short axis of tumor). 2 / 2(mm 3 )) (Figures 23 and 24), where "no treatment" indicates no treatment, "CPA" indicates CPA only, "CPA+Cont." indicates the group administered anti-human CD20 CAR-expressing T cells after CPA administration, and "CPA+7x19" indicates the group administered anti-human CD20 CAR-IL-7 / CCL19-expressing T cells after CPA administration, and † indicates the death of a mouse. Note that Figure 24 is a graph in which the values ​​on the vertical axis of the CPA+7x19 graph 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 resulted in an extremely high survival rate. Furthermore, 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 resulted in the complete disappearance of tumors. Furthermore, as shown in Figure 24, the tumor volume reached its maximum on day 10 after subcutaneous inoculation of P815-hCD20, with the minor axis being 4.86 mm to 7.25 mm and the major axis 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 demonstrated that tumors that had once grown disappeared after 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 lymphocyte count with the other anticancer drug, as in the above-described method, and then administer the anti-human CD20 CAR-IL-7 / CCL19-expressing T cells, in order to further enhance the anti-tumor activity of the CAR-expressing T cells of the present invention. This method can enhance the in vivo homeostasis of CAR-expressing T cells. [Example]

[0096] [Tumor tissue penetration effect] 5x10 on the 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 sections. One section was stained with hematoxylin and eosin (H&E), and the other section was used for immunohistochemical analysis. Immunohistochemical analysis was performed using a combination of anti-CD4 and anti-CD8 monoclonal antibodies or a combination of anti-CD3 and anti-DEC205 monoclonal antibodies as primary antibodies. Alexa Fluor™ 488-conjugated anti-rat IgG2a (green) and Alexa Fluor™ 647-conjugated anti-rat IgG2b (red) were used as secondary antibodies. Cell nuclei were stained with DAPI (blue). H&E-stained samples and immunolabeled sections were observed under a microscope at ×100 or ×200 magnification. CD4 and CD8 are T cell markers, and DEC205 is a dendritic cell marker. 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). The positive areas labeled by each fluorescent staining (CD4 staining (red), CD8 staining (green), CD3 staining (red), DEC205 staining (green), and coexistence of CD3 and DEC205 (yellow)) in the data of Figures 26(a) and (b) were quantified using the Hybrid Cell Count program (KEYENCE) and shown in Figures 27(a) and (b), respectively. In Figures 25 to 27, "no treatment" or "no treat." represents the group without treatment, "Cont." represents the group treated with anti-human CD20 CAR-expressing T cells, and 7x19 represents the group treated with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells.

[0097] (result) The results in Figure 25 show that treatment with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells resulted in the progression of necrosis (areas indicated by arrows), and areas where nuclei had disappeared were observed. Furthermore, the results in Figures 26(a) and 27(a) show that treatment with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells resulted in the infiltration of T cells into the cancer tissue, and the results in Figures 26(b) and 27(b) show that treatment with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells resulted in the infiltration of dendritic cells along with T cells into the cancer tissue. [Example]

[0098] [Tumor treatment effect of the combination of IL-7 and CCL19] 5 × 10 5 Three days after inoculation, 1 × 10 anti-human CD20 CAR-expressing T cells, anti-human CD20 CAR-IL-7-expressing T cells that express only IL-7 as an immune function promoter (without CCL19 expression), anti-human CD20 CAR-CCL19-expressing T cells that express only CCL19 as an immune function promoter (without IL-7 expression), or anti-human CD20 CAR-IL-7 / CCL19-expressing T cells that express both IL-7 and CCL19 were administered subcutaneously. 6 As a control, a group of mice was administered CAR-expressing T cells that did not express IL-7 or CCL19 intravenously. The long and short axes of the tumor 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] Anti-human CD20 CAR-IL-7-expressing T cells were obtained by preparing a pMSGV vector containing anti-human CD20 CAR-F2A-IL-7 (IL-7 expression-anti-human CD20 CAR vector) and introducing it into mouse T cells in the same manner as in "Transduction of mouse T cells" in Example 1. Similarly, anti-human CD20 CAR-CCL19-expressing T cells were obtained by preparing a pMSGV vector containing anti-human CD20 CAR-F2A-CCL19 (CCL19 expression-anti-human CD20 CAR vector) and introducing it into mouse T cells in the same manner as in "Transduction of mouse T cells" in Example 1. Each vector was prepared according to the methods in "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 in SEQ ID NO: 9 and the subsequent termination codon sequence, 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, administration of anti-human CD20 CAR-IL-7-expressing T cells or anti-human CD20 CAR-CCL19-expressing T cells resulted in tumor growth inhibition similar to or slightly lower than that of administration of Conotrol anti-human CD20 CAR-expressing T cells, whereas administration of anti-human CD20 CAR-IL-7 / CCL19-expressing T cells resulted in tumor near-disappearance. Therefore, it was revealed that although IL-7 and CCL19 alone have almost no tumor growth inhibitory effect, the combination of IL-7 and CCL19 provides an extremely high tumor growth inhibitory effect. [Example]

[0101] [ 51 Tumor cytotoxicity by Cr release assay-1] (Selection of factors that promote immune function of T cells) In the cancer tissue microenvironment, inhibitory signals are transmitted to immune cells, inhibiting the anti-tumor immune response and thereby reducing the effectiveness 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 anti-tumor effect by producing dominant-negative mutants that inhibit the action of SHP-1 and SHP-2 in T cells themselves. Therefore, we constructed vectors expressing 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 vectors expressing dominant-negative mutants of SHP1 or SHP2) A DNA fragment encoding a dominant-negative mutant of mouse SHP1 (SHP1DN) containing a mutation of the catalytic cysteine ​​residue at position 453 to serine (C453S) was prepared by site-directed mutagenesis using PCR, and a DNA fragment encoding a dominant-negative mutant of mouse SHP2 (SHP2DN) containing a mutation of the catalytic cysteine ​​residue at position 459 to serine (C459S) 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 the pMSGV vector containing the anti-human CD20 scFv CAR-F2A-MCS used in the process of constructing the IL-7 / CCL19 expression-anti-human CD20 CAR vector in Example 2 to obtain the SHP1DN expression-anti-human CD20 CAR vector and the SHP2DN expression-anti-human CD20 CAR vector, respectively. The layout of the resulting vectors is shown in Figure 29.

[0103] (Transduction of mouse T cells) The SHP1DN-expressing anti-human CD20 CAR vector and the SHP2DN-expressing 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) The cytotoxic activity of CAR-expressing T cells against tumors was measured in a standard 4-hour 51 The Cr release assay was used. P815 cells expressing human CD20 (P815-hCD20) were used as target tumor cells. The tumor cells were harvested and injected with 100 μCi Na2 51 The cells were cultured in the presence of CrO4 at 37°C for 1 hour and then washed three times. Subsequently, they 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 was 51 Cr release was measured using a TopCount scintillation counter (PerkinElmer). The percentage of specific cytotoxicity was calculated using the formula: specific cytotoxicity (%) = [(test cytotoxicity - spontaneous cytotoxicity) / (maximum cytotoxicity - spontaneous cytotoxicity)] × 100. The results are shown in Figure 30. In Figure 30(a), ○ indicates anti-human CD20 CAR-expressing T cells, and ● indicates anti-human CD20 CAR-SHP1DN-expressing T cells. In Figure 30(b), ○ indicates anti-human CD20 CAR-expressing T cells, and ● indicates anti-human CD20 CAR-SHP2DN-expressing T cells. The horizontal axis represents the effector (T cell) to target (tumor cell) ratio (E / T ratio), and the vertical axis represents specific cytotoxicity (%). Statistical significance was determined 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. [Example]

[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, "●" indicates the case where anti-FITC CAR-expressing T cells were mixed in the presence of FITC-conjugated rituximab, "○" indicates the case where anti-FITC CAR-expressing T cells were mixed in the presence of unlabeled rituximab, "■" indicates the case where anti-FITC CAR-IL-7 / CCL19-expressing T cells were mixed in the presence of FITC-conjugated rituximab, and "□" indicates the case where anti-FITC CAR-IL-7 / CCL19-expressing T cells were mixed in the presence of unlabeled rituximab.

[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 supernatant was analyzed in the same manner as above. 51Cr release was measured and the percentage of cytotoxic activity was calculated. The results are shown in Figure 32. In Figure 32, mixing with anti-human CD20 CAR-IL-7 / CCL19-expressing T cells is indicated by "●", and mixing with anti-human CD20 CAR-expressing T cells is 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 tumor cytotoxic activity per cell equivalent to that of the anti-FITC CAR-expressing T cells, and similarly, the anti-human CD20 CAR-IL-7 / CCL19-expressing T cells maintained tumor cytotoxic activity per cell at a level equivalent to that of the anti-human CD20 CAR-expressing T cells. [Example]

[0109] [In vivo survival and differentiation of CAR-expressing T cells into memory T cells] (Flow cytometry analysis) 5 × 10 5 P815-hCD20 cells were subcutaneously inoculated. On the 10th day after inoculation, cyclophosphamide (CPA, 100 mg / kg), an anticancer drug, was intraperitoneally administered. 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 intravenously administered. 21 days after administration of the CAR-expressing T cells, leukocytes were isolated from the spleen or tumor-draining lymph nodes (axillary, brachial, or inguinal). The leukocyte surface phenotypes were analyzed by flow cytometry for CD4, CD8, CD44, and CD62L. The results are shown in Figure 33. Splenic leukocytes were stimulated by culturing with mitomycin C-treated P815-hCD20 for 4 days, and T cell proliferation was examined by flow cytometry. The results are shown in Figure 34. CAR expression was confirmed using biotin-labeled protein L and APC-conjugated 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 Figure 34 indicate the percentage of protein L-positive T cells. In Figures 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 the 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 survival rate results in Figures 21 and 22, it is believed that the CAR-expressing T cells of the present invention survive efficiently in the body to which they are administered and have the ability to eliminate cancer cells and increase survival rates by becoming memory T cells, demonstrating their effectiveness in preventing cancer recurrence. [Industrial Applicability]

[0111] The CAR expression vector of the present invention can be used to generate CAR-T cells that have both viability and lymphocyte accumulation ability, as well as CAR-T cells that are resistant to immunosuppression in the cancer microenvironment, and can therefore be used in the field of cancer immunotherapy.

Claims

1. An anti-tag chimeric antigen receptor (AT-CAR) expression vector comprising a nucleic acid encoding an 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-enhancing 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 cancer cells.

3. AT-CAR-expressing T cells 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 cells according to claim 3 and a pharmaceutically 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 cells according to claim 5 or 6 and a pharmaceutically acceptable additive.

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

9. A method for producing T cells expressing AT-CAR, interleukin 7, and CCL19, the method comprising the step of introducing a nucleic acid encoding AT-CAR, a nucleic acid encoding interleukin 7, and a nucleic acid encoding CCL19 into T cells 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 cancer cells.

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 T cells expressing AT-CAR, interleukin-7, and CCL19, comprising the AT-CAR expression vector shown in (a) or (b) of the 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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