Immune active cells expressing immune function regulatory factors and expression vectors

Abstract

The present application relates to immunocompetent cells and expression vectors expressing immune function regulatory factors. The subject of the present invention is to provide immunocompetent cells expressing immune function regulatory factors of immunocompetent cells and having both proliferation ability, survival ability, and T cell aggregation ability, and to provide immunocompetent cells for preparing the immune function regulatory factor expression vectors of the immunocompetent cells. Prepare immunocompetent cells expressing cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19. The cell surface molecules that specifically recognize cancer antigens are preferably T cell receptors that specifically recognize cancer antigens, and the immunocompetent cells are preferably T cells.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201780029948.1 (PCT application number PCT / JP2017 / 010437), whose application date is March 15, 2017 and whose invention name is “Immune competent cells and expression vectors expressing immune function regulatory factors”. Technical Field

[0002] The present invention relates to immunocompetent cells expressing cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19, anticancer agents containing the immunocompetent cells, and expression vectors for preparing the immunocompetent cells. Background Art

[0003] Cancer is a disease that affects a large number of patients worldwide. Chemotherapy, radiotherapy, or surgery are commonly used for treatment. However, these treatments present numerous challenges, including side effects, partial loss of function, and difficulty treating metastases.

[0004] Therefore, in order to further maintain the high quality of life (QOL) of the patient, the development of immunotherapy has been continuously carried out in recent years. In the immunotherapy, immune cell therapy is to collect immunocompetent cells from the patient, dispose of the immunocompetent cells in a manner to improve their immune function and amplify them and then move them back into the patient's therapy. Specifically, it is known that T cells are collected from the patient, the gene encoding CAR is introduced into the T cells and amplified and then the patient's therapy is again moved back into the patient's therapy (see non-patent literature 1). The therapy is currently undergoing clinical trials around the world, and has obtained the results showing effectiveness in hematopoietic organ malignancies such as leukemia and lymphoma.

[0005] In addition, as the immune function regulatory factor of immunocompetent cells such as T cells, at least hundreds of factors such as known cytokines, chemokines, signal regulatory proteins, etc. are known. Among them, interleukin 7 (IL-7) is a cytokine necessary for the survival of T cells, and it is known that it is produced by non-hematopoietic cells such as stromal cells of bone marrow, thymus, lymphoid organs / tissues. As the T cell utilizing the function of the IL-7, the T cell (referring to patent document 1) of the chimeric cytokine receptor expressing fusion IL-7 and IL-7Rα is disclosed. However, the chimeric cytokine receptor in the T cell is limited to being expressed on the membrane surface of the T cell being imported as a fusion protein, and it is only for the cell of itself to conduct the cytokine signals such as IL-7R in a ligand-independent manner, and it is impossible to improve the function of the T cell not imported into the above-mentioned receptor.

[0006] In addition, it has been disclosed that the reason for the maintenance defect of the T cell zone in the spleen of SIRPα mutant mice is the reduced expression of CCL19, CCL21, and IL-7 (see non-patent document 2). CCL19, CCL21, and IL-7 have the function of maintaining the homeostasis of T cells in secondary lymphoid tissues (spleen, lymph nodes) (see non-patent document 3). However, the above non-patent documents 2 and 3 show the effect on non-activated T cells that exist in the T cell zone of secondary lymphoid tissue in a steady state, and do not show a direct correlation with the anti-tumor immune response. Moreover, the CCL19, CCL21, and IL-7 expressing cells in the above non-patent documents 2 and 3 are not T cells, but cells of the reticuloendothelial system present in the secondary lymphoid tissue.

[0007] On the other hand, the T cell receptor (TCR) is an antigen receptor molecule expressed on the cell membrane of T cells. It is known to exist as a heterodimer composed of α and β chains, or γ and δ chains, and activates T cells by recognizing antigen molecules bound to major histocompatibility complex (MHC) molecules.

[0008] Immunotherapy is currently being developed that utilizes the functions of these TCRs, introducing TCR genes that recognize tumor antigens expressed in cancer cells into T cells obtained from cancer patients, and then reintroducing them into the patient after expansion. Specifically, a pharmaceutical composition for treating meningioma has been disclosed that contains cells expressing a TCR that specifically recognizes WT1-expressing cells (see Patent Document 2).

[0009] While some of the aforementioned technologies have demonstrated anti-tumor effects against hematopoietic malignancies, no significant effects have been demonstrated against solid tumors. This is due to concerns about low survival efficiency of transplanted immunocompetent cells within the body and insufficient activation and localization of endogenous immunocompetent cells induced by the transplanted immunocompetent cells to tumor sites. Therefore, the development of technologies to address these issues is needed.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: International Publication No. 2013 / 123061

[0013] Patent Document 2: Japanese Patent Application Laid-Open No. 2013-116891

[0014] Non-patent literature 1: Nakazawa Yozo, Shinshu Medical Journal, 61(4): 197-203 (2013)

[0015] Non-patent document 2: SATO-HASHIMOTO M. et al., J. Immunol., 2011, Vol. 187, No. 1, 291-7

[0016] Non-patent document 3: SIEGERT S. et al., Front. Immunol., 2012, Vol. 3, Article No. 285 Summary of the Invention

[0017] Problems to be solved by the invention

[0018] The immunocompetent cells used in existing immunotherapies do not sufficiently enhance the immune-inducing effects of endogenous immunocompetent cells, their proliferation and survival abilities, or their T-cell aggregation abilities. Therefore, the present invention aims to provide immunocompetent cells that express immune function regulatory factors in immunocompetent cells and that exhibit both proliferation and survival abilities and T-cell aggregation abilities, as well as immune function regulatory factor expression vectors for use in preparing such immunocompetent cells.

[0019] Means for solving problems

[0020] The inventors of this application sought to improve cells expressing immune function regulatory factors, aiming to achieve superior immune-induction effects and anti-tumor activity in cancer immunotherapy using immunocompetent cells. In this process, they focused on cytokines, chemokines, and signaling regulatory proteins, which regulate the immune function of immunocompetent cells, and constructed vectors expressing these factors. When these expression vectors were introduced into immunocompetent cells, they discovered that these cells could be produced with superior immune-induction effects, proliferation, survival, and T cell aggregation abilities compared to existing immunocompetent cells, leading to the completion of the present invention.

[0021] That is, the present invention is disclosed as follows (1) to (9).

[0022] (1) Immunocompetent cells that express cell surface molecules that specifically recognize cancer antigens, interleukin-7 (IL-7), and CCL19.

[0023] (2) The immunocompetent cell according to (1) above, wherein the cell surface molecule that specifically recognizes a cancer antigen is a T cell receptor that specifically recognizes a cancer antigen.

[0024] (3) The immunocompetent cell according to (1) or (2) above, wherein the immunocompetent cell is a T cell.

[0025] (4) The immunocompetent cell according to any one of (1) to (3) above, wherein the cancer antigen is WT1, MART-1, NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, Glypican-3, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, mesothelin, NKG2D, P1A, GD2, or GM2.

[0026] (5) An expression vector according to any one of the following (a) to (e) for producing the immunocompetent cell according to any one of (1) to (4) above,

[0027] (a) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19;

[0028] (b) The following two expression vectors (b-1) and (b-2):

[0029] (b-1) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen,

[0030] (b-2) an expression vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19;

[0031] (c) The following two expression vectors (c-1) and (c-2):

[0032] (c-1) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding IL-7,

[0033] (c-2) an expression vector containing a nucleic acid encoding CCL19;

[0034] (d) The following two expression vectors (d-1) and (d-2):

[0035] (d-1) an expression vector containing a nucleic acid encoding IL-7,

[0036] (d-2) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding CCL19;

[0037] (e) The following three expression vectors (e-1), (e-2), and (e-3):

[0038] (e-1) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen,

[0039] (e-2) an expression vector containing a nucleic acid encoding IL-7,

[0040] (e-3) An expression vector containing a nucleic acid encoding CCL19.

[0041] (6) The expression vector according to (5) above, wherein the cell surface molecule that specifically recognizes a cancer antigen is a T cell receptor that specifically recognizes a cancer antigen.

[0042] (7) The expression vector according to (5) or (6) above, characterized in that

[0043] The nucleic acid encoding the cell surface molecule that specifically recognizes the cancer antigen, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 in the expression vector of (a) are linked via a self-cleaving peptide, or

[0044] The nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 in the expression vector of (b-2) are linked via a self-cleaving peptide, or

[0045] The nucleic acid encoding the cell surface molecule that specifically recognizes the cancer antigen and the nucleic acid encoding IL-7 in the expression vector of (c-1) are linked via a self-cleaving peptide, or

[0046] In the expression vector (d-2), the nucleic acid encoding the cell surface molecule that specifically recognizes the cancer antigen and the nucleic acid encoding CCL19 are linked via a self-cleaving peptide.

[0047] (8) The expression vector according to any one of (5) to (7) above, characterized in that it contains a nucleic acid encoding a suicide gene.

[0048] (9) An anticancer agent comprising the immunocompetent cells according to any one of (1) to (4) above and a pharmaceutically acceptable additive.

[0049] Effects of the Invention

[0050] The use of the present invention's immunocompetent cells expressing cell surface molecules that specifically recognize cancer antigens, IL-7, and CCL19 (hereinafter referred to as "IL-7×CCL19-expressing immunocompetent cells") exhibits antitumor activity and can suppress the decline in survival caused by tumors formed by cancer cells bearing antigens specifically recognized by these cell surface molecules. Furthermore, the use of the present invention's expression vectors enables the production of immunocompetent cells that exhibit both proliferation and survival abilities and the ability to recruit T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] [ Figure 1 ] is a diagram showing the genetic map of the IL-7×CCL19 expression vector.

[0052] [ Figure 2A ] is a graph showing the results of investigating the number of IL-7 / CCL19-expressing T cells.

[0053] [ Figure 2B ] is a graph showing the results of investigating the survival rate of IL-7 / CCL19-expressing T cells.

[0054] [ Figure 3 ] is a graph showing the results of a T cell migration assay using IL-7 / CCL19-expressing T cells.

[0055] [ Figure 4 ] is a diagram showing the genetic map of the IL-7×CCL19×HSV-TK expression vector.

[0056] [ Figure 5 ] is a diagram showing the genetic map of the TCR×IL-7×CCL19 expression vector.

[0057] [ Figure 6 ] is a diagram showing the genetic map of the IL-7×CCL19×eGFP expression vector.

[0058] [ Figure 7 ] is a graph showing the survival rates of untreated mice, mice administered with P1A-specific TCR / eGFP-expressing T cells, and mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells.

[0059] [ Figure 8 ] is a graph showing the results of investigating the tumor volumes of untreated mice, mice administered with P1A-specific TCR / eGFP-expressing T cells, and mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells. DETAILED DESCRIPTION

[0060] The IL-7×CCL19-expressing immunocompetent cells of the present invention are not particularly limited as long as they express cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19. They may also express other immune function regulatory factors such as IL-15, CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, CCL4, Flt3L, interferon-γ, MIP-1α, GM-CSF, M-CSF, TGF-β, and TNF-α.

[0061] Cancer antigens refer to substances such as proteins and glycolipids that are expressed at higher levels in cancer cells than in normal cells, or that are specifically expressed in cancer cells. Examples of such cancer antigens include tumor-associated antigens, cancer-testis antigens, angiogenesis-associated antigens, and epitope peptides of cancer neoantigens (neoantigens) generated by gene mutations. Specific examples include WT1, MART-1, NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, Glypican-3, and KIF20. A, survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, mesothelin, NKG2D, P1A and other proteins, GD2, GM2 and other glycolipids, but are not limited thereto.

[0062] As cell surface molecules that specifically recognize cancer antigens, cell surface receptors, artificial receptors, and adhesion factors that specifically recognize cancer antigens can be mentioned. Preferably, T cell receptors that specifically recognize cancer antigens, chimeric antigen receptors (CARs) that specifically recognize cancer antigens, etc. are given molecules that specifically recognize cancer by expressing them on the cell surface. TCR can be more preferably mentioned. As TCR, it can be a heterodimer (α·βTCR) comprising an α chain and a β chain or a heterodimer (γ·δTCR) comprising a γ chain and a δ chain that specifically recognize cancer antigens. It should be noted that if the cell surface molecules that specifically recognize cancer antigens are specific for the recognition of cancer antigens, they can also be recognized indirectly. For example, antibodies and other molecules that specifically recognize cancer antigens are administered to the subject simultaneously or continuously with the immunocompetent cells of the present invention, and by recognizing the antibodies and other molecules or the labels marked on the recognition antibodies and other molecules, the immunocompetent cells of the present invention can indirectly specifically recognize cancer antigens. Examples of antibodies that recognize antibodies include CD16, a cell surface molecule, and examples of tags that label molecules such as antibodies include FITC.

[0063] As the type of immune active cells in the IL-7×CCL19 expressing immune active cells of the present invention, any cells that participate in the immune response may be mentioned, such as T cells, natural killer cells (NK cells), B cells and other lymphocyte lineage cells, monocytes, macrophages, dendritic cells and other antigen presenting cells, neutrophils, eosinophils, basophils, mast cells and other granulocytes; preferably, T cells from mammals such as humans, dogs, cats, pigs, mice (preferably T cells from humans) can be mentioned. In addition, T cells can be obtained by isolating and purifying immune cells from body fluids such as blood and bone marrow fluid, tissues such as spleen, thymus, lymph nodes, or cancer tissues such as primary tumors, metastatic tumors, cancerous ascites, etc., or by using T cells prepared from ES cells and iPS cells. As the T cells, α·βT cells, γ·δT cells, CD8 + T cells, CD4 + T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells.

[0064] Methods for producing IL-7×CCL19-expressing immunocompetent cells of the present invention include methods in which the expression vector of the present invention described below is introduced into immunocompetent cells. Alternatively, methods include methods in which a vector expressing a cell surface molecule that specifically recognizes a cancer antigen, interleukin 7 (IL-7), and / or CCL19 is introduced into fertilized eggs, ES cells, or iPS cells, followed by induction; and methods in which a vector expressing a cell surface molecule that specifically recognizes a cancer antigen, interleukin 7 (IL-7), and / or CCL19 is further introduced, as needed, into immunocompetent cells isolated from a transgenic mammal expressing a cell surface molecule that specifically recognizes a cancer antigen by gene transfer.

[0065] The method for introducing the expression vector of the present invention described below into the above-mentioned immunocompetent cells is not particularly limited, and examples thereof include methods of introduction by known methods such as viral infection, calcium phosphate method, lipofection, microinjection, and electroporation. Preferably, introduction by viral infection is used.

[0066] Examples of viral infection methods include the following: transfecting the expression vector of the present invention and a packaging plasmid into packaging cells such as GP2-293 cells (manufactured by TAKARA BIO), Plat-GP cells (manufactured by COSMOBIO), PG13 cells (ATCC CRL-10686), and PA317 cells (ATCC CRL-9078) to prepare recombinant virus, which is then infected into immunocompetent cells. This can be performed using commercially available kits such as Retrovirus Packaging Kit Eco (manufactured by TAKARA BIO).

[0067] In addition, the immunocompetent cells of the present invention can be prepared by the following method: using known gene editing technology, a polynucleotide comprising a base sequence encoding a cell surface molecule that specifically recognizes a cancer antigen, IL-7, and CCL19 is incorporated into the genome of the cell in a manner that allows expression under the regulation of an appropriate promoter. As known gene editing technologies, technologies using endonucleases such as zinc finger nucleases, TALEN (transcription activation effector nuclease-like), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas systems can be cited. In the case of expressing other foreign proteins in the immunocompetent cells of the present invention, gene editing technology can also be used in the same manner to incorporate a polynucleotide comprising a base sequence encoding other foreign proteins into the genome of the cell in a manner that allows expression under the regulation of an appropriate promoter. Methods for incorporating a polynucleotide into the cell genome so that it can be expressed under the regulation of an appropriate promoter include: a method in which a polynucleotide to which a base sequence encoding a cell surface molecule that specifically recognizes a cancer antigen, IL-7, or CCL19 (or other proteins) is functionally linked downstream of an appropriate promoter (i.e., a polynucleotide to which a coding sequence is linked so that it can be expressed under the regulation of the promoter) is incorporated into a non-coding region of the cell genome, etc.; a method in which a polynucleotide containing a base sequence encoding a cell surface molecule that specifically recognizes a cancer antigen, IL-7, or CCL19 (or other proteins) is incorporated downstream of an endogenous promoter in the cell genome, etc. Examples of endogenous promoters include promoters such as TCRα and TCRβ.

[0068] Furthermore, the following herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9 can also be expressed in the IL-7×CCL19-expressing immunocompetent cells of the present invention.

[0069] The IL-7×CCL19-expressing immunocompetent cells of the present invention have strong proliferation, survival, and endogenous T cell aggregation capabilities due to their expression of cell surface molecules that specifically recognize cancer antigens, IL-7, and CCL19, and can be used in various adoptive immunotherapies using immunocompetent cells. Examples of adoptive immunotherapy include, but are not limited to, dendritic cell therapy, NK cell therapy, γ·δT cell therapy, α·βT cell therapy, CTL therapy, and TIL therapy. The following method can be mentioned: the expression vector of the present invention described below is introduced into immunocompetent cells collected from a patient for amplification, and then administered to the patient. Specific examples are given below, but are not limited to these. Dendritic cell therapy includes the steps of: introducing surgically removed cancer tissue or its lysate into dendritic cells differentiated from monocytes collected from the patient, and administering it to the patient; it can also include the step of introducing the expression vector of the present invention into dendritic cells. Here, epitope peptides of cancer antigen molecules can also be artificially synthesized and used instead of the above-mentioned cancer tissue or lysate. NK cell therapy includes the following steps: activating and proliferating NK cells collected from patients using various stimulating substances such as IL-2, and administering them to patients; it may also include the step of introducing the vector of the present invention into NK cells. It should be noted that by combining antibody drugs against cancer with activated NK cells, it is expected that cancer cells can be attacked efficiently. γ·δT cell therapy includes the following steps: culturing and stimulating lymphocytes collected from patients using IL-2 or zoledronic acid to proliferate γ·δT cells, and administering them to patients; it may also include the step of introducing the expression vector of the present invention into γ·δT cells. α·βT cell therapy includes the steps of culturing lymphocytes collected from patients using anti-CD3 antibodies or IL-2, administering activated α·βT cells to patients, and it may also include the step of introducing the expression vector of the present invention into α·βT cells. CTL therapy involves stimulating lymphocytes collected from the patient with cancer cells, culturing them with anti-CD3 antibodies or IL-2, and then administering them to the patient. It may also include introducing the expression vector of the present invention into the CTLs. Alternatively, antigen-presenting cells that present cancer antigen epitope peptides may be used instead of the cancer cells described above. TIL therapy involves collecting lymphocytes from cancer tissue collected from the patient, stimulating and culturing them with IL-2 or the like, and administering them to the patient. It may also include introducing the expression vector of the present invention into the lymphocytes.

[0070] The expression vector of the present invention is any one of the following (a) to (e) for preparing the IL-7×CCL19-expressing immunocompetent cells of the present invention.

[0071] (a) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19;

[0072] (b) The following two expression vectors (b-1) and (b-2):

[0073] (b-1) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen,

[0074] (b-2) an expression vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19;

[0075] (c) The following two expression vectors (c-1) and (c-2):

[0076] (c-1) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding IL-7,

[0077] (c-2) an expression vector containing a nucleic acid encoding CCL19;

[0078] (d) The following two expression vectors (d-1) and (d-2):

[0079] (d-1) an expression vector containing a nucleic acid encoding IL-7,

[0080] (d-2) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding CCL19;

[0081] (e) The following three expression vectors (e-1), (e-2), and (e-3):

[0082] (e-1) an expression vector containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen,

[0083] (e-2) an expression vector containing a nucleic acid encoding IL-7,

[0084] (e-3) an expression vector containing a nucleic acid encoding CCL19;

[0085] The expression vector of the present invention may further contain nucleic acids encoding other immune function regulatory factors such as IL-15, CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, CCL4, Flt3L, interferon-γ, MIP-1α, GM-CSF, M-CSF, TGF-β, TNF-α, etc.

[0086] The nucleic acids encoding cell surface molecules that specifically recognize cancer antigens, interleukin-7 (IL-7), and CCL19 can each be derived from mammals, preferably humans. Each of these nucleic acids can be appropriately selected depending on the cell type into which the expression vector of the present invention is introduced. Sequence information for each of these nucleic acids can be appropriately obtained by searching known literature or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0087] As the nucleic acid encoding the cell surface molecules that specifically recognize cancer antigens, preferably nucleic acids from humans can be mentioned. The nucleic acid encoding the cell surface molecules that specifically recognize cancer antigens can be a nucleic acid encoding a T cell receptor (TCR) or a nucleic acid encoding a chimeric antigen receptor (CAR), which can be a nucleic acid of natural origin or an artificially synthesized nucleic acid. It can be appropriately selected according to the type of cell into which the expression vector of the present invention is introduced, and the sequence information can be appropriately obtained by searching known documents or databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / guide / ).

[0088] Nucleic acids encoding cell surface molecules that specifically recognize cancer antigens, nucleic acids encoding IL-7, and nucleic acids encoding CCL19 can be prepared based on the base sequence information of the respective encoding nucleic acids using known techniques such as chemical synthesis and PCR amplification. It should be noted that the codons selected to encode amino acids may be altered to optimize expression of the nucleic acid in the target host cell.

[0089] The TCR in the nucleic acid encoding the TCR may be a heterodimer comprising an α chain and a β chain (α·βTCR) or a heterodimer comprising a γ chain and a δ chain (γ·δTCR). It should be noted that the nucleic acid encoding the α·βTCR includes both nucleic acid encoding the α chain and the β chain of the TCR, and the nucleic acid encoding the γ·δTCR includes both nucleic acid encoding the γ chain and the δ chain of the TCR.

[0090] Sequence information of the nucleic acid encoding the TCR can be obtained by identifying the α-chain and β-chain nucleic acids of the TCR subunits of CTLs induced with a specific antigenic peptide using methods known in the art (International Publication No. 2007 / 032255 and Morgan et al., J. Immunol., 171, 3288 (2003)). For example, PCR is preferably used to analyze TCRs. PCR primers used for analysis include, but are not limited to, the 5'-R primer (5'-gtctaccaggcattcgcttcat-3': SEQ ID NO: 3) as the 5'-end primer, the 3'-TRa-C primer (5'-tcagctggaccacagccgcagcgt-3': SEQ ID NO: 4) specific for the TCR α chain C region, the 3-TRb-C1 primer (5'-tcagaaatcctttctcttgac-3': SEQ ID NO: 5) specific for the TCR β chain C1 region, or the 3-TRβ-C2 primer (5'-ctagcctctggaatcctttctctt-3': SEQ ID NO: 6) specific for the TCR β chain C2 region. TCR derivatives can bind to target cells presenting antigenic peptides with high affinity and can mediate efficient killing of target cells presenting antigenic peptides in vivo and in vitro.

[0091] As the nucleic acid encoding the TCR, for example, a nucleic acid encoding a TCR specific for MART1 (Cancer Res. 54, 5265-5268 (1994)), a nucleic acid encoding an TCR specific for MAGE-A3 (Anticancer Res., 20, 1793-1799 (2000)), gp100-specific TCR (J. Immunol. 170, 2186-2194 (2003)), NY-ESO-1-specific TCR (J. Immunol., 174, 4415-4423 (2005)), WT1-specific TCR (Blood, 106, 470-476 (2005)), MAGE-A1-specific TCR (Int. Immunol., 8, 1463-1466 (1996)), P1A-specific TCR (Sarma, S., Y. Guo, Y. Guilloux, C. Lee, X.-F. Bai, Y. Liu. 1999. Cytotoxic T lymphocytes to anunmutated tumor antigen P1A: normal development but restrained The nucleic acid of a TCR encoding a TCR described in the aforementioned literature may be a nucleic acid encoding an antigen molecule bound to an MHC molecule (e.g., J. Exp. Med. 189:811), and may be a nucleic acid encoding a TCR having an effector function. J. Exp. Med. 189:811), and may be a nucleic acid sequence 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 a nucleic acid sequence encoding a TCR described in the aforementioned literature. Alternatively, the nucleic acid sequence may be a nucleic acid sequence encoding a TCR described in the aforementioned literature, wherein the sequence encoding the CDRs is determined, the sequence encoding the CDRs is maintained, and the sequence other than the CDR encoding sequence has an identity of 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more to a nucleic acid sequence encoding a TCR described in the aforementioned literature.

[0092] Examples of nucleic acids encoding IL-7 include base sequences encoding the amino acid sequence set forth in SEQ ID NO: 1. As long as the nucleic acid has an effect of enhancing cell proliferation or cell survival in the context of IL-7, a base sequence 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 base sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 may also be used. Examples of nucleic acids encoding CCL19 include base sequences encoding the amino acid sequence set forth in SEQ ID NO: 2. As long as the nucleic acid has an effect of enhancing cell migration in the context of CCL19, a base sequence 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 base sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 may also be used.

[0093] In addition, the expression vector of the present invention may contain a nucleic acid encoding a suicide gene. The so-called suicide gene refers to a gene with the following function: by being expressed, it directly or secondarily induces a cytotoxic substance, causing the cell death of the organism. By making the expression vector of the present invention contain a nucleic acid encoding a suicide gene, according to the treatment process of cancer, for example, when the tumor disappears, a drug that activates the suicide gene function is administered, thereby regulating the immune-competent cells in the organism. In addition, IL-7 or CCL19 are different from other cytokines and have a low possibility of causing cytokine release syndrome as a side effect or tumorigenesis of gene-transferred cells. However, since the function of the immune-competent cells introduced with the expression vector of the present invention is improved, the cytokines released when attacking the target cancer tissue may unexpectedly affect the surrounding tissues. In this case, by making the expression vector of the present invention contain a nucleic acid encoding a suicide gene, the risk of cytokine release syndrome can be effectively reduced.

[0094] Examples of suicide genes include genes encoding herpes simplex virus thymidine kinase (HSV-TK) or inducible caspase 9 as described in the following literature. Drugs that activate the functions of these genes include ganciclovir for the former and chemical induction of dimerization (CID) AP1903 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. Front Pharmacol. 2014 Nov 27; 5: 254., Minagawa K., Pharmaceuticals (Basel). 2015 May 8; 8(2): 230-49., Bole-Richard ... E., Front Pharmacol. 2015 Aug 25;6:174).

[0095] In the expression vector (a) of the present invention containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, any nucleic acid can be arranged at any upstream or downstream. Specifically, taking the case of containing a nucleic acid encoding a TCR as an example of a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, the order from upstream may be a nucleic acid encoding a TCR, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19, or a nucleic acid encoding a TCR, a nucleic acid encoding CCL19, and a nucleic acid encoding IL-7, or a nucleic acid encoding IL-7, a nucleic acid encoding CCL19, and a nucleic acid encoding TCR, or a nucleic acid encoding IL-7, a nucleic acid encoding TCR, and a nucleic acid encoding CCL19, or a nucleic acid encoding CCL19, a nucleic acid encoding TCR, and a nucleic acid encoding IL-7, or a nucleic acid encoding CCL19, a nucleic acid encoding TCR, and a nucleic acid encoding IL-7, or a nucleic acid encoding CCL19, a nucleic acid encoding TCR, and a nucleic acid encoding IL-7.

[0096] In the expression vector (b-2) of the present invention containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19, the arrangement of the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 is not particularly limited. The nucleic acid encoding CCL19 may be arranged upstream or downstream relative to the nucleic acid encoding IL-7.

[0097] In the expression vector (c-1) of the vector of the present invention containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding IL-7, the arrangement of the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and the nucleic acid encoding IL-7 is not particularly limited, and the nucleic acid encoding IL-7 can be arranged upstream or downstream relative to the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen.

[0098] In the expression vector (d-2) of the vector of the present invention containing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and a nucleic acid encoding CCL19, the arrangement of the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen and the nucleic acid encoding CCL19 is not particularly limited. The nucleic acid encoding CCL19 may be arranged upstream or downstream of the nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen.

[0099] It should be noted that the nucleic acid encoding the cell surface molecule that specifically recognizes cancer antigens, the nucleic acid encoding IL-7, and the nucleic acid encoding CCL19 can be transcribed using different promoters, or can be transcribed using a single promoter using an internal ribosomal entry site (IRES) or a self-cleaving 2A peptide.

[0100] When a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 are transcribed from a single promoter using an internal ribosome entry site (IRES) or a self-cleaving 2A peptide, any nucleic acid may be included between the nucleic acids encoding IL-7 and CCL19, when a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen is included, between the nucleic acid encoding the IL-7 and CCL19 nucleic acids, when a nucleic acid encoding an α·β TCR is included, between the nucleic acid encoding the α chain and the nucleic acid encoding the β chain, and when a nucleic acid encoding a γ·δ TCR is included, between the nucleic acid encoding the γ chain and the nucleic acid encoding the δ chain. Any nucleic acid may be included as long as each nucleic acid can be expressed, but it is preferably linked via a sequence encoding a self-cleaving peptide (2A peptide) or an IRES, preferably linked via a sequence encoding a 2A peptide. Linking using such sequences allows efficient expression of each nucleic acid.

[0101] In addition, when containing a nucleic acid encoding a suicide gene, the position of the suicide gene is not particularly limited. For example, it can be located downstream of a promoter for expressing a nucleic acid encoding a cell surface molecule that specifically recognizes a cancer antigen, a nucleic acid encoding IL-7, or a nucleic acid encoding CCL19. It can be located upstream or downstream of each of the above nucleic acids via a sequence encoding a 2A peptide or an IRES, or it can be located downstream of another promoter.

[0102] The so-called 2A peptide refers to a self-cleaving peptide derived from a virus, which has the following characteristics: it is cleaved by the endoplasmic reticulum between the GP (one residue from the C-terminus) in the amino acid sequence shown in SEQ ID NO: 7 (Szymczak et al., Expert Opin. Biol. Ther. 5(5):627-638 (2005)). Therefore, the nucleic acids incorporated before and after the 2A peptide are expressed independently in the cell.

[0103] The 2A peptide is preferably a 2A peptide derived from picornavirus, rotavirus, insect virus, aphthous virus, or trypanosome virus, and more preferably a 2A peptide (F2A) derived from picornavirus as shown in SEQ ID NO: 8.

[0104] The expression vectors of the present invention may be linear or circular, and may be non-viral vectors such as plasmids, viral vectors, or transposon-based vectors. Furthermore, these vectors may contain regulatory sequences such as promoters and terminators, and selectable marker sequences such as drug resistance genes and reporter genes. By operably placing the nucleic acid encoding IL-7 and the nucleic acid encoding CCL19 downstream of the promoter sequence, each nucleic acid can be efficiently transcribed.

[0105] Examples of such promoters include viral promoters such as the retroviral LTR promoter, the SV40 early promoter, the cytomegalovirus promoter, and the herpes simplex virus thymidine kinase promoter; and mammalian promoters such as the phosphoglycerate kinase (PGK) promoter, the Xist promoter, the β-actin promoter, and the RNA polymerase II promoter. Furthermore, tetracycline-responsive promoters induced by tetracycline and the interferon-induced Mx1 promoter can also be used. By using promoters induced by these specific substances in the expression vectors of the present invention, the induction of IL-7 and CCL19 expression can be regulated according to the course of cancer treatment.

[0106] Examples of the viral vector include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. Preferred examples include retroviral vectors, and more preferred examples include pMSGV vectors (Tamada et al., Clin Cancer Res 18: 6436-6445 (2002)) and pMSCV vectors (manufactured by 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.

[0107] To confirm that immunocompetent cells contain the expression vector of the present invention, for example, when containing a nucleic acid encoding a TCR, TCR expression can be investigated using the following methods: flow cytometry; PCR methods such as Northern blotting, Southern blotting, and RT-PCR; ELISA and Western blotting. If the expression vector of the present invention contains a marker gene, this can be confirmed by investigating the expression of the marker gene inserted into the expression vector.

[0108] When the expression vector contained in the IL-7×CCL19-expressing immunocompetent cells of the present invention contains a nucleic acid encoding TCR, the variable region of the expressed TCR is located outside the cell. By having such a TCR variable region, the TCR-expressing immunocompetent cells can recognize antigen molecules bound to MHC molecules.

[0109] The anticancer agent of the present invention is not particularly limited as long as it contains the IL-7×CCL19-expressing immunocompetent cells of the present invention and pharmaceutically acceptable additives. Examples of such additives include physiological saline, buffered physiological saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonic agents, fillers, and lubricants.

[0110] The anticancer agent of the present invention can be administered to a subject in need of cancer treatment using methods known to those skilled in the art. Examples of administration methods include intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intramedullary, and subarachnoid (cerebrospinal fluid) injection.

[0111] The amount of IL-7×CCL19-expressing immunocompetent cells of the present invention contained in the administered anticancer agent can be appropriately adjusted according to the type, location, severity of the cancer, age, weight, and condition of the subject to be treated, but preferably, 1×10 4 ~1×10 10 , preferably 1×10 5 ~1×10 9 More preferably 5×10 6 ~5×10 8 indivual.

[0112] The anticancer agent to be administered can be administered 4 times, 3 times, 2 times or once a day, every other day, every other 2 days, every other 3 days, every other 4 days, every other 5 days, once a week, every 7 days, every 8 days, every 9 days, twice a week, once a month or twice a month.

[0113] Cancers targeted by the anticancer agents of the present invention and the following cancer treatment methods may be solid tumors or blood cancers, and examples include adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, 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; and sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, Wilms' tumor, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma; blastomas, lymphomas, and leukemias.

[0114] The anticancer agent of the present invention can be used in combination with other anticancer agents. Other anticancer agents include: alkylating drugs such as cyclophosphamide, bendamustine, ifosfamide, 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, gefitinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib; proteasome inhibitors such as bortezomib, and calcineurin inhibitors such as cyclosporine and tacrolimus; anticancer antibiotics such as idarubicin, doxorubicin, and mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum preparations such as cisplatin, oxaliplatin, and carboplatin; hormone therapy drugs such as tamoxifen and bicalutamide; and immunomodulatory drugs such as interferon, nivolumab, and pembrolizumab. Preferred examples include alkylating drugs and metabolic antagonists.

[0115] Examples of methods for "using the anticancer agent of the present invention in combination with another anticancer agent" include: treating with another anticancer agent before using the anticancer agent of the present invention; using the anticancer agent of the present invention and another anticancer agent simultaneously; and treating with the anticancer agent of the present invention before using the other anticancer agent. A preferred method is treating with another anticancer agent before using the anticancer agent of the present invention. Furthermore, using the anticancer agent of the present invention in combination with another anticancer agent can further enhance the therapeutic effect of cancer while reducing the frequency or dosage of administration of each anticancer agent, thereby reducing side effects caused by each anticancer agent. Furthermore, the anticancer agent of the present invention may also contain the aforementioned other anticancer agent.

[0116] As another embodiment 1 of the present invention, the following may be mentioned: 1) a method for treating cancer, characterized in that immunocompetent cells expressing cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19 are administered to a patient in need of cancer treatment; 2) immunocompetent cells that are used as anticancer agents and express cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19; and 3) use of immunocompetent cells that express cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19 in the preparation of anticancer agents.

[0117] In addition, as another aspect 2 of the present invention, a kit for preparing immunocompetent cells expressing cell surface molecules that specifically recognize cancer antigens, interleukin 7 (IL-7), and CCL19, comprising the above-mentioned expression vector of the present invention, can be cited. The kit is not particularly limited as long as it comprises the expression vector of the present invention, and may also include instructions for preparing immunocompetent cells expressing IL-7×CCL19 of the present invention and reagents for introducing the expression vector of the present invention into immunocompetent cells.

[0118] Example 1

[0119] (Selection of immune function regulatory factors)

[0120] There are at least hundreds of molecules in the body that can regulate T cell function. Based on prior knowledge and experience, the inventors of this application first selected IL-7 and CCL19 from a large number of combinations as regulatory molecules to further enhance the immune function regulation effect in immunocompetent cells. Furthermore, rather than selecting each alone, they selected a combination of IL-7 and CCL19 to prepare a vector expressing these immune function regulatory factors in immunocompetent cells.

[0121] (Preparation of vectors expressing IL-7 and CCL19-1)

[0122] Artificially synthesized anti-FITC CAR DNA fragment (SEQ ID NO: 9) encoding an anti-FITC CAR containing an anti-FITC scFv, a mouse CD8 transmembrane region, and a mouse CD28-4-1BB-CD3δ intracellular signaling motif; an F2A-MCS DNA fragment (SEQ ID NO: 10) encoding the 2A peptide (F2A) shown in SEQ ID NO: 8 and the restriction endonuclease site (MCS) following the peptide; and an IL-7-F2A-CCL19 DNA fragment (SEQ ID NO: 11) encoding mouse IL-7 (without a stop codon) and the following F2A and mouse CCL19 (manufactured by Life Technology).

[0123] In order to prepare a vector expressing IL-7 and CCL19, the above-mentioned anti-FITC CAR DNA fragment was connected to the above-mentioned F2A-MCS DNA fragment to prepare an anti-FITC CAR-F2A-MCS construct. Next, the prepared construct was cloned into the pMSGV retroviral 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 above-mentioned IL-7-F2A-CCL19 DNA fragment was inserted into the MCS of the pMSGV vector by treatment with restriction endonucleases (NsiI and SalI) and ligation to obtain a pMSGV vector containing anti-FITC CAR-F2A-IL-7-F2A-CCL19 (IL-7×CCL19 expression vector (1)). The genetic map of the obtained vector is shown in FIG. Figure 1 In addition, as a control, the anti-FITC CAR DNA fragment was cloned into the pMSGV retroviral expression vector to prepare a pMSGV vector without IL-7 and CCL19 (control vector (1)).

[0124] (Preparation of Retrovirus Introduced with IL-7×CCL19 Expression Vector)

[0125] To transduce mouse T cells, retroviruses were prepared. The IL-7×CCL19 expression vector (1) or control vector (1) and pCL-Eco plasmid (Imgenex) were transfected into GP2-293 packaging cell lines (TAKARA BIO) using Lipofectamine 2000 or 3000 (Life Technologies). Retroviruses containing the IL-7×CCL19 expression vector (1) or control vector (1) were prepared.

[0126] DMEM supplemented with 10% FCS, 100 U / ml penicillin, and 100 mg / ml streptomycin was used as the culture medium for the GP2-293 cells. RPMI-1640 supplemented with 10% FCS, 100 U / ml penicillin, 100 mg / ml streptomycin, 50 mM 2-mercaptoethanol, and 2 mM L-glutamine was used as the culture medium for the T cells used in the examples described below.

[0127] (Transduction of mouse T cells)

[0128] For transduction of mouse T cells, 3×10 T cells from spleen and lymph nodes were transduced with immobilized anti-CD3 mAb (3 μg / ml) and IL-2 (100 IU / ml).6 The purified mouse T cells were activated for 48 hours. Next, the supernatant containing the retrovirus introduced with the IL-7×CCL19 expression vector (1) or the control vector (1) prepared above was mixed with the mouse T cells (1×10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 6 The cells were mixed with 100 cells / ml of culture medium and centrifuged at 1500 rpm for 2 hours. The cells were then cultured in the presence of IL-2 (100 IU / ml) for 6 hours. To remove the retrovirus from the culture medium, the mouse T cells were recovered and transferred to a new proliferation culture medium (RPMI) containing IL-2 (100 IU / ml). The cells were cultured for another 42 hours to obtain mouse T cells into which the IL-7×CCL19 expression vector (1) had been introduced (IL-7 / CCL19-expressing T cells (1)) or mouse T cells into which the control vector (1) had been introduced (control T cells (1)).

[0129] (Preparation of expression vectors expressing IL-7 and CCL19-2)

[0130] In the preparation of the above-mentioned IL-7×CCL19 expression vector (1), the sequence of the anti-FITC scFv region contained in the sequence shown in SEQ ID NO: 9 was replaced with the sequence of the anti-human CD20 scFv (SEQ ID NO: 12) synthesized by Life Technology based on the rituximab sequence. In addition, the pMSGV vector containing anti-human CD20 CAR-F2A-IL-7-F2A-CCL19 (IL-7×CCL19 expression vector (2)) was prepared by the same method as the above-mentioned "Preparation of an expression vector expressing IL-7 and CCL19-1". Similarly, in the preparation of the above-mentioned control vector (1), the sequence of the anti-FITC scFv region contained in the sequence shown in SEQ ID NO: 9 was replaced with the sequence of the anti-human CD20 scFv (SEQ ID NO: 12). In addition, the pMSGV vector not containing IL-7 and CCL19 (control vector (2)) was prepared by the same method as the above-mentioned "Preparation of an expression vector expressing IL-7 and CCL19-1". By the same method as above, the IL-7×CCL19 expression vector (2) or the control vector (2) was introduced into mouse T cells using retrovirus to prepare IL-7 / CCL19-expressing T cells (2) or control T cells (2).

[0131] Example 2

[0132] (Cell number and survival rate of IL-7 / CCL19-expressing T cells)

[0133] We investigated whether IL-7 and CCL19 produced by IL-7 / CCL19-expressing T cells could exert biological functions and show immune induction effects. 5 The samples of T cells (2) or control T cells were cultured for 5 days. In order to exclude the effect of human CD20 CAR on the expression of IL-7 and CCL19, the above culture was performed in the absence of antigen stimulation caused by CD20. Next, the cell number and viability were determined using trypan blue. The results are shown in Figure 2. Figure 2A 、 Figure 2B shown. Figure 2A is the number of cells, Figure 2B The black columns represent IL-7 / CCL19-expressing T cells, and the white columns represent control T cells.

[0134] (result)

[0135] like Figure 2A 、 Figure 2B As shown, the number of cells in IL-7 / CCL19-expressing T cells (2) was approximately five times higher, and the survival rate was approximately two times higher, compared to control T cells (2). Therefore, it was clarified that by using IL-7 / CCL19-expressing T cells obtained by introducing the expression vector of the present invention into T cells, IL-7 and CCL19 exerted biological functions and showed an immune-inducing effect.

[0136] Example 3

[0137] [T cell migration assay]

[0138] (T cell migration assay using IL-7 / CCL19-expressing T cells)

[0139] The migration inducing effect of CCL19 was studied by using a cell migration test performed using Transwell. For the migration of responding side T cells, a 96-well Transwell (registered trademark) chamber (manufactured by Corning Costar) was used to allow them to migrate through a polycarbonate filter with a pore size of 5 μm, and the measurement was performed. Specifically, IL-7 / CCL19 expressing T cells (1) or control T cells (1) were cultured in the lower layer of the chamber. In order to exclude the effect of FITC CAR on the expression of IL-7 and CCL19, the above culture was performed in the absence of antibody stimulation caused by FITC. Responding side T cells were prepared from spleen and lymph nodes by negative selection using MACS (manufactured by Miltenyi Biotech). Responding side T cells were labeled with CytoTell blue (manufactured by AAT Bioquest) and cultured in the upper layer for 3 hours. The migration from the upper layer to the lower layer of the chamber was investigated by flow cytometry (EC800: manufactured by Sony Corporation), and data analysis was performed using FlowJo software (manufactured by Tree Star Corporation). The results are shown in FIG. Figure 3 shown. Figure 3 In the figure, black columns represent IL-7 / CCL19-expressing T cells (1), white columns represent control T cells (1), and the vertical axis represents the absolute number of responder-side T cells that migrated to the lower chamber. Statistically significant differences were examined using the Student's t-test.

[0140] (result)

[0141] like Figure 3 As shown, IL-7 / CCL19 expressing T cells (1) caused about 1.8 times more T cells to migrate to the lower layer compared to control T cells (1). In T cell and other lymphocyte transplantation therapies, the damage to cancer cells caused by the administered T cells is certainly important, but in addition, it is also important to activate the endogenous T cells (= host-side immune cells) originally present in cancer patients and mobilize them to attack cancer cells. For this reason, from the perspective of improving the effect of immunotherapy, it is preferable not only to simply transplant lymphocytes with anti-tumor activity from the outside, but also to induce a positive interaction between the transplanted T cells and endogenous T cells by some means, so that the endogenous T cells gather locally in the cancer. By Figure 3 The results clearly indicate that IL-7 / CCL19-expressing T cells (1) have the ability to aggregate endogenous T cells, thereby inducing a positive interaction between the transplanted T cells and endogenous T cells.

[0142] In addition, by Figure 2A 、 Figure 2B 、 Figure 3The results clearly show that T cells expressing IL-7 and CCL19 proliferate effectively through IL-7, have a high survival rate, and have an important effect of aggregating T cells by CCL19, which is indispensable for immune induction, and have excellent immune induction effects. That is, it can be clearly seen that in immunocompetent cells, by expressing the two regulatory molecules "IL-7" and "CCL19", the proliferation ability, survival rate, and immune induction effect of the immunocompetent cells can be improved. Furthermore, as described above, T cells expressing IL-7 and CCL19 have both proliferation ability, survival ability, and T cell aggregation ability, thus indicating the possibility of having T cell and dendritic cell infiltration effects and tumor growth inhibition effects in cancer tissues.

[0143] Example 4

[0144] [Preparation of IL-7×CCL19×HSV-TK expression vector]

[0145] The following base sequence is cloned into the multiple cloning site of the pMSGV1 vector: a base sequence formed by tandemly linking the base sequences encoding the genes for IL-7, CCL19, and HSV-TK, which serves as a suicide gene, with the base sequence encoding the 2A peptide, which serves as a self-cleaving peptide, between them. This allows the preparation of a vector expressing IL-7, CCL19, and HSV-TK. The genetic map of the vector is shown below. Figure 4 shown.

[0146] By administering ganciclovir to a subject to whom the immunocompetent cells introduced with the IL-7×CCL19×HSV-TK expression vector prepared as described above have been administered, the immunocompetent cells in the subject can be regulated.

[0147] Example 5

[0148] [Preparation of TCR×IL-7×CCL19 expression vector]

[0149] The following base sequence is cloned into the multiple cloning site of the pMSGV1 vector: a base sequence formed by tandemly linking the base sequences encoding the genes for TCR, IL-7, and CCL19 with the base sequence encoding the 2A peptide, which is a self-cleaving peptide. This allows the preparation of a vector expressing TCR, IL-7, and CCL19. The genetic map of the vector is shown below. Figure 5 shown.

[0150] Immunocompetent cells introduced with the TCR×IL-7×CCL19 expression vector prepared as described above are capable of specifically binding not only to cancer antigens present on the surface of cancer cells, but also to complexes of cancer antigen peptides from cancer cells presented via MHC, thereby inducing specific T cells against a wider range of tumor-associated target molecules.

[0151] Example 6

[0152] [Preparation of expression vectors for IL-7, CCL19, and eGFP]

[0153] An IL-7-F2A-CCL19 DNA fragment encoding mouse IL-7 (without a stop codon), followed by F2A, and mouse CCL19 was artificially synthesized (manufactured by Life Technology).

[0154] To prepare a vector expressing IL-7, CCL19, and eGFP, the IL-7-F2A-CCL19 DNA fragment synthesized above was inserted into the MCS of the pMSGV retroviral expression vector (Tamada K et al., Clin Cancer Res 18:6436-6445 (2002)) containing the F2A-eGFP sequence by treatment with restriction endonucleases (NCOI and ECORI) and ligation. This yielded a pMSGV vector (IL-7×CCL19 expression vector (3)) containing the IL-7-F2A-CCL19-F2A-eGFP DNA fragment (SEQ ID NO: 13). The genetic map of the resulting vector is shown in FIG. Figure 6 As shown. In addition, as a control, a pMSGV vector containing eGFP but not IL-7 and CCL19 was prepared (control vector (3)). It should be noted that in sequence number 13, bases 1 to 462 are IL-7 (bases 1 to 75 are the signal sequence of IL-7), bases 463 to 537 are F2A, bases 538 to 861 are CCL19 (bases 538 to 612 are the signal sequence of CCL19), bases 868 to 942 are F2A, bases 946 to 1662 are the nucleic acid encoding eGFP, and bases 1663 to 1665 are the stop codon. In addition, the amino acid sequence corresponding to the above base sequence 13 is shown in sequence number 14. Note that, in order to use the restriction endonuclease NcoI, the 4th base thymine (t) in SEQ ID NO: 13 was replaced by guanine (g) (the 2nd amino acid phenylalanine (F) in SEQ ID NO: 14 was replaced by valine (V)).

[0155] [Preparation of T cells expressing P815 tumor antigen P1A-specific TCR, IL-7, CCL19, and eGFP]

[0156] From transgenic mice (obtained from Y.Liu, which express H-2L dSpleen cells were collected from transgenic mice expressing a TCR specific for the P815 tumor antigen P1A (Sarma, S., Y. Guo, Y. Guilloux, C. Lee, X.-F. Bai, Y. Liu. 1999. J. Exp. Med. 189: 811.) to obtain mouse T cells expressing a TCR specific for the P815 tumor antigen P1A derived from spleen cells (P1A-specific TCR-T cells). Next, retroviruses containing an IL-7×CCL19 expression vector (3) and a control vector (3) were prepared by the same method as in Example 1 and transduced into spleen cells (3×10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 6 Cells were activated at 400 μg / well for 48 hours to obtain P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells or P1A-specific TCR / eGFP-expressing T cells. Flow cytometric analysis was performed to detect eGFP as a surrogate marker to confirm transduction of each expression vector. The eGFP expression level of each T cell obtained was 70-80% in all experiments.

[0157] On day 0, 6-10 week-old male DBA / 2 mice (n=30) were subcutaneously inoculated in the flank with 5×10 5 On day 6, mice were irradiated with a sublethal dose (3-5 Gy) for preconditioning. On day 7, mice (n=10) were divided into three groups and intravenously administered with 1×10 6 P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells or P1A-specific TCR / eGFP-expressing T cells (70-80% eGFP positive for either cell type). Then, the survival rate of each mouse was analyzed, and the tumor volume of the dead mice was measured. The analysis results of the survival rate of each mouse are as follows: Figure 7 The tumor volume of the deceased mice was measured as shown in Figure 8 shown.

[0158] Figure 7In the figure, ▲ represents the results for untreated mice, ■ represents the results for mice administered with P1A-specific TCR / eGFP-expressing T cells, and ● represents the results for mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells. The horizontal axis represents the number of days (days) from subcutaneous inoculation of P815 mastocytoma, and the vertical axis represents the survival rate (%). 80% of mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells were still alive at day 60, and 50% were still alive beyond day 100. This demonstrates that the use of immunocompetent cells expressing P1A-specific TCR, IL-7, and CCL19 exerts an anti-tumor effect, suppressing the decrease in survival caused by the tumor.

[0159] in addition, Figure 8 The horizontal axis is the number of days from subcutaneous inoculation of P815 mast cell tumor (days), and the vertical axis is the tumor volume (mm 3 ).Depend on Figure 8 It was clear that the increase in tumor volume was significantly suppressed in mice administered with P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells, confirming that P1A-specific TCR / IL-7 / CCL19 / eGFP-expressing T cells have excellent anti-tumor activity and can exert a therapeutic effect on solid tumors.

[0160] Industrial applicability

[0161] The IL-7×CCL19-expressing immunocompetent cells of the present invention have the ability to proliferate, survive, and aggregate lymphocytes, and therefore can be used in the field of immunotherapy.

Claims

1. Use of a nucleic acid encoding interleukin-7 (IL-7) and a nucleic acid encoding CCL19 for introduction into immunocompetent cells expressing T cell receptors (TCRs) to prepare immunocompetent cells expressing TCRs, IL-7, and CCL19.

2. The use according to claim 1, characterized in that Nucleic acids encoding IL-7 and CCL19 are introduced into immunocompetent cells expressing TCR using expression vectors.

3. The use according to claim 2, characterized in that An expression vector containing a nucleic acid encoding IL-7 and a nucleic acid encoding CCL19 is used to introduce the nucleic acids into immunocompetent cells expressing TCR.

4. The use according to claim 2 or 3, characterized in that The expression vector contains nucleic acid encoding the suicide gene.

5. The use according to any one of claims 1 to 3, characterized in that The immunocompetent cells are lymphocytes, antigen-presenting cells, or granulocytes.

6. The use according to any one of claims 1 to 3, characterized in that The immune active cells are T cells.

7. The use according to any one of claims 1 to 3, characterized in that The TCR is a TCR that recognizes a cancer antigen, wherein the cancer antigen is WT1, MART-1, NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-A4, Glypican-3, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, mesothelin, NKG2D, P1A, GD2, or GM2.

8. A method for preparing immunocompetent cells expressing T cell receptor (TCR), interleukin 7 (IL-7), and CCL19, characterized in that: Nucleic acids encoding IL-7 and CCL19 are introduced into immunocompetent cells expressing TCR.

9. The preparation method according to claim 8, wherein Nucleic acids encoding IL-7 and CCL19 are introduced into immunocompetent cells expressing TCR using expression vectors.

10. The preparation method according to claim 8 or 9, characterized in that: The immune active cells are T cells.

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