T cell expressing CAR and CAR expression vector

Abstract

The invention relates to a T cell for expressing CAR and a CAR expression vector. Provided is an immune cell (CAR-T cell or the like) having higher antitumor activity than an immune cell (CAR-T cell or the like) that expresses CAR alone (not expressing a cytokine and / or a chemokine). A T cell provided in one aspect of the invention expresses (1) a chimeric antigen receptor (CAR), (2) at least one selected from the group consisting of interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), and interleukin-27 (IL-27), and (3) a CC chemokine ligand 19 (CCL19).

Description

[0001] This application is a divisional application of a patent application for an invention titled "T Cells Expressing CAR and CAR Expression Vectors" with an application date of August 30, 2019, an application number of 201980056592.X (international application number PCT / JP2019 / 034055). Technical Field

[0002] The present invention relates to immune cells expressing a chimeric antigen receptor (referred to as "CAR" in this specification.) that can be used for cancer immunotherapy (referred to as "CAR-expressing immune cells" in this specification.), typically T cells expressing CAR (referred to as "CAR-T cells" in this specification.) and expression vectors for producing CAR-expressing immune cells (such as CAR-T cells). More specifically, the present invention relates to CAR-expressing immune cells (such as CAR-T cells) expressing specified cytokines and chemokines and expression vectors for producing such CAR-expressing immune cells. Background Art

[0003] Although cancer immunotherapy by administering CAR-T cells has shown effectiveness in hematological malignancies such as leukemia and lymphoma, especially in solid tumors, there are cancer types and cases in which no therapeutic effect is seen due to problems such as low survival efficiency of CAR-T cells in the living body and inhibition of the activity of CAR-T cells in the tumor microenvironment caused by the tumor immune escape mechanism possessed by cancer cells. Therefore, there is a particular need for CAR-T cells with higher anti-tumor activity that show therapeutic effects in solid tumors.

[0004] Patent Document 1 and Non-Patent Document 1 describe CAR-T cells expressing IL-7 and CCL19, which have superior anti-tumor activity compared to conventional CAR-T cells. However, Patent Document 1 and Non-Patent Document 1 do not contain specific descriptions of combinations of cytokines and chemokines other than these.

[0005] Non-Patent Document 2 describes CAR-T cells expressing membrane-bound chimeric IL-15 (mbIL-15), in which signal transduction independent of CAR is improved and sustained. However, Non-Patent Document 2 does not contain specific descriptions of combinations of mbIL-15 and chemokines such as CCL19.

[0006] Non-Patent Document 3 describes that by using a fusion protein formed by connecting IL-15 and the sushi domain of IL-15Rα via a flexible linker, compared with the previous case of using IL-15 and the sushi domain of IL-15Rα in combination, it can enhance the activities in the proliferation of lymphocytes (NK cells, NK-T cells, memory CD8-positive cells), activation of dendritic cells, etc. However, Non-Patent Document 3 does not contain specific descriptions regarding the combination of IL-15 and the sushi domain of IL-15Rα with chemokines such as CCL19.

[0007] Non-Patent Document 4 describes that by transfecting a secreted fusion protein of murine IL-15 and IL-15Rα, the survival ability and proliferation ability of CD8-positive T cells are improved. However, Non-Patent Document 4 does not contain specific descriptions regarding the combination of IL-15 with chemokines such as CCL19.

[0008] Non-Patent Document 5 describes single-chain IL-27 (formed by connecting p28 and EBI3 with a flexible linker) and its therapeutic effect on inflammatory bowel disease (IBD). However, Non-Patent Document 5 does not contain specific descriptions regarding CAR-T cells, nor regarding the combination of single-chain IL-27 with chemokines such as CCL19.

[0009] Patent Document 2 discloses T cells expressing recombinant IL-7, recombinant IL-15, or a combination thereof, and describes that the expression of such cytokines improves the survival of T cells. However, Patent Document 2 does not contain specific descriptions regarding CAR-T cells, nor regarding the combination of the above-mentioned specific cytokines with chemokines such as CCL19.

[0010] Patent Document 3 describes engineered natural killer T cells containing an expression construct encoding IL-2, IL-4, IL-7, IL-15, or a combination thereof and a CAR construct. However, Patent Document 3 does not contain specific descriptions regarding the combination of the above-mentioned specific cytokines with chemokines such as CCL19.

[0011] Patent Document 4 describes CAR-T cells expressing membrane-bound cytokines such as IL-7, IL-15 (IL-15 / IL-15Rα fusion protein), and IL-21. However, Patent Document 4 does not contain specific descriptions regarding the combination of the above-mentioned specific cytokines with chemokines such as CCL19.

[0012] Patent Document 5 describes CAR-T cells targeting CD19 that express IL-15. However, Patent Document 5 does not contain specific descriptions regarding the combination of IL-15 with chemokines such as CCL19.

[0013] Non-patent Document 6 describes CAR-T cells targeting GPC3 that express IL-15 and / or IL-21. However, Patent Document 6 does not contain a specific description of the combination of the above-mentioned specific cytokines and chemokines such as CCL19.

[0014] Prior art documents

[0015] Patent documents

[0016] Patent Document 1: WO2016 / 056228

[0017] Patent Document 2: WO2007 / 037780

[0018] Patent Document 3: WO2013 / 040371

[0019] Patent Document 4: WO2014 / 186469

[0020] Patent Document 5: US2013 / 0071414

[0021] Non-patent documents

[0022] Non-patent Document 1: Adachi et al., Nature Biotechnology, VOL 36, No 4, 346-353

[0023] Non-patent Document 2: Hurton et al., Proc Natl Acad Sci., 113(48), E7788-97,2016

[0024] Non-patent Document 3: Mortier et al., J Biol Chem. 281(3), 1612-9, 2006

[0025] Non-patent Document 4: Rowley et al., Eur J Immunol. 39(2), 491-506, 2009

[0026] Non-patent Document 5: Sasaoka et al., Am J Physiol Gastrointest Liver Physiol300: G568-576

[0027] Non-Patent Document 6: Barta et al., Armored Glypican-3-Specific CAR T cells for the Immunotherapy of Hepatocellular Carcinoma, ASGCT 2018, May 2018, abstract Summary of the Invention

[0028] Problems to be Solved by the Invention

[0029] The subject of the present invention is to provide immune cells (such as CAR-T cells) with higher anti-tumor activity compared to immune cells (such as CAR-T cells) that express only CAR (without expressing cytokines and / or chemokines).

[0030] Means for Solving the Problems

[0031] There are at least several hundred kinds of molecules in the living body that can control the functions of immune cells such as T cells. The inventors of the present invention found that by expressing at least one selected from the group consisting of interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), and interleukin-27 (IL-27) as cytokines in combination with CC chemokine ligand 19 (CCL19) as a chemokine in CAR-T cells and expressing them together with CAR, the anti-tumor activity is enhanced compared to when only CAR is expressed, and in particular, the persistence and proliferation of CAR-T cells are improved, thus completing the present invention.

[0032] It should be noted that IL-15, IL-18, IL-27, and CCL19 are cytokines and chemokines that are not expressed in natural (without introducing exogenous genes) T cells present in the living body. IL-21 is a cytokine expressed in natural T cells (such as NKT cells, CD4-positive T cells) present in the living body. In the present invention, expressing the above-specified cytokines and chemokines means: by introducing exogenous genes for expressing the above-specified cytokines and chemokines into T cells, thereby expressing the above-specified cytokines and chemokines at a level higher than that of natural T cells present in the living body.

[0033] In addition, the embodiment of introducing the above-specified interleukin and chemokine into T cells together with CAR and expressing them can be extended to the embodiment of introducing them into immune cells other than T cells, such as NK cells, monocytes, macrophages, dendritic cells, etc., and expressing them.

[0034] The present invention includes at least the following inventions. [1]

[0036] A T cell that expresses:

[0037] (1) A chimeric antigen receptor (CAR);

[0038] (2) At least one selected from the group consisting of interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), and interleukin-27 (IL-27); and

[0039] (3) CC chemokine ligand 19 (CCL19). [2]

[0041] The T cell according to item 1, wherein the above (2) is IL-15. [3]

[0043] The T cell according to item 2, wherein the above IL-15 is IL-15 in a state of being linked to IL-15Rα to form a fusion protein. [4]

[0045] The T cell according to item 3, wherein the above fusion protein is a fusion protein formed by linking IL-15LSP and IL-15 (IL15 LSP ), a fusion protein formed by linking the extracellular domain of IL-15Rα and IL-15 ( s IL15 RA ), a fusion protein formed by linking IL-15 and full-length IL-15Rα ( mb IL15 RA ), or a fusion protein formed by linking IL-15Rα containing a signal peptide and a sushi domain and IL-15 ( sushi IL15). [5]

[0047] The T cell according to item 3, wherein the above fusion protein is a fusion protein formed by linking IL-15 and full-length IL-15Rα ( mb IL15 RA ), or a fusion protein formed by linking IL-15Rα containing a signal peptide and a sushi domain and IL-15 ( sushi IL15). [6]

[0049] A drug that contains the T cell described in item 1. [7]

[0051] The drug according to item 6, which is a therapeutic agent for cancer. [8]

[0053] The medicament according to item 7, wherein the cancer is melanoma, Merkel cell carcinoma, colorectal cancer, renal cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, non-small cell lung cancer, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma or leukemia. [9]

[0055] An expression vector comprising:

[0056] (1) a nucleic acid encoding a CAR;

[0057] (2) a nucleic acid encoding at least one selected from the group consisting of IL-15, IL-18, IL-21 and IL-27; and

[0058] (3) a nucleic acid encoding CCL19.

[10]

[0060] The expression vector according to item 9, wherein the above (2) is IL-15.

[11]

[0062] The expression vector according to item 10, wherein the above IL-15 is IL-15 in a state of being linked to IL-15Rα to form a fusion protein.

[12]

[0064] The expression vector according to item 11, wherein the above fusion protein is a fusion protein formed by linking IL-15LSP and IL-15 (IL15 LSP ), a fusion protein formed by linking the extracellular domain of IL-15Rα and IL-15 ( s IL15 RA ), a fusion protein formed by linking IL-15 and full-length IL-15Rα ( mb IL15 RA ) or a fusion protein formed by linking IL-15Rα containing a signal peptide and a sushi domain and IL-15 ( sushi IL15).

[13]

[0066] The expression vector according to item 11, wherein the above fusion protein is a fusion protein formed by linking IL-15 and full-length IL-15Rα ( mb IL15 RA ) or a fusion protein formed by linking IL-15Rα containing a signal peptide and a sushi domain and IL-15 ( sushiIL15).

[14]

[0068] A method for manufacturing CAR-T cells, which comprises the step of introducing the expression vector described in item 9 into T cells.

[15]

[0070] A method for treating cancer, which comprises the step of administering the T cells described in item 1 to a subject in need of cancer treatment.

[16]

[0072] According to the treatment method described in item 15, wherein the cancer is melanoma, Merkel cell carcinoma, colorectal cancer, renal cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, non-small cell lung cancer, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma or leukemia.

[17]

[0074] The T cells according to item 1 are used as an active ingredient for the treatment of cancer.

[18]

[0076] According to the T cells described in item 17, wherein the cancer is melanoma, Merkel cell carcinoma, colorectal cancer, renal cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, non-small cell lung cancer, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma or leukemia.

[0077] In this specification, the T cells of the above [1] are sometimes referred to as "the T cells of the present invention", the drug of the above [6] is referred to as "the drug of the present invention", the expression vector of the above [9] is referred to as "the expression vector of the present invention", and the manufacturing method of the CAR-T cells of the above

[14] is referred to as "the manufacturing method of the present invention".

[0078] A person skilled in the art can appropriately transform the above-described inventive methods (scopes) by referring to the entire content disclosed in this specification. For example, the drug of the present invention in [6] above can be transformed into "a method for treating cancer, which comprises the step of administering T cells expressing (1) CAR, (2) at least one selected from the group consisting of IL-15, IL-18, IL-21, and IL-27, and (3) CCL19 to a subject in need of cancer treatment", "a T cell expressing (1) CAR, (2) at least one selected from the group consisting of IL-15, IL-18, IL-21, and IL-27, and (3) CCL19, which is used as an active ingredient for treating cancer", "the use of a T cell expressing (1) CAR, (2) at least one selected from the group consisting of IL-15, IL-18, IL-21, and IL-27, and (3) CCL19 in the manufacture of a drug for treating cancer", etc.

[0079] A person skilled in the art can, by referring to the entire content disclosed in this specification, appropriately transform each invention such as the T cells of the present invention obtained by expressing a specified cytokine and chemokine together with CAR in T cells, the drug of the present invention as an associated embodiment thereof, the expression vector of the present invention, and the manufacturing method of the present invention into each invention such as immune cells other than T cells (NK cells, monocytes, macrophages, dendritic cells, etc.) obtained by expressing a specified cytokine and chemokine together with CAR in immune cells other than T cells, and the drug, expression vector, manufacturing method, etc. as associated embodiments thereof.

[0080] Advantages of the Invention

[0081] The T cells of the present invention enhance the persistence and proliferation of T cells by expressing designated cytokines (such as IL-15) and chemokines (CCL19), and thus can enhance the anti-tumor activity brought by CAR (reducing the number of residual tumor cells, increasing the production amount of IFNγ, increasing the migration and accumulation of host immune cells (T cells, dendritic cells, NK cells, etc.) to the tumor site). In addition, by using a drug containing such T cells of the present invention, the therapeutic effect of cancer can be improved. In particular, the T cells of the present invention may be involved in the activation of NK cells, and thus have the possibility of being useful in the treatment of tumors sensitive to NK cells (such as melanoma, Merkel cell carcinoma, colorectal cancer, renal cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, non-small cell lung cancer, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma, leukemia, etc.). In addition, it is expected to reduce the number of administered cells by enhancing the anti-tumor activity, thereby reducing side effects such as CRS (Cytokine Release Syndrome) and lowering the manufacturing cost.

[0082] The combination of the designated cytokines and chemokines of the present invention also exhibits the following effects: when introduced into all immune cells together with the CAR gene, that is, not only into the above-mentioned T cells but also into NK cells, monocytes, macrophages, dendritic cells, etc. and expressed, various CAR-expressing immune cells with high anti-tumor activity can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The results of analyzing the expression levels of CAR (horizontal axis) and CD8 (vertical axis) in the following T cells by flow cytometry in Example (Test Example 1) are shown. The values (%) in the figure represent the percentage of the cell population in each partition relative to all cells. "Transduction (-)" is the result for the transduction (-) T cells (non-transduced T cells) of Comparative Example 1, "conv. CAR-T" is the result for the conv.CAR-T cells (anti-human CD20 CAR-T cells) of Comparative Example 2, "15 LSP x 19 CAR-T" is the result for the IL15 LSP ×CCL19 CAR-T cells (IL15 LSP _CCL19_anti-human CD20 CAR-T cells) of Example 1-1, " s 15 RA x 19 CAR-T" is the result for Example 1-2 s IL15 RA×CCL19 CAR-T cells ( s IL15 RA _CCL19_ anti-human CD20 CAR-T cells), the result of " mb 15 RA x 19 CAR-T" is for Examples 1-3 mb IL15 RA ×CCL19 CAR-T cells ( mb IL15 RA _CCL19_ anti-human CD20 CAR-T cells), the result of " sushi 15 x 19 CAR-T" is for Examples 1-4 sushi IL15×CCL19 CAR-T cells ( sushi IL15_CCL19_ anti-human CD20 CAR-T cells), the result of "18 x 19CAR-T" is for IL-18×CCL19 CAR-T cells (IL18_CCL19_ anti-human CD20 CAR-T cells) of Example 2, the result of "21 x 19 CAR-T" is for IL-21×CCL19 CAR-T cells (IL21_CCL19_ anti-human CD20CAR-T cells) of Example 3, " sc 27 x 19 CAR-T" is for Example 4 sc IL27×CCL19 CAR-T cells ( sc IL27_CCL19_ anti-human CD20 CAR-T cells) result. (The same is true for the following Figures 2 to 6 .)

[0084] Figure 2 Show the results of measuring the secretion amounts of (A) IL-15, (B) IL-18, (C) IL-21, (D) IL-27, and (E) CCL19 in the culture supernatant of the above-mentioned various T cells by ELISA in the examples (Test Example 1).

[0085] Figure 3 Show the results of measuring the viable cell numbers of the above-mentioned various T cells 3 days, 5 days, and 7 days after activation stimulation by co-culturing with human CD20-expressing P815 mastocytoma (hCD20 / P815) in the examples (Test Example 2).

[0086] Figure 4Shows the results of histogram analysis of the staining intensity of each of the above T cells using CytoTell reagent in the example (test example 2). The peak of the histogram indicates the generation produced by cell division, and the values (%) in the pie chart represent the fractions of gating in the Thy1.2-positive T cell population (1 represents the first generation that has not divided, 2 represents the second generation after dividing once, 3 represents the third generation after dividing twice, 4 represents the fourth generation after dividing three times, and >5 represents the fifth generation and later generations after dividing four or more times.).

[0087] Figure 5 Shows the results of measuring the tumor cell cytotoxic activity of each of the above T cells against (A) target tumor cells (hCD20 / P815) and (B) control tumor cells (P815) by flow cytometry in the example (test example 3).

[0088] Figure 6 Shows the results of measuring the concentration of IFNγ in the culture supernatant of each of the above T cells when co-cultured with (A) target tumor cells (hCD20 / P815) and (B) control tumor cells (P815) in the example (test example 3).

[0089] Figure 7 Shows the configuration of genes and the like contained in the base sequence of SEQ ID NO: 1 (anti-human CD20 CAR DNA fragment).

[0090] Figure 8 Shows the configuration of genes and the like contained in the base sequence of SEQ ID NO: 3 (MCS DNA fragment).

[0091] Figure 9 Shows the configuration of genes and the like contained in the base sequence of SEQ ID NO: 4 (IL15 LSP _F2A_CCL19 DNA fragment).

[0092] Figure 10 Shows the configuration of genes and the like contained in the base sequence of SEQ ID NO: 6 ( s IL15 RA _F2A_CCL19 DNA fragment).

[0093] Figure 11 Shows the configuration of genes and the like contained in the base sequence of SEQ ID NO: 8 ( mb IL15 RA _F2A_CCL19 DNA fragment).

[0094] Figure 12 Shows the configuration of genes and the like contained in the base sequence of SEQ ID NO: 10 ( sushi IL15_F2A_CCL19 DNA fragment).

[0095] Figure 13 Shows the configuration of genes, etc. contained in the base sequence of SEQ ID NO: 12 (IL-18_F2A_CCL19 DNA fragment).

[0096] Figure 14 Shows the configuration of genes, etc. contained in the base sequence of SEQ ID NO: 14 (IL-21_F2A_CCL19 DNA fragment).

[0097] Figure 15 Shows SEQ ID NO: 16 ( sc IL27_F2A_CCL19 DNA fragment) of the configuration of genes, etc. contained in the base sequence.

[0098] Figure 16 Shows the results of measuring the anti-tumor activity (change in tumor volume) against a mouse melanoma tumor transplantation model in the Examples (Test Example 4).

[0099] Figure 17 Shows the results of measuring the anti-tumor activity (change in tumor volume) against a mouse colorectal cancer tumor model in the Examples (Test Example 4). Detailed implementation mode

[0100] In this specification, "transfection" means introducing any substance into the interior of a cell. The interior of the cell includes at least the cytoplasm and the nucleus.

[0101] "Culture" or "perform culturing" means: maintaining, proliferating, and / or differentiating cells outside the tissue or in vitro, for example, in a culture dish, culture plate, flask, or culture tank (vessel).

[0102] "Pluripotency" means: the ability to differentiate into various tissues and cells with different morphologies and functions, and also the ability to differentiate into cells of any one of the three germ layers. "Pluripotency" cannot differentiate into the blastoderm, and thus does not have the ability to form an individual. In this regard, it is different from "totipotency" which can differentiate into all tissues of an organism including the blastoderm.

[0103] "Multipotency" means: the ability to differentiate into cells of a limited number of multiple systems. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent stem cells, not pluripotent stem cells.

[0104] Examples of "stem cell" include, for example, pluripotent stem cells.

[0105] The "pluripotent stem cell" that can be used in the present invention refers to: a stem cell having the ability to differentiate into various tissues and cells with different forms and functions in an organism and also having the ability to differentiate into cells of any one of the three germ layers (endoderm, mesoderm, and ectoderm). It is not particularly limited, and examples thereof include embryonic stem cells (ESCs), embryonic stem cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells, embryonic germ cells, induced pluripotent stem cells (sometimes also referred to as "iPSCs" in this specification), and the like.

[0106] In addition, the "multipotent stem cell" that can be used in the present invention refers to: a stem cell having the ability to differentiate into cells of a limited number of multiple systems. As the "multipotent stem cell" that can be used in the present invention, examples include: dental pulp stem cells, stem cells derived from oral mucosa, hair follicle stem cells, cultured fibroblasts, somatic stem cells derived from bone marrow stem cells, and the like. Preferred pluripotent stem cells are ESCs and iPSCs.

[0107] "Induced pluripotent stem cells (iPSCs)" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, "induced pluripotent stem cells" include various types. In addition to the iPSCs established by Yamanaka et al. by introducing the four factors Oct3 / 4·Sox2·Klf4·c-Myc into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872.), Nanog-iPS cells established by screening using the expression of Nanog as an index after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007).Nature 448, 313-317.), iPS cells produced by a method without c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPS cells established by introducing six factors by a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.). In addition, induced pluripotent stem cells established by introducing the four factors OCT3 / 4·SOX2·NANOG·LIN28 produced by Thomson et al. (Yu J., Thomson JA. et al.,Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (Japanese Patent Laid-Open No. 2008-307007), etc. can also be used.In addition, any of the induced pluripotent stem cells well-known in the art described in all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No 7, 795-797) or patents (e.g., Japanese Unexamined Patent Application Publication No. 2008-307007, Japanese Unexamined Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used.

[0108] As the "induced pluripotent stem cells", various iPSC strains established by NIH, RIKEN (The Institute of Physical and Chemical Research), Kyoto University, etc. can be used. For example, in the case of human iPSC strains, the HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain of RIKEN, and the 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, etc. of Kyoto University can be mentioned. Alternatively, clinical-grade cell strains provided by Kyoto University, Cellular Dynamics International, etc., as well as research-use and clinical-use cell strains prepared using these cell strains can be used.

[0109] As "embryonic stem cells (ESC)", in the case of mouse ESC, various mouse ESC lines established by inGenious targeting laboratory, RIKEN (The Institute of Physical and Chemical Research), etc. can be used; in the case of human ESC, various human ESC lines established by NIH, RIKEN, Kyoto University, Cellartis, etc. can be used. For example, as human ESC cell lines, CHB-1 to CHB-12 lines, RUES1 line, RUES2 line, HUES1 to HUES28 lines, etc. of NIH, H1 line, H9 line of WisCell Research, KhES-1 line, KhES-2 line, KhES-3 line, KhES-4 line, KhES-5 line, SSES1 line, SSES2 line, SSES3 line, etc. of RIKEN can be used. Alternatively, clinical-grade cell lines and research-use and clinical-use cell lines prepared using these cell lines can be used.

[0110] "comprise(s) or comprising ~" means that although it indicates including the elements connected after this phrase, it is not limited thereto. Therefore, it indicates including the elements connected after this phrase, but does not indicate excluding any other elements.

[0111] "consist(s) of or consisting of ~" means that it includes all the elements connected after this phrase and is limited to these. Therefore, the phrase "consist(s) of ~" indicates that the listed elements are required or necessary, and there are substantially no other elements. "Consisting essentially of ~" means that it includes any elements connected after this phrase, and this element is limited to other elements that do not affect the activity or function specified in the present invention. Therefore, the phrase "consisting essentially of ~" indicates that the listed elements are required or necessary, and other elements are optional, and sometimes they exist and sometimes they do not exist depending on whether they affect the activity or function of the listed elements.

[0112] Regarding the specified combination of cytokines and chemokines of the present invention, by co-introducing genes into immune cells, especially T cells that undertake cellular immunity in acquired immunity, NK cells that undertake innate immunity, monocytes, macrophages, dendritic cells, etc. together with the CAR gene and expressing them, CAR-expressing immune cells with high anti-tumor activity can be provided. In other words, the gene introduction and expression of the specified combination of cytokines and chemokines of the present invention can endow further technical features to T cells (CAR-T cells), NK cells (CAR-NK cells), monocytes (CAR-monocytes), macrophages (CAR-macrophages), dendritic cells (CAR-dendritic cells), etc. that have been respectively introduced with the CAR gene and expressed.

[0113] The "immune cells" are not particularly limited as long as they are cells (so-called immune effector cells) that have the ability to inhibit target cells such as cancer cells (pathogenic cells) through a certain action mechanism. Examples include T cells that undertake cellular immunity in acquired immunity, NK cells that undertake innate immunity, monocytes, macrophages, dendritic cells, etc. In a preferred embodiment, the immune cells can be T cells. On the other hand, in another preferred embodiment, the immune cells can be cells that undertake innate immunity such as NK cells, macrophages, dendritic cells, etc. For T cells, even when the HLA types are the same, there is a certain risk of causing GVHD through allogeneic (allograft) transplantation. In contrast, it is generally considered that allogeneic NK cells, etc. do not cause GVHD. Therefore, if various HLA-type allogeneic immune cells are prepared, they can be used off-the-shelf. CAR-NK cells are described in, for example, US2016 / 0096892, Mol Ther. 25(8): 1769-1781 (2017), etc., and CAR-dendritic cells, CAR-macrophages, etc. are described in, for example, WO2017 / 019848, eLIFE. 2018e36688, etc.

[0114] Hereinafter, the present invention will be described by way of the description of an embodiment when T cells are used as a representative example of immune cells. However, the present invention is not limited to the embodiment in which the immune cells are T cells, and embodiments in which the immune cells are NK cells, monocytes, macrophages, dendritic cells, etc. are also included in the present invention. In the following description, "(CAR-)T cells" can also be appropriately replaced with "(CAR-)NK cells", "(CAR-)monocytes", "(CAR-)macrophages", "(CAR-)dendritic cells", etc.

[0115] The T cells of the present invention will be described in detail below.

[0116] The T cells of the present invention express (1) CAR, (2) at least one selected from the group consisting of IL-15, IL-18, IL-21, and IL-27, and (3) CCL19.

[0117] The T cells in the present invention can be αβ T cells, γδ T cells, CD8 + T cells, CD4 + T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells, etc., just like the generally or publicly known CAR-expressing T cells.

[0118] The T cells can be T cells isolated and purified from immune cells in body fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, or cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites. Additionally, the T cells can also be T cells induced to differentiate from induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), or other stem cells and progenitor cells under appropriate culture conditions.

[0119] The T cells can be cells of human origin or cells of mammalian origin other than human (non-human mammals). Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep, and monkeys.

[0120] (1) CAR

[0121] The CAR expressed by the T cells of the present invention is basically the same as the usual or well-known CAR, and is constructed by connecting peptides of (i) a single-chain antibody that recognizes the cell surface antigen of cancer cells, (ii) a transmembrane region, and (iii) a signal transduction region that induces the activation of T cells, with a spacer as needed.

[0122] (i) The single-chain antibody that recognizes the cell surface antigen of cancer cells is typically a single-chain variable region fragment (scFv), which is composed of a light chain variable region and a heavy chain variable region from the antigen-binding site of a monoclonal antibody that specifically binds to the antigen, and a linker peptide that connects them.

[0123] The "cell surface antigen of cancer cells" targeted by CAR can be a biomolecule specifically expressed in cancer cells and their precursor cells, a biomolecule newly observed to be expressed due to cell carcinogenesis, or a biomolecule with an increased expression level in cancer cells compared to normal cells. Such antigens are usually referred to as "tumor-associated antigens" (TAA), and examples include, but are not limited to, BCMA, B7-H3, B7-H6, CD7, CD10, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD34, CD38, CD41, CD44, CD56, CD70, CD74, CD97, CD123, CD133, CD138, CD171, CD248, CAIX, CEA, c-Met, CS1 (CD319), CSPG4, CLDN6, CLD18A2, CYP1B1, DNAM-1, GD2, GD3, GM2, GFRα4, GPC3, GPR20, GPRC5D, globoH, Gp100, GPR20, GPRC5D, EGFR, EGFR variants, EpCAM, EGP2, EGP40, FAP, FITC, HER2, HER3, HPV E6, HPV E7, hTERT, IgG κ chain, IL-11Ra, IL-13Ra2, KIT, Lewis A, Lewis Y, Legumain, LMP1, LMP2, Ly6k, LICAM, MAD-CT-1, MAD-CT-2, MAGE-A1, Melanoma-associated antigen 1, MUC1, MUC16, NA-17, NY-BR-1, NY-ESO-1, O-acetyl-GD2, h5T4, PANX3, PDGRFb, PLAC1, polysialic acid, PSCA, PSMA, RAGE1, ROR1, sLe, SSEA-4, TARP, TAG-72, TEM7R, Tn antigen, TRAIL receptor, TRP2, TSHR, alpha-fetoprotein, mesothelin, folate receptor alpha (FRα), folate receptor beta (FRβ), FBP, UPK2, VEGF-R2, WT-1.

[0124] (ii) The transmembrane region is a polypeptide that serves as a region for anchoring the CAR to the cell membrane of T cells. Examples of such transmembrane regions include those derived from BTLA, CD3ε, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, 4-1BB (CD137), CTLA-4, GITR, ICOS, LAG3, OX40, SLAMF4 (CD244, 2B4), or the α or β chain of the T cell receptor. Alternatively, a variant transmembrane region having an amino acid sequence with 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the natural amino acid sequence of the above transmembrane regions can also be used. In one embodiment of the present invention, as the transmembrane region, the transmembrane region of CD8 is preferred.

[0125] A hinge region may be linked to the transmembrane region, and the hinge region is a peptide (oligopeptide or polypeptide) composed of an arbitrary amino acid sequence and having a length of 1 to 100 amino acids, preferably 10 to 70 amino acids. Examples of such hinge regions include those derived from CD3, CD8, KIR2DS2, or IgG4, IgD, or other immunoglobulins. Alternatively, a variant hinge region having an amino acid sequence with 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology to the natural amino acid sequence of the above hinge regions can also be used. In one embodiment of the present invention, as the hinge region, the hinge region of CD8 is preferred.

[0126] (iii) The signal transduction region that induces the activation of T cells is a polypeptide that serves as a region for transmitting signals into T cells when the single-chain antibody recognizes and binds to the cell surface antigen of cancer cells. As such a signal transduction region, for example, one or more intracellular regions selected from the group consisting of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activated NK cell receptors, Toll-like receptors, B7-H3, BAFFR, BTLA, BY55 (CD160), CD2, CD3ζ, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD103, 4-1BB (CD137), CDS, CEACAM1, CRTAM, CNAM1 (CD226), DAP10, Fc receptor-associated γ chain, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS (CD278), IL2Rβ, IL2Rγ, IL7Rα, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, Ly9 (CD229), LAT, LFA-1 (CD11a / CD18), LIGHT, LTBR, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PSGL1, SELPLG (CD162), SEMA4D (CD100), SLAM (SLAMF1, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8 (BLAME), SLP-76, TNFR2, TRANCE / RANKL, VLA1, and VLA-6 can be mentioned. Alternatively, a variant signal transduction region (intracellular region) having an amino acid sequence with a homology of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the natural amino acid sequence of the above signal transduction region (intracellular region) can also be used. In one embodiment of the present invention, the signal transduction region preferably contains three intracellular regions, CD28, 4-1BB, and CD3ζ.

[0127] In the case where the signal transduction region contains a plurality of intracellular regions, the intracellular regions can be connected to each other via a linker peptide (oligopeptide or polypeptide) composed of 2 to 10 amino acids. As such a linker peptide, for example, a peptide composed of a glycine-serine continuous sequence can be mentioned.

[0128] The single-chain antibody and the transmembrane region, and the transmembrane region and the signal transduction region can each contain a spacer region, which is a peptide (oligopeptide or polypeptide) composed of an arbitrary amino acid sequence and having a length of 1 to 100 amino acids, preferably 10 to 50 amino acids. As the spacer region, for example, a peptide composed of a glycine-serine continuous sequence can be mentioned.

[0129] The amino acid sequence of the above-mentioned CAR expressed by the T cells of the present invention only needs to be an amino acid sequence suitable for the use of T cells, typically a function as an active ingredient of a drug for treating cancer. As the amino acid sequence of the single-chain antibody (i) against the cell surface antigen of cancer cells, various amino acid sequences are known, and their amino acid sequence information can be used in the present invention. Alternatively, an antibody against the cell surface antigen of cancer cells as desired can be re-produced by a conventional method, and the amino acid sequence of the new antibody (preferably the variable regions of the heavy and light chains, especially the CDRs) can be determined and its information can be used in the present invention. In addition, as the amino acid sequences of the transmembrane region (ii) and the T cell activation signal transduction region (iii) respectively, various amino acid sequences from human and non-human mammalian sources are also known and are registered in databases such as NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov / guide / ) and UniProt (The Universal Protein Resource; https: / / www.uniprot.org), so their information can be used in the present invention.

[0130] (2) Cytokines

[0131] The cytokine expressed by the T cells of the present invention is at least one selected from the group consisting of IL-15, IL-18, IL-21, and IL-27. The T cells of the present invention can express any one of the above 4 cytokines, or can express two or more.

[0132] IL-15 is a cytokine with functions related to the survival, activation, and differentiation of T cells into memory T cells, as well as the activation of NK cells. The term "IL-15" in the present invention includes not only the natural full-length IL-15 protein but also various embodiments within the scope of the function of IL-15 that maintain the effects of the present invention. That is, "IL-15" in the present invention includes not only the whole protein (polypeptide) composed of the amino acid sequence of natural IL-15 but also a part thereof (functional partial polypeptide), a variant of the whole or a part of natural IL-15 in which one or more amino acid sequences are deleted, substituted, or added with respect to the natural amino acid sequence (preferably within the range of having the homology described below), and further includes a form in which the whole or a part of natural IL-15 or a variant thereof is linked to another protein to form a fusion protein (for example, in a state where it is linked to the whole or a part of IL-15Rα to form a fusion protein). In the present invention, T cells "expressing IL-15" include expressing the above various forms of IL-15 (the whole or a part of the protein having the natural amino acid sequence or a variant thereof, which may or may not form a fusion protein form) within the range of not losing the effects of the present invention.

[0133] As IL-15, for example, the following four embodiments can be cited. All of these are well-known (refer to the aforementioned Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, and Patent Document 4, etc.). However, the IL-15 that can be used in the present invention is not limited to these specific examples, and various modified IL-15s (sometimes referred to as "modified" IL-15 in this specification) can also be used from the viewpoints of the presence or absence of signal peptides and linkers in the sequence and their types, the order of each element, and other viewpoints.

[0134] The first embodiment of IL-15: IL-15LSP / IL-15 (sometimes denoted as "IL15" in this specification). LSP ".

[0135] "IL-15LSP" is a long-chain signal peptide consisting of 29 amino acids and binds to the N-terminal side of IL-15 in the first embodiment. By expressing IL-15 of the first embodiment, the anti-tumor activity of CAR-T cells is enhanced. The term "IL-15LSP" includes not only the whole polypeptide composed of the amino acid sequence of natural IL-15LSP, but also a part thereof (functional partial polypeptide), and variants of the whole or a part of natural IL-15LSP in which one or more amino acid sequences are deleted, substituted or added with respect to the natural amino acid sequence (preferably within a range having homology as described below). It should be noted that as an example of the modified type of the first embodiment, those in which "IL-15LSP" is replaced with "IL-2SP" (signal peptide of IL-2, which can be the whole of a natural protein, a part thereof, or their variants) can be cited.

[0136] The second embodiment of IL-15: IL-15 / IL-15Rα extracellular domain (sometimes denoted as " s IL15 RA " in this specification.)

[0137] "IL-15Rα extracellular domain" refers to the part of IL-15Rα (interleukin-15 receptor α chain) excluding the transmembrane domain and the intracellular domain. The IL-15Rα extracellular domain contains a sushi domain (a motif observable in various binding proteins). The term "IL-15Rα extracellular domain" includes not only the whole polypeptide composed of the amino acid sequence of natural IL-15Rα extracellular domain, but also a part thereof (functional partial polypeptide), and variants of the whole or a part of natural IL-15Rα extracellular domain in which one or more amino acid sequences are deleted, substituted or added with respect to the natural amino acid sequence (preferably within a range having homology as described below). In the second embodiment, the IL-15Rα extracellular domain is usually bound to the C-terminal side of IL-15 via a linker (for example, a glycine-serine linker consisting of 26 amino acids). In addition, in the second embodiment, IL-2SP (IL-2 signal peptide, which can be the whole of a natural protein, a part thereof, or their variants) is usually bound to the N-terminal side of IL-15 instead of IL-15LSP. The second embodiment is a secreted type and becomes an agonist that strongly binds to IL-15Rβ (interleukin-15 receptor β chain shared with the interleukin-2 receptor) and γc (common γ chain shared as a receptor for interleukins 2, 4, 7, 9, 15, 21, etc.).

[0138] The third embodiment of IL-15: IL-15 / full-length IL-15Rα (sometimes denoted as " mbIL15 RA ”.)

[0139] "Full-length IL-15Rα" includes both the extracellular domain, transmembrane domain and intracellular domain of IL-15Rα, and in the third embodiment, it is usually bound to the C-terminal side of IL-15 via a linker (e.g., a glycine-serine linker consisting of 20 amino acids). The term "full-length IL-15Rα" includes not only the whole protein (polypeptide) composed of the amino acid sequence of natural full-length IL-15Rα, but also variants of natural full-length IL-15Rα in which one or more amino acid sequences are deleted, substituted or added with respect to the natural amino acid sequence (preferably within the range of having homology as described below). In addition, in the third embodiment, IL-2SP (which can be the whole or a part of a natural protein or their variants) is usually bound to the N-terminal side of IL-15 in place of IL-15LSP. The third embodiment is a membrane-bound type, and by expressing it, the anti-tumor activity of CAR-T cells is enhanced.

[0140] The fourth embodiment of IL-15: IL-15Rαsushi / IL-15 (sometimes denoted as " sushi IL15" in this specification).)

[0141] In the fourth embodiment, IL-15Rα containing a signal peptide and a sushi domain is usually bound to the N-terminal side of IL-15 via a linker (e.g., a linker consisting of 20 amino acids). The term "IL-15Rαsushi" also includes not only the whole polypeptide composed of the amino acid sequence of natural IL-15Rαsushi, but also a part thereof (functional partial polypeptide), and variants of the whole or a part of natural IL-15Rαsushi in which one or more amino acid sequences are deleted, substituted or added with respect to the natural amino acid sequence (preferably within the range of having homology as described below). The fourth embodiment is a secreted type.

[0142] In the present invention, from the viewpoints of the persistence and proliferation of cells shown in the following examples (test examples), as IL-15, IL-15 (the whole or a part of a protein having a natural amino acid sequence or their variants) in the form of a fusion protein with IL-15Rα (the whole or a part of a protein having a natural amino acid sequence or their variants) is preferred. For example, the second, third or fourth embodiments or their modified forms are preferred, and the third and fourth embodiments or their modified forms are more preferred.

[0143] IL-18 is a cytokine that has functions related to the activation of T cells, the activation of NK cells, and the activation of dendritic cells, etc. The term "IL-18" in the present invention includes not only the natural full-length IL-18 protein, but also various embodiments within the scope of maintaining the function of IL-18 in the effects of the present invention. That is, "IL-18" in the present invention includes not only the full length of the protein (polypeptide) composed of the amino acid sequence of natural IL-18, but also a part thereof (functional partial polypeptide), a variant of the whole or a part of natural IL-18 in which one or more amino acid sequences are deleted, substituted or added with respect to the natural amino acid sequence (preferably within the range of having the homology described below), and further includes the case where the whole or a part of natural IL-18 is linked to other proteins to form a fusion protein. In the present invention, T cells "expressing IL-18" include: expressing the above various forms of IL-18 (the whole or a part of the protein having the natural amino acid sequence or their variants, which may or may not form a fusion protein) within the range of not losing the effects of the present invention.

[0144] IL-21 is a cytokine that has functions related to the functions of CD8 + T cells and the differentiation into memory T cells, and the survival of NK cells, etc. The term "IL-21" in the present invention includes not only the natural full-length IL-21 protein, but also various embodiments within the scope of maintaining the function of IL-21 in the effects of the present invention. That is, "IL-21" in the present invention includes not only the full length of the protein (polypeptide) composed of the amino acid sequence of natural IL-21, but also a part thereof (functional partial polypeptide), a variant of the whole or a part of natural IL-21 in which one or more amino acid sequences are deleted, substituted or added with respect to the natural amino acid sequence (preferably within the range of having the homology described below), and further includes the case where the whole or a part of natural IL-21 is linked to other proteins to form a fusion protein. In the present invention, T cells "expressing IL-21" include: expressing the above various forms of IL-21 (the whole or a part of the protein having the natural amino acid sequence or their variants, which may or may not form a fusion protein) within the range of not losing the effects of the present invention.

[0145] IL-27 is a cytokine with functions related to the survival of T cells, the activation of NK cells, and the inhibition of tumor proliferation and angiogenesis. The term "IL-27" in the present invention includes not only the natural full-length IL-27 protein, but also various embodiments within the scope of maintaining the functions of IL-27 in the effects of the present invention. That is, "IL-27" in the present invention includes not only the full length of the protein (polypeptide) composed of the amino acid sequence of natural IL-27, but also a part thereof (functional partial polypeptide), a variant of the whole or a part of natural IL-27 in which one or more amino acid sequences are deleted, substituted or added with respect to the natural amino acid sequence (preferably within the range of having the homology described below), and further includes the case where the whole or a part of natural IL-27 is connected to other proteins to form a fusion protein. In the present invention, the expression of IL-27 by T cells includes: expressing the above various forms of IL-27 (the whole or a part of the protein having the natural amino acid sequence or their variants, which may or may not form a fusion protein) within the range of not losing the effects of the present invention.

[0146] As IL-27, for example, a fusion protein (single-chain protein) formed by connecting two subunits p28 and EBI3 (Epstein-Barr virus-induced gene 3) constituting IL-27 via a linker, such as a linker composed of 20 to 30 amino acids like (G 4 S) 3 and the like (refer to the aforementioned Non-Patent Document 5, etc.), or a linker composed of GSTSGSGKPGSGEGSTKG (J Immunol. 183, 6217-26 (2009)) can be cited. The terms "p28" and "EBI3" also include the whole or a part of the polypeptide having the natural amino acid sequence or their variants, respectively.

[0147] The amino acid sequences of the above-mentioned cytokines expressed by the T cells of the present invention can be amino acid sequences that are suitable on the premise of the use of the T cells (CAR-T cells) according to the present invention, typically as an active ingredient of a drug for treating cancer, and considering the effect of enhancing the anti-tumor activity brought by the CAR-T cells, including the improvement of the persistence and proliferation of the CAR-T cells. The amino acid sequences of natural IL-15, IL-18, IL-21, and IL-27 and IL-15Rα from human and non-human mammalian (such as mouse) sources are known and are also registered in databases such as NCBI and UniProt, so their information can be used in the present invention. As an example, the amino acid sequence of human natural IL-15 (full length including signal peptide and propeptide part) registered as UniProtKB-P40933 is shown as SEQ ID NO: 18, and the amino acid sequence of human natural IL-15Rα (full length including signal peptide, sushi domain, extracellular domain, etc.) registered as UniProtKB-Q13261 is shown as SEQ ID NO: 19. In the embodiments described later in which the natural amino acid sequence of mouse is replaced with the natural amino acid sequence of human in the amino acid sequences of the specified SEQ ID NOs, the amino acid sequences of the corresponding parts contained in SEQ ID NOs: 18 and 19 can be referred to. In addition, as variants of IL-15, IL-18, IL-21, and IL-27, fusion proteins with other proteins, and their modified forms are also known, and the information of their amino acid sequences can also be used in the present invention.

[0148] Regarding the variants of IL-15, IL-18, IL-21, and IL-27 respectively, as a whole or a partial region (domain), the following variants can be listed: having an amino acid sequence with a homology of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the natural amino acid sequence of each human or non-human mammalian source when replacing the signal peptide, etc. A variant having a homology within the above range with respect to the natural amino acid sequence of a certain species source sometimes corresponds to (substantially contains) the natural amino acid sequence of another species source. In addition, IL-15, IL-18, IL-21, and IL-27 can be peptides formed by replacing the signal peptide in the sequence with a known signal peptide. In one aspect of the present invention, it can be in SEQ ID NO: 4: IL15 LSP _F2A_CCL19 DNA fragment (DNA fragment #3), SEQ ID NO: 6: s IL15 RA_F2A_CCL19 DNA fragment (DNA fragment #4), SEQ ID NO: 8: mb IL15 RA _F2A_CCL19 DNA fragment (DNA fragment #5), SEQ ID NO: 10: sushi IL15_F2A_CCL19 DNA fragment (DNA fragment #6), SEQ ID NO: 12: IL-18_F2A_CCL19 DNA fragment (DNA fragment #7), SEQ ID NO: 14: IL-21_F2A_CCL19 DNA fragment (DNA fragment #8), SEQ ID NO: 16: sc In the amino acid sequence of the protein (polypeptide) expressed by the base sequence of each of the IL27_F2A_CCL19 DNA fragments (DNA fragment #9) or in SEQ ID NO: 5: containing IL15 LSP fusion protein, SEQ ID NO: 7: containing s IL15 RA fusion protein, SEQ ID NO: 9: containing mb IL15 RA fusion protein, SEQ ID NO: 11: containing sushi fusion protein containing IL15, SEQ ID NO: 13: fusion protein containing IL-18, SEQ ID NO: 15: fusion protein containing IL-21, SEQ ID NO: 17: containing sc In the amino acid sequence of each of the fusion proteins containing IL27, the amino acid sequence of natural murine IL-15, IL-18, IL-21 or IL-27 is replaced with the amino acid sequence of a variant having the above homology or the amino acid sequence of natural human IL-15, IL-18, IL-21 or IL-27 substantially equivalent to such a variant or further variants thereof.

[0149] As variants of the IL-15Rα extracellular domain, full-length IL-15Rα and IL-15Rα sushi, respectively, there may be mentioned: variants having an amino acid sequence with a homology of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more with respect to the natural amino acid sequence from human or non-human mammalian sources. In one aspect of the present invention, it is possible to use SEQ ID NO: 4: IL15 LSP _F2A_CCL19 DNA fragment (DNA fragment #3), SEQ ID NO: 6: s IL15 RA _F2A_CCL19 DNA fragment (DNA fragment #4), SEQ ID NO: 8: mb IL15 RA _F2A_CCL19 DNA fragment (DNA fragment #5), SEQ ID NO: 10:sushi In the amino acid sequence of the protein (polypeptide) expressed by the base sequence of each of the IL15_F2A_CCL19 DNA fragments (DNA fragment #6) or in SEQ ID NO: 5: the fusion protein containing IL15 LSP , SEQ ID NO: 7: containing s IL15 RA the fusion protein, SEQ ID NO: 9: containing mb IL15 RA the fusion protein, SEQ ID NO: 11: containing sushi In the amino acid sequence of each of the fusion proteins containing IL15, the amino acid sequence of the extracellular domain of murine native IL-15Rα, full-length IL-15Rα or IL-15Rα sushi is replaced with the amino acid sequence of a variant having the above homology or the amino acid sequence of the extracellular domain of human native IL-15Rα, full-length IL-15Rα or IL-15Rα sushi that is substantially equivalent to such a variant or further variants thereof.

[0150] IL-15, IL-18, IL-21 and IL-27 are preferably derived from the same species of human or non-human mammal (e.g., mouse) as the T cells used in the production of the T cells of the present invention (the receptors for IL-15, IL-18, IL-21 and IL-27 possessed by such T cells). With respect to the portions other than IL-15, IL-18, IL-21 and IL-27 in the respective fusion proteins of IL-15, IL-18, IL-21 and IL-27 (e.g., IL-15Rα), they are also preferably derived from the same species of human or non-human mammal as the T cells used in the production of the T cells of the present invention. In one aspect of the present invention, it is possible to use SEQ ID NO: 4: IL15 LSP _F2A_CCL19 DNA fragment (DNA fragment #3), SEQ ID NO: 6: s IL15 RA _F2A_CCL19 DNA fragment (DNA fragment #4), SEQ ID NO: 8: mb IL15 RA _F2A_CCL19 DNA fragment (DNA fragment #5), SEQ ID NO: 10: sushi IL15_F2A_CCL19 DNA fragment (DNA fragment #6), SEQ ID NO: 12: IL-18_F2A_CCL19 DNA fragment (DNA fragment #7), SEQ ID NO: 14: IL-21_F2A_CCL19 DNA fragment (DNA fragment #8), SEQ ID NO: 16: scThe amino acid sequence of the protein (polypeptide) expressed by the base sequence of each of the IL27_F2A_CCL19 DNA fragments (DNA fragment #9) or in SEQ ID NO: 5: the fusion protein containing IL15 LSP , SEQ ID NO: 7: containing s IL15 RA in the fusion protein, SEQ ID NO: 9: containing mb IL15 RA in the fusion protein, SEQ ID NO: 11: containing sushi the fusion protein of IL15, SEQ ID NO: 13: the fusion protein containing IL-18, SEQ ID NO: 15: the fusion protein containing IL-21, SEQ ID NO: 17: containing sc in the amino acid sequence of each of the fusion proteins of IL27, the amino acid sequence of murine native IL-15, IL-18, IL-21 or IL-27 or the amino acid sequence of the extracellular domain of IL-15Rα, full-length IL-15Rα or IL-15Rα sushi is replaced with the respective human native amino acid sequence.

[0151] (3) Chemokine

[0152] The chemokine expressed by the T cells of the present invention is CCL19. 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 mediated by its receptor CCR7. "CCL19" in the present invention includes not only the full length of the protein (polypeptide) composed of the amino acid sequence of native CCL19, but also a part thereof (functional partial polypeptide), and variants of the full length or a part of native CCL19 in which one or more amino acid sequences are deleted, substituted or added with respect to the native amino acid sequence (preferably within the range of having the homology described below).

[0153] The amino acid sequence of the above-mentioned chemokine expressed by the T cells of the present invention can be an amino acid sequence suitable under the premise of the use of the T cells (CAR) of the present invention, typically as an active ingredient of a drug for treating cancer, and considering the effect of enhancing the anti-tumor activity brought by CAR-T cells including the improvement of the persistence and proliferation of CAR-T cells. The amino acid sequences of native CCL19 from human and non-human mammalian (e.g., mouse) sources are known and registered in databases such as NCBI and UniProt, so the information thereof can be used in the present invention. In addition, the information of the amino acid sequences known as variants of CCL19 can also be used in the present invention.

[0154] Regarding variants of CCL19, for example, variants can be enumerated that, as a whole or in part, have an amino acid sequence homology of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the amino acid sequence of natural CCL19 from human or non-human mammalian sources. In one aspect of the present invention, it can be in SEQ ID NO: 4: IL15 LSP _F2A_CCL19 DNA fragment (DNA fragment #3), SEQ ID NO: 6: s IL15 RA _F2A_CCL19 DNA fragment (DNA fragment #4), SEQ ID NO: 8: mb IL15 RA _F2A_CCL19 DNA fragment (DNA fragment #5), SEQ ID NO: 10: sushi IL15_F2A_CCL19 DNA fragment (DNA fragment #6), SEQ ID NO: 12: IL-18_F2A_CCL19 DNA fragment (DNA fragment #7), SEQ ID NO: 14: IL-21_F2A_CCL19 DNA fragment (DNA fragment #8), SEQ ID NO: 16: sc In the amino acid sequence of the protein (polypeptide) expressed by the base sequence of each of IL27_F2A_CCL19 DNA fragment (DNA fragment #9), replace the amino acid sequence of natural CCL19 of mouse with the amino acid sequence of a variant having the above homology or the amino acid sequence of natural CCL19 of human substantially equivalent to such a variant or its further variant.

[0155] CCL19 is preferably derived from CCL19 or its variant of the same species as the T cell (the receptor of CCL19 possessed by this T cell) used in the production of the T cell of the present invention, from human or non-human mammals (such as mice).

[0156] The drug of the present invention will be described in detail below.

[0157] The drug of the present invention contains the above-mentioned T cell of the present invention and can further contain other components as needed. Those skilled in the art can appropriately prepare the drug of the present invention using the T cell of the present invention while considering the use (treatment target) and dosage form.

[0158] The drug of the present invention is a drug (anticancer agent) mainly targeting cancers corresponding to the cell surface antigens (cancer-specific antigens) of cancer cells targeted by the CAR expressed on the T cells of the present invention. Therefore, as long as the cancer tissue contains cancer cells expressing the antigen targeted by the CAR and a certain level of therapeutic effect can be observed using the T cells of the present invention, the type of cancer targeted by the drug of the present invention is not particularly limited. Examples of cancers targeted by the drug of the present invention include, for example, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer (e.g., melanoma, Merkel cell carcinoma), breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, non-small cell lung cancer, tracheal cancer, bronchial cancer, colon cancer, rectal cancer, small intestine cancer, colorectal cancer, gastric cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, fallopian tube cancer, nasopharyngeal cancer, etc.; cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue; sarcomas such as chondrosarcoma, Ewing's sarcoma, rhabdomyosarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma; germ cell tumors; lymphomas; leukemia, acute myeloid leukemia, multiple myeloma. Tumors sensitive to NK cells (melanoma, Merkel cell carcinoma, colorectal cancer, kidney cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, non-small cell lung cancer, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma, leukemia, etc.) are preferred. Melanoma, colorectal cancer, kidney cancer, multiple myeloma, lymphoma, and leukemia are more preferably listed.

[0159] Examples of components other than T cells that the drug of the present invention may contain include, for example, pharmaceutically acceptable additives. More specifically, examples include physiological saline, buffered saline, cell culture medium, glucose, water for injection, glycerol, ethanol, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonic agents, fillers, lubricants, etc.

[0160] The drug of the present invention can be used by administering it to a subject in need of cancer treatment (cancer patients, cancer-bearing animals, etc.) in the same manner as known CAR-expressing T cells. Examples of the administration method include injection into the tumor, intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, etc.

[0161] The amount of the T cells of the present invention contained in the medicament of the present invention can be appropriately adjusted according to the use, dosage form, target therapeutic effect, etc., and taking into account, for example, the type, location, severity of cancer, the age, weight and condition of the subject to be treated, etc. For example, the medicament of the present invention can be formulated in such a manner that usually 1×10 4 to 1×10 10 cells, preferably 1×10 5 to 1×10 9 cells, more preferably 5×10 6 to 5×10 8 T cells of the present invention are administered in one dose.

[0162] The administration interval of the medicament of the present invention is not particularly limited and can be appropriately adjusted in consideration of the amount of the T cells of the present invention administered each time, etc. For example, it can be administered independently 4 times a day, 3 times a day, 2 times a day or once a day, every other day, every 2 days, every 3 days, every 4 days, every 5 days, once a week, every 7 days, every 8 days, every 9 days, twice a week, once a month or twice a month.

[0163] The medicament (anticancer agent) of the present invention can be used in combination with known anticancer agents. Examples of anticancer agents include alkylating agents such as cyclophosphamide, bendamustine, ifosfamide, dacarbazine; metabolic antagonists such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, enocitabine; molecularly targeted drugs such as rituximab, cetuximab, trastuzumab; kinase inhibitors such as imatinib, gefitinib, erlotinib, afatinib, dasatinib, sunitinib, trametinib; proteasome inhibitors such as bortezomib; calcineurin inhibitors such as cyclosporine, tacrolimus; anticancer antibiotics such as idarubicin, doxorubicin, mitomycin C; plant alkaloids such as irinotecan, etoposide; platinum preparations such as cisplatin, oxaliplatin, carboplatin; hormone therapy agents such as tamoxifen, bicalutamide; immunomodulators such as interferon, nivolumab, pembrolizumab.

[0164] When the medicament of the present invention is used in combination with other anticancer agents, it can be in any of the following ways: (a) using the medicament of the present invention after using other anticancer agents, (b) using the medicament of the present invention and other anticancer agents simultaneously, (c) using other anticancer agents after using the medicament of the present invention. When the medicament of the present invention is used in combination with other anticancer agents, the following effects can be expected: the therapeutic effect of cancer is further improved, and the number of administrations or the dosage of the medicament of the present invention and / or other anticancer agents is reduced, thereby reducing the side effects caused by each anticancer agent.

[0165] The expression vector of the present invention will be described in detail below.

[0166] The expression vector of the present invention comprises (1) a nucleic acid encoding a CAR, (2) a nucleic acid encoding at least one selected from the group consisting of IL-15, IL-18, IL-21, and IL-27, and (3) a nucleic acid encoding CCL19.

[0167] In the present invention, the expression vector "comprising a nucleic acid encoding IL-15" not only means comprising a nucleic acid encoding the native full-length IL-15 protein, but also comprises the IL-15s in the above various embodiments. The same applies to the expression vector "comprising a nucleic acid encoding IL-18", "comprising a nucleic acid encoding IL-21", "comprising a nucleic acid encoding IL-27", and "comprising a nucleic acid encoding CCL19".

[0168] The "nucleic acid" can be any nucleic acid as long as it is a molecule polymerized from nucleotides and molecules having the same function as the nucleotides. Examples include RNA, which is a polymer of ribonucleotides, DNA, which is a polymer of deoxyribonucleotides, a polymer composed of a mixture of ribonucleotides and deoxyribonucleotides, and a nucleotide polymer containing nucleotide analogs. Further, it can be a nucleotide polymer containing nucleic acid derivatives. In addition, the nucleic acid can be single-stranded nucleic acid or double-stranded nucleic acid. In addition, double-stranded nucleic acid also includes double-stranded nucleic acid in which one strand hybridizes to the other strand under stringent conditions.

[0169] As a nucleotide analog, as long as it is a molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA in order to improve nuclease resistance or stability, to improve affinity with complementary-strand nucleic acid, to improve cell permeability, or to make it visible compared with RNA or DNA, it can be any molecule. As nucleotide analogs, they can be naturally occurring molecules or non-natural molecules. Examples include sugar moiety-modified nucleotide analogs, phosphodiester bond-modified nucleotide analogs, etc.

[0170] As a sugar moiety-modified nucleotide analogue, any nucleotide analogue can be used as long as it is a nucleotide analogue formed by attaching or substituting any chemical structure substance to a part or all of the chemical structure of the sugar of the nucleotide. As specific examples, nucleotide analogues substituted with 2'-O-methyl ribose, nucleotide analogues substituted with 2'-O-propyl ribose, nucleotide analogues substituted with 2'-methoxyethoxy ribose, nucleotide analogues substituted with 2'-O-methoxyethyl ribose, nucleotide analogues substituted with 2'-O-[2-(guanidino)ethyl] ribose, nucleotide analogues substituted with 2'-fluoro ribose, bridged structure-type artificial nucleic acids (Bridged Nucleic Acid) (BNA) having two cyclic structures by introducing a bridging structure into the sugar moiety, more specifically, locked nucleic acid (LNA) in which the oxygen atom at the 2' position is bridged to the carbon atom at the 4' position via a methylene group and ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)] can be cited. Peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxy peptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)] etc. can also be cited.

[0171] As a phosphodiester bond-modified nucleotide analogue, any nucleotide analogue can be used as long as it is a nucleotide analogue formed by attaching or substituting any chemical substance to a part or all of the chemical structure of the phosphodiester bond of the nucleotide. As specific examples, nucleotide analogues substituted with a phosphorothioate bond, nucleotide analogues substituted with an N3'-P5' phosphoramidate bond, etc. can be cited [Cell Engineering, 16, 1463-1473 (1997)] [RNAi Method and Antisense Method, Kodansha (2005)].

[0172] As a nucleic acid derivative, any molecule can be used as long as it is a molecule formed by attaching other chemical substances to the nucleic acid in order to improve nuclease resistance, to stabilize it, to improve the affinity with complementary strand nucleic acid, to improve cell permeability, or to make it visible. As specific examples, 5'-polyamine attached derivatives, cholesterol attached derivatives, steroid attached derivatives, bile acid attached derivatives, vitamin attached derivatives, Cy5 attached derivatives, Cy3 attached derivatives, 6-FAM attached derivatives, and biotin attached derivatives etc. can be cited.

[0173] The expression vector of the present invention only needs to be an expression vector that can be introduced into cells by contacting T cells or their precursors and express the specified protein (polypeptide) encoded thereby in T cells to produce the T cells of the present invention, and there is no particular limitation on what kind of embodiment this is. Those skilled in the art can design and produce an expression vector that can express the desired protein (polypeptide) in T cells.

[0174] The expression vector of the present invention can be linear or circular, and can be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector.

[0175] Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. Examples of preferred retroviral vectors include the pMSGV vector (Tamada k et al., ClinCancer Res 18:6436-6445 (2002)) and the pMSCV vector (manufactured by Takara Bio Inc.). When using a retroviral vector, the genes contained in the vector are integrated into the genome of the host cell (T cells in the present invention), and thus the genes can be expressed stably for a long time.

[0176] In one embodiment of the present invention, one expression vector contains all of the following elements: (1) a nucleic acid encoding a CAR, (2) a nucleic acid encoding at least one selected from the group consisting of IL-15, IL-18, IL-21, and IL-27, and (3) a nucleic acid encoding CCL19. In another embodiment of the present invention, the first expression vector contains one or two of the above elements (1) to (3), and the second expression vector contains the remaining elements. In another embodiment of the present invention, the first expression vector contains the above (1), the second expression vector contains the above (2), and the third expression vector contains the above (3). Therefore, the expression vector of the present invention sometimes refers to a combination (set) of two or more expression vectors according to the embodiment, that is, the combination of the above first expression vector and the second expression vector, or the combination of the above first expression vector, the second expression vector, and the third expression vector. When two or more elements are contained in one expression vector, there is no particular limitation on the order in which the above elements are arranged from the upstream side to the downstream side.

[0177] The base sequences of the three specified elements (CAR, cytokine, and chemokine) contained in the expression vector of the present invention can be designed according to which protein (polypeptide) is selected as each element so that it encodes the amino acid sequence of the protein (polypeptide). The nucleic acid (oligonucleotide) contained in the expression vector can be produced by an oligonucleotide chemical synthesis reaction or can be produced (cloned) as cDNA.

[0178] In the expression vector of the present invention, in addition to containing the sequences (genes) encoding the above-specified elements, sequences such as promoters, terminators, enhancers, start codons, stop codons, polyadenylation signals, nuclear localization signals (NLS), multiple cloning sites (MCS), etc., which are involved in the expression of each gene, can also be included as needed (independently for each expression vector in the case of a combination of two or more expression vectors as described above).

[0179] In one embodiment of the present invention, when two or more of the above three elements are contained in one expression vector, a gene encoding a self-cleaving peptide (2A peptide) or an IRES (Internal Ribozyme Entry Site) can be inserted between the respective elements.

[0180] The 2A peptide is a self-cleaving peptide derived from a virus and has the characteristic of being cleaved by the endoplasmic reticulum at a specified position (one residue from the C-terminus) in the amino acid sequence (Szymczak et al., Expert Opin. Biol. Ther. 5(5):627-638(2005)). Therefore, the nucleic acids integrated before and after the 2A peptide are expressed independently of each other in the cell. Examples of the 2A peptide include 2A peptides derived from picornavirus, rotavirus, insect virus, foot-and-mouth disease virus, or trypanosome virus.

[0181] The expression vector of the present invention may further contain nucleic acids (base sequences) encoding "functional genes" such as reporter genes (representatively genes encoding fluorescent proteins), drug selection genes, suicide genes, etc.

[0182] When using a "drug resistance gene" as a functional gene, in the method for producing the CAR-T cells of the present invention, cells transfected with the expression vector of the present invention containing the drug resistance gene can be selected by adding a specified drug to the culture medium. Examples of the drug resistance gene include kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene. Any one of these genes can be used, or two or more can be used.

[0183] When using the "gene encoding a fluorescent protein" as a functional gene, in the method for producing the CAR-T cells of the present invention, it is possible to observe T cells into which the expression vector of the present invention has been introduced using a fluorescence microscope, or to screen cells into which the expression vector of the present invention has been introduced by a flow cytometer (cell sorter). Representative examples of reporter genes include genes encoding fluorescent proteins. Examples of the "gene encoding a fluorescent protein" include, for example: blue fluorescent proteins such as Sirius, BFP, and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green (e.g., hmAG1), ZsGreen, EmGFP, EGFP, GFP2, and HyPer; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, and mBanana; orange fluorescent proteins such as KusabiraOrange (e.g., hmKO2) and mOrange; red fluorescent proteins such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, and mStrawberry; near-infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (e.g., hdKeimaRed), mRasberry, and mPlum. Any one of these genes can be used, or two or more can be used. When using two or more genes encoding fluorescent proteins, it is preferable if the emission wavelengths of their fluorescent proteins are different so as not to interfere with each other's identification.

[0184] When using a "suicide gene" as a functional gene, a medicament that activates the function of the suicide gene is administered according to the cancer treatment process, for example, at a stage when the tumor has disappeared, thereby enabling the control of the number of the T cells of the present invention in a living body. Examples of the suicide gene include genes encoding diphtheria A toxin, herpes simplex thymidine kinase (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella-zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), or inducible caspase 9. Any one of these genes can be used, or two or more can be used. Medicaments that activate the function of each suicide gene are well-known. For example, for herpes simplex thymidine kinase (HSV-TK), ganciclovir can be cited, and for inducible caspase 9, AP1903, which is a dimer-inducing compound, can be cited.

[0185] The method for producing the CAR-T cells of the present invention will be described in detail below.

[0186] The method for producing the CAR-T cells of the present invention includes a step of introducing the above-described expression vector of the present invention into T cells (hereinafter referred to as the "expression vector introduction step").

[0187] The method for introducing the expression vector into T cells can be set as a suitable method corresponding to the implementation form of the expression vector. For example, the expression vector can be introduced into T cells by a well-known method such as a virus infection method, a calcium phosphate method, a liposome transfection method, a microinjection method, or an electroporation method. The expression vector of the present invention can be prepared into a form suitable for each method by well-known means and, additionally, appropriately using a commercially available kit (in accordance with its instructions). In the expression vector introduction step, it is only necessary to bring such a preparation into contact with T cells. Usually, T cells are cultured in a medium supplemented with the preparation containing the expression vector.

[0188] In a preferred embodiment of the present invention, the expression vector of the present invention is introduced into T cells by viral infection. For example, the following methods can be cited: using commercially available kits corresponding to various viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc., transfect the vector of the present invention and the packaging vectors (plasmids) of each virus into host cells to produce recombinant viruses, and then infect T cells with the obtained recombinant viruses. As commercially available kits for viral vectors, for example, Retrovirus packaging Kit Eco (manufactured by Takara Bio Inc.) can be cited. As host cells, for example, GP2-293 cells (manufactured by Takara Bio Inc.), Plat-GP cells (manufactured by COSMO BIO Co., Ltd.), PG13 cells (ATCC CRL-10686), PA317 cells (ATCC CRL-9078), etc. can be cited.

[0189] In the method for producing the CAR-T cells of the present invention, other steps may be included before and after the expression vector introduction step. As other steps, for example, a step of culturing T cells and a step including a treatment for activating the functional gene contained in the expression vector can be cited.

[0190] The T cells to which the expression vector of the present invention is to be introduced can usually be cultured in vitro in advance by a known method. For example, T cells can be isolated and purified from body fluids such as blood and bone marrow fluid, tissues such as spleen, thymus, and lymph nodes, or cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites collected from humans or non-human animals according to a conventional method, and then the T cells are cultured with a suitable medium, and the expression vector of the present invention (a preparation containing the expression vector of the present invention) is added to the medium. Alternatively, T cells (or their precursors) can be produced from pluripotent stem cells such as iPS cells and ES cells through an appropriate differentiation induction step in advance, and the expression vector of the present invention (a preparation containing the expression vector of the present invention) is added to their medium.

[0191] In addition, when the expression vector of the present invention contains a drug resistance gene as a functional gene, in order to screen the T cells into which the expression vector has been introduced, a step of adding a drug corresponding to the used drug resistance gene to the medium and culturing the T cells can be performed after the expression vector introduction step. Those skilled in the art can appropriately adjust the use conditions of the drug corresponding to the drug resistance gene, such as the concentration in the medium, the culture time, etc.

[0192] When the expression vector of the present invention contains a gene encoding a fluorescent protein (reporter gene) as a functional gene, after the expression vector introduction step, a step of observing T cells into which the expression vector has been introduced using a fluorescence microscope or a step of screening T cells into which the expression vector has been introduced using a cell sorter can be performed. Those skilled in the art can appropriately adjust the conditions for observation using a fluorescence microscope and screening using a cell sorter, such as irradiation with light having an excitation wavelength corresponding to the fluorescent protein and detection of light having an emission wavelength.

[0193] Example

[0194] In the following examples, the CAR is a CAR targeting human CD20, specifically, an anti-human CD20 CAR composed of an anti-human CD20 scFv, a mouse CD8 transmembrane region, and a mouse CD28_4-1BB_CD3ζ intracellular signaling motif. In addition, the cytokine in the following examples is mouse IL-15 (“IL15 LSP ”, “ s IL15RA”, “ mb IL15 RA ” or “ sushi IL15”); mouse IL-18; mouse IL-21; a single-chain protein containing mouse IL-27 (p28 and EBI3) “ sc IL27”. Any one of the above. The chemokine in the following examples is mouse CCL19. After producing mouse-derived T cells expressing these CARs, cytokines, and chemokines as described below, experiments were conducted.

[0195] [Table 1]

[0196]

[0197] [Example 1-1] IL15 LSP ×CCL19 CAR-T cells

[0198] In the vector production step, first, three DNA fragments (DNA fragments 1 to 3) were artificially synthesized by step 1, and then, by step 2, one expression vector containing all the genes encoding the specified CAR, cytokine, and chemokine was produced using the above three DNA fragments. Next, in the CAR-T cell production step, first, a retroviral vector was prepared using the above expression vector by step 1, and then, the specified genes were transduced into mouse T cells using the retroviral vector preparation by step 2.

[0199] [A: Vector production step] IL15 LSP _CCL19_Anti-human CD20 CAR expression vector production

[0200] Step 1: Synthesis of DNA fragments

[0201] DNA fragment #1: DNA fragment containing anti-human CD20 CAR

[0202] A DNA fragment (DNA fragment #1) containing a base sequence encoding anti-human CD20 CAR was artificially synthesized. The anti-human CD20 CAR consists of anti-human CD20 scFv, murine CD8 transmembrane region, and murine CD28_4-1BB_CD3ζ intracellular signaling motif. The base sequence of the entire DNA fragment #1 is shown in SEQ ID NO: 1( Figure 7 ). In SEQ ID NO: 1, the bases at positions 3 to 785 encode the sequence of anti-human CD20 scFv, the bases at positions 795 to 1040 encode the sequence of murine CD8 transmembrane region, and the bases at positions 1041 to 1637 encode the sequence of murine CD28_4-1BB_CD3ζ intracellular signaling motif (wherein, the bases at positions 1041 to 1163 encode the intracellular region of murine CD28, the bases at positions 1164 to 1298 encode the intracellular region of murine 4-1BB, and the bases at positions 1299 to 1637 encode the polypeptide of the intracellular region of murine CD3ζ). The amino acid sequence of the fusion protein expressed by the base sequence at positions 3 to 1637 of DNA fragment #1 is shown in SEQ ID NO: 2.

[0203] DNA fragment #2: DNA fragment containing a stop codon and a multiple cloning site

[0204] A DNA fragment (DNA fragment #2) containing the base sequence of a stop codon and the base sequence of a multiple cloning site (MCS) was artificially synthesized. The base sequence of the entire DNA fragment #2 is shown in SEQ ID NO: 3( Figure 8 ).

[0205] DNA fragment #3: IL15 LSP and CCL19 DNA fragment

[0206] A DNA fragment (DNA fragment #3) containing the base sequence encoding 2A peptide (F2A) as a self-cleaving peptide, murine IL-2 signal peptide (IL-2SP), murine IL-15 construct “IL15 LSP ” (a fusion peptide consisting of IL-15LSP and IL-15), F2A, and murine CCL19 was artificially synthesized. The base sequence of the entire DNA fragment #3 is shown in SEQ ID NO: 4( Figure 9 ). In SEQ ID NO: 4, the bases at positions 7 to 81 encode the sequence of the first F2A, and the bases at positions 82 to 168 encode murine IL-15 LSPThe sequence from the 169th to the 567th base is the sequence encoding mouse IL-15 (except for the stop codon), the sequence from the 568th to the 642nd base is the sequence encoding the second F2A, and the sequence from the 646th to the 969th base is the sequence encoding mouse CCL19. The base sequence from the 7th to the 969th of DNA fragment #3 is expressed, and self-cleavage occurs at two F2As, and the amino acid sequence of the whole fusion protein containing IL15 LSP is shown in SEQ ID NO: 5.

[0207] Step 2: Preparation of retroviral expression vector

[0208] DNA fragment #1 and DNA fragment #2 were ligated to prepare a construct (anti-human CD20 CAR_MCS). The resulting construct was integrated into the pMSGV retroviral expression vector (Tamada k et al., Clin Cancer Res 18:6436-6445 (2002)) by treatment with restriction enzymes (Nco I and Sal I), thereby preparing a pMSGV retroviral expression vector containing anti-human CD20 CAR_MCS.

[0209] Then, a construct was prepared by ligating the base sequence from the 1337th to the 1637th of DNA fragment #1 to DNA fragment #3. The resulting construct was integrated into the pMSGV retroviral vector containing anti-human CD20 CAR_MCS by treatment with restriction enzymes (Sbf I and Sal I), thereby preparing a pMSGV retroviral expression vector containing anti-human CD20 CAR_F2A_IL15 LSP _F2A_CCL19 (IL15 LSP _CCL19_anti-human CD20 CAR expression vector).

[0210] [B: CAR-T cell manufacturing step] IL15 LSP Manufacture of CCL19_anti-human CD20 CAR-T cells

[0211] Step 1: Preparation of retrovirus

[0212] Using Lipofectamine 3000 (Thermo Fisher Scientific), the IL15 obtained through the above vector preparation step LSPThe _CCL19_-anti-human CD20 CAR expression vector was transfected into the GP2-293 packaging cell line (Takara Bio Inc.) together with the pCL-Eco plasmid (Novus biologicals). It should be noted that as the culture medium for the GP2-293 packaging cell line, DMEM supplemented with 10% heat-inactivated FBS and antibiotics was used. 48 hours after transfection, the supernatant of the culture medium of the GP2-293 packaging cell line was recovered and used as IL15 LSP Preparation of _CCL19_-anti-human CD20 CAR expression vector

[0213] Step 2: Transduction of mouse T cells

[0214] 3×10 purified mouse T cells derived from the spleen and lymph nodes of mice were activated in the presence of immobilized anti-mouse CD3 monoclonal antibody (3 μg / mL), anti-mouse CD28 monoclonal antibody (1 μg / mL), and IL-2 for 48 hours. Then, in the wells of a plate coated with 25 μg / mL of RetroNectin (registered trademark: Takara Bio Inc.), the preparation containing IL15 6 from the _CCL19_-anti-human CD20 CAR expression vector obtained in Step 1 (the supernatant of the GP2-293 cell culture medium) and the mouse T cells activated as described above were mixed, centrifuged at 1500 rpm for 2 hours, and then cultured in the presence of IL-2 for 6 hours. To remove the retrovirus from the culture medium, the mouse T cells were recovered and transferred to a new RPMI complete medium containing IL-2, and cultured for another 42 hours, thereby obtaining mouse T cells transfected with the _CCL19_-anti-human CD20 CAR expression vector expressing anti-human CD20 CAR, IL15 LSP and CCL19 (IL15 LSP _CCL19_-anti-human CD20 CAR-T cells, referred to as "IL15 LSP ×CCL19 CAR-T cells" in this specification.). LSP It should be noted that the "RPMI complete medium" used in the above culture of T cells is RPMI-1640 medium supplemented with 10% heat-inactivated FBS, antibiotics, 50 mM 2-mercaptoethanol, and 25 mM HEPES buffer. Hereinafter, as the culture medium for T cells, RPMI complete medium supplemented with other ingredients as needed will be used LSP ×CCL19 CAR-T cells".

[0215]

[0216] [Example 1-2] s IL15 RA ​(Secreted IL-15 / IL-15RA fusion protein) × CCL19 CAR-T cells

[0217] [A: Vector production steps] s IL15 RA Preparation of _CCL19_ anti-human CD20 CAR expression vector

[0218] Using the same DNA fragment #1 and DNA fragment #2 as in step 1 of Example 1-1 and the following DNA fragment #4 in place of DNA fragment #3, after constructing the construct in the same manner as in step 2 of Example 1-1, a pMSGV retroviral expression vector containing anti-human CD20CAR_F2A_ s IL15 RA _F2A_CCL19 was obtained ([[]] s IL15 RA _CCL19_ anti-human CD20 CAR expression vector).

[0219] DNA fragment #4: s IL15 RA and CCL19 DNA fragment

[0220] was synthesized to contain the base sequences encoding F2A, murine IL-2SP, murine IL-15 construct " s IL15 RA "(a fusion peptide composed of IL-15, a linker, and the extracellular domain of IL-15Rα), F2A, and murine CCL19 (DNA fragment #4). The base sequence of the entire DNA fragment #4 is shown in SEQ ID NO: 6([[]] Figure 10 ). In SEQ ID NO: 6, the bases at positions 7 to 81 encode the first F2A sequence, the bases at positions 82 to 141 encode the murine IL-2SP sequence, the bases at positions 142 to 1137 encode the sequence of s IL15 RA (wherein the bases at positions 142 to 540 encode murine IL-15 (excluding the signal sequence and stop codon), the bases at positions 541 to 618 are the linker, and the bases at positions 619 to 1137 encode the extracellular domain of murine IL-15Rα), the bases at positions 1138 to 1212 encode the second F2A sequence, and the bases at positions 1216 to 1539 encode the murine CCL19 sequence. The amino acid sequence of the entire fusion protein expressed by the bases at positions 7 to 1539 of DNA fragment #4 and self-cleaved at two F2As and containing s IL15 RA is shown in SEQ ID NO: 7.

[0221] [B: CAR-T Cell Manufacturing Steps] s IL15 RA Manufacture of _CCL19_Anti-human CD20 CAR-T Cells

[0222] As a retroviral expression vector, use the s IL15 RA _CCL19_Anti-human CD20 CAR expression vector obtained through the above vector production steps to replace IL15 LSP _CCL19_Anti-human CD20 CAR expression vector, and perform the same as the CAR-T cell manufacturing steps in Example 1-1 except for this. In step 1, a preparation containing retrovirus is obtained, and in step 2, mouse T cells expressing the anti-human CD20 CAR, s IL15 RA and each gene of CCL19 are obtained ([[]] s IL15 RA _CCL19_Anti-human CD20 CAR-T cells, denoted as s IL15 RA ×CCL19 CAR-T cells in this specification).

[0223] [Example 1-3] mb IL15 RA (Membrane-bound IL-15 / IL-15RA fusion protein)×CCL19 CAR-T cells

[0224] [A: Vector Production Steps] mb IL15 RA Production of _CCL19_Anti-human CD20 CAR Expression Vector

[0225] Use the same DNA fragment #1 and DNA fragment #2 as in step 1 of Example 1-1, and the following DNA fragment #5 instead of DNA fragment #3. After constructing the construct in the same way as in step 2 of Example 1-1, a pMSGV retroviral expression vector containing anti-human CD20CAR_F2A_ mb IL15 RA _F2A_CCL19 is obtained ([[]] mb IL15 RA _CCL19_Anti-human CD20 CAR expression vector).

[0226] DNA fragment #5: mb IL15 RA and CCL19 DNA fragment

[0227] A construct containing the coding sequences for F2A, murine IL-2SP, and murine IL-15, “ mb IL15 RA ” (a fusion peptide consisting of IL-15, a linker, and full-length IL-15Rα), F2A, and the DNA fragment with the base sequence of murine CCL19 was synthesized. The entire base sequence of this DNA fragment #5 is shown in SEQ ID NO: 8( Figure 11 ). In SEQ ID NO: 8, the bases at positions 7 to 81 encode the first F2A, the bases at positions 82 to 141 encode murine IL-2SP, and the bases at positions 142 to 1311 encode mb IL15 RA (wherein the bases at positions 142 to 540 encode murine IL-15 (excluding the signal sequence and stop codon), the bases at positions 541 to 618 are the linker, and the bases at positions 619 to 1311 encode murine IL-15Rα (full length)), the bases at positions 1312 to 1386 encode the second F2A, and the bases at positions 1390 to 1713 encode murine CCL19. The amino acid sequence of the entire fusion protein expressed from the bases at positions 7 to 1713 of DNA fragment #5 and self-cleaved at two F2A sites and containing mb IL15 RA is shown in SEQ ID NO: 9.

[0228] [B: CAR-T Cell Manufacturing Steps] mb IL15 RA _CCL19_Production of Anti-Human CD20 CAR-T Cells

[0229] As a retroviral expression vector, the mb IL15 RA _CCL19_anti-human CD20 CAR expression vector obtained through the above vector production steps was used in place of the IL15 LSP _CCL19_anti-human CD20 CAR expression vector. Otherwise, the CAR-T cell manufacturing steps of Example 1-1 were carried out in the same manner. A preparation containing retrovirus was obtained in Step 1, and murine T cells expressing the anti-human CD20 CAR, mb IL15 RA and each gene of CCL19 were produced in Step 2 ( mb IL15 RA _CCL19_anti-human CD20 CAR-T cells, which are referred to as “ mb IL15×CCL19 CAR-T cells” in this specification.).

[0230] [Example 1-4]sushi IL15 (secreted IL-15RA / IL-15 fusion protein) × CCL19 CAR-T cells

[0231] [A: Vector production steps] sushi Production of IL15_CCL19_anti-human CD20 CAR expression vector

[0232] Using the same DNA fragment 1 and DNA fragment 2 as in step 1 of Example 1-1, and the following DNA fragment #6 in place of DNA fragment #3, after constructing the construct in the same manner as in step 2 of Example 1-1, a pMSGV retroviral expression vector containing anti-human CD20 CAR_F2A_ sushi IL15_F2A_CCL19 was obtained ( sushi IL15_CCL19_anti-human CD20 CAR expression vector).

[0233] DNA fragment #6: sushi DNA fragment for IL15 and CCL19

[0234] A DNA fragment containing the base sequences encoding F2A, murine IL-15 construct " sushi IL15" (a fusion peptide consisting of the sushi domain of IL-15Rα, a linker, and IL-15), F2A, and murine CCL19 was synthesized. The base sequence of the entire DNA fragment #6 is shown in SEQ ID NO: 10 ( Figure 12 ). In SEQ ID NO: 10, the bases at positions 7 to 81 encode the sequence of the first F2A, the bases at positions 82 to 777 encode the sequence of sushi IL15 (wherein the bases at positions 82 to 375 encode the SP and sushi domain of murine IL-15Rα, the bases at positions 376 to 435 are the linker, and the bases at positions 436 to 777 encode murine IL-15 (excluding the signal sequence and propeptide)), the bases at positions 778 to 852 encode the sequence of the second F2A, and the bases at positions 856 to 1179 encode the sequence of murine CCL19. The amino acid sequence of the entire fusion protein containing sushi IL15, which is expressed from the bases at positions 7 to 1179 of DNA fragment #6 and undergoes self-cleavage at two F2As, is shown in SEQ ID NO: 11.

[0235] [B: CAR-T cell manufacturing steps] sushi Manufacture of IL15_CCL19_anti-human CD20 CAR-T cells

[0236] As the retroviral expression vector, the one obtained through the above vector production steps was usedsushi An IL15_CCL19_anti-human CD20 CAR expression vector is used to replace IL15 LSP The _CCL19_anti-human CD20 CAR expression vector is otherwise prepared in the same manner as the CAR-T cell manufacturing steps of Example 1-1. A preparation containing retrovirus is obtained in Step 1, and mouse T cells expressing the anti-human CD20 CAR, sushi IL15, and CCL19 genes contained in the retrovirus are obtained in Step 2 ( sushi IL15_CCL19_anti-human CD20 CAR-T cells, which are referred to as " sushi IL15×CCL19 CAR-T cells" in this specification.).

[0237] [Example 2] IL-18×CCL19 CAR-T cells

[0238] [A: Vector production step] Production of the IL18_CCL19_anti-human CD20 CAR expression vector

[0239] Using DNA fragment 1 and DNA fragment 2 in the same manner as in Step 1 of Example 1-1, and DNA fragment #7 as described below in place of DNA fragment #3, after constructing the construct in the same manner as in Step 2 of Example 1-1, a pMSGV retroviral expression vector containing anti-human CD20 CAR_F2A_IL-18_F2A_CCL19 (IL18_CCL19_anti-human CD20 CAR expression vector) is obtained.

[0240] DNA fragment #7: DNA fragment for IL-18 and CCL19

[0241] A DNA fragment containing the base sequences encoding the first F2A, mouse IL-18, the second F2A, and mouse CCL19 was artificially synthesized. The entire base sequence of this DNA fragment #7 is shown in SEQ ID NO: 12 ( Figure 13 ). In SEQ ID NO: 12, the bases at positions 7 to 81 encode the first F2A sequence, the bases at positions 82 to 657 encode the mouse IL-18 sequence, the bases at positions 658 to 732 encode the second F2A sequence, and the bases at positions 736 to 1059 encode the mouse CCL19 sequence. The amino acid sequence of the entire fusion protein expressed by the bases at positions 7 to 1059 of DNA fragment #7 and self-cleaved at two F2A sites is shown in SEQ ID NO: 13.

[0242] [B: CAR-T cell manufacturing step] Production of IL18_CCL19_anti-human CD20 CAR-T cells

[0243] As a retroviral expression vector, the IL18_CCL19_anti-human CD20 CAR expression vector obtained by the above vector production steps was used to replace IL15 LSP _CCL19_anti-human CD20 CAR expression vector, and the rest was carried out in the same manner as the CAR-T cell production steps in Example 1-1. A preparation containing retrovirus was obtained in Step 1, and mouse T cells expressing the genes of anti-human CD20 CAR, IL-18, and CCL19 contained in the retrovirus were obtained in Step 2 (IL18_CCL19_anti-human CD20 CAR-T cells, referred to as "IL18×CCL19 CAR-T cells" in this specification).

[0244] [Example 3] IL-21×CCL19 CAR-T cells

[0245] [A: Vector production step] Production of IL21_CCL19_anti-human CD20 CAR expression vector

[0246] Using the same DNA fragment 1 and DNA fragment 2 as in Step 1 of Example 1-1, and DNA fragment #8 as described below instead of DNA fragment #3, after constructing the construct in the same manner as in Step 2 of Example 1-1, a pMSGV retroviral expression vector containing anti-human CD20 CAR_F2A_IL-21_F2A_CCL19 (IL21_CCL19_anti-human CD20 CAR expression vector) was obtained.

[0247] DNA fragment #8: DNA fragment for IL-21 and CCL19

[0248] A DNA fragment containing the base sequences encoding the first F2A, mouse IL-21, the second F2A, and mouse CCL19 was artificially synthesized. The base sequence of the entire DNA fragment #8 is shown in SEQ ID NO: 14( Figure 14 ). In SEQ ID NO: 14, the bases at positions 7 to 81 are the sequence encoding the first F2A, the bases at positions 82 to 567 are the sequence encoding mouse IL-21, the bases at positions 568 to 642 are the sequence encoding the second F2A, and the bases at positions 646 to 969 are the sequence encoding mouse CCL19. The amino acid sequence of the entire fusion protein expressed by the bases at positions 7 to 969 of DNA fragment #8 and self-cleaved at two F2As is shown in SEQ ID NO: 15.

[0249] [B: CAR-T cell production step] Production of IL21_CCL19_anti-human CD20 CAR-T cells

[0250] As a retroviral expression vector, the IL21_CCL19_anti-human CD20 CAR expression vector obtained through the above vector production steps was used to replace IL15 LSP _CCL19_anti-human CD20 CAR expression vector, and except for this, it was carried out in the same manner as the CAR-T cell production steps of Example 1-1. A preparation containing retrovirus was obtained in Step 1, and mouse T cells expressing the genes of anti-human CD20 CAR, IL-21, and CCL19 contained in the retrovirus were obtained in Step 2 (IL21_CCL19_anti-human CD20 CAR-T cells, referred to as "IL21×CCL19 CAR-T cells" in this specification.).

[0251] [Example 4] sc IL27×CCL19 CAR-T cells

[0252] [A: Vector production step] sc Production of IL27_CCL19_anti-human CD20 CAR expression vector

[0253] Using the same DNA fragment 1 and DNA fragment 2 as in Step 1 of Example 1-1, and DNA fragment #9 as described below instead of DNA fragment #3, after constructing the construct in the same manner as in Step 2 of Example 1-1, a pMSGV retroviral expression vector containing anti-human CD20 CAR_F2A_scIL-27_F2A_CCL19 was obtained ( sc IL27_CCL19_anti-human CD20 CAR expression vector).

[0254] DNA fragment #9: sc DNA fragment for IL27 and CCL19

[0255] A DNA fragment containing the base sequences encoding the first F2A, mouse IL-27 construct "scIL27" (a fusion peptide composed of p28, linker, and EBI3), the second F2A, and CCL19 was artificially synthesized. The base sequence of the entire DNA fragment #9 is shown in SEQ ID NO: 16 ( Figure 15 ). In SEQ ID NO: 16, the bases at positions 7 to 81 are the sequence encoding the first F2A, and the bases at positions 82 to 1437 are the encoding scThe sequence of IL27 (wherein, the bases at positions 82 to 765 encode murine EBI3, the bases at positions 766 to 819 are the linker, and the bases at positions 820 to 1437 encode murine p28), the bases at positions 1438 to 1512 are the sequence encoding the second F2A, and the bases at positions 1516 to 1839 are the sequence encoding murine CCL19. The fusion protein of IL27 expressed from the bases at positions 7 to 1839 of DNA fragment #9 and self-cleaved at two F2A sites, which contains sc The amino acid sequence of the entire IL27 fusion protein is shown in SEQ ID NO: 17.

[0256] [B: CAR-T cell manufacturing steps] sc Manufacturing steps of IL27_CCL19_anti-human CD20 CAR-T cells

[0257] As the retroviral expression vector, use the sc IL27_CCL19_anti-human CD20 CAR expression vector obtained through the above vector production steps to replace the IL15 LSP _CCL19_anti-human CD20 CAR expression vector. Except for this, carry out the same as the CAR-T cell manufacturing steps in Example 1-1. In step 1, obtain a preparation containing retrovirus, and in step 2, obtain murine T cells expressing the genes of anti-human CD20 CAR, scIL27, and CCL19 contained in this retrovirus ( sc IL27_CCL19_anti-human CD20 CAR-T cells, which are referred to as " sc IL27×CCL19 CAR-T cells" in this specification.).

[0258] [Comparative Example 1] Transduced (-) T cells

[0259] Do not produce the retroviral vector and do not transfect T cells with it. In step 2 of step B in Example 1-1, add an equal amount of DMEM medium for culturing GP2-293 cells to replace the IL15 LSP _CCL19_anti-human CD20 CAR expression vector preparation. Except for this, manufacture only activated murine T cells through the same steps (referred to as "transduced (-) T cells" in this specification.).

[0260] [Comparative Example 2] conv.CAR-T cells

[0261] [A: Vector production step] Production of anti-human CD20 CAR expression vector

[0262] Proceed to the midway of Step 2 of Step A in Example 1-1 by the same procedure. Using DNA fragment 1, DNA fragment 2, and pMSGV retroviral vector, prepare a pMSGV retroviral expression vector containing anti-human CD20 CAR_MCS. Use this as a control retroviral expression vector (anti-human CD20 CAR expression vector) without co-expressing cytokines and chemokines.

[0263] [B: CAR-T cell manufacturing step] Manufacture of anti-human CD20 CAR-T cells

[0264] As the retroviral expression vector, use the anti-human CD20 CAR expression vector instead of IL15 LSP _CCL19_ anti-human CD20 CAR expression vector, and perform the same as the CAR-T cell manufacturing step in Example 1-1 except for this. Obtain a preparation containing retrovirus in Step 1, and obtain mouse T cells (anti-human CD20 CAR-T cells, referred to as "conv.CAR-T cells" in this specification) that express only the anti-human CD20 CAR contained in this retrovirus (without expressing cytokines and chemokines) in Step 2.

[0265] [Test Example 1] Confirmation of the expression levels of the introduced CAR, cytokines, and chemokines

[0266] [A: CAR expression assay by flow cytometry]

[0267] Using the T cells manufactured in the above Examples and Comparative Examples, analyze the expression level of anti-human CD20 CAR on the cell surface by flow cytometry as described below.

[0268] Note that in flow cytometry, use the flow cytometer "BD FACSCanto TM II" (BD Biosciences), and in data analysis, use FlowJo software (BD Biosciences).

[0269] Treat each T cell with biotinylated protein L (specifically binds to the κ light chain of anti-human CD20 scFv) and streptavidin conjugated with BrilliantViolet TM 421 (BV421) to detect the expression of CAR. At the same time, use an anti-mouse CD8 monoclonal antibody conjugated with allophycocyanin (APC) (BioLegend) to measure the CD8 positive rate in each T cell population. It is generally considered that CD8-positive T cells have direct cytotoxic activity. Therefore, the presence (positive rate) of CD8-positive T cells can be shown as evidence of potency.

[0270] The results are shown in Figure 1 . For the CAR-T cells of all the examples (and Comparative Example 2), it was confirmed that the anti-human CD20 CAR was expressed (positive) in more than 60% of the cells in the cell population.

[0271] [B: Measurement of the secretion amounts of cytokines and chemokines]

[0272] The culture supernatants of the respective T cells 42 hours after transduction were recovered, and the concentrations of IL-15, IL-18, IL-21, IL-27, and CCL19 were measured using commercially available ELISA kits (only IL-18 was from MBL, and the rest were from R&D systems).

[0273] The results are shown in Figure 2 . Regarding IL-15 ( Figure 2 A), IL-15 with a concentration of 250 pg / mL was detected from the culture supernatant of the CAR-T cells (15LSPx19 CAR-T) of Example 1-1. On the other hand, the concentrations of IL-15 in the culture supernatants of the CAR-T cells of Example 1-2 (s15RAx19 CAR-T), Example 1-3 (mb15RAx19 CAR-T), and Example 1-4 (sushi15x19 CAR-T) were at the same level as those of the activated T cells not transduced (transduction (-): Comparative Example 1) and the T cells expressing anti-human CD20 CAR as a control (conv. CAR-T: Comparative Example 2), and the secretion of IL-15 was not confirmed by the above ELISA kit used in Test Example 1. However, in Test Example 2 described later, Example 1-2, Example 1-3, and Example 1-4 showed a significant cell proliferation effect, and thus it can be said that these examples also express and secrete IL-15 in the same manner as Example 1-1.

[0274] Regarding IL-18 ( Figure 2 B), IL-18 with a concentration of 150 pg / mL or more was detected from the culture supernatant of the CAR-T cells (18x19 CAR-T) of Example 2. On the other hand, the secretion amount in the culture supernatant of the T cells expressing anti-human CD20 CAR as a control (conv. CAR-T: Comparative Example 2) was a trace amount at the same level as that of the activated T cells not transduced (transduction (-): Comparative Example 1).

[0275] Regarding IL-21 ( Figure 2C), IL-18 at a concentration of 400 pg / mL or more was detected in the culture supernatant of the CAR-T cells (21x19 CAR-T) of Example 3. On the other hand, the secretion levels of the T cells expressing anti-human CD20 CAR (conv. CAR-T: Comparative Example 2) as a control and the activated T cells not transduced (transduction (-): Comparative Example 1) were both below the detection limit (NotDetected).

[0276] Regarding IL-27( Figure 2 D), p28, which is a subunit of IL-27, at a concentration of 250 pg / mL or more was detected in the culture supernatant of the CAR-T cells (sc27x19 CAR-T) of Example 4. On the other hand, the secretion levels of the anti-human CD20 CAR-T cells (conv. CAR-T: Comparative Example 2) as a control and the activated T cells not transduced (transduction (-): Comparative Example 1) were at the same level (the concentration in the culture supernatant was about 50 pg / mL).

[0277] Regarding CCL19( Figure 2 E), the concentrations in the culture supernatants of the anti-human CD20 CAR-T cells (conv. CAR-T: Comparative Example 2) and the activated T cells not transduced (transduction (-): Comparative Example 1) as controls were below the detection limit, whereas the concentrations in the culture supernatants of the CAR-T cells of each example were 150 to 500 pg / mL.

[0278] [Test Example 2] Evaluation of the in vitro proliferative ability of CAR-T cells

[0279] Using the T cells prepared in the above Examples and Comparative Examples, the cell numbers and proliferative abilities of the CAR-T cells were measured as described below, and thus whether the cytokines (IL-15, IL-18, IL-21, and IL-27) produced by each T cell exerted biological functions and showed an immune induction effect was investigated.

[0280] Each T cell was stained with CytoTell TM UltraGreen (AAT Bioquest) and co-cultured in the same well with P815 mastocytoma (hCD20 / P815) recombinantly expressing human CD20 after treatment with mitomycin C for stimulation. Cells were collected 3 days, 5 days, or 7 days after the start of stimulation, and flow cytometry analysis was performed. Staining was performed with an anti-Thy1.2 monoclonal antibody conjugated with APC (eBioscience), and Thy1.2-positive cells in each T cell (lymphocyte) population were regarded as viable T cells, and the cell numbers were measured.

[0281] The results are shown in Figure 3 . The transduced (-) T cells (Comparative Example 1) did not undergo activation stimulation even when co-cultured with hCD20 / P815 cells, or the number of viable cells was 1 / 10 or less on the 3rd day of culture. On the other hand, IL15 after co-culture with hCD20 / P815 cells LSP ×CCL19 CAR-T cells (Example 1-1), s IL15 RA ×CCL19 CAR-T cells (Example 1-2), mb IL15 RA ×CCL19 CAR-T cells (Example 1-3), sushi IL15 × CCL19 CAR-T cells (Example 1-4), IL-18 × CCL19 CAR-T cells (Example 2), IL-21 × CCL19 CAR-T cells (Example 3), and scIL27 × CCL19 CAR-T cells (Example 4) all showed proliferation and maintenance of viable cell numbers equal to or higher than those of the control, namely conv.CAR-T cells (Comparative Example 2), on the 3rd day of culture. Further, when the culture was continued until the 7th day, especially s IL15 RA ×CCL19 CAR-T cells (Example 1-2), mb IL15 RA ×CCL19 CAR-T cells (Example 1-3), sushi IL15 × CCL19 CAR-T cells (Example 1-4) showed significant cell proliferation. IL15 LSP ×CCL19 CAR-T cells (Example 1-1), IL-18 × CCL19 CAR-T cells (Example 2), IL-21 × CCL19 CAR-T cells (Example 3), and scIL27 × CCL19 CAR-T cells (Example 4) maintained the same cell numbers as the control, namely conv.CAR-T cells (Comparative Example 2), from the 3rd day to the 7th day of culture.

[0282] Histogram analysis of the staining intensity using CytoTell reagent in the Thy1.2-positive cell population was performed together. The results for the cell population 5 days after the start of stimulation are shown in Figure 4 . IL15 LSP ×CCL19 CAR-T cells (Example 1-1), s IL15 RA ×CCL19 CAR-T cells (Example 1-2), mb IL15 RA ×CCL19 CAR-T cells (Example 1-3),sushi Compared with the control, namely conv.CAR-T cells (Comparative Example 2), the proportion of the cell population of the second generation (after 1 division) of IL15×CCL19 CAR-T cells (Examples 1-4), IL-18×CCL19 CAR-T cells (Example 2), and IL-21×CCL19 CAR-T cells (Example 3) was significantly reduced, and the proportion of the cell population after the fifth generation (after more than 4 divisions) increased.

[0283] From Figure 3 and Figure 4 the results, it can be seen that in terms of enhancing the survival and proliferation of CAR-T cells and exerting biological functions, the combination of the IL-15 / IL-15Rα fusion protein and CCL-19 produced in s IL15 RA ×CCL19 CAR-T cells (Examples 1-2), mb IL15 RA ×CCL19 CAR-T cells (Examples 1-3), and sushi IL15×CCL19 CAR-T cells (Examples 1-4) is particularly preferred.

[0284] [Test Example 3] Evaluation of the Tumor Cell Cytotoxic Activity of CAR-T Cells

[0285] The target antigen-specific tumor cell cytotoxic activity was studied using the T cells prepared in the above Examples and Comparative Examples.

[0286] [A: Determination of Tumor Cell Cytotoxic Activity by Flow Cytometry]

[0287] The P815 mastocytoma (hCD20 / P815) recombinantly expressed in the manner of expressing human CD20 was used as the target tumor cell, and the P815 mastocytoma (P815) not subjected to such gene recombination was used as the control tumor cell.

[0288] After collecting the above target tumor cells or control tumor cells and seeding them in a culture plate, the CAR-T cells of each Example as effector T cells (IL15 LSP ×CCL19 CAR-T cells (Examples 1-1), s IL15 RA ×CCL19 CAR-T cells (Examples 1-2), mb IL15 RA ×CCL19 CAR-T cells (Examples 1-3), sushiIL15×CCL19 CAR-T cells (Examples 1-4), IL-18×CCL19 CAR-T cells (Example 2), IL-21×CCL19 CAR-T cells (Example 3), and scIL27×CCL19 CAR-T cells (Example 4)) or the CAR-T cells of Comparative Example 2 (conv.CAR-T cells) were co-cultured for 72 hours. Effector T cells were added in such a way that the number of CAR-positive cells reached 1 / 3 of the amount of target tumor cells (E:T ratio = 1:3). Untransduced T cells were added to other T cell populations according to the seeding number of the T cells with the lowest CAR expression rate, so that the total number of seeded T cells was the same in each group. After 72 hours of co-culture, all cells were recovered and subjected to flow cytometry analysis and determination of the viable cell count. Staining was performed with anti-Thy1.2 monoclonal antibody conjugated with APC (eBioscience) and Zombie Green Fixable Viability Kit (BioLegend), and the proportions of T cells and tumor cells in the viable cell population were calculated from the Thy1.2 positive rate. The viable cell count was measured using NucleoCounter NC-200 (chemometec).

[0289] The results are shown in Figure 5 . As Figure 5 shown in A, the number of hCD20 / P815 tumor cells after 72 hours of co-culture was calculated from the Thy1.2 negative rate in viable cells. The results showed that the number of hCD20 / P815 tumor cells after co-culture with IL15 LSP ×CCL19 CAR-T cells (Example 1-1), s IL15 RA ×CCL19 CAR-T cells (Example 1-2), mb IL15 RA ×CCL19 CAR-T cells (Example 1-3), sushi IL15×CCL19 CAR-T cells (Example 1-4), IL-18×CCL19 CAR-T cells (Example 2), IL-21×CCL19 CAR-T cells (Example 3), or scIL27×CCL19 CAR-T cells (Example 4) were significantly reduced compared to transduced (-) T cells (Comparative Example 1), and further reduced compared to conv.CAR-T cells (Comparative Example 2), with the surviving tumor cells being less than 1%. On the other hand, as Figure 5 shown in B, in the case of any cell population, no reduction in tumor cells was observed in P815 cells that do not express human CD20, and no non-specific cytotoxicity against tumor cells that do not express human CD20 was confirmed.

[0290] [B: IFNγ production assay using ELISA analysis]

[0291] The interferon γ (IFNγ) produced by CAR-T cells was measured using a commercially available ELISA assay kit (R&D Company) as an indicator of the activation of target antigen-specific cytotoxic ability. The CAR-T cells (IL15 LSP ×CCL19 CAR-T cells (Example 1-1), s IL15 RA ×CCL19 CAR-T cells (Example 1-2), mb IL15 RA ×CCL19 CAR-T cells (Example 1-3), sushi IL15×CCL19 CAR-T cells (Example 1-4), IL-18×CCL19 CAR-T cells (Example 2), IL-21×CCL19 CAR-T cells (Example 3), and scIL27×CCL19 CAR-T cells (Example 4)) or the CAR-T cells of Comparative Example 2 (conv.CAR-T cells) after 4 days of co-culture. The amount of IFNγ in the culture supernatant was measured.

[0292] The results are shown in Figure 6 . As Figure 6 shown in A, in the culture supernatant of any one of the IL15 LSP ×CCL19 CAR-T cells (Example 1-1), s IL15 RA ×CCL19 CAR-T cells (Example 1-2), mb IL15 RA ×CCL19 CAR-T cells (Example 1-3), sushi IL15×CCL19 CAR-T cells (Example 1-4), IL-18×CCL19 CAR-T cells (Example 2), IL-21×CCL19 CAR-T cells (Example 3), or scIL27×CCL19 CAR-T cells (Example 4) after co-culture with hCD20-P815 cells, IFNγ of about 15 ng / mL or more higher than that of conv.CAR-T cells (Comparative Example 2) was detected. The amount of IFNγ production in the group after co-culture with transduced (-) T cells (Comparative Example 1) was below the detection limit. On the other hand, as Figure 6As shown in B, the concentration of IFNγ in the culture supernatant of each CAR-expressing cell after co-culture with P815 cells was 10 pg / mL or less.

[0293] From the results of the above Test Example 3 ( Figure 5 and Figure 6 ), it was confirmed that the T cells of the above Examples and Comparative Example 2 all showed cytotoxic activity specific to the target antigen, that is, against cells expressing human CD20.

[0294] [Test Example 4] Therapeutic effect in a mouse tumor model

[0295] Using the T cells prepared in Examples 1-3, Examples 1-4, and Comparative Example 2 above, the therapeutic effect on a mouse melanoma tumor transplantation model or a mouse colorectal cancer tumor model was studied using tumor-bearing mice as follows.

[0296] (Therapeutic effect in a mouse melanoma tumor transplantation model)

[0297] 5×10 5 mouse melanoma B16F10 (B16F10-hCD20) recombinantly expressing human CD20 were inoculated subcutaneously into C57BL / 6N mice. On the 7th day after inoculation, cyclophosphamide (CPA, 50 mg / kg) as an anticancer agent was administered intraperitoneally, and on the 10th day, 1×10 6 CAR-T cells of Example 1-3 ( mb IL15 RA xCCL19 CAR-T) or CAR-T cells of Example 1-4 ( sushi IL15xCCL19 CAR-T) or Comparative Example 2 (Conv.CAR-T) were administered intravenously. As a control group, a CAR-T untreated group that received only CPA administration and a non-treated group that received no treatment after inoculating the B16F10-hCD20 mouse melanoma were set. The tumor volume of the mice was measured twice a week.

[0298] The results of the tumor volume of the mouse melanoma tumor transplantation model are shown in Figure 16 . The horizontal axis is the number of days after inoculation with the day of subcutaneous inoculation of B16F10-hCD20 into the mouse set as day 0, and the vertical axis is the tumor volume (long diameter of the tumor × (short diameter of the tumor) 2 / 2 (mm 3 )). The standard deviation was calculated in each experimental group. "no treatment (no treatment)" is the group without treatment, "CPA" is the group that received only CPA administration, "CPA+Conv." is the group that received Comparative Example 2 (Conv.CAR-T) after administering CPA, "CPA+ mb IL15RA "x19" is the group of CAR-T cells ( mb IL15 RA xCCL19 CAR-T) of Examples 1-3 after administration of CPA, "CPA + sushi IL15 RA x19" is the group of CAR-T cells ( sushi IL15xCCL19 CAR-T) of Examples 1-4 after administration of CPA.

[0299] As Figure 16 shown, compared with when administering Comparative Example 2 (Conv.CAR-T), the non-treatment group (no treatment), and when administering only CPA, in the case of administering the CAR-T cells ( mb IL15 RA xCCL19 CAR-T) of Examples 1-3 and the CAR-T cells ( sushi IL15 RA xCCL19 CAR-T) of Examples 1-4, a reduction effect of tumor volume was confirmed. Therefore, it can be known that the CAR-T cells ( mb IL15 RA xCCL19 CAR-T) of Examples 1-3 and the CAR-T cells ( sushi IL15xCCL19 CAR-T) of Examples 1-4 have excellent anti-tumor activity in the mouse melanoma tumor model.

[0300] (Therapeutic effect in mouse colorectal cancer tumor transplantation model)

[0301] 5 × 10 5 of mouse colorectal cancer MC38 (MC38-hCD20) recombinantly expressed in a manner expressing human CD20 were inoculated subcutaneously into C57BL / 6N mice. On the 7th day after inoculation, cyclophosphamide (CPA, 50 mg / kg) as an anticancer agent was administered intraperitoneally, and on the 10th day, 1 × 10 6 of the CAR-T cells ( mb IL15 RA xCCL19 CAR-T) of Examples 1-3 or the CAR-T cells ( sushi IL15xCCL19 CAR-T) of Examples 1-4 or Comparative Example 2 (Conv.CAR-T) were administered intravenously. As a control group, a CAR-T untreated group that only received CPA administration and a non-treatment group that did not receive any treatment after inoculating MC38-hCD20 mouse colorectal cancer were set. The tumor volume of the mice was measured twice a week.

[0302] The results of the tumor volume of the mouse colorectal cancer tumor transplantation model are shown inFigure 17 。The horizontal axis represents the number of days after inoculation, with the day of subcutaneous inoculation of MC38-hCD20 into the mouse set as day 0, and the vertical axis represents the tumor volume (long diameter of the tumor × (short diameter of the tumor) 2 / 2 (mm 3 )). The standard deviation was calculated for each experimental group. "no treatment" is the group without treatment, "CPA" is the group treated only with CPA, "CPA+Conv." is the group treated with CPA followed by the administration of Comparative Example 2 (Conv.CAR-T), "CPA+ mb IL15 RA x19" is the group treated with CPA followed by the administration of the CAR-T cells of Examples 1-3( mb IL15 RA xCCL19 CAR-T), "CPA+ sushi IL15 RA x19" is the group treated with CPA followed by the administration of the CAR-T cells of Examples 1-4( sushi IL15xCCL19 CAR-T).

[0303] As Figure 17 shown, compared with the administration of Comparative Example 2 (Conv.CAR-T), the non-treatment group (no treatment), and the group treated only with CPA, a tumor volume reduction effect was confirmed in the administration of the CAR-T cells of Examples 1-3( mb IL15 RA xCCL19 CAR-T) and the CAR-T cells of Examples 1-4( sushi IL15xCCL19 CAR-T). Therefore, it can be seen that the CAR-T cells of Examples 1-3( mb IL15 RA xCCL19 CAR-T) and the CAR-T cells of Examples 1-4( sushi IL15xCCL19 CAR-T) also have excellent anti-tumor activity in a mouse colorectal cancer model.

[0304] In the above-described examples and test examples, T cells derived from mice, cytokines containing natural amino acid sequences of mice (such as fusion proteins in which IL-15 is linked to IL-15Rα, etc.), and chemokines (CCL19) were used, and in vivo tests on mice were also conducted. However, those skilled in the art can understand that in embodiments related to mammals other than mice, preferably humans, that is, when using T cells derived from humans, cytokines containing natural amino acid sequences of humans (such as fusion proteins in which IL-15 is linked to IL-15Rα, etc.), and chemokines (CCL19), and when conducting in vivo tests on humans, the present invention can also be implemented and the effects of the present invention can be exerted.

[0305] For example, the fusion protein containing IL15 implemented (produced and used) using the natural amino acid sequence of the mouse shown in SEQ ID NO: 5 LSP can be implemented using the amino acid sequences of positions 1 to 29 (signal peptide part) and positions 49 to 162 (IL-15 part) of the human natural amino acid sequence of IL-15 shown in SEQ ID NO: 18, and can also be implemented using a nucleic acid having a base sequence encoding such an amino acid sequence.

[0306] The fusion protein containing s IL15 RA implemented using the natural amino acid sequence of the mouse shown in SEQ ID NO: 7 can be implemented using the amino acid sequences of positions 49 to 162 (IL-15 part) of the human natural amino acid sequence of IL-15 shown in SEQ ID NO: 18, and the amino acid sequences of positions 31 to 205 (extracellular domain of IL-15Rα) of the human natural amino acid sequence of IL-15Rα shown in SEQ ID NO: 19, and can also be implemented using a nucleic acid having a base sequence encoding such an amino acid sequence.

[0307] The fusion protein containing mb IL15 RA implemented using the natural amino acid sequence of the mouse shown in SEQ ID NO: 9 can be implemented using the amino acid sequences of positions 49 to 162 (IL-15 part) of the human natural amino acid sequence of IL-15 shown in SEQ ID NO: 18, and the amino acid sequences of positions 1 to 267 (full-length IL-15Rα) of the human natural amino acid sequence of IL-15Rα shown in SEQ ID NO: 19, and can also be implemented using a nucleic acid having a base sequence encoding such an amino acid sequence.

[0308] The fusion protein containing sushiThe fusion protein of IL15 can be implemented using the amino acid sequence from position 49 to position 162 (IL-15 part) in the human natural amino acid sequence of IL-15 shown in SEQ ID NO: 18, and the amino acid sequence from position 31 to position 95 (sushi domain) in the human natural amino acid sequence of IL-15Rα shown in SEQ ID NO: 19. Additionally, it can be implemented using a nucleic acid having a base sequence encoding such an amino acid sequence.

[0309] Industrial Applicability

[0310] The CAR-T cells of the present invention with excellent persistence and proliferation are suitable for manufacturing drugs for treating cancers and the like. Additionally, the expression vector of the present invention is suitable for manufacturing such CAR-T cells.

[0311] Sequence Listing Free Text

[0312] SEQ ID NO: 1: Base sequence of the DNA fragment (DNA fragment #1) containing the anti-human CD20 CAR

[0313] SEQ ID NO: 2: Amino acid sequence of the fusion protein expressed by the base sequence from position 3 to position 1637 of DNA fragment #1

[0314] SEQ ID NO: 3: Base sequence of the MCS DNA fragment (DNA fragment #2) encoding the stop codon and restriction enzyme sites

[0315] SEQ ID NO: 4: IL15 LSP Base sequence of the _F2A_CCL19 DNA fragment (DNA fragment #3)

[0316] SEQ ID NO: 5: Amino acid sequence of the entire fusion protein expressed by the base sequence from position 7 to position 969 of DNA fragment #3 and self-cleaving at two F2As and containing IL15 LSP

[0317] SEQ ID NO: 6: s IL15 RA Amino acid sequence of the _F2A_CCL19 DNA fragment (DNA fragment #4)

[0318] SEQ ID NO: 7: Amino acid sequence of the entire fusion protein expressed by the base sequence from position 7 to position 1539 of DNA fragment #4 and self-cleaving at two F2As and containing s IL15 RA

[0319] SEQ ID NO: 8: mb IL15 RA Base sequence of the _F2A_CCL19 DNA fragment (DNA fragment #5)

[0320] Sequence No. 9: Expressed by the base sequence from the 7th to the 1713th positions of DNA fragment #5, self-cleaving at two F2A, and containing mb IL15 RA the amino acid sequence of the whole fusion protein

[0321] Sequence No. 10: sushi The base sequence of the IL15_F2A_CCL19 DNA fragment (DNA fragment #6)

[0322] Sequence No. 11: Expressed by the base sequence from the 7th to the 1179th positions of DNA fragment #6, self-cleaving at two F2A, and containing sushi the amino acid sequence of the whole fusion protein of IL15

[0323] Sequence No. 12: The base sequence of the IL-18_F2A_CCL19 DNA fragment (DNA fragment #7)

[0324] Sequence No. 13: The amino acid sequence of the whole fusion protein expressed by the base sequence from the 7th to the 1059th positions of DNA fragment #7 and self-cleaving at two F2A

[0325] Sequence No. 14: The base sequence of the IL-21_F2A_CCL19 DNA fragment (DNA fragment #8)

[0326] Sequence No. 15: The amino acid sequence of the whole fusion protein expressed by the base sequence from the 7th to the 969th positions of DNA fragment #8 and self-cleaving at two F2A

[0327] Sequence No. 16: sc The IL27_F2A_CCL19 DNA fragment (DNA fragment #9)

[0328] Sequence No. 17: Expressed by the base sequence from the 7th to the 1839th positions of DNA fragment #9, self-cleaving at two F2A, and containing sc the amino acid sequence of the whole fusion protein of IL27

[0329] Sequence No. 18: The amino acid sequence of the natural human IL-15 (full length including the signal peptide and propeptide part) registered as UniProtKB-P40933

[0330] Sequence No. 19: The amino acid sequence of the natural human IL-15Rα (full length including the signal peptide, sushi domain, extracellular domain, etc.) registered as UniProtKB-Q13261

Claims

1. A T cell that expresses: (1) a chimeric antigen receptor (CAR); (2) at least one selected from the group consisting of interleukin-18 (IL-18), interleukin-21 (IL-21), and interleukin-27 (IL-27); and (3) CC chemokine ligand 19 (CCL19).

2. A pharmaceutical composition comprising the T cell according to claim 1.

3. The pharmaceutical composition according to claim 2, which is a therapeutic agent for cancer.

4. The pharmaceutical composition according to claim 3, wherein, the cancer is melanoma, Merkel cell carcinoma, colorectal cancer, renal cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, osteosarcoma, multiple myeloma, lymphoma, or leukemia.

5. An expression vector comprising: (1) a nucleic acid encoding a CAR; (2) a nucleic acid encoding at least one selected from the group consisting of IL-18, IL-21, and IL-27; and (3) a nucleic acid encoding CCL19.

6. A method for manufacturing a CAR-T cell, which includes the step of introducing the expression vector according to claim 5 into a T cell.

7. Use of the T cell according to claim 1 in the preparation of a pharmaceutical composition for treating cancer.

8. The use according to claim 7, wherein, the cancer is melanoma, Merkel cell carcinoma, colorectal cancer, renal cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, cholangiocarcinoma, neuroblastoma, osteosarcoma, multiple myeloma, lymphoma, or leukemia.

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