EA045607B1 - CAR-EXPRESSING T-CELLS AND CAR-EXPRESSING VECTOR - Google Patents

Description

Field of technology

The present invention relates to immune cells that express chimeric antigen receptor (hereinafter referred to as CAR) (these cells are hereinafter referred to as CAR-expressing immune cells) and which can be used for cancer immunotherapy, namely, CAR-expressing T cells (hereinafter referred to as CAR -T cells), and to an expression vector to produce 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 predefined cytokines and chemokines, and an expression vector for producing these cells.

Prerequisites for creating an invention

Cancer immunotherapy administered by CAR T cells has been shown to be effective in the treatment of hematopoietic malignancies such as leukemia and lymphoma, however, there are types of cancer and cases of cancer in which therapeutic effect is not observed due to such problems such as low survival efficiency of CAR-T cells in vivo and inhibition of CAR-T cell activity in the microenvironment of cancer tumors through the mechanism of cancer cell escape from immune surveillance, especially in solid cancer tumors. Therefore, the need to obtain CAR-T cells that provide a therapeutic effect and have higher antitumor activity, especially in solid cancer tumors, remains relevant.

Patent Document 1 and Non-Patent Document 1 disclose CAR-T cells that express IL-7 and CCL19 and have higher antitumor activity than conventional CAR-T cells. However, Patent Document 1 and Non-Patent Document 1 do not specifically describe other combinations of cytokines and chemokines.

Non-Patent Document 2 discloses CAR-T cells that have enhanced persistence independent of CAR signaling and express membrane-bound chimeric IL-15 (mbIL-15). However, Non-Patent Document 2 does not specifically describe combinations of mbIL-15 with chemokines such as CCL19.

Non-Patent Document 3 states that the use of a fusion protein containing IL-15 linked to the IL-15Rasushi domain via a flexible linker can impart higher activity in the proliferation of lymphocytes (such as NK cells, NK-T cells and CD8-positive memory cells), in the activation of dendritic cells, etc. compared to the standard combined use of IL-15 domain and IL-15Rasushi. However, Non-Patent Document 3 does not specifically describe combinations of the IL-15 and IL-15 Rasushi domain with chemokines such as CCL19.

Non-Patent Document 4 states that transfection of murine IL-15 and IL-15Ra secretory fusion protein increases the viability and proliferation of CD8-positive T cells. However, Non-Patent Document 4 does not specifically describe combinations of IL-15 with chemokines such as CCL19.

Non-Patent Document 5 describes single chain IL-27 (p28 and EBI3 linked by a flexible linker) and its effect in the treatment of inflammatory bowel disease (IBD). However, Non-Patent Document 5 does not specifically describe CAR-T cells and combinations of single-chain IL-27 with chemokines such as CCL19.

Patent Document 2 discloses T cells expressing recombinant IL-7, recombinant IL-15, or combinations thereof, and states that expression of such cytokines improves the survival of T cells. However, Patent Document 2 does not specifically describe CAR-T cells and combinations of specific cytokines with chemokines such as CCL19.

Patent Document 3 discloses modified natural killer T cells containing an expression construct encoding IL-2, IL-4, IL-7, IL-15, or combinations thereof, and a CAR construct. However, Patent Document 3 does not specifically describe combinations of specific cytokines with chemokines such as CCL19.

Patent Document 4 discloses CAR-T cells expressing membrane-bound cytokines such as IL-7, IL-15 (IL-15/IL-15Ra fusion protein) and IL-21. However, Patent Document 4 does not specifically describe combinations of specific cytokines with chemokines such as CCL19.

Patent Document 5 discloses CAR-T cells expressing IL-15 and targeting CD19. However, Patent Document 5 does not specifically describe combinations of IL-15 with chemokines such as CCL19.

Non-Patent Document 6 discloses CAR-T cells expressing IL-15 and/or IL-21 and targeting GPC3. However, Patent Document 6 does not specifically describe combinations of specific cytokines with chemokines such as CCL19.

List of cited documents

Patent documents.

Patent Document 1: WO 2016/056228.

Patent document 2: WO 2007/037780.

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Patent document 3: WO 2013/040371.

Patent document 4: WO 2014/186469.

Patent Document 5: US 2013/0071414.

Non-patent documents.

Non-Patent Document 1: Adachi et al., Nature Biotechnology, VOL 36, No. 4, 346-353.

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

Non-Patent Document 3: Mortier et al., J Biol Chem. 281(3), 1612-9, 2006.

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

Non-Patent Document 5: Sasaoka et al., Am J Physiol Gastrointest Liver Physiol 300: G568-576.

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.

The essence of the invention

Technical problem

The purpose of the present invention is to obtain immune cells (such as CAR-T cells) having higher antitumor activity than immune cells (such as CAR-T cells) expressing only CAR (not expressing cytokines and/or chemokines).

The solution of the problem

In vivo, there are at least several hundred molecules that can regulate the functions of immune cells such as T cells. The present inventors have discovered that antitumor activity is enhanced by CAR-T cells expressing at least one cytokine selected from the group consisting of interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21) and interleukin-27 (IL-27), which are cytokines, in combination with CC-19 chemokine ligand (CCL19), which is a chemokine, in combination with CAR, compared with cells expressing CAR alone , and in particular, the persistence and proliferation of CAR-T cells is increased, and on this basis the present invention can be implemented.

TL-15, IL-18, IL-27, and CCL19 are cytokines and chemokines that are not expressed by native T cells (that are not supplied with exogenous genes) in vivo. IL-21 is a cytokine expressed by natural T cells in vivo (such as NK T cells and CD4 positive T cells). In the present invention, expression of a predetermined cytokine and chemokine means expression of a predetermined cytokine and chemokine at higher levels than in natural T cells in vivo when genes expressing predetermined exogenous cytokine and chemokine are introduced into the T cells.

In addition, the embodiment of gene transfer and expression of the above-mentioned predefined interleukins and chemokine together with CAR in T cells can be expanded to the embodiment of gene transfer and expression in immune cells other than T cells, such as NK cells, monocytes, macrophages and dendritic cells.

The present invention includes at least the following aspects.

1. T cell expressing:

(1) chimeric antigen receptor (CAR);

(2) at least one cytokine 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) CC chemokine ligand 19 (CCL19).

2. A T cell according to claim 1, wherein (2) is IL-15.

3. A T cell according to claim 2, wherein IL-15 binds to IL-15Ra to form a fusion protein.

4. The T cell according to claim 3, wherein the fusion protein is a fusion protein containing IL15LSP linked to IL-15 (IL15 _lsp ), a fusion protein containing the extracellular domain of IL-15Ra linked to IL-15 ( _s IL15 _ra ), a fusion protein containing IL-15 linked to full-length IL-15Ra ( _mb IL15 _RA ), or a fusion protein containing IL-15Ra including a signal peptide and a sushi domain linked to IL15 ⁽ sushi ^IL-15) .

5. The T cell according to claim 3, wherein the fusion protein is a fusion protein comprising IL-15 linked to full-length IL-15Ra ( _mb IL15 _RA ), or a fusion protein containing IL-15Ra including a signal peptide and a sushi domain , associated with IL-15 _sushi IL-15).

6. A medicinal product containing a T cell according to claim 1.

7. The drug according to claim 6, wherein the drug is a therapeutic agent against cancer.

8. The medicinal product according to claim 7, wherein the cancer is melanoma, Merkel cell cancer, 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 bowel cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia , multiple myeloma, lymphoma or leukemia.

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9. Expression vector containing:

(1) a nucleic acid encoding a CAR;

(2) a nucleic acid encoding at least one cytokine selected from the group consisting of IL-15, IL-18, IL-21 and IL-27; and (3) a nucleic acid encoding CCL19.

10. The expression vector according to claim 9, wherein (2) is IL-15.

11. An expression vector according to claim 10, wherein IL-15 binds to IL-15Ra to form a fusion protein.

12. The expression vector of claim 11, wherein the fusion protein is a fusion protein containing IL-15LSP associated with IL-15 (IL15 _lsp ), a fusion protein containing the extracellular domain of IL15Ra associated with IL-15 ( _S IL15 _RA ) , a fusion protein containing IL-15 linked to full-length IL15Ra ( _mb IL15 _RA ), or a fusion protein containing IL-15Ra including a signal peptide and a sushi domain linked to IL-15 ( _sushi IL-15).

13. The expression vector of claim 11, wherein the fusion protein is a fusion protein comprising IL-15 linked to full-length IL-15Ra ( _mb IL15 _RA ), or a fusion protein containing IL15Ra including a signal peptide and a sushi domain linked to IL-15 ( _sushi IL-15).

14. A method for producing a CAR-T cell, including the step of introducing an expression vector according to claim 9 into the T cell.

15. A method for treating cancer, comprising the step of administering a T cell according to claim 1 to an individual in need of treatment for cancer.

16. The method of treatment according to claim 15, wherein the cancer is melanoma, Merkel cell cancer, 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 bowel cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia , multiple myeloma, lymphoma or leukemia.

17. A T cell according to claim 1 for use as an active component for the treatment of cancer.

18. The T cell according to claim 17, wherein the cancer is melanoma, Merkel cell cancer, 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 bowel cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma or leukemia.

Further, the T cell according to the above claim 1 is also called the T cell according to the invention, the drug according to the above claim 6 is also called the drug according to the invention, the expression vector according to the above claim 9 is also called the expression vector according to the invention, and The method for producing a CAR-T cell according to the above item 14 is also called the production method according to the invention.

One skilled in the art may appropriately convert the embodiments (categories) of the invention in light of the content substantially disclosed herein. For example, the drug according to the invention according to the above claim 6 can be converted into a method for treating cancer, including the step of administering a T cell expressing: (1) CAR; (2) at least one cytokine selected from the group consisting of IL-15, IL-18, IL-21 and IL-27; and (3) CCL19 to an individual in need of cancer treatment; A T cell expressing: (1) CAR; (2) at least one cytokine selected from the group consisting of IL-15, IL-18, IL-21 and IL-27; and (3) CCL19 for use as an active component for the treatment of cancer, and the use of T cells expressing: (1) CAR; (2) at least one cytokine selected from the group consisting of IL-15, IL-18, IL-21 and IL-27; and (3) CCL19 for the production of a drug for the treatment of cancer.

One skilled in the art can appropriately transform a variant of the T cell of the invention, which can be produced so that it expresses a predetermined cytokine and chemokine along with a CAR, and corresponding variants thereof, such as a drug of the invention, an expression vector of the invention, and method of producing according to the invention, in a variant of immune cells other than T cells (such as NK cells, monocytes, macrophages and dendritic cells), predefined cytokines and chemokines, together with CARs and their corresponding variants, such as drug expression vector and method of production, taking into account the contents generally disclosed in this application.

Preferred Effects of the Invention

The persistence and proliferation of T cells according to the invention are enhanced by the T cell expressing a predetermined cytokine (such as IL-15) and a chemokine (CCL19). Therefore, the antitumor activity of CARs may be enhanced (e.g., reducing the number of residual tumor cells, increasing the amount of IFNy produced and

- 3 045607 increased migration and accumulation of host immune cells (such as T cells, dendritic cells, NK cells) at the tumor site). In addition, the therapeutic effect against cancer can be improved by using the T cell-containing drug of the invention. In particular, the T cell according to the invention can participate in the activation of NK cells and, therefore, can be used for the treatment of NK cell-sensitive carcinomas (such as melanoma, Merkel cell cancer, colorectal cancer, kidney cancer, breast, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, non-small cell lung cancer, head and neck cancer, small bowel cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, sarcoma Ewing's, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma or leukemia). In addition, since the enhancement of antitumor activity reduces the number of cells administered, effects such as a reduction in side effects such as CRS (Cytokine Release Syndrome) and a reduction in production costs are expected.

The combination of predefined cytokines and chemokine according to the invention has such an effect and effect that it is possible to obtain various CAR-expressing immune cells with high antitumor activity even when the genes are transferred and expressed together with the CAR gene in immune cells as a whole, that is, not only in T cells, as mentioned above, but also in NK cells, monocytes, macrophages and dendritic cells.

Brief description of drawings

In fig. 1 shows the results of analysis of the expression level of CAR (horizontal axis) and CD8 (vertical axis) in the T cells mentioned below in the example (Experimental Example 1) using flow cytometry. The numerical value (%) in this figure indicates the percentage of the cell population in each compartment relative to the total number of cells. Transduction (-) means non-transduced T cells of Comparative Example 1 (T cells without transduction), transformed CART means transformed CAR-T cells of Comparative Example 2 (CAR-T cells against human CD20), 15 _LSP x19 CAR-T means IL15 _LSP χCCL19-CAR-T cells of Example 1-1 (T cells containing CAR IL15 _LSP _CCL19 against human CD20), _S 15 _RA x19 CAR-T means _S IL15 _RA xCCL19CAR-T cells of Example 1-2 (T -cells containing CAR _S IL15 _{RA_CCL19} against human CD20), _mb 15 _RA x19 CAR-T means _mb IL15 _RA χCCL19-CAR-T cells of example 1-3 (T cells containing CAR _mb IL15 _{RA_CCL19} against human CD20 ), _sushi 15x19 CAR-T means _sushi IL15χCCL19-CAR-T cells of example 1-4 (T cells containing CAR _sushi IL15_CCL19 against human CD20), 18x19 CAR-T means IL-18χCCL19-CAR-T cells of example 2 (T -cells containing CAR IL18_CCL19 against human CD20), 21x19 CAR-T means IL-21χCCL19-CAR-T cells of Example 3 (T cells containing CAR IL21_CCL19 against human CD20), and _sc 27x19 CAR-T means _sc IL27xCCL19CAR- Example 4 T cells (T cells containing CAR _sc IL27_CCL19 against human CD20). This also applies to Figures 2-6 below.

In fig. 2 shows the results of measuring the amount of (A) IL-15, (B) IL-18, (C) IL-21, (D) IL-27 and (E) CCL19 secreted into the T cell culture supernatant in the example (in experimental example 1) using ELISA.

In fig. 3 shows the results of measuring the number of live cells in T cells 3 days, 5 days and 7 days after stimulation of activation by co-culture with P815 mastocytoma expressing human CD20 (hCD20/P815) in the example (in Experimental Example 2).

In fig. 4 shows in the form of a histogram the results of the analysis of the intensity of staining with the CytoTell reagent in T cells in the example (in Experimental Example 2). The peak of the histogram corresponds to the number of generations resulting from cell division, and the numerical value (%) in the ring plot indicates the percentage of gated fractions in the population of Thy1.2-positive T cells (1 represents the first generation without division, 2 represents the second generation after one division, 3 represents the third generation after two divisions, 4 represents the fourth generation after three divisions, >5 represents the fifth and subsequent generations after four or more divisions).

In fig. 5 shows the results of determining the cytotoxic activity of T cells (A) in relation to target tumor cells (hCD20/P815) and (B) to control tumor cells (P815) in the example (in experimental example 3) using flow cytometry.

In fig. Figure 6 shows the results of determining the concentration of IFNy in the T cell culture supernatant during co-cultivation of (A) with target tumor cells (hCD20/P815) and (B) with control tumor cells (P815) in the example (in experimental example 3).

In fig. 7 shows the arrangement of genes and the like contained in the nucleotide sequence of SEQ ID NO: 1 (anti-human CD20 CAR DNA fragment).

In fig. 8 shows the arrangement of genes and the like contained in the nucleotide sequence of SEQ ID NO: 3 (MCS DNA fragment).

In fig. 9 shows the location of genes and the like contained in the nucleotide sequence of SEQ ID NO: 4 (DNA fragment IL15 _L sp_F2A_CCL19).

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In fig. 10 shows the location of genes, etc. contained in the nucleotide sequence

SEQ ID NO: 6 (DNA fragment sIL15ra_F2A_CCL19).

In fig. 11 shows the location of genes, etc. contained in the nucleotide sequence

SEQ ID NO: 8 (DNA fragment _mb IL15 _{RA_F2A_CCL19} ).

In fig. 12 shows the location of genes and the like contained in the nucleotide sequence of SEQ ID NO: 10 (DNA fragment _sushl IL15_F2A_CCL19).

In fig. 13 shows the location of genes and the like contained in the nucleotide sequence of SEQ ID NO: 12 (DNA fragment IL-18_F2A_CCL19).

In fig. 14 shows the arrangement of genes and the like contained in the nucleotide sequence of SEQ ID NO: 14 (DNA fragment IL-21_F2A_CCL19).

In fig. 15 shows the location of genes and the like contained in the nucleotide sequence of SEQ ID NO: 16 (DNA fragment _sc IL27_F2A_CCL19).

In fig. 16 presents the results of determining antitumor activity (changes in tumor volume) for a mouse melanoma transplant model in the example (in experimental example 4).

In fig. 17 shows the results of determining the antitumor activity (change in tumor volume) for a mouse model of colorectal cancer in the example (in Experimental Example 4).

Description of Embodiments of the Invention

As used herein, the term transfection means the introduction of any substance into a cell. A cell includes at least a cytoplasm and a nucleus.

Culture or cultivation means the maintenance, proliferation and/or differentiation of cells outside tissues or outside the body, for example, in a dish, petri dish, flask or culture tank.

Pluripotency means the ability to differentiate into tissues and cells having different morphologies and different functions, and the ability to differentiate into cells of any lineage of the three germ layers.

Pluripotency is different from totipotency, which means the ability to differentiate into any tissue of the body, including blastocysts, that is, pluripotency does not mean the ability to differentiate into blastocysts and therefore does not have the ability to form living organisms.

Multipotency means the ability to differentiate into cells of a limited number of lineages. For example, mesenchymal stem cells, hematopoietic stem cells and neural stem cells are multipotent, but not pluripotent.

Examples of stem cells include pluripotent stem cells.

Pluripotent stem cells that can be used in the present invention mean stem cells that can differentiate into tissues and cells having different morphologies and functions of a living organism, and have the ability to differentiate into cells of any lineage of the three germ layers (endoderm, mesoderm and ectoderm) . Examples thereof include, but are not limited to, embryonic stem cells (ESC), embryonic stem cells derived from cloned embryos obtained by nuclear transplantation; sperm stem cells; embryonic germ cells and induced pluripotent stem cells (hereinafter also called iPSC).

Moreover, multipotent stem cells that can be used in the present invention refer to stem cells that have the ability to differentiate into cells of a limited number of lineages. Examples of multipotent stem cells that can be used in the present invention are somatic stem cells derived from dental pulp stem cells, oral mucosal stem cells, hair follicle stem cells, cultured fibroblasts and bone marrow stem cells. Preferred pluripotent stem cells are ESCs and iPSCs.

Induced pluripotent stem cells (iPSC) refer to cells obtained by introducing a specific factor (nuclear reprogramming factor) into mammalian somatic cells or undifferentiated stem cells and reprogramming the cells. Currently, various types of induced pluripotent stem cells are known. In addition to iPSCs obtained by introducing the four factors Oct3/4, Sox2, Klf4 and c-Myc into mouse fibroblasts as described by Yamanaka et al. (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), iPSCs derived from human cells and obtained by introducing the same four factors into human fibroblasts can also be used (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872.), Nanog-iPS cells obtained by introducing four factors and then sorting the cells by expressing Nanog as an index (Okita, K., Ichisaka, T., and Yamanaka, S. (2007) Nature 448, 313-317), iPS cells obtained by a method excluding c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106); and iPS cells obtained by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May; 8 (5): 409-12, Okita K et al. Stem Cells. 31 (3):

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458-66). In addition, induced pluripotent stem cells obtained by Thomson et al. by introducing the four factors OCT3/4, SOX2, NANOG and LIN28 can also be used (Yu J., Thomson JA. Et al., Science (2007) 318: 1917 -1920.); induced pluripotent stem cells obtained by Daley, et al. (Park IH, Daley GQ. Et al., Nature (2007) 451: 141-146); induced pluripotent stem cells obtained by Sakurada, et al. (JP 2008-307007), etc. In addition to the above cells, any of the induced pluripotent stem cells known to those skilled in the art and described in all published articles (such as Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol 3, can also be used Issue 5, 568-574; Kim JB., Scholer HR., Et al., Nature, (2008) 454, 646-650; and Huangfu D., Melton, DA., Et al., Nature Biotechnology, (2008) 26, No. 7, 795-797) or in patents (such as JP 2008-307007, JP 2008-283972, US 2008-2336610, US 2009-047263, WO 2007069666, WO 2008-118220, WO 2008-12 4133, WO 2008-151058, WO 2009-006930, WO 2009-006997 and WO 2009-007852).

Various iPSC lines created by specialists from the NIH, the Institute of Physical and Chemical Research (RIKEN), Kyoto University, etc. can be used as induced pluripotent stem cells. Examples of human iPSC lines include the HiPSRTKEN-1A line, the HiPS-RTKEN-2A line, the HiPS-RIKEN-12A line, and the Nips-B2 line from RIKEN, as well as the 253G1 line, the 201B7 line, the 409B2 line, the 454E2 line, the 606A1 line, and the 610B1 line. and line 648A1 of Kyoto University. Alternatively, clinically valuable cell lines provided by Kyoto University, Cellular Dynamics International or the like, and cell lines for research and clinical applications obtained using such cell lines can be used.

As embryonic stem cells (ESCs), murine ESCs, such as various murine ESC lines created in a special laboratory of inGenious, RIKEN (Institute for Physical and Chemical Research), etc., as well as human ESCs, such as various human ESC lines created at NIH, Institute of Physical and Chemical Research, Kyoto University and Cellartis. Thus, for example, human ESC lines can be used, such as the START-1 - START-12 lines, the RUES1 line, the RUES2 line and the HUES1 -HUES28 lines from MH, the H1 line and the H9 line from WisCell Research, and the KhES-1 line. KhES-2 line, KhES-3 line, KhES-4 line, KhES-5 line, SSES1 line, SSES2 line and SSES3 line from RIKEN. Alternatively, clinically valuable cell lines and cell lines for research and clinical use derived from such cell lines may be used.

The term contains or containing indicates inclusion of, but is not limited to, the elements listed behind the term. Accordingly, the inclusion of the elements listed behind this term is intended, but the presence of any other elements is not excluded. The term consists of or consisting of means inclusion of and is limited to all elements listed behind the term. Accordingly, the term consists of or consists of indicates that the listed elements are necessary or indispensable, and other elements are essentially absent. The term consists essentially of, or consists essentially of, indicates the inclusion of any element following this term, and other elements are limited to elements that do not affect the activity or effect of the element specified in this application. Accordingly, the term consists essentially of, or consists essentially of, indicates that the listed elements are necessary or indispensable, but other elements may be optionally selected, and these elements may be present or absent in depending on whether these other elements influence the activity or function of the listed elements.

The combination of predefined cytokines and chemokine according to the invention can provide CAR-expressing immune cells with high antitumor activity due to gene transfer to immune cells, and in particular to T cells responsible for cell-mediated acquired immunity, and NK cells, monocytes, macrophages and dendritic cells, which are responsible for natural immunity along with the CAR gene and express these genes. In other words, gene transfer and expression of a combination of a predetermined cytokine and chemokine according to the invention can impart additional specific properties to T cells (CAR-T cells), NK cells (CAR-NK cells), monocytes (CAR-monocytes), macrophages (CARmacrophages) and dendritic cells (CAR-dendritic cells), into which the CAR gene is transferred and expressed.

Immune cells have no specific limitations, provided that they are cells capable of damaging target cells (pathogenic cells), such as cancer cells, through a specific mechanism of action (so-called effector immune cells). Examples of immune cells are T cells, which are responsible for cell-mediated acquired immunity, and NK cells, monocytes, macrophages, and dendritic cells, which are responsible for natural immunity. In one preferred embodiment of the invention, the immune cells may be T cells. In another preferred embodiment of the invention, it

- 6 045607 murine cells can be cells responsible for natural immunity, such as NK cells, macrophages and dendritic cells. It is believed that although T cells are associated with a high risk of developing graft-versus-host disease (GVHD) in allogeneic transplantation (allograft), even in the case of HLA type matching, however, allo-NK cells do not cause GVHD. Accordingly, by obtaining various alloimmune cells with corresponding HLA types, the use of a commercially available drug becomes possible. CAR-NK cells are described, for example, in US patent 2016/0096892, Mol. Ther. 25 (8): 1769-1781 (2017), etc., and dendritic CAR cells, CAR macrophages, etc. described, for example, in WO 2017/019848, eLIFE. 2018 e36688 et al.

Next, the present invention will be described with reference to embodiments thereof in which T cells are used as a representative example of immune cells. However, the present invention is not limited only to embodiments in which the immune cells are T cells, and the present invention also includes embodiments in which the immune cells are NK cells, monocytes, macrophages, dendritic cells or the like. In the following description, CAR-T cells can be appropriately interpreted as CAR-NK cells, CARmonocytes, CAR macrophages, dendritic CAR cells, or the like.

Next, the T cell according to the invention will be described in detail.

The T cell according to the invention expresses: (1) CAR; (2) at least one cytokine selected from the group consisting of IL-15, IL-18, IL-21 and IL-27; and (3) CCL19.

The T cells of the invention may be any T cells, such as αβ T cells, γδ T cells, CD8 ⁺ T cells, CD4 ⁺ T cells, tumor infiltrating T cells, T cells memory cells, naïve T cells and NK-T cells, that is, T cells expressing common or known CARs.

The T cell can be isolated and purified from immune cells infiltrating body fluids such as blood and bone marrow fluid, tissues such as spleen, thymus, lymph nodes, or cancerous tissues such as primary tumors, metastatic tumors, and cancerous ascites . In addition, the T cell can be obtained by culturing induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), other stem cells, progenitor cells, and the like. under appropriate conditions and thereby induce and differentiate such cells into T cells.

T cells can be derived from humans or from non-human mammals (non-human mammals). Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, pigs, cows, horses, sheep and monkeys.

(1) CAR.

The CAR expressed by a T cell according to the invention mainly consists of peptides at the sites of (i) a single chain antibody that recognizes a cancer cell surface antigen, (ii) a transmembrane domain, and (iii) a signaling domain that induces T cell activation, wherein the peptides, if necessary, are linked through spacers, like conventional or known CARs.

The single chain antibody (i) that recognizes a cancer cell surface antigen is typically a single chain variable fragment (scFv) consisting of a light chain variable region and a heavy chain variable region derived from the antigen binding sites of the monoclonal antibody that specifically binds the antigen and a linker peptide that binds such regions.

The cancer cell surface antigen targeted by the CAR may be a biomolecule that is specifically expressed in cancer cells and their progenitor cells; a biomolecule that has been previously expressed by cell malignancy; or a biomolecule that is overexpressed in cancer cells compared to normal cells. Such an antigen is commonly referred to as tumor associated antigen (TAA), and examples may include 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, GFRa4, GPC3, GPR20, GPRC5D, globoH, Gp100, GPR20, GPRC5D, EGFR, EGFR variant, EpCAM, EGP2, EGP40, FAP, FITC, HER2, HER3, HPV E6, HPV E7, hTERT, IgGK chain, IL-11Ra, IL-13Ra2, KIT, Lewis A antigen, Lewis Y antigen, 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, Oacetyl-GD2, h5T4, PaNX3, PDGRFb, PLAC1, polysialic acid, PSCA, PSMA, RAGE1, ROR1, sLe, SSEA-4, TARP, TAG-72 , TEM7R, Tn antigen, TRAIL receptor, TRP2, TSHR, α-fetoprotein, mesothelin, folate receptor a (FRa), folate receptor β (FRe), FBP, UPK2, VEGF-R2 and WT1, but not limited to these examples .

Transmembrane domain (ii) is a polypeptide that serves as a region for anchoring CAR to the cell membrane of T cells. Examples of the transmembrane domain may include BTLA, CD3s, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137,

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CD154, 4-1BB (CD137), CTLA-4, GITR, ICOS, LAG3, OX40, SLAMF4 (CD244, 2B4), or transmembrane domains derived from the α- or β-chain of the T-cell receptor. Alternatively, mutant transmembrane domains having an amino acid sequence that is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more may also be used. , 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the natural amino acid sequence of the above transmembrane domain. In one embodiment of the invention, the CD8 transmembrane domain is preferred as such a transmembrane domain.

Attached to the transmembrane domain may be a hinge region, which is a peptide (oligopeptide or polypeptide) consisting of any amino acid sequence having a length of from 1 to 100 amino acids, and preferably from 10 to 70 amino acids. Examples of the hinge region may include hinge regions derived from CD3, CD8, KIR2DS2 or IgG4, IgD or other immunoglobulins. Alternatively, mutant hinge regions may also be used having an amino acid sequence that is 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 homologous to the natural amino acid sequences of the above hinge regions. In one embodiment of the invention, the CD8 hinge region is preferred as such a hinge region.

The signaling domain (iii), which induces T cell activation, is a polypeptide that serves as a region for signaling within T cells when a single chain antibody recognizes and binds to a cancer cell surface antigen. Examples of a signaling domain may include one or more intracellular domains selected from the group consisting of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activated NK cell receptors, decoy-like receptors, B7-HC , BAFFR, BTLA, BY55 (CD160), CD2, CD3Z, CD4, CD7, CD8a, CD8p, 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), IL2Re, IL2Ry, IL7Ra 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. Alternatively, mutant signaling domains (intracellular domains) having an amino acid sequence that is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, may also be used. 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the natural amino acid sequence of the above-mentioned signaling domain (intracellular domain). In one embodiment of the invention, the signaling domain preferably comprises three intracellular domains: CD28, 41BB and CD3Z.

If the signaling domain contains multiple intracellular domains, the intracellular domains can be linked to each other via linker peptides (oligopeptides or polypeptides) consisting of 2-10 amino acids. Examples of such linker peptides may include a peptide consisting of a continuous glycine-serine sequence.

Between each single chain antibody and transmembrane domain and each transmembrane domain and signal domain, a spacer region may be present, which is a peptide (oligopeptide or polypeptide) consisting of any amino acid sequence having a length of from 1 to 100 amino acids, and preferably from 10 to 50 amino acids . Examples of a spacer region may include a peptide consisting of a contiguous glycineserine sequence.

The amino acid sequence of the above-mentioned CAR expressed by a T cell according to the invention can be suitably determined depending on the use of the T cell, and generally, its functions as an active component of a drug for the treatment of cancer. As the amino acid sequence of the single chain antibody (i) against a cell surface antigen of cancer cells, various known amino acid sequences can be used in the present invention, and information about such amino acid sequences can also be used in the present invention. Alternatively, a new antibody against a cell surface antigen of target cancer cells can be produced by conventional methods, and the information obtained by determining the amino acid sequence of the new antibody (preferably the heavy chain and light chain variable regions, and in particular the CDRs) can be used in the present invention . Additionally, various amino acid sequences derived from humans and non-human mammals are known as amino acid

- 8 045607 sequences of the transmembrane domain (ii) and T cell activation signaling domain (iii), and such amino acid sequences are also registered in databases such as the database

NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/guide/) and

UniProt (Universal Protein Resource; https://www.uniprot.org). Therefore, such information can also be used in the present invention.

(2) Cytokine.

The cytokine expressed by the T cell of the invention is at least one cytokine selected from the group consisting of IL-15, IL-18, IL-21 and IL-27. The T cell of the invention may express any of the above four cytokines or two or more of them.

IL-15 is a cytokine having functions related to T cell survival and activation, differentiation into memory T cells, NK cell activation, and the like. In the present invention, the term IL-15 includes not only the full-length natural IL-15 protein, but also various variants thereof, provided that the functions of IL-15 in relation to the action and effect of the invention are maintained. That is, in the present invention, IL-15 includes not only a full-length protein (polypeptide) consisting of the amino acid sequence of natural IL-15, but also a part thereof (functional partial polypeptide) and variants of all or part of natural IL-15, in which one or more amino acid sequences have been deleted, replaced or added to the native amino acid sequence (preferably within a range having the homology described below), and in addition, full-length native IL-15 or a portion thereof or variants thereof linked to other proteins to form a fusion protein (eg, associated with full-length IL-15Ra or part thereof and forming a fusion protein). In the present invention, the term T cell expresses IL-15 means that the T cell expresses IL-15 in various forms as described above (as a full-length protein or a portion thereof, having a natural amino acid sequence or variants thereof that may be present, or may not be present, in the form of hybrid proteins), provided that the action and effect according to the invention are maintained.

Examples of IL-15 include the following four embodiments of the invention. All of them are known (see above, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4 and Patent Document 4). However, the IL-15 that can be used in the present invention is not limited to these specific examples, and various IL-15s modified according to the presence or absence and types of signal peptides and linkers in the sequences can also be used; the order of the elements and other aspects (hereinafter these IL-15 will also be referred to as modified IL-15).

First variant of IL-15: IL-15LSP/IL-15 (hereinafter also referred to as IL15 _lsp ).

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 of the invention. Expression of IL-15 according to the first embodiment of the invention enhances the antitumor activity of CAR-T cells. The term IL-15LSP also includes not only the entire polypeptide consisting of the amino acid sequence of natural IL-15LSP, but also a part thereof (functional partial polypeptide) and variants of full-length or parts of natural IL-15LSP in which one or more amino acid sequences have been deleted, replaced or added to a naturally occurring amino acid sequence (preferably within a range having the homology described below). As an example of a modified type of the first embodiment, there may be mentioned IL-15LSP, which has been replaced by IL-2SP (IL-2 signal peptide, which may be a full-length natural protein or a part thereof or variants thereof, for example IL15LSP).

Second variant of IL-15: extracellular domain IL-15/IL-15Ra (hereinafter also called SIL15RA).

The term extracellular domain of IL-15Ra means the portion of IL-15Ra (interleukin 15 receptor a chain) excluding the transmembrane domain and the intracellular domain. The extracellular domain of IL15Ra includes a sushi domain (a motif common to various binding proteins). The term extracellular domain of IL-15Ra also includes not only the full-length polypeptide consisting of the amino acid sequence of the natural extracellular domain of IL-15Ra, but also a part thereof (a functional partial polypeptide) and variants of the full-length natural IL-15Ra or a part thereof in which one or more amino acids sequences have been deleted, replaced or added to the natural amino acid sequence (preferably within the range having the homology described below). In a second embodiment, the extracellular domain of IL-15Ra is typically linked to the N-terminal side of IL-15 via a linker (eg, a 26 amino acid glycine-serine linker). In the second option, IL-2SP (IL-2 signal peptide, which can be the full-length natural protein or part or variants thereof) typically binds to the N-terminal side of IL-15 instead of IL-15LSP. The second option is of the secretory type and is an agonist that binds tightly to IL-15RP (the β chain of the interleukin 15 receptor, which is common to the interleukin 2 receptor) and yc (the γ chain, which is common to the receptor). interleukin 2, 4, 7, 9, 15, 21 or the like).

Third variant of IL-15: IL-15/full-length IL-15Ra (hereinafter also called mb ^IL15 RA ⁾ .

Full-length IL-15Ra includes an extracellular domain, a transmembrane domain, and an intracellular domain of IL-15Ra, and typically binds to the C-terminal side of IL-15 via a linker (eg, a 20 amino acid glycine-serine linker) in a third mode. The term full-length IL-15Ra also includes not only the entire protein (polypeptide) consisting of the amino acid sequence of full-length natural IL-15Ra, but also variants of full-length natural IL-15Ra in which one or more amino acid sequences have been deleted, replaced or added to the natural amino acid sequence (preferably within a range having the homology described below). In addition, IL-2SP (which may be a full-length natural protein or a portion thereof or variants thereof) typically binds to the N-terminal side of IL-15 instead of IL-15LSP also in the third embodiment of the invention. The third embodiment of the invention is of the membrane-bound type, and its expression enhances the antitumor activity of CAR-T cells.

Fourth variant of IL-15: IL-15Rasushi/IL-15 (hereinafter also called _sushi IL15).

In a fourth embodiment, IL-15Ra containing a signal peptide and a sushi domain is typically linked to the N-terminal side of IL-15 via a linker (eg, a 20 amino acid linker). The term IL-15Rasushi also includes not only the full-length polypeptide consisting of the amino acid sequence of natural IL-15Rasushi, but also a portion thereof (functional partial polypeptide) and variants of full-length natural IL-15Rasushi or a portion thereof in which one or more amino acid sequences have been deleted. replaced or added to the natural amino acid sequence (preferably within the range having the homology described below). The fourth option is of the secretory type.

In the present invention, IL-15 is preferably IL-15 (full-length protein or a portion of a protein having a natural amino acid sequence or variants thereof) in the form of a fusion protein with IL-15Ra (a full-length protein or a portion thereof having a natural amino acid sequence or variants thereof) ) taking into account the persistence and proliferation of cells, as described below in the examples (in the experimental examples). For example, the above-mentioned second, third or fourth embodiments or modified types thereof are preferred, and the above-mentioned third and fourth embodiments or modified types thereof are more preferred.

IL-18 is a cytokine having functions associated with T cell activation, NK cell activation, dendritic cell activation, and the like. In the present invention, the term IL-18 includes not only the full-length natural IL-18 protein, but also various variants thereof, provided that the functions of IL-18 in relation to the action and effect of the invention are maintained. That is, in the present invention, IL-18 includes not only a full-length protein (polypeptide) consisting of the amino acid sequence of natural IL-18, but also a part thereof (functional partial polypeptide) and variants of full-length or parts of natural IL-18, in which one or more amino acid sequences have been deleted, replaced or added to the native amino acid sequence (preferably within a range having homology described below), and in addition, full-length native IL-18 or a portion thereof or variants thereof linked to other proteins to form a fusion squirrel. In the present invention, the term expresses IL-18, when referring to a T cell, includes expression of IL-18 in various forms as described above (as a full-length protein or a portion thereof, having a natural amino acid sequence or variants thereof that may be present , or may not be present, in the form of hybrid proteins), provided that the action and effect according to the invention will be maintained.

IL-21 is a cytokine having functions associated with CD8 ⁺ T cell functions, CD8 ⁺ T cell differentiation into memory T cells, NK cell survival, and the like. In the present invention, the term IL-21 includes not only the full-length natural IL-21 protein, but also various variants thereof, provided that the functions of IL-21 in relation to the action and effect of the invention are maintained. That is, in the present invention, IL-21 includes not only a full-length protein (polypeptide) consisting of the amino acid sequence of natural IL-21, but also a part thereof (functional partial polypeptide) and variants of full-length natural IL-21 or a part thereof in which one or more amino acid sequences have been deleted, replaced or added to the native amino acid sequence (preferably within a range having the homology described below), and, in addition, full-length native IL-21 or a portion thereof linked to other proteins to form a fusion protein. In the present invention, the term express IL-21, when referring to a T cell, includes expression of IL-21 in various forms as described above (as a full-length protein or a portion thereof, having a natural amino acid sequence or variants thereof that may be present , or may not be present, in the form of hybrid proteins), provided that the action and effect according to

- 10 045607 invention.

IL-27 is a cytokine having functions associated with T cell survival, NK cell activation, inhibition of tumor proliferation and angiogenesis, and the like. In the present invention, the term IL-27 includes not only the full-length natural IL-27 protein, but also various variants thereof, provided that the functions of IL-27 in relation to the action and effect of the invention are maintained. That is, in the present invention, IL-27 includes not only a full-length protein (polypeptide) consisting of the amino acid sequence of natural IL-27, but also a part thereof (functional partial polypeptide) and variants of full-length or part of natural IL-18, in which one or a plurality of amino acid sequences have been deleted, replaced or added to the native amino acid sequence (preferably within a range having the homology described below), and, in addition, full-length native IL-27 or a portion thereof linked to other proteins to form a fusion protein. In the present invention, the term express IL-27, when referring to a T cell, includes expression of IL-27 in various forms as described above (as a full-length protein or a portion thereof, having a natural amino acid sequence or variants thereof that may be present , or may not be present, in the form of hybrid proteins), provided that the action and effect according to the invention will be maintained.

Examples of IL-27 include a fusion protein (single chain protein) where p28 and EBI3 (Epstein-Barr virus-induced gene 3), which are the two subunits that make up IL-27, are linked by a linker, for example, a linker consisting of 20-30 amino acids such as (G ₄ S) ₃ (see above, for example, Non-Patent Document 5), or a linker consisting of GSTSGSGKPGSGEGSTKG (J. Immunol. 183, 6217-26 (2009)). Each of the terms p28 and EBI3 also includes all or part of a polypeptide with a natural amino acid sequence or variants thereof.

The amino acid sequences of the above-mentioned cytokines expressed by the T cell according to the invention can be suitably determined depending on the use of the T cell (CAR-T cell) according to the invention, and usually depending on its functions as an active component of a drug for treatment cancer, taking into account the influence of CAR-T cells on enhancing antitumor activity, including increasing the persistence and proliferation of CAR-T cells. The amino acid sequences of naturally occurring IL-15, IL-18, IL-21, IL-27, and IL-15Ra derived from humans and non-human mammals (eg, mice) are known and have also been reported in databases such as NCBI and UniProt, and therefore, information about these sequences can also be used in the present invention. For example, SEQ ID NO: 18 is the amino acid sequence of natural human IL-15 (full-length IL-15, including signal peptide and propeptide portions) registered as UniProtKBP40933, and SEQ ID NO: 19 is the amino acid sequence of natural human IL-15 15Ra (full-length IL-15Ra, including signal peptide, sushi domain, extracellular domain, etc.), registered as UniProtKB-Q13261. In the embodiment described below, in which the natural mouse amino acid sequence has been replaced by the natural human amino acid sequence in the amino acid sequence with a predetermined sequence identification number, reference may be made to the amino acid sequences of the corresponding portions included in SEQ ID NOs: 18 and 19. In addition, , various variants, fusion proteins with other proteins and modified types thereof IL-15, IL-18, IL-21, IL-27 and IL-15Ra are known and information about their amino acid sequences can also be used in the present invention.

Examples of IL-15, IL-18, IL-21, and IL-27 variants include variants in which the amino acid sequence is 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, in total or in a certain region (in a domain) is homologous to their natural amino acid sequences derived from humans or non-human mammals, for example, and in the case where the signal peptide has been replaced, as shown below, it is homologous to the region excluding the signal peptide. As a variant having homology in this range with a naturally occurring amino acid sequence originating from one species, a naturally occurring amino acid sequence originating from another species (substantially included) can be used. In addition, the signal peptide in the sequence of each of IL-15, IL-18, IL-21 and IL-27 can be replaced by a known signal peptide. In one aspect of the present invention, the amino acid sequence of native murine IL-15, IL-18, IL-21 or IL-27 can be replaced by the amino acid sequence of a variant having such homology; the amino acid sequence of native human IL-15, IL-18, IL-21 or IL-27, which is essentially applicable to such a variant or an additional variant thereof in the amino acid sequences of proteins (polypeptides) expressed by the nucleotide sequences of SEQ ID NO: 4: DNA fragment IL15 _lsp _F2A_CCL19 (DNA fragment #3), SEQ ID NO: 6: DNA fragment

- 11 045607 sIL15r _{A_F2A_CCL19} (DNA fragment #4), SEQ ID NO: 8: DNA fragment _m bIL15RA_F2A_CCL19 (DNA fragment #5), SEQ ID NO: 10: DNA fragment _SUS h,IL15_F2A_CCL19 (DNA fragment #6), SEQ ID NO: 12: DNA fragment IL-18F2A_CCL19 (DNA fragment #7), SEQ ID NO: 14: DNA fragment IL21F2A_CCL19 (DNA fragment #8), and SEQ ID NO: 16: DNA fragment scIL27_F2A_CCL19 (DNA fragment #9), or in the amino acid sequences of a fusion protein containing SEQ ID NO: 5: IL15 _lsp , a fusion protein containing SEQ ID NO: 7: SIL15RA, a fusion protein containing SEQ ID NO: 9: _mb IL15RA, a fusion protein containing SEQ ID NO: 11: _sushi IL15, a fusion protein containing SEQ ID NO: 13: IL-18, a fusion protein containing SEQ ID NO: 15: IL-21, and a fusion protein containing SEQ ID NO: 17: scIL27.

Examples of IL-15Ra extracellular domain, full-length IL-15Ra, and IL-15Rasushi variants include variants having an amino acid sequence that is 80% or greater, 85% or greater, 90% or greater, 91% or greater, 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 homologous to their natural amino acid sequences originating from humans or non-human mammals. In one aspect of the present invention, the amino acid sequence of the native murine extracellular domain of IL-15Ra, full-length IL-15Ra and IL-15Rasushi can be replaced by the amino acid sequence of a variant having such homology; the amino acid sequence of the natural human extracellular domain of IL-15Ra, full-length IL-15Ra or IL-15Rasushi, which are essentially applicable to such a variant, or an additional variant thereof, in the amino acid sequences of proteins (polypeptides) expressed by the nucleotide sequences of SEQ ID NO: 4: DNA fragment IL15 _lsp _F2A_CCL19 (DNA fragment #3), SEQ ID NO: 6: DNA fragment _s IL15 _rA _F2A_CCL19 (DNA fragment #4), SEQ ID NO: 8: DNA fragment _mb IL15RA_F2A_CCL19 (DNA fragment #5), SeQ ID NO: 10: DNA fragment _{sushi_IL15_F2A_CCL19} (DNA fragment #6), or in the amino acid sequences of a fusion protein containing SEQ ID NO: 5: IL15 _lsp , a fusion protein containing SEQ ID NO: 7: _s IL15 _rA , a fusion protein containing SEQ ID NO: 9: _mb IL15 _RA , and a fusion protein containing SEQ ID NO: 11: _sushi IL15.

IL-15, IL-18, IL-21 and IL-27 are preferably derived from humans or non-human mammals (eg mice) of the same type from which the T cells (or IL-15 receptors, IL- 18, IL-21 and IL-27 T cells) used to produce T cells according to the invention. The non-cytokine portions of IL-15, IL-18, IL-21, and IL-27 (eg, IL-15Ra) contained in the IL-15, IL-18, IL-21, and IL-27 fusion proteins, respectively, also preferably originate from a human or non-human mammal of the same type from which the T cells used to produce the T cells of the invention originate. In one aspect of the present invention, the amino acid sequence of natural murine IL-15, IL-18, IL-21 or IL-27 or the amino acid sequence of the extracellular domain of IL-15Ra, full-length IL15Ra or IL-15Rasushi can be replaced with their natural human amino acid sequence in amino acid sequences of proteins (polypeptides) expressed by nucleotide sequences SEQ ID NO: 4: DNA fragment IL15 _lsp _F2A_CCL19 (DNA fragment #3), SEQ ID NO: 6: DNA fragment _s IL15 _rA _F2A_CCL19 (DNA fragment #4), SEQ ID NO : 8: DNA fragment _mb IL15 _{RA_F2A_CCL19} (DNA fragment #5), SEQ ID NO: 10: DNA fragment _sushi IL15_F2A_CCL19 (DNA fragment #6), SEQ ID NO: 12: DNA fragment IL-18_F2A_CCL19 (DNA fragment #7) , SEQ ID NO: 14: DNA fragment IL-21_F2A_CCL19 (DNA fragment #8), and SEQ ID NO: 16: DNA fragment _sc IL27_F2A_CCL19 (DNA fragment #9), or in the amino acid sequences of a fusion protein containing SEQ ID NO: 5 : IL15 _lsp , a fusion protein containing SEQ ID NO: 7: _s IL15 _rA , a fusion protein containing SEQ ID NO: 9: _mb IL15 _RA , a fusion protein containing SEQ ID NO: 11: _sushi IL15, a fusion protein containing SEQ ID NO: 13: IL-18, a fusion protein comprising SEQ ID NO: 15: IL-21, and a fusion protein comprising SEQ ID NO: 17: _sc IL27.

(3) Chemokine.

The chemokine expressed by the T cell of the invention is CCL19. CCL19 is mainly produced by dendritic cells and lymph node macrophages and has the function of inducing trafficking of T cells, B cells and mature dendritic cells through their receptor CCR7. That is, in the present invention, CCL19 includes not only a full-length protein (polypeptide) consisting of the amino acid sequence of natural CCL19, but also a part thereof (functional partial polypeptide) and variants of full-length natural CCL19 or a part thereof in which one or more amino acid sequences have been deleted, replaced or added to the natural amino acid sequence (preferably within the range having the homology described below).

The amino acid sequence of the above-mentioned chemokine expressed by the T cell according to the invention can be suitably determined depending on the use of the T cell (CAR-T cell) according to the invention, and generally depending on its function as an active component of a drug for treating cancer with taking into account the influence of CAR-T cells on the enhancement

- 12 045607 antitumor activity, including increasing the persistence and proliferation of CAR-T cells. The amino acid sequences of native CCL19 originating from humans or non-human mammals (eg mice) are known and also registered in databases such as NCBI and UniProt, and therefore their information can also be used in the present invention. In addition, the present invention can also use information about the amino acid sequences of known CCL19 variants.

Examples of CCL19 variants include variants in which the amino acid sequence is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more more than, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, throughout or in a defined region, homologous to the amino acid sequences of natural CCL19 of human or mammalian origin , except for a person. In one aspect of the present invention, the amino acid sequence of native murine CCL19 may be replaced by the amino acid sequence of a variant having such homology with the amino acid sequence of native human CCL19 that is substantially applicable to such variant or an additional variant thereof in the amino acid sequences of proteins (polypeptides) expressed by the nucleotide sequences SEQ ID NO: 4: DNA fragment IL15 _{lsp_F2A_CCL19} (DNA fragment #3), SEQ ID NO: 6: DNA fragment _s IL15 _{RA_F2A_CCL19} (DNA fragment #4), SEQ ID NO: 8: DNA fragment _mb IL15 _{RA_F2A_CCL19} ( DNA fragment #5), SEQ ID NO: 10: DNA fragment _sushl IL15_F2A_CCL19 (DNA fragment #6), SEQ ID NO: 12: DNA fragment IL-18_F2A_CCL19 (DNA fragment #7), SEQ ID NO: 14: DNA fragment IL21_F2A_CCL19 (DNA fragment #8), and SEQ ID NO: 16: DNA fragment _sc IL27_F2A_CCL19 (DNA fragment #9).

CCL19 preferably is derived from a human or non-human mammal (eg, mouse) of the same type from which the T cell used to produce the T cell of the invention (CCL19 T cell receptor) or variants thereof is derived.

The following is a detailed description of the medicinal product according to the invention.

The medicament according to the invention contains the T cell according to the invention as described above, and may also contain other components if necessary. Those skilled in the art can properly prepare the medicament of the invention using the T cells of the invention, taking into account the purpose of use (target of treatment) and dosage form.

The drug of the invention is generally a drug (anticancer agent) for treating cancer associated with a cancer cell surface antigen (cancer-specific antigen) targeted by a CAR expressed by a T cell of the invention. Accordingly, the type of cancer targeted by a drug according to the invention is not particularly limited, provided that cancer cells expressing the antigen targeted by the CAR are present in the cancer tissue and that a certain level of therapeutic effect will be achieved with using T cells according to the invention. Examples of cancers that may be targeted by a drug of the invention may include cancers such as adenocarcinoma, squamous cell carcinoma, squamous cell adenocarcinoma, undifferentiated cancer, large cell cancer, small cell cancer, skin cancer (eg, melanoma and Merkel cell cancer), cancer breast, prostate cancer, bladder cancer, vaginal cancer, neck cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, non-small cell lung cancer, cancer trachea, bronchial cancer, colon cancer, rectal cancer, small intestine cancer, colorectal cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, fallopian tube cancer and nasopharyngeal cancer; bone cancer, cartilage cancer, adipose tissue cancer, muscle cancer, vascular tissue cancer and hematopoietic tissue cancer; sarcomas such as chondrosarcoma, Ewing's sarcoma, rhabdomyosarcoma, malignant hemangioendothelioma, malignant tumor, osteosarcoma and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pulmonary pleural blastoma and retinoblastoma; embryonic cell tumors; lymphoma; leukemia, acute myeloid leukemia and multiple myeloma. Preferred examples include NK cell-sensitive carcinomas (such as melanoma, Merkel cell cancer, 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 bowel cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia , multiple myeloma, lymphoma and leukemia). More preferred examples include melanoma, colorectal cancer, kidney cancer, multiple myeloma, lymphoma and leukemia.

Examples of components that may be contained in the medicament of the invention, other than T cells, include pharmaceutically acceptable additives, and more particularly, saline, buffered saline, cell culture media, dextrose, water for injection, glycerol, ethanol, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonic agents, fillers and lubricants.

The drug of the invention can be administered to an individual in need of cancer treatment (eg, cancer patients and animals with cancer) in the same manner as is used to administer known CAR-expressing T cells. Examples of the route of administration include intratumoral, intravenous, intraarterial, intramuscular, subcutaneous and intraperitoneal injections.

The number of T cells according to the invention to be contained in the medicinal product according to the invention can be adjusted accordingly, for example, depending on the purpose of use, dosage form, intended therapeutic effect, and the like. taking into account the type, location and severity of cancer, age, body weight and condition of the individual being treated, etc. Thus, for example, a medicament according to the invention may be formulated such that the T cells according to the invention are administered in an amount of typically 1 x 10 ⁴ to 1 x 101°, preferably 1 x 10 ⁵ to 1 x 109, and more preferably 5 x 10 ⁶ to 5 x 1° ⁸ one dose.

The interval between administrations of the drug of the invention is not particularly limited, and can be suitably adjusted based on the number of T cells of the invention administered at one time and the like. The drug according to the invention can be administered independently, for example, 4 times, 3 times, twice or once a day, every other day, every 3 days, every 4 days, every 5 days, every 6 days, once per week, every 8 days, every 9 days, every 1° days, twice a week, once a month or twice a month.

The drug according to the invention (anticancer agent) can be used in combination with known anticancer agents. Examples of anticancer agents may include alkylating drugs such as cyclophosphamide, bendamustine, iosfamide and dacarbazine; antimetabolites such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine and enocytabine; molecular targeting drugs such as rituximab, cetuximab and trastuzumab; kinase inhibitors such as imatinib, gefitinib, erlotinib, afatinib, dasatinib, sunitinib and trametinib; proteasome inhibitors such as bortezomib; calcineurin inhibitors such as cyclosporine and tacrolimus; antitumor antibiotics such as idarubicin and doxorubicin, mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum-based drugs such as cisplatin, oxaliplatin and carboplatin; hormonal therapeutic drugs such as tamoxifen and bicalutamide; and immunoregulatory drugs such as interferon, nivolumab and pembrolizumab.

The use of a medicinal product according to the invention in combination with an additional anti-cancer agent can be carried out in any form: (a) using an additional anti-cancer agent and then a medicinal product according to the invention, (b) using a medicinal product according to the invention and an additional anti-cancer agent simultaneously, or (c) using a drug according to the invention and then an additional anticancer agent. If the drug of the invention is used in combination with an additional anticancer agent, the therapeutic response to cancer is expected to be greater and the side effects caused by the anticancer drugs may be reduced by reducing the number of administrations or dose of the drug of the invention and/or another anticancer agent.

The following is a detailed description of the expression vector according to the invention.

The expression vector of the invention contains: (1) a nucleic acid encoding a CAR; (2) a nucleic acid encoding at least one cytokine selected from the group consisting of IL-15, IL-18, IL-21 and IL-27; and (3) a nucleic acid encoding CCL19.

In the present invention, the term contains a nucleic acid encoding IL-15, if it refers to an expression vector, includes a nucleic acid encoding not only the full-length natural IL-15 protein, but also IL-15 in various forms, as described above. In the case of an expression vector, the terms contains a nucleic acid encoding IL-18, contains a nucleic acid encoding IL-21, contains a nucleic acid encoding IL-27, or contains a nucleic acid encoding CCL19 have the same meaning.

Nucleic acid can be any molecules obtained by polymerization of nucleotides and molecules having functions equivalent to nucleotides. Examples may include RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; a polymer containing a mixture of ribonucleotides and deoxyribonucleotides, and a nucleotide polymer containing nucleotide analogues. In addition, the nucleic acid may be a nucleotide polymer containing nucleic acid derivatives. In addition, the nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. In addition, double-stranded nucleic acid also includes double-stranded nucleic acid in which one strand hybridizes to another strand under stringent conditions.

Nucleotide analogues can be any molecule with modification of a ribonucleotide, deoxyribonucleotide, RNA or DNA to increase nuclease resistance, stabilization, increase affinity for complementary nucleic acid strands, increase cell permeability or improve cell visualization compared to RNA or DNA. Nucleotide analogs can be natural molecules or non-natural molecules. Examples of such analogs include sugar-modified nucleotide analogs and phosphodiester-modified nucleotide analogs.

Sugar-modified nucleotide analogues can be any analogue obtained by adding any chemical structural entity or substituting any chemical structural entity for part or all of the sugar chemical structure of the nucleotide. Specific examples of such analogs may include nucleotide analogs with a 2'-O-methyl ribose substitution; nucleotide analogues with substitution for 2'-O-propyl-ribose; nucleotide analogs with substitution for 2'-methoxyethoxyribose; nucleotide analogues with substitution for 2'-O-methoxyethyl ribose; nucleotide analogs with substitution for 2'-O-[2(guanidium)ethyl]ribose; nucleotide analogues with 2'-fluororibose substitution; cross-linked artificial nucleic acids having two cyclic structures as a result of the introduction of cross-linked structures into the sugar moiety (bridged nucleic acids) (BNA), and more specifically, blocked artificial nucleic acids in which an oxygen atom is in the 2' position and a carbon atom is in the 4' position -position were cross-linked with methylene (locked nucleic acids) (LNA), and artificial nucleic acids cross-linked with ethylene (ethylene bridged nucleic acids) (ENA) [Nucleic Acid Research, 32, e175 (2004)] and, in addition , peptide-containing nucleic acids (PNAs) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acids (OPNA) [J. Am. Chem. Soc, 123, 4653 (2001)] and peptide ribonucleic acids (PRNA) [J. Am. Chem. Soc, 122, 6900 (2000)].

Phosphodiester linkage modified nucleotide analogues can be any analogue obtained by adding any chemical or substituting any chemical for part or all of the chemical structure of the phosphodiester linkage of a nucleotide. Specific examples of these analogs may include nucleotide analogs with replaced phosphorothioate bonds, and nucleotide analogs with replaced N3'-P5' phosphoramidite bonds [Cell Technology, 16, 1463-1473 (1997)] [RNAi and antisense method, Kodansha Ltd. (2005)].

Nucleic acid derivatives can be any molecules made by adding another chemical to nucleic acids to increase nuclease resistance, stabilization, increase affinity for complementary strands of nucleic acids, increase cell permeability, or improve cell visualization compared to nucleic acids. Specific examples of such analogues may include 5'-polyamine added derivatives, cholesterol added derivatives, steroid added derivatives, bile acid added derivatives, vitamin added derivatives, Cy5 added derivatives, Cy3 added derivatives, 6-added derivatives. FAM, and derivatives with added biotin.

An expression vector of the invention must be capable of producing only a T cell of the invention by contacting the T cell or its precursor, introducing it into the cell, and expressing a predetermined protein (polypeptide) encoded by the vector in the T cell. This embodiment of the invention is not particularly limited. Those skilled in the art can independently design and produce an expression vector capable of expressing a desired protein (polypeptide) in a T cell.

The expression vector of the invention may be linear or cyclic and may be a non-viral vector such as a plasmid, a viral vector or a transposon vector.

Examples of the viral vector may include a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. Preferred examples of the retroviral vector may include the pMSGV vector (Tamada K. et al., ClinCancer Res 18: 6436-6445 (2002)) and the pMSCV vector (available from Takara Bio Inc). In the case of using a retroviral vector, the genes contained in the vector are incorporated into the genome of the host cell (T cells according to the invention), and therefore the genes can be stably expressed over a long period of time.

In one embodiment of the invention, a single expression vector contains all of the elements of (1) a nucleic acid encoding a CAR, (2) a nucleic acid encoding at least one cytokine 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 invention, the first expression vector contains one or two of the above elements (1)-(3), and the second expression vector contains the remaining element. In another embodiment of the invention, the first expression vector contains the above element (1), the second expression vector contains the above element (2), and the third expression vector contains the above element (3). Accordingly, an expression vector according to the invention may mean a combination (set) of a plurality of expression vectors, that is, a combination of the above-mentioned first and second expression vectors or a combination of the above-mentioned first, second and third expression vectors, depending on the option. In the case where one expression vector contains multiple elements, the order of these elements from left to right has no particular restrictions.

The nucleotide sequences of the predefined three elements (CAR, cytokine and chemokine) contained in the expression vector according to the invention can be designed to encode the amino acid sequences of proteins (polypeptides) respectively corresponding to the proteins (polypeptides) selected as elements. The nucleic acids (oligonucleotides) contained in the expression vector can be obtained by chemical synthesis of oligo-DNA or can be obtained (cloned) as cDNA.

The expression vector of the invention may contain (in the case of the above combinations of multiple expression vectors, each of them independently may contain) sequences of promoters, terminators, enhancers, start codons, stop codons, polyadenylation signals, nuclear localization signals (NLS), multiple cloning sites (MCS), etc., which participate in the expression of each gene, if necessary, in addition to the sequences (genes) encoding the above-mentioned predefined elements.

In one embodiment of the invention, if one expression vector contains two or more of the above three elements, then a gene encoding a self-cleaving peptide (2A peptide) or an IRES (internal ribozyme binding site) can be inserted between these elements.

Peptide 2A is a self-cleaving peptide derived from a virus and has the feature that a predetermined position (one residue position from the C-terminus) in the amino acid sequence is cleaved in the endoplasmic reticulum (Szymczak et al., Expert Opin. Biol. Ther, 5 (5): 627-638 (2005). Therefore, genes integrated before and after peptide 2A will be expressed in the cell independently of each other. Examples of peptide 2A include peptides derived from picornavirus, rotavirus, insect virus, aphthovirus, or trypanosome virus.

The expression vector of the invention may further contain nucleic acids (nucleotide sequences) encoding functional genes, such as a reporter gene (usually a gene encoding a fluorescent protein), a drug selection gene and a suicide gene.

In the case of using a drug resistance gene as a functional gene, cells into which the expression vector of the invention containing the drug resistance gene is introduced can be selected by adding a predetermined drug to the medium in the method for producing CAR-T cells according to invention. Examples of the drug resistance gene include a kanamycin resistance gene, an ampicillin resistance gene, and a puromycin resistance gene. In this case, one of these genes or two or more of them can be used.

In the case of using a gene encoding a fluorescent protein as a functional gene, T cells introduced with an expression vector according to the invention can be observed on a fluorescence microscope, or cells introduced with an expression vector according to the invention can be sorted on a flow cytometer (cell sorter) in the production method CAR-T cells according to the invention. A typical example of a reporter gene is a gene encoding a fluorescent protein. Examples of a gene encoding a fluorescent protein include genes encoding proteins that fluoresce in the blue range of the spectrum, such as Sirius, BFP and EBFP; proteins that fluoresce in the blue range of the spectrum, such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan and CFP; proteins that fluoresce in the green range of the spectrum, such as TurboGFP, AcGFP, TagGFP, Azami-Green (for example, hmAG1), ZsGreen, EmGFP, EGFP, GFP2 and HyPer; proteins that fluoresce in the yellow range of the spectrum, such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow and mBanana; proteins that fluoresce in the orange range of the spectrum, such as KusabiraOrange (for example, hmKO2) and mOrange; proteins that fluoresce in the red range of the spectrum, such as TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2 and mStrawberry; and near-infrared fluorescent proteins such as TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (eg hdKeimaRed), mRasberry and mPlum. In this case, any of these genes or two or more of them can be used. In the case of using two or more genes encoding fluorescent proteins, these fluorescent proteins should preferably emit at different wavelengths, which will allow them to be visualized accordingly.

In the case of using a suicide gene as a functional gene, the number of T cells according to the invention in vivo can be controlled by administering a drug that activates the function of the suicide gene, depending on the course of cancer treatment, for example, at the stage when

- 16 045607 when the tumor disappeared. Examples of a suicide gene include the gene encoding diphtheria toxin A, herpes simplex virus 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 (XducGPRT) or inducible caspase 9. In this case, any one of these genes or two or more of them. Drugs that activate the function of each suicide gene are known, and examples include ganciclovir for herpes simplex virus thymidine kinase (HSV-TK) and AP1903, which is a dimer-inducing compound for inducible caspase 9.

Next, a method for producing a CAR-T cell according to the invention will be described in detail. The method for producing CAR-T cells according to the invention includes the step of introducing an expression vector according to the invention into a T cell as described above (which will hereinafter be referred to as the step of introducing an expression vector).

The method of introducing an expression vector into a T cell may be suitable for a variant of the expression vector. For example, the expression vector can be introduced into a T cell by known methods such as the virus infection method, the calcium phosphoric acid method, the lipofection method, the microinjection method, and the electroporation method. The expression vector of the invention can be prepared in a form suitable for use in each method using known means and using commercially available kits as appropriate (in accordance with the instructions provided with the kit). At the expression vector injection stage, T cells can be cultured by contacting the drug with the T Cells, typically in a medium containing the drug including the expression vector.

In a preferred embodiment of the invention, the expression vector according to the invention is introduced into T cells by viral infection. Thus, for example, a method can be used in which the vector of the invention and the packaging vector (plasmid) of each virus are transfected into a host cell using a commercially available kit corresponding to each viral vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector and an adeno-associated a viral vector to produce a recombinant virus, after which the T cells are infected with the resulting recombinant virus. Examples of a commercially available viral vector kit include the Eco retrovirus packaging kit (available from Takara Bio Inc). Examples of the host cell include GP2-293 cells (available from Takara Bio Inc.), Plat-GP cells (available from Cosmo Bio Co., Ltd.), PG13 cells (ATCC CRL-10686), and PA317 cells (ATCC CRL9078).

The method for producing CAR-T cells according to the invention may further include other steps carried out before and after the step of introducing the expression vector. Examples of other steps include a step of culturing T cells and a step involving initiation of the functioning of a functional gene contained in the expression vector.

T cells into which the expression vector of the invention is introduced are usually pre-cultured in vitro in a known manner. For example, T cells can be isolated and purified in a conventional manner from physiological fluids such as blood and bone marrow fluid; tissues such as the spleen, thymus and lymph nodes; or cancerous tissues such as primary tumors, metastatic tumors and cancerous ascites, which are taken from human or non-human animals and then cultured in an appropriate medium, and an expression vector (a preparation containing an expression vector) can be added to this medium according to invention. Alternatively, T cells (or their precursors) can be previously obtained from iPS cells, ES cells and other pluripotent stem cells at the appropriate stage of inducing differentiation, and an expression vector (a preparation containing an expression vector) can be added to this medium according to invention.

Moreover, in case the expression vector according to the invention contains a drug resistance gene as a functional gene, the step of culturing T cells with the addition of a drug corresponding to the drug resistance gene used in the medium can be carried out after the step of introducing the expression vector for sorting T cells with an introduced expression vector. Those skilled in the art can appropriately adjust the conditions for use of the drug corresponding to the drug resistance gene, such as the concentration in the medium and the culture period.

If the expression vector according to the invention contains a gene encoding a fluorescent protein (reporter gene) as a functional gene, then the stage of observing T cells with the introduced expression vector on a fluorescent microscope, the stage of sorting T cells with the introduced expression vector on a cell sorter and etc. can be carried out after the stage of introducing the expression vector. Those skilled in the art can adjust the fluorescence microscope observation and cell sorter selection conditions accordingly,

- 17 045607 such as irradiation with light at the excitation wavelength and detection of light radiation at the emission wavelength corresponding to the fluorescent protein.

Examples

In the following examples, the CAR is a CAR targeting human CD20, and in particular, an anti-human CD20 CAR consisting of an anti-human CD20 scFv, a mouse CD8 transmembrane domain, and a mouse intracellular signaling motif CD28_41BB_CD3Z. Additionally, in the following examples, the cytokine is any of the cytokines such as murine IL-15 (IL15 _lsp , SIL15RA, mbIL 15RA or _sushi IL15; murine IL-18; murine IL-21; or murine IL-27 containing single chain _sc protein IL27 (p28 and EBI3) In the following examples, the chemokine is murine CCL19. Mouse-derived T cells expressing CAR, cytokine and chemokine were prepared and tested as follows.

Example/Comparative Example CAR Cytokine Chemokin Example 1-1 scFv vs. human CD20 IL 1 5 _L sp (mouse) CCL19 (mouse) Example 1-2 scFv vs. human CD20 _S IL1 5 _R a (mouse) CCL19 (mouse) Example 1-3 scFv vs. human CD20 _mb IL15 _RA (mouse) CCL19 (mouse) Example 1-4 scFv vs. human CD20 _SUS hiIL15 (mouse) CCL19 (mouse) Example 2 scFv vs. human CD20 IL-18 (mouse) CCL19 (mouse) Example 3 scFv vs. human CD20 IL-21 (mouse) CCL19 (mouse) Example 4 scFv vs. human CD20 _SC IL27 (mouse) CCL19 (mouse) Comparative example 1 - - - Comparative example 2 scFv vs. human CD20 - -

Example 1-1. IL15 _LSP xCCL19-CAR T cells.

In the vector production step, three types of DNA fragments (DNA fragments 1-3) were first artificially synthesized in step 1, and then one expression vector containing all the genes encoding the predefined CAR, cytokine and chemokine was produced using the three types of fragments DNA in step 2. Then in the CAR-T cell production step, a retroviral vector was first prepared using the expression vector in step 1, and then the predefined genes were transduced into mouse T cells using the retroviral vector preparation in step 2.

A: vector obtaining stage. Generation of CAR expression vector IL15LSP_CCL19 against human CD20.

Stage 1: synthesis of DNA fragments.

DNA fragment #1: DNA fragment containing CAR against human CD20.

A DNA fragment (DNA fragment #1) containing a nucleotide sequence encoding an anti-human CD20 CAR, consisting of an anti-human CD20 scFv, a mouse CD8 transmembrane domain, and a mouse CD28_4-1BB_CD3Z intracellular signaling motif, was synthesized artificially. SEQ ID NO: 1 (Fig. 7) is the nucleotide sequence

- 18 045607 total DNA fragment #1. In SEQ ID NO: 1, bases at positions 3-785 form the sequence encoding the anti-human CD20 scFv, bases at positions 795-1040 form the sequence encoding the transmembrane domain of mouse CD8, bases at positions 1041-1637 form the sequence encoding the intracellular signaling motif mouse CD28_41BB_CD3Z (among them, bases at positions 1041-1163 encode the intracellular domain of mouse CD28, bases at positions 1164-1298 encode the intracellular domain of mouse 4-1BB, and bases at positions 1299-1637 encode a polypeptide in the intracellular domain of mouse CD3Z). SEQ ID NO: 2 is the amino acid sequence of the fusion protein expressed by the nucleotide sequence at positions 3-1637 of DNA fragment #1.

DNA fragment #2: DNA fragment containing stop codons and multiple cloning sites.

A DNA fragment (DNA fragment #2) containing the nucleotide sequence of stop codons and the nucleotide sequence of multiple cloning sites (MCS) was artificially synthesized. SEQ ID NO: 3 (FIG. 8) is the nucleotide sequence of the entire DNA fragment #2.

DNA fragment #3: DNA fragment for IL15 _lsp and CCL19.

A DNA fragment (DNA fragment #3) containing the nucleotide sequence encoding peptide 2A (F2A), which is a self-cleaving peptide, murine IL-2 signal peptide (IL-2SP), a construct of murine IL-15 IL15lsp (a hybrid peptide consisting of IL -15LSP and IL-15), F2A and murine CCL19, were synthesized artificially. SEQ ID NO: 4 (FIG. 9) is the nucleotide sequence of the entire DNA fragment #3. In SEQ ID NO: 4, bases at positions 7-81 form the first F2A coding sequence, bases at positions 82-168 form the murine IL-15 _lsp coding sequence, bases at positions 169-567 form the murine IL-15 coding sequence (excluding stop codons), bases at positions 568-642 form the sequence encoding the second F2A, and bases at positions 646-969 form the sequence encoding mouse CCL19. SEQ ID NO: 5 is the amino acid sequence of the full-length fusion protein, which is expressed by nucleotide sequence at positions 7-969 of DNA fragment #3, self-cleaves at two F2A sites and contains IL15 _lsp .

Stage 2: obtaining a retroviral expression vector.

DNA fragment #1 and DNA fragment #2 were linked to each other to produce the construct (CAR_MCS vs. human CD20). The resulting construct was incorporated into the retroviral expression vector pMSGV (Tamada K. et al., Clin Cancer Res 18: 6436-6445 (2002)) by treatment with restriction enzymes (Nco I and Sal I) to obtain the retroviral expression vector pMSGV containing anti-human CAR_MCS CD20.

The nucleotide sequences at positions 1337-1637 of DNA fragment #1 and DNA fragment #3 were then linked to produce a construct. The resulting construct was incorporated into the retroviral vector pMSGV containing CAR_MCS against human CD20 by treatment with restriction enzymes (Sbf I and Sal I) to obtain the retroviral expression vector pMSGV containing CAR_F2A_IL15 _lsp _F2A_CCL19 against human CD20 (expression vector CAR IL15 _lsp _CCL19 against human CD20) .

B: stage of obtaining CAR-T cells. Generation of CAR-T cells containing CAR_IL15 _lsp _CCL19 against human CD20.

Stage 1: obtaining a retrovirus.

Using Lipofectamine 3000 (available from Thermo Fisher SCIENTIFIC KK), the CAR IL15 _lsp _CCL19 anti-human CD20 expression vector from the vector production step was transferred into the GP2-293 packaging cell line (Takara Bio Inc.) along with plasmid pCLEco (Novus Biologicals , LLC). DMEM supplemented with 10% deactivated FBS and antibiotics was used as the culture solution for the packaging cell line GP2-293. 48 hours after transfection, the supernatant of the culture solution of the packaging cell line GP2-293 was collected to obtain the CAR IL15 _lsp _CCL19 anti-human CD20 expression vector preparation.

Step 2: transduction of murine T cells.

3x106 murine T cells isolated and purified from mouse spleen and lymph nodes were activated for 48 h in the presence of immobilized anti-mouse CD3 monoclonal antibody (3 μg/ml), anti-mouse CD28 monoclonal antibody (1 μg/ml) and IL- 2. Then, the preparation containing the anti-human CD20 CAR IL15 _lsp _CCL19 expression vector (GP2-293 cell culture supernatant) obtained in step 1 and mouse T cells activated as described above were mixed in the wells of a plate in which 25 μg was immobilized /ml retronectin (registered trademark: Takara Bio Inc.) followed by centrifugation at 1500 rpm for 2 hours, and the mixture was cultured for 6 hours in the presence of IL-2. To remove the retrovirus from the culture solution, mouse T cells were collected and transferred into a new complete

- 19 045607 RPMI medium containing IL-2 and then cultured for 42 hours to obtain murine T cells (T cells containing CAR IL15 _{lsp_CCL19} anti-human CD20 and hereinafter referred to as

IL15 _lsp χCCL19-CAE.-T cells) containing the introduced CAR expression vector

IL15 _lsp _CCL19 anti-human CD20, which expresses CAR against human CD20,

IL15 _lsp and CCL19.

The complete RPMI medium used for culturing T cells was RPMI-1640 medium supplemented with deactivated 10% FBS, antibiotics, 50 mM 2mercaptoethanol, and 25 mM HEPES buffer. Complete RPMI medium, to which the components described separately were also added when necessary, was used as described below as a culture solution for T cells.

Example 1-2. SIL15RA (secretory IL-15/IL-15RA fusion protein)χCCL19-CAR-T cells.

A: vector obtaining stage.

Generation of CAR _S IL15 _RA _CCL19 expression vector against human CD20.

Using the same DNA fragment #1 and DNA fragment #2 as used in step 1 of Example 1-1, and DNA fragment #4 as described below instead of DNA fragment #3, a construct was prepared in the same manner as described in step 2 of Example 1-1, to obtain a retroviral expression vector pMSGV containing CAR_F2A_ _s IL15 _ra _F2A_CCL19 against human CD20 (CAR_ _s IL15 _ra _CCL19 anti-human CD20 expression vector).

DNA fragment #4: DNA fragment for SIL15RA and cCl19.

A DNA fragment (DNA fragment #4) was synthesized containing the nucleotide sequence encoding F2A, mouse IL-2SP, the mouse IL-15 construct SIL15RA (a hybrid peptide consisting of IL-15, a linker and the extracellular domain of IL-15Ra), F2A and mouse CCL19. SEQ ID NO: 6 (FIG. 10) is the nucleotide sequence of full length DNA fragment #4. In SEQ ID NO: 6, the bases at positions 7-81 form the first F2A coding sequence, the bases at positions 82-141 form the murine IL-2SP coding sequence, the bases at positions 142-1137 form the SIL15RA coding sequence (among them, bases at positions 142-540 encode murine IL-15 (excluding signal sequences and stop codons), bases at positions 541-618 serve as a linker, bases at positions 619-1137 encode the extracellular domain of murine IL-15Ra, bases at positions 1138-1212 form the coding sequence for the second F2A, and bases at positions 1216-1539 form the coding sequence for murine CCL19 SEQ ID NO: 7 is the amino acid sequence of the full-length fusion protein that is expressed by the nucleotide sequence at positions 7-1539 of DNA fragment #4 , self-cleaves at two F2A sites and contains S ^IL1 5RA.

B: CAR-T cell production stage. Generation of T cells containing CAR _S IL15 _RA _CCL19 against human CD20.

The retrovirus-containing drug was produced in step 1 in the same manner as in the CAR-T cell production step in Example 1-1, except that the CAR _S IL15 _{RA_CCL19} anti-human CD20 expression vector obtained in the above-mentioned production step vector was used as a retroviral expression vector instead of the CAR _S IL15 _RA _CCL19 anti-human CD20 expression vector to obtain murine T cells expressing the CAR anti-human CD20, SIL15RA and CCL19 genes contained in the retrovirus (T cells containing CAR SIL15RA_CCL19 anti-human CD20 cells, hereafter referred to as _S IL15 _RA χCCL19-CAR-T cells) in stage 2.

Example 1-3. _mb IL15 _RA (membrane-bound IL-15/IL-15RA fusion protein)x CCL19-CAR-T cells.

A: vector obtaining stage.

Generation of CAR _mb IL15 _RA _CCL19 expression vector against human CD20.

Using the same DNA fragment #1 and DNA fragment #2 as used in step 1 of Example 1-1, and DNA fragment #5 as described below instead of DNA fragment #3, a construct was prepared in the same manner as described in step 2 of Example 1-1, to obtain a retroviral expression vector pMSGV containing _{CAR_F2A_mb} IL15 _{RA_F2A_CCL19} anti-human CD20 (CAR_mb _{IL15 RA_CCL19} anti-human CD20 expression vector).

DNA fragment #5: DNA fragment for _mb IL15 _RA and CCL19.

A DNA fragment was synthesized containing the nucleotide sequence encoding F2A, mouse IL-2SP, the mouse IL-15 _mb IL15 _RA construct (a hybrid peptide consisting of IL15, a linker and full-length IL-15Ra), F2A and mouse CCL19. SEQ ID NO: 8 (FIG. 11) is the nucleotide sequence of full length DNA fragment #5. In SEQ ID NO: 8, the bases at positions 7-81 form the sequence encoding the first F2A, the bases at positions 82-141 form the sequence encoding murine IL-2SP, the bases at positions 1421311 form the sequence encoding _mb IL 15 _RA (among them , bases at positions 142-540 encode murine IL-15 (excluding signal sequences and stop codons), bases at positions 541-618 serve as a linker, bases at positions 619-1311 encode murine IL-15Ra ( full-length), bases at positions 1312-1386 form the sequence encoding the second F2A, and bases at positions 1390-1713 form the sequence encoding murine CCL19. SEQ ID NO: 9 is the amino acid sequence of the full-length fusion protein that is expressed by the nucleotide sequence at positions 7 -1713 DNA fragment #5, self-cleaves at two F2A sites and contains _mb IL15 _RA .

B: CAR-T cell production stage.

Generation of T cells containing CAR _mb IL15 _RA _CCL19 against human CD20.

The retrovirus-containing drug was produced in step 1 in the same manner as in the CAR-T cell production step in Example 1-1, except that the CAR _mb IL15 _{RA_CCL19} anti-human CD20 expression vector obtained in the above-mentioned production step vector was used as a retroviral expression vector instead of the CAR IL15 _lsp _CCL19 anti-human CD20 expression vector to obtain murine T cells expressing the CAR anti-human CD20, _mb IL15 _RA and CCL19 genes contained in the retrovirus (T cells containing CAR _mb IL15 _RA _CCL19 against human CD20, hereafter referred to as _mb IL15 _RA χCCL19-CAR T cells) in stage 2.

Example 1-4. _sushi IL15 (IL-15RA/IL-15 secretory fusion protein)χCCL19-CAR-T cells.

A: vector obtaining stage.

Generation of CAR _sushi IL15_CCL19 anti-human CD20 expression vector.

Using the same DNA fragment #1 and DNA fragment #2 as used in step 1 of Example 1-1, and DNA fragment #6 as described below instead of DNA fragment #3, a construct was prepared in the same manner as described in step 2 of Example 1-1, to obtain a retroviral expression vector pMSGV containing CAR_F2A_ _sushi IL15_F2A_CCL19 anti-human CD20 (CAR_ _sushi IL15_CCL19 anti-human CD20 expression vector).

DNA fragment #6: DNA fragment for _sushi IL15 and CCL19.

A DNA fragment was synthesized containing the nucleotide sequence encoding F2A, the mouse IL-15 _sushi construct IL15 (a hybrid peptide consisting of the IL-15Rasushi domain, linker and IL-15), F2A and mouse CCL19. SEQ ID NO: 10 (FIG. 12) is the nucleotide sequence of full length DNA fragment #6. In SEQ ID NO: 10, the bases at positions 781 form the first F2A coding sequence, the bases at positions 82-777 form _{the sushi} IL15 coding sequence (among them, the bases at positions 82-375 encode the SP and sushi domain of mouse IL-15Ra, bases at positions 376-435 serve as a linker, bases at positions 436-777 encode murine IL-15 (excluding signal sequences and propeptides), bases at positions 778-852 form the sequence encoding the second F2A, and bases at positions 856- 1179 form the coding sequence for murine CCL19 SEQ ID NO: 11 is the amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-1179 of DNA fragment #6, self-cleaves at two F2A sites and contains _sushi IL15.

B: production stage.

CAR-T cells. Generation of T cells containing CAR _sushi IL15_CCL19 against human CD20.

The retrovirus-containing drug was produced in step 1 in the same manner as in the CAR-T cell production step in Example 1-1, except that the CAR _sushi IL15_CCL19 anti-human CD20 expression vector obtained in the above-mentioned vector production step was used as a retroviral expression vector instead of the CAR IL-15 _lsp _CCL19 anti-human CD20 expression vector to obtain murine T cells expressing the CAR anti-human CD20, _sushi IL15 and CCL19 genes contained in the retrovirus (T cells containing CAR. _sushi IL15_CCL19 against human CD20, hereafter referred to as _sushi IL15χCCL19-CAR-T cells) in stage 2.

Example 2. IL-18χCCL19-CAR-T cells.

A: Stage of obtaining a vector.

Generation of CAR IL-18_CCL19 anti-human CD20 expression vector.

Using the same DNA fragment #1 and DNA fragment #2 as used in step 1 of Example 1-1, and DNA fragment #7 as described below instead of DNA fragment #3, a construct was prepared in the same manner as described in step 2 of Example 1-1, to obtain a retroviral expression vector pMSGV containing CAR_F2A_IL-18_F2A_CCL19 anti-human CD20 (anti-human CD20 CAR_IL-18_CCL19 expression vector).

DNA fragment #7: DNA fragment for IL-18 and CCL19.

A DNA fragment was artificially synthesized containing the nucleotide sequence encoding the first F2A, mouse IL-18, the second F2A and mouse CCL19. SEQ ID NO: 12 (FIG. 13) is the nucleotide sequence of full-length DNA fragment #7. In SEQ ID NO: 12, the bases at positions 7-81 form the sequence encoding the first F2A, the bases at positions 82-657 form the sequence encoding murine IL-18, the bases at positions 657-732 form the sequence encoding the second F2A, and the bases in positions

- 21 045607

736-1059 form the coding sequence for murine CCL19. SEQ ID NO: 13 is the amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-1059 of DNA fragment #7, and self-cleaves at two F2A sites.

B: production stage.

CAR-T cells. Generation of T cells containing CAR IL-18_CCL19 against human CD20.

The retrovirus-containing drug was prepared in step 1 in the same manner as in the CAR-T cell production step in Example 1-1, except that the anti-human CD20 CAR expression vector IL18_CCL19 obtained in the above-mentioned vector preparation step was used as a retroviral expression vector instead of the CAR IL15 _lsp _CCL19 anti-human CD20 expression vector to produce murine T cells expressing the CAR anti-human CD20, IL-18 and CCL19 genes contained in the retrovirus (T cells containing CAR IL18_CCL19 anti-human CD20, hereafter called IL-18χCCL19-CAR-T cells) in stage 2.

Example 3. IL-21χCCL19-CAR-T cells.

A: vector obtaining stage.

Generation of CAR IL-21_CCL19 anti-human CD20 expression vector.

Using the same DNA fragment #1 and DNA fragment #2 as used in step 1 of Example 1-1, and DNA fragment #8 as described below instead of DNA fragment #3, a construct was prepared in the same manner as described in step 2 of example 1-1, to obtain a retroviral expression vector pMSGV containing CAR_F2A_IL-21_F2A_CCL19 against human CD20 (expression vector CAR_IL-21_CCL19 against human CD20).

DNA fragment #8: DNA fragment for IL-21 and CCL19.

A DNA fragment was artificially synthesized containing the nucleotide sequence encoding the first F2A, mouse IL-21, the second F2A and mouse CCL19. SEQ ID NO: 14 (FIG. 14) is the nucleotide sequence of full-length DNA fragment #8. In SEQ ID NO: 14, the bases at positions 7-81 form the sequence encoding the first F2A, the bases at positions 82-567 form the sequence encoding murine IL-21, the bases at positions 658-642 form the sequence encoding the second F2A, and the bases at positions 646-969 form the coding sequence for murine CCL19. SEQ ID NO: 15 is the amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-969 of DNA fragment #8, and self-cleaves at two F2A sites.

B: CAR-T cell production stage.

Generation of T cells containing CAR IL21_CCL19 against human CD20.

The retrovirus-containing drug was produced in step 1 in the same manner as in the CAR-T cell production step in Example 1-1, except that the anti-human CD20 CAR IL-21_CCL19 expression vector obtained in the above-mentioned vector production step , was used as a retroviral expression vector instead of the CAR IL15 _lsp _CCL19 anti-human CD20 expression vector to obtain murine T cells expressing the CAR anti-human CD20, IL-21 and CCL19 genes contained in the retrovirus (T cells containing anti-human CAR IL21_CCL19 CD20 cells, hereafter referred to as IL-21χCCL19-CAR-T cells) in stage 2.

Example 4. _SC IL27χCCL19-CAR-T cells.

A: vector obtaining stage.

Generation of the CAR _sc expression vector IL27_CCL19 against human CD20.

Using the same DNA fragment #1 and DNA fragment #2 as used in step 1 of Example 1-1, and DNA fragment #9 as described below instead of DNA fragment #3, a construct was prepared in the same manner as described in step 2 of example 1-1, to obtain a retroviral expression vector pMSGV containing CAR_F2A_ _sc IL-27_F2A_CCL19 against human CD20 (expression vector CAR_ _sc IL27_CCL19 against human CD20).

DNA fragment #9: DNA fragment for SCIL27 and cCl19.

A DNA fragment was artificially synthesized containing a nucleotide sequence encoding the first F2A, the mouse IL-27 construct SCIL27 (a hybrid peptide consisting of p28, a linker and EBI3), the second F2A and CCL19. SEQ ID NO: 16 (FIG. 15) is the nucleotide sequence of full-length DNA fragment #9. In SEQ ID NO: 16, the bases at positions 781 form the first F2A coding sequence, the bases at positions 82-1437 form the SCIL27 coding sequence (among them, the bases at positions 82-765 encode EBI3, the bases at positions 766-819 serve in as a linker, bases at positions 820-1437 encode murine p28), bases at positions 1438-1512 form the sequence encoding the second F2A, and bases at positions 1516-1839 form the sequence encoding murine CCL19. SEQ ID NO: 17 is the amino acid sequence of the full-length fusion protein, which is expressed by nucleotide sequence at positions 7-1839 of DNA fragment #9, self-cleaves at two F2A sites and contains ScIL27.

B: CAR-T cell production stage.

Generation of T cells containing CAR _sc IL27_CCL19 against human CD20.

The retrovirus-containing drug was produced in step 1 in the same manner as in the CAR-T cell production step in Example 1-1, except that the anti-human CD20 CAR _sc IL27_CCL19 expression vector obtained in the above-mentioned vector production step was used as a retroviral expression vector instead of the CAR IL-15 _lsp _CCL19 anti-human CD20 expression vector to obtain murine T cells expressing the CAR anti-human CD20, _SC IL27 and CCL19 genes contained in the retrovirus (T cells containing anti-human CAR SCIL27_CCL19 CD20 cells, hereafter referred to as _SC IL27χCCL19-CAR-T cells) in stage 2.

Comparative Example 1: T cells without transduction(-).

Mouse T cells that were only activated (hereinafter referred to as non-transduced (-) T cells) were obtained by the same method, except that retroviral vector production and transfection into T cells using the retroviral vector were not performed, and Instead of preparing the anti-human CD20 CAR IL15 _lsp _CCL19 expression vector preparation at stage 2, in Example 1-1, an equivalent amount of DMEM medium used for culturing GP2-293 cells was added.

Comparative Example 2: Transformed CAR-T cells.

A: Stage of obtaining a vector.

Preparation of anti-human CD20 CAR expression vector.

A retroviral expression vector pMSGV containing CAR_MCS against human CD20 was prepared using DNA fragment 1, DNA fragment 2 and the retroviral vector pMSGV according to the procedure described in the middle of stage 2 of stage A of Example 1-1. This vector was used as a control retroviral expression vector (anti-human CD20 CAR expression vector) that does not express cytokines and chemokines.

B: CAR-T cell production stage.

Generation of T cells containing CAR against human CD20.

The retrovirus preparation was produced in Step 1 in the same manner as in the CAR-T cell production step in Example 1-1, except that the anti-human CD20 CAR expression vector was used as the retroviral expression vector instead of the anti-human CD20 expression vector. vector CAR IL-15 _lsp _CCL19 anti-human CD20 to produce murine T cells expressing only CAR anti-human CD20 contained in a retrovirus (not expressing cytokines and chemokines) (T cells containing CAR anti-human CD20, hereinafter referred to as converted CAR- T cells) at stage 2.

Experimental example 1.

Confirmation of the expression level of the administered CAR, cytokine and chemokine.

A: assessment of CAR expression level by flow cytometry.

Using the T cells obtained in the above Examples and Comparative Examples, the cell surface expression level of anti-human CD20 CAR was analyzed by flow cytometry as described below.

A BD FACSCantoTM II flow cytometer (BD Biosciences) was used for flow cytometry, and FlowJo computer software (BD Biosciences) was used for data analysis.

T cells were treated with biotin-labeled protein L (which specifically binds to the light chain of the anti-human CD20 scFv) and Brilliant Violet™ 421-linked streptavidin (BV421) to detect CAR expression. At the same time, the number of CD8-positive cells in each T-cell population was assessed using an anti-mouse CD8 allophycocyanin-linked (APC) monoclonal antibody (BioLegend). Since CD8-positive T cells are generally believed to have direct cytotoxic activity, the presence (positive indicator) of CD8-positive T cells can confirm their activity.

In fig. 1 shows the results. It was confirmed that 60% or more of the cell population of CART cells in all examples (and in Comparative Example 2) expressed CAR against human CD20 (ie, were positive for this CAR).

B: assessment of the amount of secreted cytokines and chemokines.

T cell culture supernatants were collected 42 h post-transduction, and concentrations of IL-15, IL-18, IL-21, IL-27, and CCL19 were then determined using a commercially available ELISA kit (available from MBL for IL-18 and R&D Systems for others).

In fig. Figure 2 shows the results. In the case of IL-15 (Fig. 2A), IL-15 was detected at a concentration of 250 pg/ml in the supernatant of the CAR-T cell culture of Example 1-1 (15LSPx19 CAR-T). Meanwhile, the concentrations of IL-15 in the CAR-T cell culture supernatants of Example 1-2 (s15RAx19 CAR-T), Example 1-3 (mb15RAx19 CAR-T) and Example 1-4 (sushi 15x19 CAR-T) were similar concentrations for non-transduced activated T cells (no transduction (-): comparative example 1) and T cells, ex

- 23 045607 pressing CARs against human CD20 (transformed CAR-T: comparative example 2) and used as a control, while no secretion of IL-15 was observed using the ELISA kit used in experimental example 1. However, since example 1-2, Example 1-3 and Example 1-4 showed excellent cell proliferation effect in Experimental Example 2, which will be described below, it should be noted that IL-15 was expressed and secreted in these Examples as well as in Example 1-1 .

In the case of IL-18 (Fig. 2B), IL-18 was detected at a concentration of 150 pg/ml or more in the supernatant of the CAR-T cell culture of Example 2 (18x19 CAR-T). Meanwhile, the amount secreted from T cells expressing anti-human CD20 CAR (transduced CAR-T: comparative example 2) and used as a control in the culture supernatant was as small as in the case of non-transduced activated T cells ( without transduction (-): comparative example 1).

In the case of IL-21 (Fig. 2C), IL-21 was detected at a concentration of 400 pg/ml or more in the CAR-T cell culture supernatant of Example 3 (21x19 CAR-T). Meanwhile, the amount secreted from T cells expressing anti-human CD20 CAR (transduced CAR-T: Comparative Example 2) and used as a control was below the detection limit (not detected), as in the case of non-transduced activated T cells (without transduction(-): comparative example 1).

In the case of IL-27 (Fig. 2D), p28, which is a subunit of IL-27, was detected at a concentration of 250 pg/ml or more from the CAR-T cell culture supernatant of Example 4 (sc27x19 CAR-T). Meanwhile, the amount secreted from T cells containing anti-human CD20 CAR (transduced CAR-T: Comparative Example 2) and used as a control was the same as the amount secreted from non-transduced activated T cells (no transduction ( -): Comparative Example 1) (at a concentration of approximately 50 pg/ml in the culture supernatant).

In the case of CCL19 (Fig. 2E), although the concentrations in culture supernatants of T cells containing anti-human CD20 CAR (transduced CAR-T: comparative example 2) and used as a control, and non-transduced activated T cells (no transduction (- ): Comparative Example 1) were below the detection limit, however, the concentrations in the CAR-T cell culture supernatant of these examples ranged from 150 to 500 pg/ml.

Experimental example 2. Assessment of the proliferative capacity of CAR-T cells in vitro.

Using T cells prepared as described above in the Examples and Comparative Examples, an assay was performed to determine whether each cytokine produced by the T Cells (IL-15, IL-18, IL-21, and IL-27) biological functions and immune-inducing effects by measuring the number and proliferative capacity of CAR-T cells as outlined below.

T cells were stained with CytoTellTM UltraGreen (AAT Bioquest) and then cocultured in the same well as P815 mastocytoma (hCD20/P815), treated with mitomycin C, and genetically transformed to express human CD20, followed by stimulation. After culture for 3 days, 5 days, or 7 days after the start of stimulation, cells were harvested and analyzed by flow cytometry. Cells were stained with APC-linked anti-Thy1.2 monoclonal antibody (eBioscience), and the number of Thy1.2-positive cells in the T cell (lymphocyte) population was counted as the number of surviving T cells.

In fig. 3 presents the results. Non-transduced (-) T cells (Comparative Example 1) were not stimulated to activate even when co-cultured with hCD20/P815 cells, and the number of living cells was 1/10 or less on day 3 of culture. Meanwhile, on the 3rd day of culture, it was confirmed that the level of live cell proliferation was equivalent to or higher than that of the transformed CAR-T cells (Comparative Example 2) used as control and was maintained in T cells containing CAR IL15 _LSP xCCL19 (example 1-1), in T cells containing CAR _S IL15 _RA xCCL19 (example 1-2), in T cells containing CAR _mb IL15 _RA xCCL19 (example 1-3), in T cells containing CAR _sushi IL15xCCL19 (Example 1-4), in T cells containing CAR IL-18x CCL19 (Example 2), in T cells containing CAR IL-21xCCL19 (Example 3), and in T cells containing CAR _SC IL27xCCL19 (Example 4), which were co-cultured with hCD20/P815 cells. Additionally, if culture was continued until day 7, CAR _S IL15 _RA xCCL19 T cells (Example 1-2), CAR _mb IL15 _RA xCCL19 CAR T cells (Example 1-3), and CAR T cells _sushi IL15xCCL19 (example 1-4) generally maintained a high level of proliferation. T cells containing CAR IL-15 _lsp xCCL19 (Example 1-1), T cells containing CAR IL18xCCL19 (Example 2), T cells containing CAR IL-21xCCL19 (Example 3), and T cells containing The IL27xCCL19 CAR _SC (Example 4) maintained a cell number equivalent to the number of converted CAR-T cells (Comparative Example 2) used as a control on culture days 3-7.

In addition, histograms were constructed based on the analysis of the intensity of staining with the CytoTell reagent in the population of Thy1.2-positive cells. In fig. Figure 4 shows the results for each cell population 5 days after the start of stimulation. In T cells containing CAR IL15 _LSP xCCL19 (example 1-1), in T cells containing CAR _S IL15 _RA xCCL19 (example 1-2), in T cells containing CAR _mb IL15 _RA x CCL19 (example 1 -3), in T cells containing CAR _sushi IL15xCCL19 (example 1-4), in T cells,

- 24 045607 containing CARIL-18xCCL19 (example 2), in T cells containing CARIL-21 xCCL19 (example 3), the number of cell populations of the second generation (after one division) decreased significantly, and the number of cell populations after the fifth generation (after four or more divisions) increased compared to the transformed CAR-T cells (Comparative Example 2) used as a control.

Based on the results presented in Fig. 3 and fig. 4, it was revealed that combinations of IL-15/IL-15Ra and CCL19 fusion protein produced in T cells containing CAR SIL15 _RA xCCL19 (Example 12) in T cells containing CAR _mb IL15 _RA xCCL19 (Example 1- 3), and in T cells containing CAR _sushi IL15xCCL19 (Example 1-4), are particularly preferred for promoting CAR-T cell survival and proliferation and biological functions.

Experimental example 3. Evaluation of the antitumor cytotoxic activity of CAR-T cells.

Using T cells obtained as described above in the Examples and Comparative Examples, antitumor cytotoxic activity specific to the target antigen was evaluated.

A: assessment of antitumor cytotoxic activity using flow cytometry.

P815 mastocytomas (hCD20/P815), genetically transformed to express human CD20, were used as target tumor cells, and P815 mastocytomas (P815), which were not genetically transformed as above, were used as control tumor cells.

After collecting target tumor cells or control tumor cells and seeding them on a culture plate, CAR-T cells of examples (T cells containing CAR IL15 _LSP xCCL19 (Example 1-1), T cells containing CAR _S IL15 _RA x CCL19 (example 1-2), T cells containing CARm _b IL15 _RA xCCL19 (example 1-3), T cells containing CAR _sushi IL15xCCL19 (example 1-4), T cells containing CAR IL-18x CCL19 (Example 2), T cells containing CAR IL-21xCCL19 (Example 3), and T cells containing CAR _SC IL27xCCL19 (Example 4)), or CAR-T cells of Comparative Example 2 (transformed CAR-T cells) were added as effector T cells, and these cells were co-cultured for 72 hours. Effector T cells were added so that the number of CAR-positive cells was 1/3 of the target tumor cells (E:T ratio = 1 :3), and the total numbers of T cells seeded were chosen to be the same in each group by adding, according to the number of T cells seeded with the lowest level of CAR expression, non-transduced T cells, to the other T cells. cells. After coculture for 72 h, all cells were collected, analyzed by flow cytometry, and the number of living cells was determined. Cells were stained with APC-linked anti-Thy1.2 monoclonal antibody (eBioscience) and the Zombie Green Fixable Viability Kit (BioLegend), and the number of T cells and tumor cells in the living cell population was calculated from the number of Thy1 .2-positive cells. The number of living cells was determined using NucleoCounter NC-200 (Chemometec).

In fig. Figure 5 presents the results. As shown in FIG. 5A, the number of hCD20/P815 tumor cells after co-culture for 72 hours was calculated based on the level of Thy1.2-negative cells in living cells. As a result, the number of hCD20/P815 tumor cells co-cultured with CAR IL15 _LSP xCCL19 T cells (Example 1-1), CAR _S IL15 _RA xCCL19 T cells (Example 1-2), T cells containing CAR _mb IL15 _RA xCCL19 (example 1-3), T cells containing CARs _ushi IL15xCCL19 (example 1-4), T cells containing CAR IL-18xCCL19 (example 2), T cells containing CAR IL- 21x CCL19 (Example 3), and scIL27xCCL19 CAR-containing T cells (Example 4) were significantly reduced compared to non-transduced (-) T cells (Comparative Example 1), and also decreased compared to transduced CAR-T cells (Comparative Example 2), and the survival rate of tumor cells was 1% or less. Meanwhile, no reduction in the number of tumor cells, P815 cells not expressing human CD20, was observed in either group, as shown in FIG. 5B, and there was no evidence of cytotoxicity that is not specific to tumor cells not expressing human CD20.

B: assessment of IFNy production by ELISA assay.

As an index of activation of target antigen-specific cytotoxicity, the level of interferon γ (IFNγ) production by CAR-T cells was determined using a commercially available ELISA assay kit (R&D). CAR-T cells examples (T cells containing CAR IL15 _LSP xCCL19 (example 1-1), T cells containing CAR _S IL15 _RA xCCL19 (example 1-2), T cells containing CAR _mb IL15 _RA xCCL19 (example 1-3), T cells containing CAR _sushi IL15xCCL19 (example 1-4), T cells containing CAR IL-18xCCL19 (example 2), T cells containing CAR IL-21xCCL19 (example 3), and Cells containing CAR _SC IL27xCCL19 (Example 4)) or CAR-T cells of Comparative Example 2 (transformed CAR-T cells) were co-cultured with P815 cells (control tumor cells) or hCD20-P815 cells (target tumor cells ) to determine the quantity

- 25 045607

IFNy in culture supernatant 4 days after coculture.

In fig. Figure 6 shows the results. As shown in Figure 6A, the level of IFNy was higher than in the transformed CAR-T cells (Comparative Example 2), and approximately 15 ng/ml or more IFNy was detected in the culture supernatant of T cells containing CAR IL15 _lsp xCCL19 (Example 1 -1), T cells containing CAR _S IL15 _RA x CCL19 (example 1-2), T cells containing CAR _mb IL15 _RA xCCL19 (example 1-3), T cells containing CAR _sushi IL15xCCL19 (example 1 -4), T cells containing CAR IL-18xCCL19 (Example 2), T cells containing CAR IL-21xCCL19 (Example 3), and T cells containing CAR scIL27xCCL19 (Example 4), which were co-cultured with the cells hCD20-P815. The level of IFNy produced in groups co-cultured with non-transduced (−) T cells (Comparative Example 1) was below the detection limit. Meanwhile, the concentration of IFNy in the culture supernatant of CAR-expressing cells co-cultured with P815 cells was 10 pg/ml or less, as shown in FIG. 6B.

From the above results of Experimental Example 3 (FIG. 5 and FIG. 6), it was confirmed that all T cells of Examples and Comparative Example 2 were cells expressing human CD20, that is, they had target antigen-specific cytotoxic activity .

Experimental Example 4: Therapeutic effect on a mouse tumor model.

The therapeutic effect of T cells obtained as described in Example 1-3, Example 1-4 and Comparative Example 2 in a murine melanoma tumor graft model or in a murine colorectal cancer model was evaluated using the cancer-bearing mice described below. tumor.

Therapeutic effect in a murine melanoma transplant model.

5x105 B16F1O murine melanoma cells genetically transformed to express human CD20 (B16F10-hCD20) were inoculated subcutaneously into C57BL/6N mice. The anticancer agent, cyclophosphamide (CPA, 50 mg/kg), was administered intraperitoneally on day 7 postinoculation, and 1x106 CAR-T cells of Example 1-3 (T cells containing CAR _mb IL15 _RA xCCL19), CAR-T cells of Example 1-4 (T cells containing CAR _sushi IL15xCCL19) or cells of comparative example 2 (transformed CAR-T cells) were administered intravenously on the 10th day. As control groups, a group was selected that was not treated with CAR-T and was administered only CPA, and a group that was inoculated with B16F10-hCD20 murine melanoma and was subsequently left untreated. Tumor volume in mice was measured twice a week.

In fig. 16 shows the results of tumor volume determination in a murine melanoma tumor graft model. The horizontal axis shows the number of days after inoculation, where the day when mice were subcutaneously inoculated with B16F10-hCD20 was taken as day 0, and the vertical axis shows tumor volume (large tumor diameter)χ(small tumor diameter) ² /2 (mm ³ )). The standard deviation was calculated for each experimental group. The term no treatment refers to the group that received no treatment, the term CPA refers to the group that received only CPA, and the term CPA+Conv. means the group that was administered CPA and then the cells of Comparative Example 2 (transformed CAR-T cells), the term CPA+ _mb IL15 _RA x19 means the group that was administered CPA and then the CAR-T cells of Example 1-3 (T -cells containing CAR _mb IL15 _RA xCCL19), and the term CPA+ _sushi IL15x19 means the group that was administered CPA and then administered CAR-T cells of Example 1-4 (T cells containing CARsushiIL15xCCL19).

As shown in FIG. 16, the effect of reducing tumor volume was confirmed in the groups that were administered CAR-T cells of Example 1-3 (T cells containing CAR _mb IL15 _RA xCCL19) and CAR-T cells of Example 1-4 (T cells containing CARsushiIL15xCCL19) compared with the groups that received the cells of Comparative Example 2 (transformed CAR-T cells), the untreated groups (no treatment), and the groups that received only CPA. Accordingly, it was found that the CAR T cells of Example 1-3 (T cells containing CAR _mb IL15 _RA xCCL19) and CAR T cells of Example 1-4 (T cells containing CAR sushiIL15xCCL19) had excellent antitumor activity in mice with the melanoma.

Therapeutic effect in a murine colorectal cancer transplant model.

5x105 murine colorectal cancer MC38 (MC38-hCD20) cells genetically transformed to express human CD20 were subcutaneously inoculated into C57BL/6N mice. The anticancer agent, cyclophosphamide (CPA, 50 mg/kg), was administered intraperitoneally on day 7 postinoculation, and 1x106 CAR-T cells of Example 1-3 (T cells containing CAR _mb IL15 _RA xCCL19), CAR-T cells of Example 1-4 (T cells containing CAR _sushi IL15xCCL19) or cells of Comparative Example 2 (transformed CAR-T cells) were administered intravenously on the 10th day. As control groups, a group was selected that was not treated with CAR-T and was administered only CPA, and a group that was inoculated with MC38-hCD20 murine colorectal cancer and was subsequently not treated. Tumor volume in mice was measured twice a week.

In fig. 17 shows the results of tumor volume determination in a murine colorectal cancer graft model. The horizontal axis shows the number of days after inoculation, where the day when mice were subcutaneously inoculated with MC38-hCD20 was taken as day 0, and the vertical axis shows tumor volume (large tumor diameter) x (small tumor diameter) ² /2 (mm ³ )). The standard deviation was calculated for each experimental group. The term no treatment refers to the group that received no treatment, the term CPA refers to the group that received only CPA, and the term CPA+Conv. means the group that was administered CPA and then the cells of Comparative Example 2 (transformed CAR-T cells), the term CPA+ _mb IL15 _RA x19 means the group that was administered CPA and then the CAR-T cells of Example 1-3 (T -cells containing CAR _mb IL15 _RA xCCL19), and the term CPA+ _sushi IL15x19 means the group that was administered CPA and then administered CAR-T cells of Example 1-4 (T cells containing CAR _sushi IL15xCCL19).

As shown in FIG. 17, the effect of reducing tumor volume was confirmed in the groups that were administered CAR-T cells of Example 1-3 (T cells containing CAR _mb IL15 _RA xCCL19) and CAR-T cells of Example 1-4 (T cells containing CARsushiIL15xCCL19) compared with the groups that received the cells of Comparative Example 2 (transformed CAR-T cells), the untreated groups (no treatment), and the groups that received only CPA. Accordingly, it was found that the CAR-T cells of Example 1-3 (T cells containing CAR _mb IL15 _RA xCCL 19) and CAR-T cells of Example 1-4 (T cells containing CAR sushiIL15xCCL19) had excellent antitumor activity in mice with a model of colorectal cancer.

In the above examples and experimental examples, murine T cells, a cytokine (fusion protein with IL-15 linked to IL-15Ra or the like) and a chemokine (CCL19), each containing a native murine amino acid sequence, were used and in vivo tests in mice. However, it will be apparent to those skilled in the art that in embodiments of the invention also related to mammals other than mice, and preferably to humans, that is, in the case of human T cells, the cytokine (IL-15 fusion protein associated with IL-1 15Ra or the like) or a chemokine (CCL19), each of which contains a human natural amino acid sequence or when performing in vivo tests on humans, the present invention can be carried out in the same manner for the purpose of detecting the action and effect of the invention.

Thus, for example, a fusion protein containing IL-15 _lsp that was prepared (produced and used) using the native murine amino acid sequence shown in SEQ ID NO: 5 can be produced using the amino acid sequences at positions 1-29 (part signal peptide) and 49-162 (part of IL-15) in the natural human amino acid sequence for IL-15, as shown in SEQ ID NO: 18, or using nucleic acids having a nucleotide sequence encoding such amino acid sequences.

A fusion protein containing IL15Ra _s , which was produced using the native murine amino acid sequence shown in SEQ ID NO: 7, can be produced using the amino acid sequence at positions 49-162 (part of IL-15) in the native human amino acid sequence for IL -15, as shown in SEQ ID NO: 18, and using the amino acid sequence at positions 31-205 (extracellular domain of IL-15Ra) in the natural human amino acid sequence for IL-15Ra, as shown in SEQ ID NO: 19, or with using nucleic acids having a nucleotide sequence encoding such amino acid sequences.

A fusion protein containing mbIL15RA, which was produced using the native murine amino acid sequence shown in SEQ ID NO: 7, can be produced using the amino acid sequence at positions 49-162 (part of IL-15) in the native human amino acid sequence for IL-15. 15, as shown in SEQ ID NO: 18, and using the amino acid sequence at positions 1-267 (full-length IL-15Ra) in the natural human amino acid sequence for IL-15Ra, as shown in SEQ ID NO: 19, or using nucleic acid acids having a nucleotide sequence encoding such amino acid sequences.

A fusion protein containing _sushi IL15, which was produced using the native murine amino acid sequence shown in SEQ ID NO: 11, can be prepared using the amino acid sequence at positions 49-162 (part of IL-15) in the native human amino acid sequence for IL -15, as shown in SEQ ID NO: 18, and using the amino acid sequence at positions 31-95 (sushi domain) in the natural human amino acid sequence for IL-15Ra, as shown in SEQ ID NO: 19, or using nucleic acids having a nucleotide sequence encoding such amino acid sequences.

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Claims (14)

Industrial Application The CAR-T cells of the invention, which have excellent persistence and proliferation, are suitable for preparing a drug for treating cancer or the like. In addition, the expression vector according to the invention is suitable for the production of CAR-T cells. List of sequences in free format. SEQ ID NO: 1: Nucleotide sequence of the DNA fragment containing the anti-human CD20 CAR (DNA fragment #1). SEQ ID NO: 2: Amino acid sequence of the fusion protein expressed by the nucleotide sequence at positions 3-1637 of DNA fragment #1. SEQ ID NO: 3: Nucleotide sequence of the MCS DNA fragment encoding stop codons and restriction enzyme sites (DNA fragment #2). SEQ ID NO: 4: Nucleotide sequence of DNA fragment IL15lsp_F2A_CCL19 (DNA fragment #3). SEQ ID NO: 5: The amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-969 of DNA fragment #3, self-cleaves at two F2A sites and contains IL15lsp. SEQ ID NO: 6: Amino acid sequence of DNA fragment sIL15ra_F2A_CCL19 (DNA fragment #4). SEQ ID NO: 7: The amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-1539 of DNA fragment #4, self-cleaves at two F2A sites and contains SIL15RA. SEQ ID NO: 8: Nucleotide sequence of DNA fragment mbIL15RA_F2A_CCL19 (DNA fragment #5). SEQ ID NO: 9: The amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-1713 of DNA fragment #5, self-cleaves at two F2A sites and contains mbIL15RA. SEQ ID NO: 10: Nucleotide sequence of DNA fragment sushiIL15_F2A_CCL19 (DNA fragment #6). SEQ ID NO: 11: The amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-1179 of DNA fragment #6, self-cleaves at two F2A sites and contains sushiIL15. SEQ ID NO: 12: Nucleotide sequence of DNA fragment IL-18_F2A_CCL19 (DNA fragment #7). SEQ ID NO: 13: The amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-1059 of DNA fragment #7, and self-cleaves at two F2A sites. SEQ ID NO: 14: Nucleotide sequence of DNA fragment IL-21_F2A_CCL19 (DNA fragment #8). SEQ ID NO: 15: The amino acid sequence of the full-length fusion protein that is expressed by the nucleotide sequence at positions 7-969 of DNA fragment #8, and self-cleaves at two F2A sites. SEQ ID NO: 16: DNA fragment scIL27_F2A_CCL19 (DNA fragment #9). SEQ ID NO: 17: The amino acid sequence of the full-length fusion protein, which is expressed by the nucleotide sequence at positions 7-1839 of DNA fragment #9, self-cleaves at two F2A sites and contains SCIL27. SEQ ID NO: 18: Amino acid sequence of native human IL-15 (full length, including signal peptide and propeptide portions), registered as UniProtKB-P40933. SEQ ID NO: 19: Amino acid sequence of native human IL-15Ra (full length, including signal peptide, sushi domain, extracellular domain, etc.), registered as UniProtKB-Q13261. CLAIM (1) a nucleic acid encoding a CAR; (1) chimeric antigen receptor (CAR); 1. T cell expressing: (2) a nucleic acid encoding an IL-15 fusion protein; and (3) a nucleic acid encoding CCL19. 2. The T cell of claim 1, wherein the fusion protein is a fusion protein containing IL-15LSP linked to IL-15 (IL15 _lsp ), a fusion protein containing the extracellular domain of IL-15Ra linked to IL-15 (sIL15 _RA ), a fusion protein containing IL-15 linked to full-length IL-15Ra ( _mb IL15 _RA ), or a fusion protein containing IL-15Ra including a signal peptide and a sushi domain linked to IL-15 ( _sushi IL15). - 28 045607 (2) interleukin-15 (IL-15) fusion protein; and (3) CC chemokine ligand 19 (CCL19). 3. The T cell of claim 2, wherein the fusion protein is a fusion protein comprising IL-15 linked to full-length IL-15Ra ( _mb IL15 _RA ) or a fusion protein comprising IL-15Ra including a signal peptide and a sushi domain , associated with IL-15 ( _sush jIL15). 4. Pharmaceutical composition containing a T cell according to claim 1. 5. Pharmaceutical composition according to claim 4 for use in the treatment of cancer. 6. A pharmaceutical composition for use according to claim 5, wherein the cancer is melanoma, Merkel cell cancer, 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 bowel cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma or leukemia. 7. Expression vector containing: 8. The expression vector of claim 7, wherein the fusion protein is a fusion protein containing IL-15LSP linked to IL-15 (IL15 _lsp ), a fusion protein containing the extracellular domain of IL-15Ra linked to IL-15 ( _s ILI5 _RA ), a fusion protein containing IL-15 linked to full-length IL-15Ra ( _mb ILI5 _RA ), or a fusion protein containing IL-15Ra including a signal peptide and a sushi domain linked to IL15 (sus _b iIL15). 9. The expression vector of claim 7, wherein the fusion protein is a fusion protein containing IL-15 linked to full-length IL-15Ra ( _mb ILI5 _RA ), or a fusion protein containing IL-15Ra including a signal peptide and a sushi domain, associated with IL-15 ( _sush jIL15). 10. A method for producing a CAR-T cell, comprising the step of introducing the expression vector according to claim 7 into the T cell. 11. A method for treating cancer, comprising the step of administering the T cell according to claim 1 to an individual in need of treatment for cancer. 12. The method of treatment according to claim 11, where the cancer is melanoma, Merkel cell cancer, 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 bowel cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia , multiple myeloma, lymphoma or leukemia. 13. Use of a T cell according to claim 1 for the preparation of a pharmaceutical composition for the treatment of cancer. 14. Use according to claim 13, wherein the cancer is melanoma, Merkel cell cancer, colorectal cancer, kidney cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, non-small cell cancer lung, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma or leukemia. -