Method for producing genetically modified immunocompetent cells

Abstract

The present disclosure addresses the problem of providing a novel method for producing immunocompetent cells in which a retrovirus vector is used to perform transduction.　Carried out in the present invention is, for example, a method for producing genetically modified immunocompetent cells, said method comprising a transduction step for performing transduction to an immunocompetent cell with a retrovirus vector, said method substantially not comprising an expansion culturing step after the transduction step or comprising an expansion culturing step of 24 hours or less after the transduction step.

Description

Method for manufacturing genetically modified immune cells

[0001] This disclosure relates to a method for producing genetically modified immune cells.

[0002] In recent years, genetically modified immune cells such as T cells (hereinafter referred to as genetically modified immune cells) have been used in the treatment of malignant tumors. For example, Patent Document 1 discloses T cells that express chimeric antigen receptor (CAR), interleukin 7 (IL-7), and chemokine (C-C motif) ligand 19 (CCL19).

[0003] International Publication No. 2016 / 056228

[0004] Genetically modified immune cells are typically manufactured using a method that includes activating the immune cells, introducing foreign genes into the activated immune cells using a vector, and expanding the culture of the genetically modified immune cells. Generally, expanding the culture of the immune cells for a sufficient period after gene introduction is considered a necessary step in order to produce a therapeutically effective amount of immune cells.

[0005] One embodiment of this disclosure aims to provide a novel method for producing immune cells by transduction using a retroviral vector.

[0006] This disclosure includes the following aspects:

[0007] <1> A method for producing genetically modified immune cells, comprising a transduction step in which immune cells are transduced using a retroviral vector, and substantially not comprising an expansion culture step after the transduction step.

[0008] <2> The method for producing the retroviral vector according to <1>, wherein the retroviral vector includes at least one exogenous gene selected from the group consisting of genes for expressing cytokines, genes for expressing chemokines, and genes for expressing cell surface molecules that recognize malignant tumor antigens.

[0009] <3> The production method according to <2>, wherein the cell surface molecule is at least one selected from the group consisting of a chimeric antigen receptor (CAR) and a T cell receptor (TCR).

[0010] <4> The production method according to <2> or <3>, wherein the cytokine is at least one selected from the group consisting of IL-7, IL-15, and IL-21.

[0011] <5> The production method according to any one of <2> to <4>, wherein the chemokine is at least one of CCL19 and CCL21.

[0012] <6> The production method according to any one of <1> to <5>, wherein the genetically modified immune cell is at least one selected from the group consisting of a T cell, an NK cell, a B cell, a monocyte, a macrophage, a dendritic cell, a neutrophil, an eosinophil, a basophil, and a mast cell.

[0013] <7> The production method according to any one of <1> to <6>, wherein the proportion of stem cell memory immune cells in the CAR(+), CD8(+) cell group of the produced genetically modified immune cell is 60% or more.

[0014] <8> The production method according to any one of <1> to <7>, wherein the PD-1 expression rate in the CD4(+) cell group of the produced genetically modified immune cell is 10% or more.

[0015] <9> The production method according to any one of <1> to <8>, wherein the PD-1 expression rate in the CD8(+) cell group of the produced genetically modified immune cell is 4% or more.

[0016] <10> The production method according to any one of <1> to <9>, wherein the TIGIT expression rate in the CD4(+) cell group or the CD8(+) cell group of the produced genetically modified immune cell is 5% or more.

[0017] <11> The production method according to any one of <1> to <10>, wherein the LAG3 expression rate in the CD4(+) cell group or the CD8(+) cell group of the produced genetically modified immune cell is 5% or more.

[0018] <12> A manufacturing method according to any one of <1> to <11>, comprising an activation step of activating the immune cells before the transduction step, wherein the activation time is 24 hours or more and 120 hours or less.

[0019] <13> A pharmaceutical composition for cancer treatment comprising: (1) gene-modified immune cells transduced into immune cells by a retroviral vector, wherein (1) the gene-modified immune cells having a PD-1 expression rate of 10% or more in a CD4(+) cell group; (2) the gene-modified immune cells having a PD-1 expression rate of 4% or more in a CD8(+) cell group; (3) the gene-modified immune cells having a TIGIT expression rate of 5% or more in a CD4(+) cell group or a CD8(+) cell group; or (4) the gene-modified immune cells having a LAG3 expression rate of 5% or more in a CD4(+) cell group or a CD8(+) cell group; and a pharmaceutically acceptable additive.

[0020] This disclosure provides a novel method for producing immune cells by transduction using retroviral vectors.

[0021] The upper left of Figure 1 (1A) shows the CAR expression rate results for anti-GM2 CAR-IL-7 / CCL19 expressing T cell A in the example. The upper right of Figure 1 (1B) shows the CAR expression rate results for anti-GM2 CAR-IL-7 / CCL19 expressing T cell B in the example. The lower left of Figure 1 (1C) shows the CAR expression rate results for anti-GM2 CAR-IL-7 / CCL19 expressing T cell a in the example. The lower right of Figure 1 (1D) shows the CAR expression rate results for anti-GM2 CAR-IL-7 / CCL19 expressing T cell b in the example. The upper left of Figure 2 (2A) shows the CAR expression rate results for anti-EGFRviii CAR-IL-7 / CCL19 expressing T cell A in the example. The upper right of Figure 2 (2B) shows the CAR expression rate results for anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell B in the example. The lower left of Figure 2 (2C) shows the CAR expression rate results for anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell a in the example. The lower right of Figure 2 (2D) shows the CAR expression rate results for anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell b in the example. The left of Figure 3 (3A) shows the percentage of CAR(+) cells in the CD4(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell A produced in the example. The right side of Figure 3 (3B) shows the proportion of CD4(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19-expressing T cell A produced in the example. The left side of Figure 4 (4A) shows the proportion of CAR(+) cells in the CD4(+) cell group of anti-GM2 CAR-IL-7 / CCL19-expressing T cell B produced in the example. The right side of Figure 4 (4B) shows the proportion of CD4(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19-expressing T cell B produced in the example. The left side of Figure 5 (5A) shows the percentage of CAR(+) cells in the CD4(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cells a prepared in the example.The right side of Figure 5 (5B) shows the proportion of CD4(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19-expressing T cell a produced in the example. The left side of Figure 6 (6A) shows the proportion of CAR(+) cells in the CD4(+) cell group of anti-GM2 CAR-IL-7 / CCL19-expressing T cell b produced in the example. The right side of Figure 6 (6B) shows the proportion of CD4(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19-expressing T cell b produced in the example. The left side of Figure 7 (7A) shows the proportion of CAR(+) cells in the CD4(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell A produced in the example. The right side of Figure 7 (7B) shows the proportion of CD4(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell A produced in the example. The left side of Figure 8 (8A) shows the proportion of CAR(+) cells in the CD4(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell B produced in the example. The right side of Figure 8 (8B) shows the proportion of CD4(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell B produced in the example. The left side of Figure 9 (9A) shows the proportion of CAR(+) cells in the CD4(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell a produced in the example. The right side of Figure 9 (9B) shows the proportion of CD4(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell a produced in the example. The left side of Figure 10 (10A) shows the percentage of CAR(+) cells in the CD4(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells b prepared in the example.The right side of Figure 10 (10B) shows the proportion of CD4(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell b produced in the example. The left side of Figure 11 (11A) shows the proportion of CAR(+) cells in the CD8(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell A produced in the example. The right side of Figure 11 (11B) shows the proportion of CD8(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD8(+) CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell A produced in the example. The left side of Figure 12 (12A) shows the proportion of CAR(+) cells in the CD8(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell B produced in the example. The right side of Figure 12 (12B) shows the proportion of CD8(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD8(+) CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell B produced in the example. The left side of Figure 13 (13A) shows the proportion of CAR(+) cells in the CD8(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell a produced in the example. The right side of Figure 13 (13B) shows the proportion of CD8(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD8(+)CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell a produced in the example. The left side of Figure 14 (14A) shows the proportion of CAR(+) cells in the CD8(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell b produced in the example. The right side of Figure 14 (14B) shows the proportion of CD8(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD8(+)CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell b produced in the example. The left side of Figure 15 (15A) shows the percentage of CAR(+) cells in the CD8(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell A prepared in the example.The right side of Figure 15 (15B) shows the proportion of CD8(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD8(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell A produced in the example. The left side of Figure 16 (16A) shows the proportion of CAR(+) cells in the CD8(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell B produced in the example. The right side of Figure 16 (16B) shows the proportion of CD8(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD8(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell B produced in the example. The left side of Figure 17 (17A) shows the proportion of CAR(+) cells in the CD8(+) cell group of anti-EGFRviiiii CAR-IL-7 / CCL19 expressing T cell a prepared in the example. The right side of Figure 17 (17B) shows the proportion of CD8(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD8(+) CAR(+) cell group of anti-EGFRviiiii CAR-IL-7 / CCL19 expressing T cell a prepared in the example. The left side of Figure 18 (18A) shows the proportion of CAR(+) cells in the CD8(+) cell group of anti-EGFRviiiii CAR-IL-7 / CCL19 expressing T cell b prepared in the example. The right side of Figure 18 (18B) shows the proportion of CD8(+), CAR(+), CD45RA(+), and human CCR7(+) cells in the CD8(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell b produced in the example. The left side of Figure 19 (19A) shows the PD-1 expression rate in the CD4(+), CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell B produced in the example. The right side of Figure 19 (19B) shows the PD-1 expression rate in the CD4(+), CAR(+) cell group of anti-GM2 CAR-IL-7 / CCL19 expressing T cell b produced in the example. The left side of Figure 20 (20A) shows the PD-1 expression rate in the CD8(+) and CAR(+) cell groups within anti-GM2 CAR-IL-7 / CCL19 expressing T cell B prepared in the example.The right side of Figure 20 (20B) shows the PD-1 expression rate in the CD8(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19-expressing T cell b prepared in the example. The left side of Figure 21 (21A) shows the TIGIT expression rate in the CD4(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19-expressing T cell B prepared in the example. The right side of Figure 21 (21B) shows the TIGIT expression rate in the CD4(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19-expressing T cell b prepared in the example. The left side of Figure 22 (22A) shows the TIGIT expression rate in the CD8(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19-expressing T cell B prepared in the example. The right side of Figure 22 (22B) shows the TIGIT expression rate in the CD8(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19-expressing T cell b prepared in the example. The left side of Figure 23 (23A) shows the LAG3 expression rate in the CD4(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19-expressing T cell B prepared in the example. The right side of Figure 23 (23B) shows the LAG3 expression rate in the CD4(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19-expressing T cell b prepared in the example. The left side of Figure 24 (24A) shows the LAG3 expression rate in the CD8(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19-expressing T cell B prepared in the example. Figure 24 (right, 24B) shows the LAG3 expression rate in the CD8(+) and CAR(+) cell group in anti-GM2 CAR-IL-7 / CCL19 expressing T cells b prepared in the example. Figure 25 shows the results of the ATP assay in the example, measuring the antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells targeting GM2-expressing tumor cells. Figure 26 shows the results of the ATP assay in the example, measuring the antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells targeting tumor cells that do not express GM2. Figure 27 shows the results of the in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the example.Figure 28 shows the results of the in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the examples. Figure 29 shows the results of the in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the examples. Figure 30 shows the results of the in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the examples. Figure 31 shows the results of the in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the examples. Figure 32 shows the results of the in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the examples. Figure 33 shows the results of IFN-γ production by anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the example. Figure 34 shows the results of in vivo antitumor activity by anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the example. Figure 35 shows the results of in vivo antitumor activity by anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the example. Figure 36 shows the results of in vivo antitumor activity by anti-GM2 CAR-IL-7 / CCL19 expressing T cells in the example. Figure 37 shows the number of CD3-positive cells in the mouse spleen 90 days after administration of anti-GM2 CAR-IL-7 / CCL19 expressing T cells. Figure 38 shows the number of CD4-positive CAR-T cells and CD8-positive CAR-T cells in mouse spleens 90 days after administration of anti-GM2 CAR-IL-7 / CCL19 expressing T cells. Figure 39 shows the proportion of CD4(+) and CD8(+) cells in anti-GM2 CAR-IL-7 / CCL19 expressing T cell B. Figure 40 shows the CAR expression level in the CD4(+) cell group. Figure 41 shows the CAR expression level in the CD8(+) cell group. Figure 42 shows the proportion of CD4(+) and CD8(+) cells in anti-GM2 CAR-IL-7 / CCL19 expressing T cell b. Figure 43 shows the CAR expression level in the CD4(+) cell group. Figure 44 shows the CAR expression level in the CD8(+) cell group.Figure 45(1A) shows the CAR expression rate of anti-GM2 CAR-expressing T cell D, Figure 45(1B) shows the CAR expression rate of anti-GM2 CAR-IL-7 / CCL19-expressing T cell C, Figure 45(1C) shows the CAR expression rate of anti-GM2 CAR-expressing T cell d, and Figure 45(1D) shows the CAR expression rate of anti-GM2 CAR-IL-7 / CCL19-expressing T cell c. Figure 46 shows the IL-7 concentration in the culture supernatant. Figure 47 shows the CCL19 concentration in the culture supernatant. Figure 48 shows the proportion of human CD45RA(+) and human CCR7(+) cells in the CD4(+) CAR(+) cell population. Figure 49 shows the proportion of human CD45RA(+) and human CCR7(+) cells in the CD8(+) CAR(+) cell population. Figure 50 shows the IL-7Rα expression levels of anti-GM2 CAR-IL-7 / CCL19 expressing T cell C, anti-GM2 CAR-IL-7 / CCL19 expressing T cell c, anti-GM2 CAR expressing T cell D, and anti-GM2 CAR expressing T cell d. Figure 51 shows the results of in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cell C and others in the examples. Figure 52 shows the results of in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cell C and others in the examples. Figure 53 shows the results of IFN-γ concentration in the supernatant after in vitro culture of anti-GM2 CAR-IL-7 / CCL19 expressing T cell C and others in the examples. Figure 54 shows the results of in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, etc., in the examples. Figure 55 shows the results of in vitro antitumor activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, etc., in the examples. Figure 56 shows the results of immunostaining analysis of T cells infiltrating the tumor site. Figure 57 shows the results of immunostaining analysis of T cells infiltrating the tumor site. Figure 58 shows the results of immunofatigue resistance test-1. Figure 59 shows the results of immunofatigue resistance test-1. Figure 60 shows the results of immunofatigue resistance test-1. Figure 61 shows the results of immunofatigue resistance test-1. Figure 62 shows the results of immunofatigue resistance test-2. Figure 63 shows the results of immunofatigue resistance test-2. Figure 64 shows the results of immunofatigue resistance test-3. Figure 65 shows the results of the residual T cell count in relapse suppression trial-1.Figure 66 shows the results for the percentage of CCR7-positive cells among CAR-positive and CAR-negative residual T cells in relapse suppression study-1. Figure 67 shows the results of the rechallenge study against GM2-positive tumor Lu-135 in relapse suppression study-2. Figure 68 shows the average tumor volume results for each group. Figure 69 shows the results of the rechallenge study against GM2-negative SW480 in relapse suppression study-2. Figure 70 shows the results for residual T cells in the spleen on Day 182. Figure 71 shows the results of the in vivo antitumor activity of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell E in the example. Figure 72 shows the results of the in vivo antitumor activity of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell e in the example. Figure 73 shows the proportion of CD8(+) and CAR(+) cells in anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell F. Figure 74 shows the proportion of CD8(+) and CAR(+) cells in anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell f. Figure 75 shows the proportion of human CD45RA(+) and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell F. Figure 76 shows the proportion of human CD45RA(+) and human CCR7(+) cells in the CD4(+) CAR(+) cell group of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell f. Figure 77 shows the proportion of human CD45RA(+) and human CCR7(+) cells in the CD8(+) CAR(+) cell population of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell F. Figure 78 shows the proportion of human CD45RA(+) and human CCR7(+) cells in the CD8(+) CAR(+) cell population of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell f. Figure 79 shows the results of the immune fatigue resistance test-4. Figure 80 shows the results of the in vivo antitumor activity of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell F and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell f in the example.Figure 81 shows the results of the in vivo antitumor activity of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f in the examples.

[0022] <Definitions> In this specification, "comprise" is used to mean "consist of". "Comprise" means that it may include components other than those that are included, while "consist of" means that it does not essentially include components other than those that are included. "Consisting only of" means that it does not include components other than those that are included.

[0023] In this specification, the "~" indicating a numerical range is used to mean that the numbers before and after it are included as the lower and upper limits. In this specification, in numerical ranges described in stages, the upper or lower limit stated in one numerical range may be replaced with the upper or lower limit of another numerical range described in stages. Also, in this specification, in numerical ranges described, the upper or lower limit of that numerical range may be replaced with the values ​​shown in the examples.

[0024] In this specification, “subject” can be a mammal, for example, a rodent (e.g., mouse and rat), a dog, a cat, a cow, a horse, a pig, or a primate (e.g., monkey, gorilla, orangutan, bonobo, chimpanzee, and human), for example, a human.

[0025] In this specification, "substantially does not include a bulk culture step" means that bulk culture is not performed with the intention of doing so, and if the immune cells are inevitably bulk cultured, this is included in "substantially does not include a bulk culture step". Bulk culture of immune cells usually refers to a process that is carried out for 3 to 7 days, but in this specification, "bulk culture step" refers to a step in which a foreign gene is introduced into immune cells, and then the transduced immune cells are cultured for more than 24 hours, and the number of cells is more than double that of before culture. The following steps are not included in the above bulk culture step: a step in which a foreign gene is introduced into immune cells, and then the transduced immune cells are cultured for more than 24 hours, and the number of cells is no more than double that of before culture; a step in which transduced immune cells are cultured for less than 24 hours, and the number of cells is more than double that of before culture; and a step in which a foreign gene is introduced into immune cells, and then the transduced immune cells are cultured for less than 24 hours, and the number of cells is no more than double that of before culture.

[0026] In this specification, "immune cells" refers to cells that perform immune functions in living organisms. Examples of immune cells include lymphocytes such as T cells, natural killer cells (NK cells), and B cells; antigen-presenting cells such as monocytes, macrophages, and dendritic cells; and granulocytes such as neutrophils, eosinophils, basophils, and mast cells. In a preferred embodiment, the immune cells are T cells. For information on NK cells expressing CAR, see, for example, US2016 / 0096892, Mol Ther. 25(8): 1769-1781 (2017), and for information on dendritic cells expressing CAR, macrophages expressing CAR, etc., see, for example, WO2017 / 019848, eLIFE. 2018 Jun 4:7:e36688, etc.

[0027] In this specification, "retroviral vector" refers to a vector derived from a retrovirus. Here, a retrovirus is an RNA virus that reverse transcribes its own genomic RNA into a linear double-stranded DNA copy and then covalently integrates its own genomic DNA into the host genome. In non-dividing cells, it does not have the process of covalent integration into the host genome and is therefore unable to be integrated. Examples of retroviral vectors include pMSGV vectors and pMSC vectors. In this disclosure, "retrovirus" does not include "lentiviruses."

[0028] In this specification, "lentivirus" refers to a virus belonging to the retroviridae family that can be introduced into non-dividing cells and possesses the tat and rev viral replication regulatory genes. Examples of lentiviruses include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). In this specification, "lentiviral vector" refers to a vector derived from the above-mentioned lentivirus. A lentiviral vector may or may not possess the above-mentioned viral replication regulatory genes. For example, a lentiviral vector may not possess tat and only possess rev. The origin of retroviral vectors and lentiviral vectors can be determined by examining their sequences.

[0029] In this specification, “stem cell memory immune cells” refers to undifferentiated immune cells, which, in order of increasing degree of undifferentiation, are naive immune cells, stem cell memory immune cells, and central memory immune cells. In this disclosure, stem cell memory immune cells refer to CD45RA(+)CCR7(+) cells.

[0030] In this specification, "cancer" means a malignant tumor.

[0031] In this specification, polypeptides, polynucleotides, vectors, and cells may be in an isolated state. That is, the polypeptides, polynucleotides, vectors, and cells described herein may be isolated polypeptides, isolated polynucleotides, isolated vectors, and isolated cells.

[0032] In this specification, “isolation” means separating a component from at least one other component present in the system.

[0033] Pluripotency refers to the ability to differentiate into various tissues and cells with different forms and functions, and to differentiate into cells of any of the three germ layers. Pluripotency is distinguished from totipotency, which can differentiate into all tissues of the living organism, including the blastodisc, in that pluripotency cannot differentiate into the blastodisc and therefore cannot form an individual.

[0034] "Pluripotency" refers to the ability to differentiate into multiple, limited numbers of cell lineages. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent, but not pluripotent.

[0035] Examples of "stem cells" include pluripotent stem cells.

[0036] In this disclosure, "pluripotent stem cell" refers to a stem cell that can differentiate into various tissues and cells with different forms and functions in the living body, and has the ability to differentiate into any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of such stem cells, though not limited to them, include embryonic stem cells (ESCs), embryonic stem cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells, embryonic germ cells, and induced pluripotent stem cells (sometimes referred to as "iPSCs" in this specification).

[0037] Furthermore, the term "pluripotent stem cell" as used in this disclosure refers to stem cells that have the ability to differentiate into a limited number of cell lineages. Examples of "pluripotent stem cells" usable in this invention include dental pulp stem cells, oral mucosa-derived stem cells, hair follicle stem cells, cultured fibroblasts, and somatic stem cells derived from bone marrow stem cells. Preferred pluripotent stem cells are ESCs and iPSCs.

[0038] "Induced pluripotent stem cells (iPSCs)" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including iPSCs established by Yamanaka et al. by introducing four factors—Oct3 / 4, Sox2, Klf4, and c-Myc—into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPS cells established by selecting cells based on Nanog expression after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPS cells produced by methods that do not include c-Myc (Nakagawa M, Yamanaka S., et al. iPS cells established by introducing six factors using a virus-free method (Nature Biotechnology, (2008) 26, 101-106) can also be used. (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.) In addition, induced pluripotent stem cells established by introducing four factors—OCT3 / 4, SOX2, NANOG, and LIN28—created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (Japanese Patent Publication No. 2008-307007) can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No 7, Any induced pluripotent stem cells known in the art described in 795-797) or patents (e.g., JP 2008-307007, JP 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used.

[0039] Various iPSC lines established by the NIH (National Institutes of Health), RIKEN (Riken Institute of Physical and Chemical Research), Kyoto University, and others can be used as "induced pluripotent stem cells." For example, human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2, and Kyoto University's 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, and 648A1. Alternatively, clinical-grade cell lines provided by Kyoto University, Cellular Dynamics International, etc., as well as research and clinical cell lines created using those cell lines, may be used.

[0040] For embryonic stem cells (ESCs), various mouse ESC strains established by inGenious Targeting Laboratory, RIKEN (the Institute of Physical and Chemical Research), etc., are available, while various human ESC strains established by the NIH, RIKEN, Kyoto University, and Cellartis are available. For example, human ESC strains that can be used include NIH's CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strain, etc., WisCell Research's H1 strain, H9 strain, RIKEN's KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. Alternatively, clinical-grade cell lines and research and clinical cell lines created using those cell lines may also be used.

[0041] <Method for producing genetically modified immune cells> In one embodiment, the method for producing genetically modified immune cells according to the disclosure includes a transduction step in which immune cells are transduced using a retroviral vector, and substantially does not include a culture step after the transduction step.

[0042] According to one embodiment of this disclosure, a method for producing genetically modified immune cells that exhibit excellent antitumor activity can be provided.

[0043] Genetically modified immune cells are required to be manufactured in a short period of time from the standpoint of manufacturing cost and manufacturing efficiency. Furthermore, from the standpoint of enabling early initiation of treatment for patients who require genetically modified immune cells, it is also required that they be manufactured in a short period of time. According to one embodiment of this disclosure, a manufacturing method is provided that enables the production of genetically modified immune cells in a short period of time and at low cost.

[0044] Genetically modified immune cells are sometimes required to exert their anti-cancer effects early after administration to a patient. According to one embodiment of this disclosure, it is possible to provide a manufacturing method that enables the production of genetically modified immune cells that exert anti-tumor activity against tumor cells early on, or a manufacturing method that enables the production of genetically modified immune cells that tend to exert anti-cancer effects rapidly after administration to a patient. According to one embodiment of this disclosure, it is possible to provide a manufacturing method that can extend the remaining time of genetically modified immune cells in the body after administration to a patient. Furthermore, according to one embodiment of this disclosure, it is possible to provide a method for producing genetically modified immune cells that have resistance to stimulation by tumor cells, etc., and have sustained anti-tumor activity. Moreover, according to one embodiment of this disclosure, it is possible to provide a method for producing genetically modified immune cells that can produce genetically modified immune cells that are effective against cancer recurrence.

[0045] The method for producing genetically modified immune cells according to this disclosure may further include an activation step of activating the immune cells before the transduction step. The method for producing genetically modified immune cells according to this disclosure may further include a recovery step of recovering the genetically modified immune cells after the transduction step. The method for producing genetically modified immune cells according to this disclosure may include a step of freezing the transduced immune cells within 12 hours after the recovery step. The method for producing genetically modified immune cells according to this disclosure may include a step of adding a pharmaceutically acceptable additive within 12 hours after the recovery step. The method for producing genetically modified immune cells according to this disclosure may include a step of adding a pharmaceutically acceptable additive and freezing within 12 hours after the recovery step.

[0046] In the CAR(+) and CD4(+) cell population of genetically modified immune cells produced by the manufacturing method of this disclosure, the proportion of stem cell memory immune cells is preferably 35% or more, more preferably 50% or more, even more preferably 60% or more, particularly preferably 65% ​​or more, most preferably 70% or more, and may also be 75% or more or 80% or more. In the CAR(+) and CD8(+) cell population of genetically modified immune cells produced by the manufacturing method of this disclosure, the proportion of stem cell memory immune cells is even more preferably 60% or more, particularly preferably 65% ​​or more, most preferably 70% or more, and may also be 75% or more or 80% or more. Generally, if the proportion of stem cell memory immune cells is high enough to be within the above preferred ranges, the proportion of effector memory immune cells will relatively decrease, and therefore, the toxic activity when co-cultured with cancer cells is thought to decrease. However, it is surprising that the genetically modified immune cells produced by the manufacturing method of the present invention can exhibit excellent toxic activity against cancer cells despite having a high proportion of stem cell memory immune cells. By setting the proportion of stem cell memory immune cells to within the preferred numerical range described above, the toxic activity of the genetically modified immune cells produced by the manufacturing method of this disclosure tends to be further improved. Furthermore, by setting the proportion of stem cell memory immune cells to within the preferred numerical range described above, the onset of anticancer effects after administration of the genetically modified immune cells produced by the manufacturing method of this disclosure tends to be accelerated. The upper limit of the above proportion is not particularly limited and can be 99% or less. In this specification, the above proportion is determined by analysis by flow cytometry. Details of the analysis method are described in the examples.

[0047] The PD-1 expression rate in the CD4(+) cell group of genetically modified immune cells produced is preferably 10% or higher, more preferably 15% or higher, and even more preferably 20% or higher. The upper limit of the expression rate is not particularly limited, but it can be 30% or less. The PD-1 expression rate in the CD8(+) cell group of genetically modified immune cells produced is preferably 4% or higher, more preferably 5% or higher, and even more preferably 8% or higher. The upper limit of the expression rate is not particularly limited, but it can be 20% or less. The PD-1 expression rate in the CD4(+) or CD8(+) cell group of genetically modified immune cells produced by a method including a wide-batch culture step is approximately 0.5 to 2%. In this disclosure, the PD-1 expression rate is measured by the method described in Example <PD-1, TIGIT, and LAG3 Expression Rates>.

[0048] The TIGIT expression rate in the CD4(+) or CD8(+) cell population of genetically modified immune cells produced is preferably 5% or higher, more preferably 7% or higher, and even more preferably 10% or higher. The upper limit of the expression rate is not particularly limited, but it can be 20% or less. The TIGIT expression rate in the CD4(+) or CD8(+) cell population of genetically modified immune cells produced by a method including a wide-batch culture step is approximately 1-5%. In this disclosure, the TIGIT expression rate is measured by the method described in Example <PD-1, TIGIT and LAG3 expression rates>.

[0049] The LAG3 expression rate in the CD4(+) or CD8(+) cell population of genetically modified immune cells produced is preferably 5% or higher, more preferably 7% or higher, and even more preferably 10% or higher. The upper limit of the expression rate is not particularly limited, but it can be 20% or less. The LAG3 expression rate in the CD4(+) or CD8(+) cell population of genetically modified immune cells produced by a method including a wide-area culture step is approximately 1-5%. In this disclosure, the LAG3 expression rate is measured by the method described in Example <PD-1, TIGIT and LAG3 Expression Rate>.

[0050] The expression rate of IL-7Rα (interleukin-7 receptor α chain) in the manufactured genetically modified immune cells is preferably 65% ​​or higher, more preferably 70% or higher, and even more preferably 75% or higher. The upper limit of the expression rate is not particularly limited, but it can be 90% or lower. In this disclosure, the IL-7Rα expression rate is measured by the method described in the examples.

[0051] The genetically modified immune cells produced in this disclosure maintain high expression rates of PD-1, TIGIT, and LAG3 by essentially not undergoing large-scale culture.

[0052] The following describes each step included in the method for producing genetically modified immune cells as disclosed herein.

[0053] (Transduction Step) The method for producing genetically modified immune cells according to this disclosure includes a transduction step in which immune cells are transduced using a retroviral vector. The retroviral vector used may be one type or two or more types.

[0054] In this disclosure, the transduction step is performed by a viral infection method. The viral infection method can be carried out by conventionally known methods, for example, by transfecting host cells with a vector containing the desired foreign gene and packaging vectors (plasmids) for each virus using a corresponding commercially available kit to produce a recombinant viral vector, and then infecting immune cells with the obtained recombinant viral vector. Infection of immune cells with a recombinant viral vector (recombinant retroviral vector) can be carried out by culturing the immune cells in a medium supplemented with the recombinant virus. The medium supplemented with the recombinant virus can be centrifuged as needed. Alternatively, multiple media supplemented with the recombinant viral vector may be used to carry out the infection of immune cells with the recombinant virus in multiple steps. An example of a commercially available viral vector kit is the Retrovirus Packaging Kit Eco. Examples of host cells include GP2-293 cells, Plat-GP cells, PG13 cells (ATCC CRL-10686), PA317 cells (ATCC CRL-9078), etc.

[0055] In this disclosure, the retroviral vector may contain foreign genes. In one embodiment, examples of foreign genes include a gene for expressing a cell surface molecule that recognizes a malignant tumor antigen, a gene for expressing a cytokine, and a gene for expressing a chemokine. In one embodiment, the gene for expressing a cell surface molecule that recognizes a malignant tumor antigen is a nucleic acid encoding a polypeptide that constitutes a cell surface molecule that recognizes a malignant tumor antigen. In one embodiment, the gene for expressing a cytokine is a nucleic acid encoding a cytokine. In one embodiment, the gene for expressing a chemokine is a nucleic acid encoding a chemokine. The retroviral vector may contain one foreign gene or two or more foreign genes. Furthermore, the retroviral vector may contain two or more genes for expressing cell surface molecules (for example, cell surface molecules that recognize different malignant tumor antigens). Furthermore, the retroviral vector may contain two or more genes for expressing cytokines. Furthermore, the retroviral vector may contain two or more genes for expressing chemokines.

[0056] If there are multiple foreign genes, all of them may be contained in a single retroviral vector, or each of them may be contained in a separate retroviral vector, or some of the multiple foreign genes may be contained in a single retroviral vector and the rest in separate retroviral vectors. When a single retroviral vector contains multiple foreign genes, there is no particular limitation on the order in which these foreign genes are arranged from upstream to downstream.

[0057] Furthermore, the retroviral vector may include a promoter, a packaging signal, a primer binding site, and one or two long terminal repeats (LTRs). The promoter may be an inducible promoter.

[0058] In one embodiment, the transduction step is performed by simultaneously or stepwise introducing a retroviral vector containing genes for expressing cell surface molecules and a retroviral vector containing genes for expressing cytokines and chemokines into immune cells. In another embodiment, the transduction step is performed by simultaneously or stepwise introducing a retroviral vector containing genes for expressing cell surface molecules and cytokines and a retroviral vector containing genes for expressing cell surface molecules and chemokines into immune cells. In yet another embodiment, the transduction step is performed by simultaneously or stepwise introducing a retroviral vector containing genes for expressing cell surface molecules and cytokines and a retroviral vector containing genes for expressing cytokines and chemokines into immune cells. In another embodiment, the transduction step is carried out by simultaneously or stepwise introducing into immune cells a retroviral vector containing genes for expressing cell surface molecules and genes for expressing chemokines, and a retroviral vector containing genes for expressing cytokines and genes for expressing chemokines. In another embodiment, the transduction step is carried out by simultaneously or stepwise introducing into immune cells a retroviral vector containing genes for expressing cell surface molecules and genes for expressing cytokines, and a retroviral vector containing genes for expressing chemokines. In another embodiment, the transduction step is carried out by simultaneously or stepwise introducing into immune cells a retroviral vector containing genes for expressing cell surface molecules and genes for expressing chemokines, and a retroviral vector containing genes for expressing cytokines.In another embodiment, the transduction step is performed by simultaneously or stepwise introducing into immune cells a retroviral vector containing genes for expressing cell surface molecules, a retroviral vector containing genes for expressing cytokines, and a retroviral vector containing genes for expressing chemokines.

[0059] In yet another embodiment, the transduction step is carried out by pre-preparing immune cells that express a cell surface molecule that recognizes a desired malignant tumor antigen, and simultaneously or stepwise introducing a retroviral vector containing a gene for cytokine expression and a retroviral vector containing a gene for chemokine expression into the immune cells. In yet another embodiment, the transduction step is carried out by pre-preparing immune cells that express a cell surface molecule that recognizes a desired malignant tumor antigen, and introducing a retroviral vector containing a gene for cytokine expression and a gene for chemokine expression into the immune cells.

[0060] The malignant tumor antigens recognized by cell surface molecules are not particularly limited, as long as they are biomolecules specifically expressed on cancer cells and their precursor cells, biomolecules whose expression is newly observed due to the carcinogenesis of cells, or biomolecules whose expression levels are increased in cancer cells compared to normal cells. Such antigens are also called "tumor-associated antigens" (TAAs). Examples of TAAs 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, and CS1 (CD319). , CSPG4, CLDN6, CLD18A2, CYP1B1, DNAM-1, GD2, GD3, GM2, GFRα4, GPC3, GPR20, GPRC5D, globoH, Gp10 0, GPR20, GPRC5D, EGFR (EGFRviii etc.), EGFRvariant, EpCAM, EGP2, EGP40, FAP, FITC, HER2, HER3, HPV E6, HPV E7, hTERT, IgG κ chain, IL-11Ra, IL-13Ra2, KIT, Lewis A, Lewis Y, Legumain, LMP1, LMP2, Ly6k, LICAM, MAD-CT-1, MAD-CT-2, MAGE-A1, Melanoma-associated antigen 1, MUC1, MUC16, NA-17, NY-BR-1, NY-ESO-1, O-acetyl-GD2, h5T4, PANX3, PDGRFb, PLAC1, Polysialic Examples of such substances include, but are not limited to, acid, PSCA, PSMA, RAGE1, ROR1, sLe, SSEA-4, TARP, TAG-72, TEM7R, Tn antigen, TRAIL receptor, TRP2, TSHR, α-fetoprotein, mesothelin, folate receptor α (FRα), folate receptor β (FRβ), FBP, UPK2, VEGF-R2, WT-1, etc.

[0061] Examples of cell surface molecules include cell surface receptors, artificial receptors, and adhesion molecules that directly or indirectly recognize malignant tumor antigens. A cell surface molecule that specifically recognizes malignant tumor antigens may only function to directly or indirectly bind to the malignant tumor antigen, thereby positioning the genetically modified immune cells obtained by the manufacturing method described herein near malignant tumor cells. However, to further enhance the cancer treatment effect, it may also have the function of triggering intracellular signaling that activates the immune response of immune cells. In one embodiment, the cell surface molecule that specifically recognizes malignant tumor antigens may be an antibody or antibody fragment that specifically recognizes malignant tumor antigens. Here, the antibody or antibody fragment is not limited to IgM, IgD, IgG, IgA, IgE, etc., but may also be a small molecule antibody such as Fab or scFv. Examples of cell surface molecules that specifically recognize malignant tumor antigens include T cell receptors (TCRs) that specifically recognize malignant tumor antigens and CARs that specifically recognize malignant tumor antigens. TCR is an example of the above cell surface receptor, CAR is an example of the above artificial receptor, and antibodies (Fab, Fab', F(ab') ２ (Including low-molecular-weight antibodies such as scFv) can be considered an example of the adhesion factors mentioned above. Of course, as long as they specifically recognize malignant tumor antigens, adhesion factors may be molecules other than antibodies, such as sugar chains and aptamers. Furthermore, cell surface molecules that specifically recognize cancer antigens may recognize them indirectly, as long as their recognition of cancer antigens is specific. For example, by administering molecules such as antibodies that specifically recognize cancer antigens to a target simultaneously or consecutively with the immune cells of the present invention, the immune cells can indirectly and specifically recognize cancer antigens by recognizing the antibody molecules or by recognizing tags labeled on the antibody molecules. Examples of cell surface molecules that recognize antibodies include CD16, CD19, CD20, etc., and examples of tags that label antibody molecules include FITC, etc.

[0062] Antibodies generally have a structure in which two heavy chains (H chains) and two light chains (L chains), stabilized by a pair of disulfide bonds, are associated. The heavy chain consists of a heavy chain variable region (VH), heavy chain constant regions (CH1, CH2, CH3), and a hinge region located between CH1 and CH2, while the light chain consists of a light chain variable region (VL) and a light chain constant region (CL). Among these, the variable region fragment (Fv) consisting of VH and VL is directly involved in antigen binding and is the region that gives diversity to the antibody. Furthermore, the antigen-binding region consisting of VL, CL, VH, and CH1 is called the Fab region, and the region consisting of the hinge region, CH2, and CH3 is called the Fc region. Of the variable regions, the region that directly contacts the antigen changes particularly greatly and is called the complementarity-determinating region (CDR). The region of an immunoglobulin that is relatively less prone to mutation, other than the CDRs, is called the framework region (FR). The variable regions of the light and heavy chains each contain three CDRs, which are called heavy chain CDR1-3 (HCDR1-3) and light chain CDR1-3 (LCDR1-3), respectively, starting from the N-terminus. Antibodies can be human chimeric antibodies, humanized antibodies, or human antibodies. Human chimeric antibodies can be produced by replacing the constant region of a non-human antibody with the constant region of a human antibody. Humanized antibodies can be produced by replacing the regions of a non-human antibody other than the six CDRs with the corresponding regions of a human antibody. Human antibodies can be produced using animals (e.g., mice, rabbits, etc.) in which at least the heavy chain variable region of the immunoglobulin has been replaced with the corresponding region of a human gene locus. If the constant region is non-human, a human antibody can be obtained by replacing the constant region with the amino acid sequence of a human antibody. Antibodies can generally bind selectively or specifically to their antigens.

[0063] CDRs can be determined, for example, based on the numbering by Kabat et al. (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH.), Chothia, AbM, contact, IMGT, Aho, Martin (Enhanced Chothia), etc. In this way, heavy chain CDRs 1-3 and light chain CDRs 1-3 can be estimated based on the amino acid sequences of the heavy chain variable region and light chain variable region, respectively, using conventional methods.

[0064] In this specification, "TCR" refers to an antigen receptor molecule expressed on the cell membrane of a T cell. TCRs exist as heterodimers consisting of alpha and beta chains, or gamma and delta chains, and are known to activate T cells by recognizing antigen molecules bound to the major histocompatibility complex (MHC) molecule. TCRs may be heterodimers consisting of alpha and beta chains (alpha-beta TCRs) or heterodimers consisting of gamma and delta chains (gamma-delta TCRs), as long as they specifically recognize malignant tumor antigens. TCRs may be endogenous or exogenous (recombinant TCRs). Sources of T cells expressing endogenous TCRs and T cells into which exogenous TCRs are introduced include, but are not limited to, tumor-infiltrating lymphocytes (TILs), tumor-associated lymph nodes, peripheral blood lymphocytes, pleural fluid lymphocytes, and ascites fluid lymphocytes. Methods for isolating T cells expressing TCRs with specific antigen-binding properties include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that alter cell density; magnetic separation using antibody-coated magnetic beads; affinity chromatography (e.g., affinity chromatography using negative selection); cytotoxic agents linked to or used in combination with monoclonal antibodies (including, but not limited to, complement and cytotoxins); panning with antibodies bound to solid matrices such as plates and chips; elutriation; selective proliferation by antigen stimulation; and separation using MHC-antigen complexes. Transgenic animals, such as transgenic mice modified to express specific TCRs, have also been developed.

[0065] As TCR, MART1-specific TCR (Cancer Res. 54, 5265-5268 (1994)) and MAGE-A3-specific TCR (Anticancer Res., 20, 1793-1799 (2000)), gp100-specific TCR (J. Immunol. 170, 2186-2194 (2003)), NY-ESO-1-specific TCR (J. Immunol., 174, 4415-442 3 (2005)), WT1-specific TCR (Blood, 106, 470-476 (2005)), MAGE-A1-specific TCR (Int. Immunol., 8, 1463-1466 (1996)), P1A-specific TCR (Sarma, S., Y. Guo, Y. TCRs such as MAGE-A10-specific TCR, AFP-specific TCR, CT-83-specific TCR, KRAS (including variants, i.e., mKRAS)-specific TCR, MAGE-A4-specific TCR, Epha2-specific TCR, BCMA-specific TCR, 5T4-specific TCR, PRAME-specific TCR, and HA-1-specific TCR have been reported, and the nucleic acid sequences encoding them have also been reported in the above-mentioned literature. (Guilloux, C. Lee, X.-F. Bai, Y. Liu. 1999. Cytotoxic T lymphocytes to an unmutated tumor antigen P1A: normal development but restrained effector function. J. Exp. Med. 189:811.) When the cell surface molecule that specifically recognizes a malignant tumor antigen is a TCR, the nucleotide sequence of the nucleic acid encoding the TCR may have, for example, 80% or more, more specifically 85% or more, more specifically 90% or more, more specifically 95% or more, or more specifically 98% or more sequence identity with the nucleotide sequence encoding the TCR described in the above-mentioned literature, as long as it can recognize the target antigen molecule and activate T cells. In this disclosure, the sequence identity of the amino acid sequence and the sequence identity of the nucleotide sequence can be evaluated using default parameters, for example, the BLAST® (registered trademark, National Library of Medicine) program.Alternatively, the nucleotide sequence of the nucleic acid encoding the TCR may be a nucleotide sequence that maintains the nucleotide sequence encoding the CDR in the nucleotide sequence encoding the TCR described in the above-mentioned literature, and has a sequence identity of 60% or more, more specifically 70% or more, more specifically 80% or more, more specifically 90% or more, or more specifically 95% or more, in the nucleotide sequence of the region other than the nucleotide sequence encoding the TCR described in the above-mentioned literature.

[0066] The nucleotide sequence of a TCR varies depending on the antigen specificity of the TCR. Therefore, T cells expressing a TCR that binds to a desired antigen may be isolated, and the nucleotide sequence of that TCR may be analyzed. For example, the nucleotide sequence of a nucleic acid encoding a TCR that specifically recognizes a particular antigen can be obtained by analyzing the nucleotide sequences encoding the alpha and beta chains as TCR subunits of antitumor-active T cells (CTLs) induced using a specific antigen, using methods known in the art (International Publication No. 2007 / 032255, and Morgan et al., J Immunol, 171, 3288 (2003)). When analyzing the nucleotide sequence of a TCR, the nucleotide sequences encoding each chain may be amplified and analyzed using PCR. PCR primers may, for example, be a 5'-R primer (5'-gtctaccaggcattcgcttcat-3': SEQ ID NO: 5) as a 5'-side primer, and a 3'-TRa-C primer (5'-tcagctggaccacagcccgcagcgt-3': SEQ ID NO: 6) specific to the TCR alpha chain C region, a 3'-TRb-C1 primer (5'-tcagaaatcctttctcttgac-3': SEQ ID NO: 7) specific to the TCR beta chain C1 region, or a 3'-TRbeta-C2 primer (5'-ctagccctctgggaatcctttctctt-3': SEQ ID NO: 8) specific to the TCR beta chain C2 region, but are not limited to these. Antigen-specific TCRs can bind with high affinity to target cells presenting antigens (e.g., peptides). Furthermore, by appropriately selecting the type of immune-responsive cell, they can mediate the efficient killing of target cells presenting antigen peptides.

[0067] In this specification, "CAR" is a chimeric protein comprising an antigen-binding domain (e.g., a single-chain antibody) that recognizes cell surface antigens of malignant tumor cells and an activation signaling domain that induces activation of immune cells. CAR can be introduced into immune cells and expressed on the cell surface. Immune cells expressing CAR can target specific antigens. A single-chain antibody is typically a single-chain variable region fragment (scFv) consisting of a light chain variable region and a heavy chain variable region derived from the antigen-binding site of a monoclonal antibody that specifically binds to malignant tumor antigens, and a linker peptide that links them. In one embodiment, CAR comprises an scFv, a transmembrane domain, and an activation signaling domain. The scFv and each domain can be linked via spacers. The scFv, transmembrane domain, and activation signaling domain are preferably, but not limited to, derived from human proteins.

[0068] Examples of scFv include oligonucleotides or polypeptides that include a light chain variable region and a heavy chain variable region derived from the antigen-binding site of a monoclonal antibody, with a linker peptide located between the light chain variable region and the heavy chain variable region. ScFv can be produced by known methods. For example, a mouse or other organism may be inoculated with an antigen, lymphoid tissue may be collected, an antibody gene library may be prepared, and a nucleotide sequence encoding an antibody that recognizes a malignant tumor antigen may be obtained by direct antibody cloning, based on which a single-chain antibody may be designed. Alternatively, a hybridoma may be prepared using the collected lymphoid tissue, a hybridoma encoding an antibody that recognizes a malignant tumor antigen may be identified to obtain a monoclonal antibody, and a single-chain antibody may be designed based on its sequence information. Alternatively, a single-chain antibody library may be prepared based on a naive antibody library prepared from B cells of a healthy person, an antibody library prepared from B cells of a cancer patient with antiserum exhibiting high neutralizing activity against malignant tumor antigens, etc., and scFv that recognize malignant tumor antigens may be selected by displaying this library using phage display.

[0069] Transmembrane domains are polypeptides that fix CARs to the cell membrane of immune cells such as T cells. Examples of transmembrane domains include BTLA, CD3ε, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, 4-1BB (CD137), CTLA-4, GITR, ICOS, LAG3, OX40, SLAMF4 (CD244, 2B4), and transmembrane domains derived from the α or β chains of T cell receptors. The transmembrane domain may also be a mutant transmembrane domain 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 identical to the natural amino acid sequence of the transmembrane domain described above. In one embodiment, the transmembrane domain is preferably a CD8 transmembrane domain.

[0070] The transmembrane domain may be linked to a hinge domain, which is a peptide (oligopeptide or polypeptide) consisting of any amino acid sequence and having a length of 1 to 100 amino acids, preferably 10 to 70 amino acids. Examples of hinge domains include those derived from CD3, CD8, KIR2DS2, IgG4, IgD, and other immunoglobulins. It is also possible to use mutant hinge 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, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the natural amino acid sequence of the above-mentioned hinge domain. In one embodiment, the hinge domain is a CD8 hinge domain. Examples of hinge domains include the amino acid sequences described in SEQ ID NOs: 20 and 21.

[0071] The activation signaling domain, which induces the activation of immune cells, is a polypeptide that transmits signals into immune cells when a single-chain antibody recognizes and binds to the cell surface antigen of malignant tumor cells. The activation signaling domains include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activated NK cell receptors, Toll-like receptors, B7-H3, BAFFR, BTLA, BY55 (CD160), CD2, CD3ζ, CD4, CD7, CD8α, CD8β, CD11a, CD11b, CD11c, CD11d, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD103, 4-1BB (CD137), CDS, CEACAM1, CRTAM, CNAM1 (CD226), DAP10, and Fc. Receptor-associated γchain, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICOS (CD278), IL2Rβ, IL2Rγ, IL7Rα, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGA X, ITGB1, ITGB2, ITGB7, KIRDS2, Ly9 (CD229), LAT, LFA-1 (CD11a / CD18), LIGHT, LTBR, ​​NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF 1) One or more intracellular domains selected from the group consisting of 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, MyD88, FLT3, VLA1, and VLA-6 can be listed.The activation signaling domain may also be a mutant signaling domain (intracellular domain) 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 identical to the natural amino acid sequence of the activation signaling domain (intracellular domain) described above. In one embodiment, the activation signaling domain preferably includes two or more intracellular domains selected from the group consisting of CD28, 4-1BB, and CD3ζ. In one embodiment, the activation signaling domain includes the intracellular domains of CD3ζ and CD28, but does not include the intracellular domain of 4-1BB. In another embodiment, the activation signaling domain includes the intracellular domains of CD3ζ and 4-1BB, but does not include the intracellular domain of CD28. In yet another embodiment, the activation signaling domain includes the intracellular domains of CD3ζ, CD28, and 4-1BB.

[0072] If a signaling domain includes multiple intracellular domains, these intracellular domains may be linked to each other via linker peptides (oligopeptides or polypeptides) consisting of 2 to 10 amino acids. Examples of linker peptides include peptides consisting of a continuous glycine-serine sequence. More specific examples include, but are not limited to, linker 15 (sequence number 14) and linker 25 (sequence number 15).

[0073] The antigen-binding domain and the transmembrane domain, and the transmembrane domain and the activation signaling domain, may each be linked via a spacer, which is a peptide (oligopeptide or polypeptide) consisting of any amino acid sequence and having a length of 1 to 100 amino acids, preferably 10 to 50 amino acids. Examples of spacers include peptides consisting of a continuous glycine-serine sequence.

[0074] Information on the nucleotide sequences encoding polypeptides such as antigen-binding domains, transmembrane domains, hinge domains, and activation signaling domains in CARs can be obtained as appropriate by searching publicly available literature and databases such as NCBI (www.ncbi.nlm.nih.gov / guide / ).

[0075] For example, information on the nucleotide sequences encoding the CD28, 4-1BB, and CD3ζ polypeptides in the activation signaling domain can be obtained by searching databases such as NCBI. For human CD28, the one registered with Genbank number: NM_006139.2 (updated May 10, 2014) can be given as an example, and the amino acid sequence of the activation signaling domain can be the amino acid sequence described in SEQ ID NO: 17. For human 4-1BB, the one registered with Genbank number: NM_001561.5 (updated March 16, 2014) can be given as an example, and the amino acid sequence of the activation signaling domain can be the amino acid sequence described in SEQ ID NO: 18. For human CD3ζ, the one registered with Genbank number: NM_000734.3 (updated August 12, 2014) can be given as an example, and the amino acid sequence of the activation signaling domain can be the amino acid sequence described in SEQ ID NO: 19.

[0076] Furthermore, information on the nucleotide sequences encoding the polypeptides of the transmembrane domains of human CD8 or human CD28 can be obtained by searching databases such as NCBI. An example of a nucleotide sequence encoding the polypeptide of human CD8 is the one registered with Genbank number: NM_001768.6 (updated May 10, 2014). Additionally, examples of amino acid sequences of the transmembrane domains of human CD8 or human CD28 include those described in SEQ ID NO: 16 and SEQ ID NO: 26, respectively.

[0077] Furthermore, information on the base sequence encoding the polypeptide of the antigen-binding domain (e.g., a single-chain antibody) can also be obtained by producing a monoclonal antibody that recognizes the target cell surface antigen, determining the amino acid sequence of such a monoclonal antibody using a known method such as the Edman method, and then obtaining the information based on that amino acid sequence. Methods for producing monoclonal antibodies include using hybridomas, transforming a host with an expression vector containing the antibody gene using genetic engineering techniques, and immunizing transgenic animals with the desired antigen.

[0078] In the transduction process, the retroviral vector may contain genes for cytokine expression. Conventionally known cytokines can be appropriately selected as cytokines. Examples of such cytokines include IL-7, IL-15, IL-18, IL-21, and IL-27. Among these cytokines, from the viewpoint of antitumor activity, at least one selected from the group consisting of IL-7, IL-15, and IL-21 is preferred, at least one selected from the group consisting of IL-7 and IL-15 is more preferred, and IL-7 is even more preferred. IL-7 is a type of cytokine produced by non-hematopoietic cells such as stromal cells in bone marrow, thymus, and lymphoid organs / tissues.

[0079] The retroviral vector may contain one or more genes for expressing cytokines. The cytokines are preferably derived from the same animal species (human or non-human mammal) as the immune cells into which the foreign genes are introduced (the cytokine receptors on those immune cells). The amino acid sequences of various natural cytokines from humans and non-human mammals are publicly known and registered in databases such as NCBI (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov / guide / ) and UniProt (The Universal Protein Resource; www.uniprot.org), and this information can also be used in this disclosure. Furthermore, GenBank may be used; an example of the amino acid sequence of human IL-7 is the one registered as GenBank number: NM_000880.3 (Sequence ID 9).

[0080] In this specification, the term "cytokine" includes not only natural full-length proteins but also proteins (polypeptides) that have been modified in various ways, such as to maintain or enhance their function as cytokines. That is, in this specification, a cytokine may be any of the following: (a) a whole or a part of a protein (polypeptide) consisting of a natural amino acid sequence (functional partial polypeptide); (b) a variant in which one or more amino acid sequences are deleted, substituted or added to the natural amino acid sequence; or (c) a fusion protein in which a full-length protein or partial polypeptide is linked at the N-terminus or C-terminus to another protein (e.g., a signal peptide, a subunit of a receptor protein, etc.). Examples of the variant in (b) above include those having an amino acid sequence in which the identity with respect to the amino acid sequence of a natural cytokine 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, either in full length or as a partial region (domain).

[0081] In the transduction process, the retroviral vector may contain a gene for expressing a chemokine. Any conventionally known chemokine can be appropriately selected. Examples of such chemokines include CCL19 and CCL21, with CCL19 being preferred. CCL19 is a type of cytokine, mainly produced by dendritic cells and macrophages in lymph nodes, and has the function of inducing migration of T cells, B cells, and mature dendritic cells via its receptor, the CC chemokine receptor 7 (CCR7).

[0082] The retroviral vector may contain one or more genes for expressing chemokines. The chemokines are preferably derived from the same animal species (human or non-human mammal) as the immune cells into which the foreign genes are introduced (the chemokine receptors on those immune cells). The amino acid sequences of various natural chemokines from humans and non-human mammals are publicly known and registered in databases such as NCBI (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov / guide / ) and UniProt (The Universal Protein Resource; www.uniprot.org), and this information can also be used in this disclosure. Furthermore, GenBank may be used; an example of the amino acid sequence of human CCL19 is the one registered as GenBank number: NM_006274.2 (Sequence ID 10).

[0083] In this specification, the term "chemokine" includes not only natural full-length proteins but also proteins (polypeptides) that have been modified in various ways, such as to retain or enhance their function as chemokines. In other words, in this specification, a chemokine may be either (a) a whole or a part of a protein (polypeptide) consisting of a natural amino acid sequence (functional partial polypeptide), or (b) a variant in which one or more amino acid sequences are deleted, substituted, or added to the natural amino acid sequence. Examples of variants in (b) above include those having an amino acid sequence in which the full-length or partial region (domain) has an identity of 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the amino acid sequence of a natural chemokine.

[0084] Furthermore, IL-7 and CCL19 possess signal peptides, which are removed in mature proteins. For example, in the amino acid sequence of human IL-7 described in SEQ ID NO: 9, the sequence from positions 1 to 25 corresponds to the signal peptide. Similarly, in the amino acid sequence of human CCL19 described in SEQ ID NO: 10, the sequence from positions 1 to 21 corresponds to the signal peptide. IL-7 and CCL19 may also be variants of the above-mentioned natural proteins. Examples of IL-7 variants include the following: (6a) A polypeptide consisting of an amino acid sequence having 70% or more sequence identity (homology) with the amino acid sequence of natural IL-7 (e.g., SEQ ID NO: 9), and having the function of promoting the immune function of immune cells. (6b) A polypeptide consisting of an amino acid sequence in which one or more amino acids are mutated from the amino acid sequence of natural IL-7 (e.g., SEQ ID NO: 9), and having the function of promoting the immune function of immune cells. Examples of CCL19 variants include the following. (7a) A polypeptide comprising an amino acid sequence having 70% or more sequence identity (homologousity) with the amino acid sequence of natural CCL19 (e.g., SEQ ID NO: 10), and having the function of promoting the immune function of immune cells. (7b) A polypeptide comprising an amino acid sequence in which one or more amino acids are mutated from the amino acid sequence of natural CCL19 (e.g., SEQ ID NO: 10), and having the function of promoting the immune function of immune cells. In this specification, "function of promoting the immune function of immune cells" means a function that maintains and / or promotes the survival, proliferation, antitumor activity, migration activity, and infiltration activity into tumor tissue of immune cells.

[0085] In (6a) and (7a) above, sequence identity is not particularly limited as long as it is 70% or more, but is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more. In (6b) and (7b) above, "multiple" can be, for example, 2 to 30, preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5. Also, "mutation" may be deletion, substitution, addition, or insertion, or a combination thereof. Furthermore, the IL-7 and CCL19 mutants may have the signal peptide of these proteins changed to another signal peptide, or the signal peptide may be removed. Preferably, the IL-7 and CCL19 mutants have a signal peptide of a secreted protein and are secreted extracellularly.

[0086] The retroviral vector preferably contains genes for expressing IL-7 and CCL19. The resulting immune cells tend to be resistant to stimulation by tumor cells and other pathogens, and possess sustained antitumor activity. Furthermore, these immune cells tend to be effective against cancer recurrence.

[0087] The exogenous gene can be composed of nucleic acids (polynucleotides) having a base sequence that encodes a desired protein or polypeptide to be expressed in immune cells, such as the CARs, cytokines, and chemokines mentioned above. A person skilled in the art can design and produce an expression vector capable of expressing the desired protein (polypeptide) in immune cells. The nucleic acids contained in the expression vector may be produced by chemical DNA synthesis reactions or by production as cDNA (cloning).

[0088] In addition to the base sequence encoding the desired protein or polypeptide (foreign gene), a retroviral vector may optionally include sequences such as promoters, terminators, enhancers, start codons, stop codons, polyadenylation signals, nuclear localization signals (NLS), and multicloning sites (MCS) that are involved in the expression of each foreign gene. Furthermore, if a single retroviral vector contains multiple foreign genes, a self-cleaving peptide (e.g., 2A peptide) or a gene encoding an IRES (Internal Ribozyme Entry Site) may be inserted between each foreign gene. The promoter may be an inducible promoter. Retroviral vectors also contain reporter genes (e.g., genes encoding fluorescent proteins of various colors), drug selection genes (e.g., kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene), suicide genes (e.g., diphtheria A toxin, herpes simplex thymidine kinase (HSV-TK), carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD), cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR), purine nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella-zoster virus thymidine kinase (VZV-TK), xanthine-guanine phosphoribosyltransferase (XGPRT), inductive caspase 9 (Inductive caspase) 9) may contain nucleic acids (base sequences) that encode "functional genes," such as genes that encode such genes.

[0089] Conventionally known peptides can be used as 2A peptides. Examples include P2A (ATNFSLLKQAGDVEENNPGP: SEQ ID NO: 11), E2A (QCTNYALLKLAGDVESNPGP: SEQ ID NO: 12), F2A (GSGVKQTLNFDLLKLAGDVESNPGP: SEQ ID NO: 2), and T2A (EGRGSLLTCGDVEENNPGP: SEQ ID NO: 13).

[0090] In this specification, "nucleic acid" refers to any molecule formed by the polymerization of nucleotides and molecules having equivalent functions to nucleotides. Examples include RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; polymers of a mixture of ribonucleotides and deoxyribonucleotides; and nucleotide polymers containing nucleotide analogs. Furthermore, nucleotide polymers containing nucleic acid derivatives may also be used. Nucleic acids may be single-stranded or double-stranded. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes to the other under stringent conditions.

[0091] As nucleotide analogs, any molecule may be a ribonucleotide, deoxyribonucleotide, RNA, or DNA that has been modified to improve or stabilize nuclease resistance compared to RNA or DNA, increase affinity with complementary nucleic acids, increase cell permeability, or make them visible. Nucleotide analogs can be naturally occurring or unnatural molecules, for example, sugar-modified nucleotide analogs (e.g., nucleotide analogs substituted with 2'-O-methylribose, nucleotide analogs substituted with 2'-O-propylribose, nucleotide analogs substituted with 2'-methoxyethoxyribose, nucleotide analogs substituted with 2'-O-methoxyethylribose, nucleotide analogs substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogs substituted with 2'-fluororibose, bridged nucleotide acids (BNA), locked nucleotide acids (LNA), ethylene-bridged nucleotide acids (ENA) Examples include Acid, peptide nucleic acids (PNA), oxypeptide nucleic acids (OPNA), peptide ribonucleic acid (PRNA), and phosphate diester bond-modified nucleotide analogs (e.g., nucleotide analogs substituted with phosphorothioate bonds, nucleotide analogs substituted with N3'-P5' phosphoamide bonds).

[0092] As nucleic acid derivatives, any molecule obtained by adding another chemical substance to nucleic acid to improve nuclease resistance, stabilize it, increase affinity with complementary nucleic acid chains, increase cell permeability, or make it visible can be used. Specific examples 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-FAM-added derivatives, biotin-added derivatives, and the like.

[0093] Any of the immune cells listed above can be used, but from the viewpoint of antitumor activity, lymphocytes are preferred, T cells or natural killer cells (NK cells) are more preferred, and T cells are even more preferred. The T cells are not particularly limited and may be any T cells such as αβT cells, γδT cells, CD8+ T cells, C4+ T cells, tumor-infiltrating T cells, naive T cells, stem cell memory T cells, central memory T cells, NK T cells, regulatory T cells, etc. In one embodiment, the immune cells are a cell population mainly consisting of T cells, and may also include other cells besides T cells. The proportion of T cells to the total number of cells in the above cell population is preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, particularly preferably 80% or more, and most preferably 90%.

[0094] T cells can generally be collected from living organisms as peripheral blood mononuclear cells (PBMCs) and leukocyte apheresis, for example, from immune cells infiltrating bodily fluids such as blood and bone marrow; tissues such as the spleen, thymus, lymph nodes, and liver; or cancerous tissues such as primary tumors, metastatic tumors, and malignant ascites. In this disclosure, T cells may be specific T cells isolated and purified from a population of collected immune cells, or they may be T cells contained within a cell population (e.g., PBMCs) without isolation. Furthermore, T cells may be obtained by differentiating induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), or other stem cells, progenitor cells, etc., into T cells by culturing them under appropriate conditions.

[0095] From the viewpoint of introduction efficiency, the transduction time may be 1 hour or more, 3 hours or more, 5 hours or more, 7 hours or more, preferably 10 hours or more, more preferably 15 hours or more, 16 hours or more, 17 hours or more, even more preferably 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, or 25 hours or more. From the viewpoint of the production efficiency of immune cells, the transduction time may be 100 hours or less, 80 hours or less, 70 hours or less, 60 hours or less, 55 hours or less, preferably 48 hours or less, more preferably 40 hours or less, even more preferably 36 hours or less, 35 hours or less, 34 hours or less, 33 hours or less, 32 hours or less, 31 hours or less, or 30 hours or less. The transduction time is preferably 10 hours or more and 48 hours or less, more preferably 15 hours or more and 40 hours or less, and even more preferably 15 hours or more and 36 hours or less.

[0096] The transduction process time refers to the time it takes for immune cells to be infected with the retroviral vector in a culture vessel such as a retroviral preload plate. Infection with the retroviral vector may be performed multiple times, in which case the transduction time refers to the total viral infection time.

[0097] In the transduction process, recombinant human fibronectin fragments may be added to the culture medium in addition to the retroviral vector. This helps to co-localize the target immune cells and the viral vector, thereby increasing the efficiency of gene transfer in the transduction of immune cells by the retroviral vector. For example, Retronectin® can be used as the recombinant human fibronectin fragment.

[0098] In the transduction process, the culture method, culture medium, and other culture conditions (temperature, atmosphere, cell density, etc.) can be set by appropriately modifying known conventional methods.

[0099] The culture vessel in the transduction process is not particularly limited, and can be appropriately selected from plates, dishes, petri dishes, flasks, bags, bottles, tanks (culture tanks), etc. according to the culture scale. For example, a closed automatic culture device such as CliniMACS Prodigy (trademark) can be used.

[0100] In the transduction process, known or common media used for culturing immune cells can be used, and necessary components can be appropriately supplemented to the medium.

[0101] Examples of the medium in the transduction process include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, modified MEM zinc option, IMDM, 199 medium, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, Fisher medium, and other various commercially available products for T cell culture (e.g., CTS TM OpTmizer TM T-Cell Expansion Basal Medium, CTS TM OpTmizer TM Pro Serum Free Medium, CTS TM OpTmizer TM Pro, CTS TM OpTmizer TM T-Cell Expansion Supplement). Any one of these media can be used alone, or two or more of them can be used in combination.

[0102] The culture medium may be serum-containing, serum-free, or xeno-free. To prevent contamination by components from other animal species, the serum may be derived from the same animal as the cells being cultured. Serum-free media refer to media that do not contain unprocessed or unpurified serum and therefore may include media containing purified blood-derived components or animal tissue-derived components (such as growth factors). The medium may or may not contain any serum substitute. Serum substitutes may include materials appropriately containing albumin (such as lipid-rich albumin, bovine albumin, recombinant albumin, or humanized albumin, plant starch, dextran, protein hydrolysates, etc.), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol (α-monothioglycerol, MTG), or equivalents thereof. Commercially available materials such as knockout serum replacement (KSR), chemically defined lipid concentrated, and GlutaMAX may also be used. The culture medium may be a serum-free medium (SFM) suitable for cell development. For example, the medium may contain B-27® supplement, Xenofree B-27® supplement, NS21® supplement, GS21® supplement, or a combination thereof, at concentrations effective for producing T cells from 3D cell aggregates.

[0103] The culture medium may contain one or more substances selected from the group consisting of biotin; DL-alpha-tocopherol acetate; DL-alpha-tocopherol; vitamins such as vitamin A (acetate); BSA (bovine serum albumin) or human albumin, fatty acid-free fraction V; catalase; human recombinant insulin; human transferrin; proteins such as superoxide dismutase; corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced form); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and T3 (triiodo-L-thyronine); and PSG (penicillin, streptomycin, and L-glutamine). The culture medium may also contain externally added ascorbic acid or its derivatives (e.g., ascorbic acid 2-phosphate: PAA). Each culture medium may contain one or more substances added from an external source, selected from the group consisting of fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antibiotics, antioxidants, 2-mercaptoethanol, pyruvate, buffers, and inorganic salts.

[0104] The culture medium may contain cytokines added from an external source. Examples of cytokines include FLT3 ligand (FLT3L), interleukin-7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-3, IL-4, IL-6, IL-12, IL-15, IL-18, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, and midkines. These cytokines may be used individually or in combination of two or more. A preferred cytokine is IL-2.

[0105] (Culturing Process) The method for producing genetically modified immune cells according to the present disclosure may include the steps of introducing a foreign gene into immune cells, culturing the transduced immune cells for more than 24 hours and culturing them so that the number of cells is no more than twice that of the cells before culturing, culturing the transduced immune cells for 24 hours or less and culturing them so that the number of cells is more than twice that of the cells before culturing, or culturing the transduced immune cells for 24 hours or less and culturing them so that the number of cells is no more than twice that of the cells before culturing. However, from the viewpoint of the degree of antitumor activity, early manifestation of antitumor effect, early manifestation of anticancer effect, manufacturing cost, and manufacturing efficiency, if the method includes the step of culturing transduced immune cells after introducing a foreign gene for the purpose of proliferating immune cells, the culturing time is preferably 12 hours or less, more preferably 8 hours or less, even more preferably 4 hours or less, and particularly preferably 1 hour or less. Furthermore, from the same viewpoint as above, if the process includes a step of culturing transfected immune cells after introducing foreign genes for the purpose of proliferating immune cells, it is preferable that the culture step results in a cell number of 1.5 times or less compared to before culture, more preferably 1 time or less, and even more preferably 0.5 times or less. Moreover, it is most preferable that the process does not include any of the above steps of culturing transfected immune cells after introducing foreign genes into immune cells. The step of culturing transfected immune cells after introducing foreign genes can be carried out, for example, by placing the immune cells after the transfection step into a culture plate filled with culture medium. The time for culturing the transfected immune cells refers to the time from when the immune cells after the transfection step are placed into the culture plate until they are collected. The culture conditions, etc., in the step of culturing the transfected immune cells can be the same as those described for the activation step described later, and can be appropriately selected from the examples provided. However, the culture vessels, culture medium, and additives used in the process of culturing the transduced immune cells do not necessarily have to be the same as those used in the activation process and can be changed as appropriate.

[0106] (Activation Step) The method for producing genetically modified immune cells according to this disclosure may include a step of activating the immune cells. The activation step is performed before the transduction step. In this specification, "activating immune cells" means providing sufficient stimulation to induce detectable immune cells. "Stimulating substance" refers to a material that can activate immune cells, and may be, but is not limited to, proteins, amino acids, nucleic acids, cells, etc. In this specification, "activated immune cells" refers to immune cells that are undergoing cell division.

[0107] Preferably, at least one of a CD3 agonist or a CD28 agonist can be used as a stimulant to activate immune cells, but it is not limited to these. One type of stimulant may be used, or multiple types may be used in combination.

[0108] In this specification, "CD3 agonist" and "CD28 agonist" refer to substances that bind to CD3 and CD28 (receptors) as agonists, respectively, and stimulate T cells for activation. Typical CD3 agonists are anti-CD3 antibodies, and typical CD28 agonists are anti-CD28 antibodies, but it is also possible to use substances other than antibodies that have similar effects (for example, T-CEP (T-cell expansion protein (Matus et al., JCI insight, 2020, vol.5, issue 22, 1-14)) and PHA (phytohemagglutinin (Weng et al., J.Ethnophararmacol, 2002, 83(1-2):79-85))) as CD3 agonists and CD28 agonists. Anti-CD3 antibodies and anti-CD28 antibodies may be monoclonal or polyclonal antibodies, but monoclonal antibodies are preferred. The anti-CD3 antibody and anti-CD28 antibody may be fragmented.

[0109] In the activation step, an irritant may be used in combination with a recombinant human fibronectin fragment. This helps to co-localize the target immune cells and the viral vector, thereby increasing gene efficiency in retroviral vector-mediated transduction of immune cells. For example, retronectin® can be used as the recombinant human fibronectin fragment.

[0110] The concentration of the irritant is not particularly limited as long as it is sufficient to stimulate the surface molecules of immune cells, thereby transmitting signals into the immune cells and inducing activation. For example, when using anti-CD3 antibodies and / or CD28 antibodies, the final antibody concentrations can be set to 0.1 to 20 μg / mL. Also, when using a matrix of mobile polymer chains carrying anti-CD3 and anti-CD28 antibodies (e.g., TransACT), the final antibody concentrations can be set to 10 to 300 ng / mL. The molar ratio of anti-CD3 antibodies to anti-CD28 antibodies can be 1:5 to 5:1, preferably 1:2 to 2:1.

[0111] From the viewpoint of transduction efficiency in the transduction process, the activation time may be 2 hours or more, 5 hours or more, 7 hours or more, 10 hours or more, 15 hours or more, preferably 24 hours or more, more preferably 36 hours or more, 45 hours or more, 46 hours or more, 47 hours or more, and even more preferably 48 hours or more. From the viewpoint of the production efficiency of immune cells, the activation time may be 300 hours or less, 200 hours or less, 180 hours or less, preferably 120 hours or less, more preferably 100 hours or less, even more preferably 80 hours or less, 79 hours or less, 78 hours or less, 77 hours or less, 76 hours or less, 75 hours or less, 74 hours or less, 73 hours or less, 72 hours or less, 71 hours or less, and 70 hours or less. The activation time is preferably 24 hours or more and 120 hours or less, more preferably 36 hours or more and 100 hours or less, and even more preferably 48 hours or more and 80 hours or less.

[0112] The activation step begins when the culture of immune cells is started in a culture medium containing an irritant (including plates coated with the irritant). The activation step can be terminated by replacing the culture medium containing the irritant with a culture medium that does not contain the irritant, or by diluting the culture medium containing the irritant to a level where activation of immune cells does not occur. In this disclosure, when the activation step is terminated by diluting the culture medium, the activation time does not include the time spent culturing immune cells in the diluted culture medium. For example, when terminating the activation step using TransACT by diluting the culture medium, the activation step can be terminated by diluting to 70% or less (preferably 50% or less).

[0113] From the viewpoint of transduction efficiency and the efficiency of producing immune cells, the ratio of transduction time to activation time (transduction time / activation time) is preferably 0.1 to 0.8, more preferably 0.2 to 0.7, and even more preferably 0.3 to 0.6.

[0114] From the viewpoint of transduction efficiency and the efficiency of producing immune cells, the sum of activation time and transduction time is preferably 40 to 200 hours, more preferably 50 to 150 hours, and even more preferably 60 to 120 hours.

[0115] In the activation process, the culture method, culture medium, and other culture conditions (temperature, atmosphere, cell density, etc.) can be set by appropriately modifying conventionally known methods. In the activation process, the cell density at seeding is, for example, 1.0 x 10⁻⁶. ５ ~1.0 x 10 ７ It can be expressed as cells / mL, preferably 2.0 x 10 ５ ~6.0x10 ６ It can be expressed as cells / mL.

[0116] The culture vessel used in the activation step is not particularly limited, and any of the examples provided in the transduction step can be appropriately selected and used.

[0117] In one embodiment, the irritant, for example, a CD3 agonist and / or a CD28 agonist, is supported on a matrix of mobile polymer chains.

[0118] In this specification, the definition and embodiments of “mobile polymer chain matrix” (hereinafter, “mobile matrix”) are the same as those described in the aforementioned Patent Document 1 (International Publication No. 2014 / 048920), and reference to the said Patent Document is permitted in this disclosure. The mobile matrix may be made of collagen, purified protein, purified peptide, polysaccharide, glycosaminoglycan, or extracellular matrix composition. Examples of polysaccharides include cellulose ether, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan gum, guar gum, and alginate. Examples of other polymers include polyester, polyether, polyacrylate, polyacrylamide, polyamine, polyethyleneimine, polyquaternium polymer, polyphosphozene, polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, block copolymer, and polyurethane. Preferably, the mobile matrix is ​​a polymer of dextran.

[0119] In one embodiment, the irritant, for example, a CD3 agonist and / or a CD28 agonist, is supported on beads. Specific examples include magnetic beads such as Dynabeads Human T-Activator CD3 / CD28.

[0120] CD3 agonists and / or CD28 agonists are “supported,” or “attached,” to such mobile matrices or beads. Substances (CD3 agonists and / or CD28 agonists) can be attached to or coupled to mobile matrices by various methods known and available in the art. Attachment may be covalent or non-covalent, electrostatic, or hydrophobic, and may be achieved by various attachment means, such as chemical, mechanical, enzymatic, or other means that allow the substance to stimulate cells. For example, antibodies may be attached directly to the matrix or beads, or indirectly via anti-isotype antibodies. Another example is attachment via protein A or protein G, or other nonspecific antibody-binding molecules, attached to the matrix or beads. Another example is attachment of substances to the matrix or beads by chemical means, such as crosslinking to the matrix or beads.

[0121] One embodiment of the present disclosure is a DNA-based T-cell activator (Keskar et al., J immunother, 2020, vol.43, no.8, 231-235) in which an irritant, for example, a CD3 agonist and / or CD28 agonist, is conjugated with CD3 and CD28 antibodies via complementary oligonucleotides to a single-stranded DNA scaffold.

[0122] (Recovery Step) The method for producing genetically modified immune cells according to this disclosure may include a recovery step of transduced immune cells after the transduction step or after culturing the transduced immune cells. From a quality viewpoint, the recovery step is preferably carried out within 360 minutes, more preferably within 180 minutes, and even more preferably within 60 minutes after the transduction step or after culturing the transduced immune cells. Centrifugation can be used as a recovery method. Furthermore, as described above, a pharmaceutically acceptable additive described later may be added within 12 hours after the recovery step.

[0123] (Freezing step) The method for producing genetically modified immune cells according to this disclosure may involve freezing the recovered immune cells after the recovery step. The freezing is preferably carried out within 12 hours, more preferably within 6 hours, and even more preferably within 3 hours after the recovery step. When freezing the recovered immune cells, conventionally known cryoprotective substances or pharmaceutically acceptable additives described later may be added before freezing. Examples of freezing temperatures include -20°C and -80°C.

[0124] (Pharmaceutical composition for cancer treatment) The pharmaceutical composition for cancer treatment of the present disclosure is a genetically modified immune cell obtained by transduction of immune cells using a retroviral vector, comprising: (1) the genetically modified immune cell having a PD-1 expression rate of 10% or more in a CD4(+) cell group; (2) the genetically modified immune cell having a PD-1 expression rate of 4% or more in a CD8(+) cell group; (3) the genetically modified immune cell having a TIGIT expression rate of 5% or more in a CD4(+) cell group or a CD8(+) cell group; or (4) the genetically modified immune cell having a LAG3 expression rate of 5% or more in a CD4(+) cell group or a CD8(+) cell group; and a pharmaceutically acceptable additive. Details regarding immune cells, genetically modified immune cells, etc., have been described above and are therefore omitted here. Furthermore, the genetically modified immune cell can be produced by the manufacturing method described above.

[0125] Cancers that can be targeted by the cancer treatment pharmaceutical composition include, for example, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer (e.g., melanoma, Merkel cell carcinoma), breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, cervical cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, non-small cell lung cancer, tracheal cancer, bronchial cancer, colon cancer, rectal cancer, small intestine cancer, colorectal cancer, stomach cancer, and gastric cancer. Cancers such as pharyngeal cancer, gallbladder cancer, testicular cancer, ovarian cancer, fallopian tube cancer, and nasopharyngeal cancer; cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue; sarcomas such as chondrosarcoma, Ewing's sarcoma, rhabdomyosarcoma, malignant hemangioendothelioma, malignant shunma, osteosarcoma, and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreaticoblastoma, pleuropulmonary blastoma, and retinoblastoma; germ cell tumors; lymphoma; leukemia, acute myeloid leukemia, and multiple myeloma. Preferably, cancers that are sensitive to NK cells include melanoma, Merkel cell carcinoma, colorectal cancer, kidney cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, liver cancer, lung cancer, non-small cell lung cancer, head and neck cancer, small intestine cancer, prostate cancer, bladder cancer, rectal cancer, pancreatic cancer, Ewing's sarcoma, rhabdomyosarcoma, nasopharyngeal cancer, esophageal cancer, biliary tract cancer, neuroblastoma, osteosarcoma, acute myeloid leukemia, multiple myeloma, lymphoma, leukemia, etc. More preferably, melanoma, colorectal cancer, kidney cancer, multiple myeloma, lymphoma, and leukemia.

[0126] Pharmaceutically acceptable additives include, more specifically, physiological saline, buffered physiological saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonic agents, fillers, and lubricants.

[0127] The cancer treatment pharmaceutical composition can be administered to subjects requiring cancer treatment (cancer patients, tumor-bearing animals, etc.) using methods similar to those used for known genetically modified immune cells (e.g., CAR-T cells). Administration methods include injection into tumors, veins, arteries, muscles, subcutaneously, and within the abdominal cavity.

[0128] The amount of genetically modified immune cells contained in a pharmaceutical composition for cancer treatment can be appropriately adjusted depending on the application, dosage form, and desired therapeutic effect, taking into consideration factors such as the type, location, and severity of cancer, as well as the age, weight, and condition of the patient receiving treatment. For example, in a single dose of the pharmaceutical, the amount of genetically modified immune cells is typically 1 × 10⁶. ４ ~1 x 10 １０ pieces, preferably 1 x 10 ５ ~1 x 10 ９ 10, more preferably 5 x 10 ６ ~5 x 10 ８ It can be formulated for individual administration. Furthermore, genetically modified immune cells (e.g., T cells expressing CAR, IL-7, and CCL19) produced by the manufacturing method of this disclosure can be expected to have a therapeutic effect with a smaller dose than the conventional doses mentioned above. For example, in a single administration of a cancer treatment pharmaceutical composition, the genetically modified immune cells can be expressed as 1 × 10⁶ cells. ３ ~1 x 10 ９ pieces, preferably 1 x 10 ４ ~1 x 10 ８ pieces, more preferably 1 x 10 ５ ~1 x 10 ７ It can be formulated for individual administration.

[0129] The administration interval of the cancer treatment pharmaceutical composition is not particularly limited and can be adjusted as appropriate, taking into consideration the amount of genetically modified immune cells administered at one time. For example, it can be administered independently four times, three times, twice or once a day, every other day, every two days, every three days, every four days, every five days, once a week, every seven days, every eight days, every nine days, twice a week, once a month, or twice a month.

[0130] If a pharmaceutical composition for cancer treatment is intended for cancer treatment, it can be used in combination with known anticancer agents. Examples of anticancer agents include alkylating agents such as cyclophosphamide, bendamustine, ifosfamide, and dacarbazine; antimetabolites such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enocitabine; molecularly targeted drugs such as rituximab, cetuximab, and trastuzumab; and kinase inhibitors such as imatinib, gefetinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib. Examples include: proteasome inhibitors such as bortezomib; calcineurin inhibitors such as cyclosporine and tacrolimus; anticancer antibiotics such as idarubidine and doxorubicin mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum-based drugs such as cisplatin, oxaliplatin, and carboplatin; hormone therapy drugs such as tamoxifen and bicardamide; and immunosuppressants such as interferon, nivolumab, and pembrolizumab.

[0131] The present disclosure will be explained below with reference to examples, but the present disclosure is not limited to the following examples.

[0132] [Example 1] Preparation of anti-GM2 CAR (scFv sequence of anti-GM2 CAR) The scFv sequence of anti-GM2 CAR was designed. Specifically, a DNA fragment of VL-linker 15-VH (SEQ ID NO: VL15VH) was synthesized.

[0133] First, we prepared an existing CAR-IL-7 / CCL19 vector by inserting CAR-IL-7-F2A-CCL19-F2A-HSV-tk, which is created by flanking F2A (SEQ ID NO: 2) in a third-generation CAR construct consisting of scFv, a human CD8 transmembrane domain, and a human CD28-4-1BB-CD3ζ intracellular activation signaling domain, and then linking it with human IL-7-F2A-human CCL19-F2A-HSV-tk, into a pMSGV1 retrovirus expression vector (Tamada k et al., Clin Cancer Res 18:6436-6445(2012)). The scFv region of CAR in the above-mentioned CAR-IL-7 / CCL19 vector was replaced with a synthesized anti-GM2 scFv DNA fragment (SEQ ID NO: 1) by restriction enzyme treatment and ligation to create an IL-7 / CCL19 expression-anti-GM2 CAR vector (SEQ ID NO: 3).

[0134] (Preparation of retroviral vectors with IL-7 / CCL19 expression-anti-GM2 CAR vector) Retroviral vectors were prepared for gene transfer into T cells. Retroviral vectors with the IL-7 / CCL19 expression-anti-GM2 CAR vector were prepared by transfecting GP2-293 packaging cell lines with the above-mentioned IL-7 / CCL19 expression-anti-GM2 CAR vector and p-Ampho plasmid. The supernatant containing the retroviral vector was collected 48 hours after transfection.

[0135] (T cell activation and transduction) Peripheral blood mononuclear cells collected from the blood of healthy donors were subjected to T cell activation on a plate immobilized with anti-CD3 monoclonal antibody and retronectin (registered trademark), together with IL-2, at 37°C and 5% CO2. ２The cells were cultured in an incubator for two days (activation time 48 hours) (activation step). On the first day after the start of culture, 500 μl of the supernatant containing the retroviral vector into which the IL-7 / CCL19 expression-anti-GM2 CAR vector prepared as described above was added to each well of an untreated well plate pre-coated with retronectin, and retroviral preload plates were prepared by centrifugation at 2000 g for 2 hours. A total of two plates were prepared, washed with 1.5% BSA / PBS after centrifugation, and stored at 4°C until use. On the second day of culture, the activated cells were collected from the above plates and prepared as a cell suspension. This cell suspension was added to the retroviral preload plate and incubated at 37°C in the presence of IL-2 and 5% CO2. ２ The cells were cultured in an incubator for 24 hours, followed by the first retrovirus infection. The seeded cell count was 4 × 10⁶. ５ cells / well ~ 1.2 x 10 ６ Cells were divided into cells per well. The following day (day 4 of culture), the cell solution from each well was transferred to a second virus preload plate that had been stored, centrifuged at 500g for 1 minute, and then cultured at 37°C for 4 hours to perform a second infection (transduction step), and T cells A (anti-GM2 CAR-IL-7 / CCL19 expressing T cells A) were obtained by transduction with a retroviral vector into which an IL-7 / CCL19 expressing-anti-GM2 CAR vector had been introduced (total transduction time: 28 hours).

[0136] Anti-GM2 CAR-IL-7 / CCL19 expressing T cells B were obtained in the same manner as in the production of anti-GM2 CAR-IL-7 / CCL19 expressing T cells A, except that the activation process was changed to 3 days (72 hours).

[0137] Furthermore, after the transduction step in the production of anti-GM2 CAR-IL-7 / CCL19 expressing T cells A, the cell suspension in each well is diluted fourfold with a new culture medium containing IL-2 and transferred to a new cell culture plate at 37°C and 5% CO2. ２The cells were cultured in an incubator for three days (expansion culture step). This yielded T cells a (anti-GM2 CAR-IL-7 / CCL19 expressing T cells a) transduced with a retroviral vector into which an IL-7 / CCL19 expressing anti-GM2 CAR vector had been introduced. The cell count more than doubled before and after the 72-hour expansion culture step, indicating that this expansion culture step was effectively an expansion culture step.

[0138] Anti-GM2 CAR-IL-7 / CCL19 expressing T cells b were obtained in the same manner as in the production of anti-GM2 CAR-IL-7 / CCL19 expressing T cells a, except that the activation process was changed to 3 days (72 hours). Similar to T cells a, the cell count more than doubled before and after the 72-hour expansion culture process, indicating that this expansion culture process was substantially effective.

[0139] [Example 2] Preparation of anti-EGFRviiii CAR (Sequence of anti-EGFRviiii CAR) The sequence of the anti-EGFRviiii CAR was designed. Specifically, a DNA fragment of a second-generation CAR construct (SEQ ID NO: 22) consisting of anti-EGFRviiii VL-linker 25-VH (SEQ ID NO: 4), a human CD28 transmembrane domain, and a human CD28-CD3ζ intracellular signaling domain was synthesized. In addition, a DNA fragment of a second-generation CAR construct (SEQ ID NO: 24) consisting of anti-EGFRviiii VL-linker 25-VH, a human CD8 transmembrane domain, and a human 4-1BB-CD3ζ intracellular signaling domain was synthesized.

[0140] (Preparation of an anti-EGFRviii CAR expression vector expressing IL-7 / CCL19 and HSV-tk) First, an existing CAR-IL-7 / CCL19 vector was prepared by inserting CAR-IL-7-F2A-CCL19-F2A-HSV-tk, which is formed by flanking F2A in the CAR construct and then linking it with human IL-7-F2A-human CCL19-F2A-HSV-tk, into a pMSGV1 retrovirus expression vector (Tamada k et al., Clin Cancer Res 18:6436-6445(2012)). The CAR construct portion in the above-mentioned CAR-IL-7 / CCL19 vector was replaced by restriction enzyme treatment and ligation with a DNA fragment of a second-generation CAR (SEQ ID NO: 22) consisting of synthesized anti-EGFRviiii scFv, a human CD28 transmembrane domain, and a human CD28-CD3ζ intracellular signaling domain, to create the IL-7 / CCL19 expression-anti-EGFRviiii CAR vector (SEQ ID NO: 23).

[0141] (Construction of retroviruses with IL-7 / CCL19 expression-anti-EGFRviiii CAR vector) Retroviruses were constructed for gene transfer into T cells. Retroviruses with the IL-7 / CCL19 expression-anti-EGFRviiii CAR vector were constructed by transfecting GP2-293 packaging cell lines with the aforementioned IL-7 / CCL19 expression-anti-EGFRviiii CAR vector and p-Ampho plasmid. The supernatant containing the retrovirus was collected 48 hours after the start of transfection.

[0142] (T cell activation and transduction) Peripheral blood mononuclear cells collected from the blood of healthy donors were subjected to T cell activation on a plate immobilized with anti-CD3 monoclonal antibody and retronectin (registered trademark), together with IL-2, at 37°C and 5% CO2. ２The cells were cultured in an incubator for 3 days (activation time 72 hours) (activation step). On the second day after the start of culture, 500 μl of the supernatant containing the retrovirus into which the IL-7 / CCL19 expression-anti-EGFRviiii CAR vector prepared above had been introduced was added to each well of a pre-coated retronectin-coated, untreated well plate, and retrovirus preload plates were prepared by centrifugation at 2000 g for 2 hours. Two plates were prepared in total, washed with 1.5% BSA / PBS after centrifugation, and stored at 4°C until use. On the second day of culture, the activated cells were collected from the above plates and prepared as a cell suspension. This cell suspension was added to the retrovirus preload plate and incubated at 37°C in the presence of IL-2 and 5% CO2. ２ The cells were cultured in an incubator for 24 hours, followed by the first retrovirus infection. The seeded cell count was 4 × 10⁶. ５ cells / well ~ 1.2 x 10 ６ Cells were divided into cells per well. The following day (day 4 of culture), the cell solution from each well was transferred to a second virus preload plate that had been stored, centrifuged at 500g for 1 minute, and then cultured at 37°C for 4 hours to perform a second infection (transduction step), and T cells A (anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells A) were obtained by transduction with a retroviral vector into which an IL-7 / CCL19 expressing anti-EGFRviiii CAR vector had been introduced (total transduction time: 28 hours).

[0143] The CAR construct portion of the existing CAR-IL-7 / CCL19 vector was replaced by restriction enzyme treatment and ligation with a DNA fragment of a second-generation CAR (SEQ ID NO: 24) consisting of synthesized anti-EGFRviiii scFv, a human CD8 transmembrane domain, and a human 4-1BB-CD3ζ intracellular signaling domain to create an IL-7 / CCL19 expression-anti-EGFRviiii CAR vector (SEQ ID NO: 25). Anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells B were obtained in the same manner as anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells A.

[0144] Furthermore, after the transduction step in the production of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells A, the cell suspension in each well is diluted fourfold with a new culture medium containing IL-2 and transferred to a new cell culture plate at 37°C and 5% CO2. ２ The cells were cultured in an incubator (expansion culture step). Peripheral blood mononuclear cells were cultured for four days from the start of culture (expansion culture time 96 hours), thereby obtaining T cells a transduced with the IL-7 / CCL19-expressing anti-EGFRviiii CAR vector (anti-EGFRviiii CAR-IL-7 / CCL19-expressing T cells a). The cell count more than doubled before and after the 96-hour expansion culture step, indicating that this expansion culture step was effectively an expansion culture step.

[0145] Furthermore, after the transduction step in the production of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells B, 1 ml of the cell suspension from each well is transferred to a new cell culture plate, diluted fourfold with a new culture medium containing IL-2, and cultured at 37°C in 5% CO2. ２ The cells were cultured in an incubator (expansion culture step). Peripheral blood mononuclear cells were cultured for 4 days from the start of culture (expansion culture time 96 hours), thereby obtaining T cells b (anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells b) into which the IL-7 / CCL19 expressing-anti-EGFRviiii CAR vector had been introduced. Similar to T cells a, the cell number more than doubled before and after the 96-hour expansion culture step, indicating that this expansion culture step was effectively an expansion culture step.

[0146] <Measurement of CAR expression rate by flow cytometry> (Flow cytometry analysis) The expression level of CARs that recognize GM2 as an antigen was analyzed by flow cytometry.

[0147] (Results) The results are shown in Figures 1(1A) to (1D). Figure 1(1A) shows the results for anti-GM2 CAR-IL-7 / CCL19 expressing T cells A, Figure 1(1B) shows the results for anti-GM2 CAR-IL-7 / CCL19 expressing T cells B, Figure 1(1C) shows the results for anti-GM2 CAR-IL-7 / CCL19 expressing T cells a, and Figure 1(1D) shows the results for anti-GM2 CAR-IL-7 / CCL19 expressing T cells b. The numbers in the figures represent the percentage of each population. As shown in Figures 1(1A) to (1D), it was found that the presence or absence of an expansion culture step in the production of anti-GM2 CAR-IL-7 / CCL19 expressing T cells did not affect the CAR expression rate of the genetically modified immune cells produced.

[0148] Similarly, the CAR expression rate was analyzed for anti-EGFRviiiii CAR-IL-7 / CCL19 expressing T cells. The results are shown in Figures 2(2A) to (2D). Figure 2(2A) shows the results for anti-EGFRviiiii CAR-IL-7 / CCL19 expressing T cells A, Figure 2(2B) shows the results for anti-EGFRviiiii CAR-IL-7 / CCL19 expressing T cells B, Figure 2(2C) shows the results for anti-EGFRviiiii CAR-IL-7 / CCL19 expressing T cells a, and Figure 2(2D) shows the results for anti-EGFRviiiii CAR-IL-7 / CCL19 expressing T cells b. The numbers in the figures represent the percentage of each population. As shown in Figures 2(2A) to (2D), it was found that anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells obtained without performing the expansion culture process had a higher CAR expression rate compared to anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells obtained by performing the expansion culture process.

[0149] <Phenotype Analysis - 1> Flow cytometry analysis was performed to determine the proportion of CAR(+) cells in the CD4(+) cell population of the prepared anti-GM2 CAR-IL-7 / CCL19 expressing T cell A. The result was 89.8%, as shown in Figure 3 (3A). Next, the CD4(+) CAR(+) cells were stained by reacting them with anti-human CD45RA monoclonal antibody and anti-human CCR7 monoclonal antibody. Flow cytometry analysis was performed to determine the proportion of human CD45RA(+) and human CCR7(+) cells in the CD4(+) CAR(+) cell population. The result was 82.8%, as shown in Figure 3 (3B).

[0150] Phenotype analysis was similarly performed on anti-GM2 CAR-IL-7 / CCL19 expressing T cells B, anti-GM2 CAR-IL-7 / CCL19 expressing T cells a, anti-GM2 CAR-IL-7 / CCL19 expressing T cells b, anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells A, anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells B, anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells a and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells b. The results for each are shown in Figures 4(4A), (4B) to 10(10A), (10B), and summarized in Table 1. Compared to CAR-IL-7 / CCL19-expressing T cells obtained through the expansion culture process, CAR-IL-7 / CCL19-expressing T cells obtained without the expansion culture process had a higher proportion of human CD45RA(+) and human CCR7(+) cells, as well as a higher proportion of stem cell memory cells.

[0151]

[0152] <Phenotype Analysis - 2> Flow cytometry analysis was performed on the prepared anti-GM2 CAR-IL-7 / CCL19 expressing T cell A to determine the proportion of CAR(+) cells within the CD8(+) cell population. The result was 95.9%, as shown in Figure 11 (11A). Next, the CD8(+) CAR(+) cells were stained by reacting them with anti-human CD45RA monoclonal antibody and anti-human CCR7 monoclonal antibody. Flow cytometry analysis was performed to determine the proportion of human CD45RA(+) and human CCR7(+) cells within the CD8(+) CAR(+) cell population. The result was 92.8%, as shown in Figure 11 (11B).

[0153] Phenotype analysis was similarly performed on anti-GM2 CAR-IL-7 / CCL19 expressing T cells B, anti-GM2 CAR-IL-7 / CCL19 expressing T cells a, anti-GM2 CAR-IL-7 / CCL19 expressing T cells b, anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells A, anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells B, anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells a and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells b. The results for each are shown in Figures 12(12A), (12B) to 18(18A), (18B), and summarized in Table 2. Compared to CAR-IL-7 / CCL19-expressing T cells obtained through the expansion culture process, CAR-IL-7 / CCL19-expressing T cells obtained without the expansion culture process had a higher proportion of human CD45RA(+) and human CCR7(+) cells, as well as a higher proportion of stem cell memory cells.

[0154]

[0155] <PD-1, TIGIT, and LAG3 Expression Rates> First, CD4(+) and CD8(+) cell groups were obtained from anti-GM2 CAR-IL-7 / CCL19 expressing T cell B by flow cytometry. The proportions of CD4(+) and CD8(+) cell groups are shown in Figure 39. Next, the CAR expression levels in the CD4(+) cell group and the CD8(+) cell group were measured. The results are shown in Figures 40 and 41. Furthermore, the expression levels of PD-1, TIGIT, and LAG3 in the CD4(+) and CAR(+) cell groups were measured by flow cytometry. The results are shown in Figure 19A, Figure 21A, and Figure 23A. Similarly, the expression levels of PD-1, TIGIT, and LAG3 in the CD8(+) and CAR(+) cell groups were measured by flow cytometry. The results are shown in Figure 20, 20A, Figure 22, and Figure 24, 24A.

[0156] Similar to anti-GM2 CAR-IL-7 / CCL19 expressing T cell B, CD4(+) and CD8(+) cell populations were obtained for anti-GM2 CAR-IL-7 / CCL19 expressing T cell b by flow cytometry. The proportions of CD4(+) and CD8(+) cell populations are shown in Figure 42. Next, the CAR expression levels in the CD4(+) cell population and the CD8(+) cell population were measured. The results are shown in Figures 43 and 44. Furthermore, the expression levels of PD-1, TIGIT, and LAG3 in the CD4(+) and CAR(+) cell populations were measured by flow cytometry. The results are shown in Figure 19B, Figure 21B, and Figure 23B. Similarly, the expression levels of PD-1, TIGIT, and LAG3 in the CD8(+) and CAR(+) cell populations were measured by flow cytometry. The results are shown in Figure 20, 20B, Figure 22, 22B, and Figure 24, 24B. Compared to CAR-IL-7 / CCL19-expressing T cells obtained by performing the expansion culture process, CAR-IL-7 / CCL19-expressing T cells obtained without performing the expansion culture process showed higher expression rates of PD-1, TIGIT, and LAG3.

[0157] <Measurement of cytotoxic activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells targeting tumor cells by ATP assay> ATP assays were performed with various ET ratios (Effector target ratios) of anti-GM2 CAR-IL-7 / CCL19 expressing T cells against tumor cells. In order to measure the cytotoxic activity of anti-GM2 CAR-IL-7 / CCL19 expressing T cells B and anti-GM2 CAR-IL-7 / CCL19 expressing T cells b against tumor cells, InVITRO cytotoxicity test-1 and InVITRO cytotoxicity test-2 were performed.

[0158] <In VITRO cytotoxicity test - 1> (co-culture test) CAR-positive cells 5 x 10 ５ CAR-T cell solutions were prepared by suspending anti-GM2 CAR-IL-7 / CCL19-expressing T cells B and anti-GM2 CAR-IL-7 / CCL19-expressing T cells b in tumor cell medium to a concentration of cells / mL. Alternatively, activated T cells were used for the same preparation. The activated T cells used included activated T cells that had not undergone the expansion culture process and activated T cells that had undergone the expansion culture process for 4 days. The activation time for both was 72 hours. Here, activated T cells refer to activated T cells that have not been transduced with foreign genes. CAR-T cell solutions or activated T cells, serially diluted in tumor cell medium, were added at a rate of 100 μL / well to well plates containing tumor cells, to prepare E:T ratios of 0:1, 0.01:1, 0.03:1, 0.1:1, 0.3:1, or 1:1. The above well plate was heated to 37°C and 5 vol% CO2. ２ CO settings ２ The samples were placed in an incubator and co-cultured for 48 hours. ２ The tumor cell culture medium was removed from the well plates taken from the incubator and washed with PBS. An equal volume of CellTiter-Glo™ Reagent (100 μL / well) was added to the wells of the co-culture plate, and after shaking for 10 minutes, the luminescence signal was measured using a plate reader to evaluate the number of viable cells. The results are shown in Figure 25.

[0159] Furthermore, the same test was performed using the GM2-negative SW480 cell line instead of the GM2-positive MSTO211H GFP-Luc cell line. The results are shown in Figure 26. In Figures 25 and 26, anti-GM2 CAR-IL-7 / CCL19 expressing T cells B are indicated as 72h-0d GM2(3rd)7x19 CAR-T, anti-GM2 CAR-IL-7 / CCL19 expressing T cells b are indicated as 72h-4d GM2(3rd)7x19 CAR-T, activated T cells that did not undergo the expansion culture process are indicated as 72h-0d Act-T, and activated T cells that underwent the expansion culture process for 4 days are indicated as 72h-4d Act-T.

[0160] As shown in Figure 25, anti-GM2 CAR-IL-7 / CCL19 expressing T cells showed stronger cytotoxic activity against GM2-positive cancer cells compared to activated T cells. Furthermore, CAR-IL-7 / CCL19 expressing T cells B, obtained without the expansion culture process, showed stronger cytotoxic activity compared to CAR-IL-7 / CCL19 expressing T cells b, obtained by the expansion culture process.

[0161] <In VITRO Cytotoxicity Test - 2> (Co-culture test) The real-time cytotoxicity assay of CAR-T cells against cancer cells was evaluated by measuring the impedance (electrical resistance) of adherent cancer cells using xCELLigence®. 2 × 10⁶ GM2-positive tumor cell lines Y-MESO-8A, MSTO211H GFP Luc, or A549 Luc were placed in a plate with electrodes accumulated at the bottom of the wells. ４ Cells were seeded individually. One day after seeding, anti-GM2 CAR-IL-7 / CCL19 expressing T cells B and anti-GM2 CAR-IL-7 / CCL19 expressing T cells b were seeded in wells containing each GM2-positive tumor cell line, with an ET ratio of 1:1 or 0.2:1. Co-culture was then carried out for 7 days at 37°C. Changes in the number of remaining cancer cells during co-culture with CAR-T cells were monitored by measuring electrical resistance (CELL Index). The results for A549 Luc are shown in Figure 27, the results for Y-MESO-8A in Figure 28, and the results for MSTO211H GFP Luc in Figure 29.

[0162] Furthermore, the cytotoxic activity against cancer cells by co-culturing with CAR-T cells was also evaluated using xCELLigence®. The cytotoxic activity results for A549 Luc are shown in Figure 30, the cytotoxic activity results for Y-MESO-8A are shown in Figure 31, and the cytotoxic activity results for MSTO211H GFP Luc are shown in Figure 32. In Figures 27 and 30, the case with only GM2-positive tumor cell lines is represented by A10, A11, and A12; the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells B and GM2-positive tumor cell lines with an ET ratio of 1:1 is represented by C1, C2, and C3; the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells B and GM2-positive tumor cell lines with an ET ratio of 0.2:1 is represented by D1, D2, and D3; the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells b and GM2-positive tumor cell lines with an ET ratio of 1:1 is represented by G1, G2, and G3; and the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells b and GM2-positive tumor cell lines with an ET ratio of 0.2:1 is represented by H1, H2, and H3. In Figures 28 and 31, the case with only GM2-positive tumor cell lines is represented by B10 and B11, the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells B and GM2-positive tumor cell lines with an ET ratio of 1:1 is represented by C4, C5, and C6, the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells B and GM2-positive tumor cell lines with an ET ratio of 0.2:1 is represented by D4, D5, and D6, the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells b and GM2-positive tumor cell lines with an ET ratio of 1:1 is represented by G4, G5, and G6, and the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells b and GM2-positive tumor cell lines with an ET ratio of 0.2:1 is represented by H4, H5, and H6.In Figures 29 and 32, the case with only GM2-positive tumor cell lines is represented by C10, C11, and C12; the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells B and GM2-positive tumor cell lines with an ET ratio of 1:1 is represented by C7, C8, and C9; the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells B and GM2-positive tumor cell lines with an ET ratio of 0.2:1 is represented by D7, D8, and D9; the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells b and GM2-positive tumor cell lines with an ET ratio of 1:1 is represented by G7, G8, and G9; and the case with anti-GM2 CAR-IL-7 / CCL19-expressing T cells b and GM2-positive tumor cell lines with an ET ratio of 0.2:1 is represented by H7, H8, and H9.

[0163] Figures 27 to 32 show that CAR-IL-7 / CCL19-expressing T cells obtained without the expansion culture process exhibited higher cytotoxic activity compared to those obtained with the expansion culture process. While it is generally predicted that CAR-IL-7 / CCL19-expressing T cells obtained without the expansion culture process would have lower cytotoxic activity due to the higher proportion of stem cell memory cells compared to those obtained with the expansion culture process, this result was unexpected. Furthermore, as can be seen from Figures 27 to 32, compared to CAR-IL-7 / CCL19-expressing T cells obtained by performing the expansion culture process, CAR-IL-7 / CCL19-expressing T cells obtained without performing the expansion culture process show a faster decrease in impedance values ​​after seeding, indicating a faster rate of cancer cell death. Additionally, the time it takes to achieve nearly 100% cancer cell elimination is shorter, indicating that these cells exert cytotoxic activity early on.

[0164] <In VITRO Cytokine Production Test> Anti-GM2 CAR-IL-7 / CCL19 expressing T cells B or anti-GM2 CAR-IL-7 / CCL19 expressing T cells b were mixed with the GM2-positive tumor cell line MSTO211H GFP Luc at an ET ratio of 1:1 or 0.3:1 and cultured for 48 hours. The concentration of IFN-γ produced in the culture supernatant was measured by ELISA. The results are shown in Figure 33. A commercially available IFN-γ ELISA kit was used for the above measurement. In Figure 33, anti-GM2 CAR-IL-7 / CCL19 expressing T cells B are shown as 72h-0d CAR-T, anti-GM2 CAR-IL-7 / CCL19 expressing T cells b are shown as 72h-4d CAR-T, activated T cells that did not undergo the expansion culture process are shown as 72h-0d Act-T, and activated T cells that underwent the expansion culture process for 4 days are shown as 72h-4d Act-T.

[0165] Figure 33 shows that CAR-IL-7 / CCL19-expressing T cells obtained without performing the expansion culture process had a higher IFN-γ production capacity compared to CAR-IL-7 / CCL19-expressing T cells obtained by performing the expansion culture process.

[0166] <In VIVO Antitumor Activity Test> (Administration of anti-GM2 CAR-IL-7 / CCL19 expressing T cells to tumor model mice) Human small cell lung cancer cell line Lu-135 was administered to immunodeficient mice, NOD / SCID / IL2rgKO (NSG), in a dose of 5.0 × 10⁻¹⁶. ６ The mice were inoculated subcutaneously with 3.0 × 10¹⁶ cells to create a GM2-expressing tumor model mouse. Three days after subcutaneous inoculation, 3.0 × 10¹⁶ cells were obtained. ５ Individual anti-GM2 CAR-IL-7 / CCL19 expressing T cells B were administered intravenously to mice. Subsequently, the tumor volume of the model mice was measured and summarized in Figure 34. The horizontal axis represents the number of days after inoculation of the tumor cells into the mice (the day of inoculation was considered day 0), and the vertical axis represents the tumor volume (long axis of the tumor × short axis of the tumor). ２ / 2 (mm) ３ )) indicates.

[0167] Except for changing anti-GM2 CAR-IL-7 / CCL19 expressing T cells B to anti-GM2 CAR-IL-7 / CCL19 expressing T cells b, the tumor volume increase or decrease was observed in the same manner as above, and the results are summarized in Figure 35. In addition, the dose of anti-GM2 CAR-IL-7 / CCL19 expressing T cells b was changed to 1.0 × 10⁻⁶. ７ The number of tumors was changed to one, and the increase or decrease in tumor volume was observed and summarized in Figure 36.

[0168] Figures 34 to 36 show that CAR-IL-7 / CCL19-expressing T cells B, obtained without performing the expansion culture process, suppress tumor volume increase compared to CAR-IL-7 / CCL19-expressing T cells b, obtained by performing the expansion culture process. In particular, comparing Figure 34 and Figure 36, it was found that even though the dose of CAR-IL-7 / CCL19-expressing T cells b was more than 30 times the dose of CAR-IL-7 / CCL19-expressing T cells B, the effect of suppressing tumor volume increase by administering CAR-IL-7 / CCL19-expressing T cells B was superior. Furthermore, these results indicate that the antitumor activity per cell of CAR-IL-7 / CCL19-expressing T cells B is higher than that per cell of CAR-IL-7 / CCL19-expressing T cells b.

[0169] The number of CD3-positive cells, CD4-positive CAR-T cells, and CD8-positive CAR-T cells in the spleens of mice administered anti-GM2 CAR-IL-7 / CCL19-expressing T cells B was measured 85 to 95 days later and is shown in Figures 37 and 38. Similarly, the number of CD3-positive cells, CD4-positive CAR-T cells, and CD8-positive CAR-T cells in the spleens of mice administered anti-GM2 CAR-IL-7 / CCL19-expressing T cells b was measured and is shown in Figures 37 and 38. As shown in Figure 38, the number of CD4-positive CAR-T cells and CD8-positive CAR-T cells was below the detection limit (indicated as N.D. in Figure 38). In Figures 37 and 38, the results of administering anti-GM2 CAR-IL-7 / CCL19 expressing T cells B are labeled as 72h-0d, and the results of administering anti-GM2 CAR-IL-7 / CCL19 expressing T cells b are labeled as 72h-4d.

[0170] NOG-ΔMHC mice were inoculated with the human small cell lung cancer cell line Lu-135 in 5 × 10⁻¹⁵ doses. ６ 3 × 10¹ units were inoculated subcutaneously into mice to create a GM2-expressing tumor model mouse. Three days after subcutaneous inoculation, 3 × 10¹ units were observed. ５ 3 x 10⁶ anti-GM2 CAR-IL-7 / CCL19 expressing T cells B or anti-GM2 CAR-IL-7 / CCL19 expressing T cells b were administered intravenously to mice. ５ Immunostaining was used to analyze tumor-invasive T cells from anti-GM2 CAR-IL-7 / CCL19-expressing T cells b and anti-GM2 CAR-IL-7 / CCL19-expressing T cells B administered with 1 dose. The results showed significant invasion of both CD4-positive and CD8-positive cells in anti-GM2 CAR-IL-7 / CCL19-expressing T cells B, while almost no tumor-invasive T cells were observed in anti-GM2 CAR-IL-7 / CCL19-expressing T cells b (Figures 56 and 57). Furthermore, it was revealed that CCR7-positive cells were more frequently infiltrating the tumor in CAR-IL-7 / CCL19-expressing T cells B.

[0171] [Example 3] Production of anti-GM2 CAR-IL-7 / CCL19 expressing T cells The activation time was set to 72 hours, and anti-GM2 CAR-IL-7 / CCL19 expressing T cells C were produced in the same manner as in Example 1. In addition, an expansion culture step was performed in the same manner as for anti-GM2 CAR-IL-7 / CCL19 expressing T cells a in Example 1, and anti-GM2 CAR-IL-7 / CCL19 expressing T cells c were produced. The number of cells more than doubled before and after the expansion culture step, indicating that the expansion culture step was substantially an expansion culture step.

[0172] Anti-GM2 CAR-expressing T cells D were prepared in the same manner as anti-GM2 CAR-IL-7 / CCL19-expressing T cells C, except that human IL-7 and human CCL19 were not expressed. Furthermore, an expansion culture process was performed to prepare anti-GM2 CAR-expressing T cells d that do not express human IL-7 and human CCL19.

[0173] <Measurement of CAR expression rate by flow cytometry> (Flow cytometry analysis) The expression level of CARs that recognize GM2 as an antigen was analyzed by flow cytometry.

[0174] (Results) The results are shown in Figures 45(1A) to (1D). Figure 45(1A) shows the results for anti-GM2 CAR-expressing T cells D, Figure 45(1B) shows the results for anti-GM2 CAR-IL-7 / CCL19-expressing T cells C, Figure 45(1C) shows the results for anti-GM2 CAR-expressing T cells d, and Figure 45(1D) shows the results for anti-GM2 CAR-IL-7 / CCL19-expressing T cells c. The numbers in the figures represent the percentage of each population. As shown in Figures 45(1A) to (1D), it was found that the presence or absence of the expansion culture process in manufacturing does not affect the CAR expression rate of the genetically modified immune cells produced.

[0175] <Measurement of IL-7 and CCL19 concentrations in culture supernatant> The concentrations of IL-7 and CCL19 in the supernatant immediately before harvesting anti-GM2 CAR-IL-7 / CCL19 expressing T cells C and c, prepared as described above, were measured using an ELISA kit. The results are shown in Figures 46 and 47 (in the figures, anti-GM2 CAR-IL-7 / CCL19 expressing T cells C are referred to as T cell C, and anti-GM2 CAR-IL-7 / CCL19 expressing T cells c are referred to as T cell c. The same applies hereafter). The concentrations of IL-7 and CCL19 in the supernatant at the time of cell harvesting were lower in anti-GM2 CAR-IL-7 / CCL19 expressing T cells C than in anti-GM2 CAR-IL-7 / CCL19 expressing T cells c. Anti-GM2 CAR-IL-7 / CCL19 expressing T cells C were harvested without undergoing a wide-culture process, and the number of cells per well at the time of harvesting was small. Therefore, it is presumed that the IL-7 and CCL19 concentrations were lower compared to anti-GM2 CAR-IL-7 / CCL19 expressing T cells c.

[0176] <Phenotype Analysis - 1> Using the same method as described above, flow cytometry analysis was performed to determine the proportion of human CD45RA(+) and human CCR7(+) cells in the CD4(+) CAR(+) cell population for anti-GM2 CAR-IL-7 / CCL19 expressing T cell C, anti-GM2 CAR-IL-7 / CCL19 expressing T cell c, anti-GM2 CAR expressing T cell D, and anti-GM2 CAR expressing T cell d. The results are summarized in Table 3 and shown in Figure 48 (in the figure, anti-GM2 CAR expressing T cell D is indicated as T cell D, and anti-GM2 CAR d is indicated as T cell d. The same applies hereafter). Furthermore, using the same method as described above, flow cytometry analysis was performed to determine the proportion of human CD45RA(+) and human CCR7(+) cells in the CD8(+) CAR(+) cell population for anti-GM2 CAR-IL-7 / CCL19 expressing T cell C, anti-GM2 CAR-IL-7 / CCL19 expressing T cell c, anti-GM2 CAR expressing T cell D, and anti-GM2 CAR expressing T cell d. The results are summarized in Table 4 and shown in Figure 49.

[0177]

[0178]

[0179] <Measurement of IL-7Rα Expression Levels> Using the same method as described above, the expression levels of IL-7Rα in anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, anti-GM2 CAR expressing T cells D, and anti-GM2 CAR expressing T cells d were measured by flow cytometry analysis. The results are shown in Figure 50. Comparing anti-GM2 CAR-IL-7 / CCL19 expressing T cells c and anti-GM2 CAR expressing T cells d, it can be seen that the expression level of IL-7Rα is lower in anti-GM2 CAR-IL-7 / CCL19 expressing T cells c. On the other hand, comparing anti-GM2 CAR-IL-7 / CCL19 expressing T cells C and anti-GM2 CAR expressing T cells D, it can be seen that the difference is minor. This suggests that in anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, IL-7Rα expression was suppressed due to prolonged exposure to high concentrations of IL-7 produced by autocrine secretion during expansion culture, whereas in anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, a high level of IL-7Rα expression was maintained because the cells were exposed to relatively low concentrations of IL-7 for only a short time. Therefore, it is suggested that the present invention makes it possible to produce T cells that maintain responsiveness to IL-7 and are rich in early memory phenotypes that are considered to have a strong antitumor effect.

[0180] <In VITRO Cytotoxicity Test - 1> Anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, anti-GM2 CAR expressing T cells D, or anti-GM2 CAR expressing T cells d were co-cultured with the GM2-positive MSTO211H GFP Luc cell line or the GM2-negative SW480 cell line using the same method as described above, and the luminescence signal was measured to evaluate the number of remaining tumor cells. The results are shown in Figures 51 and 52, respectively. In addition, the supernatant after co-culture was collected and the concentration of IFN-γ was measured by ELISA, and the results are shown in Figure 53. Compared to anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, anti-GM2 CAR-IL-7 / CCL19 expressing T cells c showed higher cytotoxic activity and greater IFN-γ secretion. Similarly, compared to anti-GM2 CAR expressing T cells d, anti-GM2 CAR expressing T cells D showed higher cytotoxic activity and greater IFN-γ secretion.

[0181] <In VITRO Cytotoxicity Test - 2> Anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, anti-GM2 CAR expressing T cells D, or anti-GM2 CAR expressing T cells d, along with MSTO211H GFP Luc or A549 Luc, were evaluated in real time by measuring the impedance (electrical resistance) of adhered cancer cells using xCELLigence® in the same manner as described above. Changes in the number of remaining cancer cells in co-culture were monitored by measuring the electrical resistance (CELL Index). The results for MSTO211H GFP Luc are shown in Figure 54, and the results for A549 Luc are shown in Figure 55. Compared to anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, anti-GM2 CAR-IL-7 / CCL19 expressing T cells c were found to exhibit higher cytotoxic activity and exert cytotoxic activity earlier. Furthermore, compared to anti-GM2 CAR expressing T cells d, anti-GM2 CAR expressing T cells D were found to exhibit higher cytotoxic activity and exert cytotoxic activity earlier.

[0182] <Immune fatigue resistance test - 1> 3 x 10 ５ A number of anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, anti-GM2 CAR expressing T cells D, or anti-GM2 CAR expressing T cells d, and 1 × 10 ５ Co-culture with one MSTO211H GFP Luc cell line was initiated, and every 3-4 days, 1 x 10 MSTO211H GFP Luc cell line was added. ５ The cells were added, and co-culture was continued for 20 days. Changes in the number of remaining tumor cells and T cells during this period were observed and summarized in Figures 58 and 59. The black arrows in the figures indicate the time when the MSTO211H GFP Luc cell line was added. Up to Day 6, after the completion of the two tumor cell additions, anti-GM2 CAR-IL-7 / CCL19 expressing T cells C and anti-GM2 CAR expressing T cells D had fewer remaining tumor cells than anti-GM2 CAR-IL-7 / CCL19 expressing T cells c and anti-GM2 CAR expressing T cells d, respectively. After the third addition of tumor cells, the number of residual tumor cells increased in anti-GM2 CAR-expressing T cells D, anti-GM2 CAR-IL-7 / CCL19-expressing T cells c, and anti-GM2 CAR-expressing T cells d, while the number of residual tumor cells remained low in anti-GM2 CAR-IL-7 / CCL19-expressing T cells C, indicating that high antitumor activity was maintained.

[0183] Correlating with these results, anti-GM2 CAR-IL-7 / CCL19 expressing T cells C and anti-GM2 CAR expressing T cells D showed superior proliferation to anti-GM2 CAR-IL-7 / CCL19 expressing T cells c and anti-GM2 CAR expressing T cells d, respectively, until Day 6, after which the proliferation of anti-GM2 CAR expressing T cells D decreased. On the other hand, anti-GM2 CAR-IL-7 / CCL19 expressing T cells C showed a sustained proliferative response against tumor cells. These results were observed in both CD4-positive and CD8-positive T cell fractions (Figures 60 and 61). In Figure 60, the lines for T cells c and T cells d overlap with the horizontal axis.

[0184] <Immune Fatigue Resistance Test - 2> The expression rates of PD-1 and TIGIT in anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, anti-GM2 CAR expressing T cells D, or anti-GM2 CAR expressing T cells d were measured at Day 0, Day 10, and Day 17 during the co-culture period of Immunune Fatigue Resistance Test - 1, and are summarized in Figures 62 and 63. At Day 0, the expression levels of PD-1 and TIGIT in anti-GM2 CAR-IL-7 / CCL19-expressing T cells C and D were higher than those in anti-GM2 CAR-expressing T cells c and d, respectively. However, these levels decreased with repeated addition of tumor cells, and after the fifth addition, they were lower than those of anti-GM2 CAR-IL-7 / CCL19-expressing T cells c and d.

[0185] <Immune Fatigue Resistance Test - 3> During the co-culture period of Immunoe Fatigue Resistance Test - 1, at time points Day 3, Day 6, Day 10, Day 13, Day 17, and Day 20, the IL-7 concentration in the supernatant of co-cultured anti-GM2 CAR-IL-7 / CCL19 expressing T cells C or anti-GM2 CAR-IL-7 / CCL19 expressing T cells c was measured by ELISA and summarized in Figure 64. In the figure, anti-GM2 CAR-IL-7 / CCL19 expressing T cells C are indicated as Swift 7x19, and anti-GM2 CAR-IL-7 / CCL19 expressing T cells c are indicated as Standard 7x19. At all time points, anti-GM2 CAR-IL-7 / CCL19-expressing T cells C showed higher levels of IL-7 than anti-GM2 CAR-IL-7 / CCL19-expressing T cells c. Furthermore, while the IL-7 concentration in anti-GM2 CAR-IL-7 / CCL19-expressing T cells c decreased significantly after the third addition of tumor cells, it was found that a high concentration was maintained in anti-GM2 CAR-IL-7 / CCL19-expressing T cells C. From these results, it is presumed that anti-GM2 CAR-IL-7 / CCL19-expressing T cells C, by being rich in stem cell memory and central memory fractions, have enhanced cell proliferation in the early stages of culture, and that the sustained high levels of IL-7 production during repeated culture with tumor cells leads to long-term cell proliferation and sustained antitumor activity.

[0186] <Recurrence Prevention Test - 1> In the above In VIVO antitumor activity test, 3 × 10 ５ Mice in which tumors were eradicated by administering 1 x 10⁶ anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, and 1 x 10⁶ mice. ７The spleens of mice in which tumors were eradicated by administering anti-GM2 CAR-IL-7 / CCL19-expressing T cells c were removed on Day 49, and the number of remaining T cells was confirmed (Figure 65). As a result, the group administered anti-GM2 CAR-IL-7 / CCL19-expressing T cells c had more CD3-positive cells remaining compared to the group administered anti-GM2 CAR-IL-7 / CCL19-expressing T cells c, and this trend was observed in both CAR-negative and CAR-positive T cells (Figure 65). Furthermore, in a similar experiment, the proportion of CCR7-positive cells among the remaining CAR-positive and CAR-negative T cells was shown to be higher in the anti-GM2 CAR-IL-7 / CCL19-expressing T cell c group compared to the anti-GM2 CAR-IL-7 / CCL19-expressing T cell c group (Figure 66). Based on the above, it was suggested that in the group treated with anti-GM2 CAR-IL-7 / CCL19 T cells, many memory T cells remained in the mouse body for a long period, indicating an excellent inhibitory effect on tumor recurrence.

[0187] <Recurrence Prevention Test - 2> In the above In VIVO antitumor activity test, 3 × 10 ５ Mice in which tumors were eradicated by administering 1 x 10⁶ anti-GM2 CAR-IL-7 / CCL19 expressing T cells C, and 1 x 10⁶ mice. ７ In mice in which tumors were eradicated by administration of anti-GM2 CAR-IL-7 / CCL19 expressing T cells c, GM2-positive tumors Lu-135 were rechallenged on Day 60. The rejection rate in the anti-GM2 CAR-IL-7 / CCL19 expressing T cell c administration group was 40%, compared to approximately 27% in the anti-GM2 CAR-IL-7 / CCL19 expressing T cell c administration group (Figure 67). Furthermore, when comparing the average tumor volume in each group, tumor growth was significantly suppressed in the anti-GM2 CAR-IL-7 / CCL19 expressing T cell c administration group compared to the anti-GM2 CAR-IL-7 / CCL19 expressing T cell c administration group (Figure 68). In the rechallenge of GM2-negative SW480, no significant difference in tumor growth was observed between the two groups (Figure 69). In summary, anti-GM2 CAR-IL-7 / CCL19 expressing T cells C have not only potent tumor therapeutic effects but also long-term tumor recurrence suppression effects.

[0188] To confirm that the long-term survival of anti-GM2 CAR-IL-7 / CCL19 expressing T cells C is not due to tumorigenesis, 3 × 10⁶ anti-GM2 CAR-IL-7 / CCL19 expressing T cells C were subjected to a study. ５ Mice that rejected tumors after being administered 1x10⁻¹ doses, and anti-GM2 CAR-IL-7 / CCL19 expressing T cells c were compared at 1x10⁻¹⁶ doses. ７ Mice that rejected the tumor after being administered 1 dose were observed for an extended period, and the number of remaining T cells in the spleen was measured on Day 182 (Figure 70). The number of remaining T cells in the group administered anti-GM2 CAR-IL-7 / CCL19-expressing T cells C was significantly reduced compared to Day 49, and was at almost the same level as the group administered anti-GM2 CAR-IL-7 / CCL19-expressing T cells c. From these results, autonomous proliferation was not observed in the long-term surviving anti-GM2 CAR-IL-7 / CCL19-expressing T cells C, and it was confirmed that the long-term survival of anti-GM2 CAR-IL-7 / CCL19-expressing T cells C was not due to tumor formation.

[0189] [Example 4] Production of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells The activation time was set to 72 hours, and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells E were produced in the same manner as in Example 2. The signal transduction domain was the signal transduction domain in human 4-1BB-CD3ζ cells. In addition, an expansion culture step was performed in the same manner as for anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells a in Example 2, to produce anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells e. The number of cells more than doubled before and after the expansion culture step, indicating that the expansion culture step was substantially an expansion culture step.

[0190] <In VIVO Antitumor Activity Test> The tumor therapeutic effect was investigated using anti-EGFRviii CAR-IL-7 / CCL19 expressing T cells E or anti-EGFRviii CAR-IL-7 / CCL19 expressing T cells e and U87MG EGFRviii, which is an EGFRviii-positive tumor. (Administration of anti-EGFRviii CAR-IL-7 / CCL19 expressing T cells to tumor model mice) U87MG EGFRviii cell line was administered to NOG-ΔMHC mice in a dose of 3.5 × 10⁻⁶ ５EGFRviiii-expressing tumor model mice were created by subcutaneously inoculating mice with 1 × 10⁶ cells. Ten days after subcutaneous inoculation, 1 × 10⁶ cells were observed. ６ One anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell E or anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell e was administered intravenously to mice. Subsequently, the tumor volume of the model mice was measured using the same method as described above, and the results are summarized in Figures 71 and 72. In the figures, anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell E is indicated as T cell E, and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell e is indicated as T cell e. In the in vivo antitumor activity test, the expansion culture period for CAR-IL-7 / CCL19 expressing T cell e was set to 4 days.

[0191] As a result, a significantly higher tumor therapeutic effect was observed only in the anti-EGFRviii CAR-IL-7 / CCL19 expressing T cell group E (Figures 71 and 72). These results demonstrate that the excellent antitumor effect of the present invention is due to a universally applicable mechanism independent of the target molecule or CAR construct of CAR, and that it can be observed even in established tumors 10 days after inoculation.

[0192] [Example 5] Production of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells The activation time was set to 72 hours, and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F were produced in the same manner as in Example 2. The signal transduction domain was the signal transduction domain in human 4-1BB-CD3ζ cells. In addition, an expansion culture step was performed in the same manner as for anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells a in Example 2, and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f were produced. The number of cells more than doubled before and after the expansion culture step, indicating that the expansion culture step was substantially an expansion culture step.

[0193] <Phenotype Analysis> Flow cytometry analysis was performed to determine the percentage of CD8(+) and CAR(+) cells in the prepared anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F and anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f. The results were 35.6% and 35.2%, respectively, as shown in Figures 73 and 74.

[0194] Next, CD4(+) CAR(+) cells were stained by reacting them with anti-human CD45RA monoclonal antibody and anti-human CCR7 monoclonal antibody. Flow cytometry analysis was used to determine the proportion of human CD45RA(+) and human CCR7(+) cells in the CD4(+) CAR(+) cell population. The results were 38.6% and 17.8%, respectively, as shown in Figures 75 and 76.

[0195] Next, CD8(+) CAR(+) cells were stained by reacting them with anti-human CD45RA monoclonal antibody and anti-human CCR7 monoclonal antibody. Flow cytometry analysis was used to determine the proportion of human CD45RA(+) and human CCR7(+) cells in the CD8(+) CAR(+) cell population. The results were 69.1% and 30.4%, respectively, as shown in Figures 77 and 78.

[0196] <Immune fatigue resistance test - 4> Anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F or anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f and the U87MG EGFRviiii cell line were each subjected to 1 × 10⁻¹⁶ tests. ５ The number of cells was adjusted to 1, and co-culture was started under conditions of E:T = 1:1. On the third day after the start of co-culture, anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F or anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f were harvested and 1 × 10⁻⁶ were collected. ５The U87MG EGFRviiiii cell line was seeded into new culture wells. This procedure was repeated every 3-4 days for 21 days. This experiment was conducted using T cells from two donors (n=2). Changes in the number of residual tumor cells and T cells during this period were observed and summarized in Figure 79. The black arrows in the figure indicate the point in time when the U87MG EGFRviiiii cell line was added.

[0197] Anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F showed superior proliferation compared to anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f.

[0198] <In Vivo Antitumor Activity Test> The tumor therapeutic effect was investigated using anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F or anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f, and U87MG EGFRviiii, an EGFRviiii-positive tumor. (Intravenous administration of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells in a subcutaneous tumor model) U87MG EGFRviiii cell line was administered to NOG-ΔMHC mice in a dose of 3.5 × 10⁻⁶ ５ EGFRviiii-expressing tumor model mice were created by subcutaneously inoculating mice with 1 × 10⁶ cells. Ten days after subcutaneous inoculation, 1 × 10⁶ cells were observed. ６ One anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell F or anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell f was administered intravenously to mice. After administration, the mice were observed and their survival rate was determined (number of mice: 10). As a result, the survival rate at 88 days for mice administered anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell F was 70%. The results are shown in Figure 80. On the other hand, the survival rate at 88 days for mice administered anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cell f was 0%.

[0199] (Intracerebroventricular administration of anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells in an intracranial tumor model) U87MG EGFRviiii cell line was administered to NOG-ΔMHC mice in a dose of 5.0 × 10⁻¹⁴. ５The EGFRviiii-expressing tumor model mouse was created by inoculating the mouse skull with 2 × 10¹⁶ cells. Seven days after inoculation, 2 × 10¹⁶ cells were inoculated. ５ One anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F were administered intravenously to mice, and they were observed for 247 days. As a result, the survival rate at 247 days for mice administered anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F was 40%. The results are shown in Figure 81. On the other hand, when anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f were used, the survival rate at 50 days was 0%. The results are shown in Figure 81. From this, it can be seen that anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F have a higher tumor treatment effect than anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells f. Furthermore, 40% of the mice treated with anti-EGFRviiii CAR-IL-7 / CCL19 expressing T cells F survived for more than 200 days, suggesting that the tumor was completely rejected.

Claims

1. A method for producing genetically modified immune cells, comprising a transduction step in which immune cells are transduced using a retroviral vector, and substantially not comprising an expansion culture step after the transduction step.

2. The method for producing a product according to claim 1, wherein the retroviral vector comprises at least one exogenous gene selected from the group consisting of genes for expressing cytokines, genes for expressing chemokines, and genes for expressing cell surface molecules that recognize malignant tumor antigens.

3. The method for producing a cell surface molecule according to claim 2, wherein the cell surface molecule is at least one selected from the group consisting of chimeric antigen receptors (CARs) and T cell receptors (TCRs).

4. The manufacturing method according to claim 2 or 3, wherein the cytokine is at least one selected from the group consisting of IL-7, IL-15, and IL-21.

5. The manufacturing method according to any one of claims 2 to 4, wherein the chemokine is at least one of CCL19 and CCL21.

6. The method for producing a genetically modified immune cell according to any one of claims 1 to 5, wherein the genetically modified immune cell is at least one selected from the group consisting of T cells, NK cells, B cells, monocytes, macrophages, dendritic cells, neutrophils, eosinophils, basophils, and mast cells.

7. The manufacturing method according to any one of claims 1 to 6, wherein the proportion of stem cell memory immune cells in the CAR(+) and CD8(+) cell population of the manufactured gene-modified immune cells is 60% or more.

8. The manufacturing method according to any one of claims 1 to 7, wherein the PD-1 expression rate in the CD4(+) cell population of the genetically modified immune cells produced is 10% or more.

9. The manufacturing method according to any one of claims 1 to 8, wherein the PD-1 expression rate in the CD8(+) cell population of the genetically modified immune cells produced is 4% or more.

10. The manufacturing method according to any one of claims 1 to 9, wherein the TIGIT expression rate in the CD4(+) cell population or CD8(+) cell population of the genetically modified immune cells produced is 5% or more.

11. The manufacturing method according to any one of claims 1 to 10, wherein the LAG3 expression rate in the CD4(+) cell population or CD8(+) cell population of the genetically modified immune cells produced is 5% or more.

12. The manufacturing method according to any one of claims 1 to 11, comprising an activation step of activating the immune cells prior to the transduction step, wherein the activation time is 24 hours or more and 120 hours or less.

13. A pharmaceutical composition for cancer treatment comprising: (1) genetically modified immune cells transduced into immune cells by a retroviral vector, wherein (1) the genetically modified immune cells have a PD-1 expression rate of 10% or more in a CD4(+) cell group; (2) the genetically modified immune cells have a PD-1 expression rate of 4% or more in a CD8(+) cell group; (3) the genetically modified immune cells have a TIGIT expression rate of 5% or more in a CD4(+) cell group or a CD8(+) cell group; or (4) the genetically modified immune cells have a LAG3 expression rate of 5% or more in a CD4(+) cell group or a CD8(+) cell group; and a pharmaceutically acceptable additive.

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