Chimeric cytokine receptor

Abstract

To simultaneously improve therapeutic efficacy and reduce side effects in an adoptive immunotherapy, the present invention provides a novel technique for enhancing functions such as proliferation ability of immune cells to be used in the adoptive immunotherapy and imparting an activity to capture a cytokine, which may induce side effects, to the immune cells.　Provided is a chimeric cytokine receptor that contains a ligand binding region on the N-terminal side and a T cell-activation region on the C-terminal side, wherein: the ligand binding region comprises the cytokine binding region of a cytokine receptor; the T cell-activation region contains the transmembrane and intracellular domains of IL-7 (interleukin-7) receptor α chain; and the transmembrane domain has an insert selected from the group consisting of (a) the amino acid sequence represented by SEQ ID NO: 2 between the positions 243 and 244 in the amino acid sequence represented by SEQ ID NO: 1, (b) the amino acid sequence represented by SEQ ID NO: 3 between the positions 241 and 242 in the amino acid sequence represented by SEQ ID NO: 1, (c) the amino acid sequence represented by SEQ ID NO: 4 between the positions 244 and 245 in the amino acid sequence represented by SEQ ID NO: 1, (d) the amino acid sequence represented by SEQ ID NO: 5 between the positions 244 and 245 in the amino acid sequence represented by SEQ ID NO: 1, and (e) the amino acid sequence represented by SEQ ID NO: 6 between the positions 246 and 247 in the amino acid sequence represented by SEQ ID NO: 1.

Description

[Technical Field]

[0001] The present invention relates to a chimeric cytokine receptor and a method for producing chimeric antigen receptor (CAR)-transformed cells having long-lasting cytotoxic activity. [Background technology]

[0002] Adoptive immunotherapy is a treatment method that involves collecting immune cells from a donor, such as the patient themselves, culturing, stimulating, manipulating, and / or proliferating them outside the body, and then introducing the cells into the patient. Examples of adoptive immunotherapy include tumor-infiltrating lymphocyte therapy, TCR gene-modified T cell therapy, and chimeric antigen receptor-T (CAR-T) cell therapy for cancer patients.

[0003] Among these, CAR-T cell therapy is a method that involves introducing a chimeric antigen receptor (CAR) into T cells, which is a single-chain antibody that recognizes cancer cell surface antigens, combined with a transmembrane domain and a signaling domain of a co-stimulatory molecule involved in T cell activation. CAR-T cell therapy aims to kill tumor cells by introducing the CAR gene encoding the CAR into T cells collected from the patient and then returning them to the patient.

[0004] Currently, CAR-T cell therapy targeting the CD19 molecule is approved in Europe, the United States, and Japan for the treatment of B-cell leukemia and lymphoma. Its response rate is extremely high, with reports indicating remission in over 80% of refractory cases. Furthermore, development of CAR-T cell therapies targeting other antigens and cancer types is progressing, and the market is expected to expand in the future.

[0005] Adoptive immunotherapy is a promising treatment that could potentially cure relapsed and refractory tumors, but it has yet to demonstrate sustained therapeutic effects in many cancers, with the exception of a few types. One reason why sustained therapeutic effects are not obtained in CAR-T cell therapy is that the functions of the therapeutic cells, such as their proliferative capacity, are weakened.

[0006] Furthermore, side effects such as cytokine release syndrome and neurotoxic syndrome are also problematic in adoptive immunotherapy. In cytokine release syndrome associated with CAR-T cell therapy, cytokines such as IL-6 are released from macrophages activated by GM-CSF secreted from CAR-T cells, leading to hypotension and multiple organ failure (Non-Patent Literature 1, 2). In neurotoxic syndrome, cytokines such as IL-1β are released from macrophages activated by CAR-T cells, causing aphasia, mental disorders, seizures, cerebral edema, etc. (Non-Patent Literature 3, 4).

[0007] Therefore, in adoptive immunotherapy, such as CAR-T cell therapy, there are two challenges: improving treatment efficacy and reducing side effects. However, it is known that in adoptive immunotherapy, there is a correlation between improved treatment efficacy and the frequency and grade of side effects. Therefore, it is thought that increasing treatment efficacy will increase side effects, and conversely, reducing side effects will decrease treatment efficacy, making it difficult to achieve both improved treatment efficacy and reduced side effects simultaneously.

[0008] Therefore, new technologies are needed in adoptive immunotherapy to achieve both improved therapeutic efficacy and reduced side effects. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Maude SL, et al., N Engl J Med, 2018, 378(5):439-448. [Non-Patent Document 2] Sachdeva M,, et al., J Biol Chem., 2019, 294(14):5430-5437. [Non-Patent Document 3] Giavridis T, et al., Nat Med., 2018, 24(6):731-738. [Non-Patent Document 4] Norelli M., et al., Nat Med, 2018, 24(6):739-748.

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a new technique for enhancing functions such as proliferation ability in immune cells used for adoptive immunotherapy and imparting the activity of capturing cytokines that can cause side effects to immune cells in order to achieve both improved therapeutic effects and reduced side effects in adoptive immunotherapy.

Means for Solving the Problems

[0011] In order to solve the above problems, the present inventors prepared a chimeric cytokine receptor based on a plurality of cytokine receptors as a new artificial receptor that enables both enhancement of functions such as proliferation ability and cytokine capture in CAR-T cells. This chimeric cytokine receptor is composed of a cytokine binding region for cytokine capture on the N-terminal side and a T cell activation region based on the constitutively active IL-7 (interleukin-7) receptor α chain on the C-terminal side. The present inventors found that in CAR-T cells into which the chimeric cytokine receptor was introduced together with a chimeric antigen receptor (CAR), functions such as proliferation ability and cytotoxic activity were enhanced, and that the CAR-T cells acquired the activity of capturing extracellular cytokines. Furthermore, the present inventors also found that CAR-T cells into which the chimeric cytokine receptor of the present invention was introduced together with CAR exhibited long-term survival ability and a long-term sustained antitumor effect in vivo.

[0012] On the other hand, when another chimeric cytokine receptor based on a constitutively active CD28 receptor, which is known to contribute to enhancing functions such as the proliferative ability and cytotoxic activity of T cells, similar to the constitutively active IL-7 receptor α chain, was prepared, the above effects were not obtained. Therefore, it was found that it is important for obtaining the above effects that the T cell activation region of the chimeric cytokine receptor is based on the constitutively active IL-7 receptor α chain rather than the constitutively active CD28 receptor.

[0013] The present invention is based on the above research results and provides the following. (1) A chimeric cytokine receptor comprising: a ligand binding region on the N-terminal side and a T cell activation region on the C-terminal side, wherein the ligand binding region consists of the cytokine binding region of a cytokine receptor, the T cell activation region includes the transmembrane domain and the intracellular domain of the IL-7 (interleukin-7) receptor α chain, the transmembrane domain is (a) the amino acid sequence shown in SEQ ID NO: 2 inserted between positions 243 and 244 in the amino acid sequence shown in SEQ ID NO: 1, (b) the amino acid sequence shown in SEQ ID NO: 3 inserted between positions 241 and 242 in the amino acid sequence shown in SEQ ID NO: 1, (c) the amino acid sequence shown in SEQ ID NO: 4 inserted between positions 244 and 245 in the amino acid sequence shown in SEQ ID NO: 1, (d) the amino acid sequence shown in SEQ ID NO: 5 inserted between positions 244 and 245 in the amino acid sequence shown in SEQ ID NO: 1, and (e) the amino acid sequence shown in SEQ ID NO: 6 inserted between positions 246 and 247 in the amino acid sequence shown in SEQ ID NO: 1 and the chimeric cytokine receptor into which any one selected from the group consisting of is inserted. (2) The chimeric cytokine receptor according to (1), wherein the cytokine receptor is selected from the group consisting of IL-6 (interleukin-6) receptor, IL-1 (interleukin-1) receptor type 2, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α chain, and GM-CSF receptor β chain. (3) The chimeric cytokine receptor according to (2), wherein the cytokine receptor is an IL-6 receptor and further comprises a ligand-binding domain for gp130 (glycoprotein 130) at its N-terminal side. (4) A chimeric cytokine receptor according to any one of (1) to (3), wherein one or more motifs selected from the group consisting of a JAK-binding motif, a STAT3-related motif, and a STAT5 / PI3K-related motif contained in the intracellular domain have a mutation that suppresses its activity. (5) The chimeric cytokine receptor according to (4), wherein the mutation is Y449F, M452L, or Y456F in the amino acid sequence shown in Sequence ID No. 1. (6) A nucleic acid encoding a chimeric cytokine receptor as described in any of (1) to (5). (7) A gene expression vector containing the nucleic acids described in (6) in an expressible state. (8) A host cell containing the gene expression vector described in (7). (9) The host cell according to (8), further comprising a CAR expression vector containing a nucleotide sequence encoding a chimeric antigen receptor (CAR) in an expressible state. (10) A host cell according to (8) or (9), further comprising an IL-1 receptor type 2 expression vector containing a nucleotide sequence encoding the full-length IL-1 receptor type 2 in an expressible state. (11) A host cell described in any of (8) to (10), which is an immune cell. (12) The host cell according to (11), wherein the immune cell is a T cell, an NK cell, or a macrophage. A cell preparation comprising the host cells described in any of (13)(8) to (12). (14) A method for producing chimeric antigen receptor (CAR)-transformed cells having long-lasting cytotoxic activity, comprising the steps of: isolating peripheral blood mononuclear cells from peripheral blood derived from a subject; and introducing the gene expression vector described in claim 7 and the CAR expression vector into the peripheral blood mononuclear cells isolated in the step, wherein the CAR expression vector contains a nucleotide sequence encoding a chimeric antigen receptor (CAR) in an expressible state. This specification includes the disclosures of Japanese Patent Application No. 2021-106789, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0014] By introducing the chimeric cytokine receptor of the present invention into immune cells used in adoptive immunotherapy, their functions, such as proliferative capacity, can be enhanced. Furthermore, immune cells into which the chimeric cytokine receptor of the present invention has been introduced can capture cytokines from outside the cell. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the structure of the IL-7 receptor α chain. In the figure, SP indicates the signal peptide. [Figure 2] This figure shows the structures of the chimeric cytokine receptors IL6R-ca7R and GP130-IL6R-ca7R. Figure 2A shows the structure of IL6R-ca7R. Figure 2B shows the structure of GP130-IL6R-ca7R. In the figures, SP represents the signal peptide, and PPCL represents the constitutively active insertion mutation, the four amino acid residue Pro-Pro-Cys-Leu (SEQ ID NO: 2). [Figure 3] Flow cytometry plots showing the cell surface expression of IL6RA, IL6R-ca7R, and / or GP130-IL6R-ca7R receptors in CAR-T cells and control cells 5 days after retroviral introduction of the IL6R-ca7R gene or GP130-IL6R-ca7R gene. The numbers indicated by "IL6RA+" in the figures represent the percentage of cells determined to be positive based on anti-IL6RA antibody. [Figure 4]This figure shows the results of flow cytometry analysis of the dynamics of receptor internalization at various time points after administration of recombinant IL-6 to GP130-IL6R-ca7R-introduced CAR-T cells or control cells. [Figure 5] This figure shows the quantitative results of flow cytometry. Figure 5A shows the fluorescence intensity detected by anti-IL6RA antibody at each time point after IL-6 administration in GP130-IL6R-ca7R-transformed CAR-T cells or control cells. Figure 5B shows the percentage of positive cells detected by anti-IL6RA antibody. The results show the mean value of an experiment with n=4, and the error bars show the standard deviation. The P values ​​shown in the figure indicate statistical significance by one-way ANOVA. [Figure 6] This figure shows the results of measuring the cytokine (IL-6) scavenging ability of each CAR-T cell. Figure 6A shows the experimental procedure. Figure 6B shows the results of adding IL-6 to each CAR-T cell and measuring the IL-6 concentration in the supernatant. Figure 6C shows the results measured using the same method as in Figure 6B for "control_cell", "IL6R-ca7R_cell", and "GP130-IL6R-ca7R_cell". In addition, the results of measuring the IL-6 concentration in the supernatant after adding IL-6 to the culture supernatant isolated from each CAR-T cell are shown for "control_sup", "IL6R-ca7R_sup", and "GP130-IL6R-ca7R_sup". The results show the mean values ​​from an experiment with n=3, and the error bars show the standard deviation. The P values ​​shown in the figure indicate statistical significance by one-way ANOVA. [Figure 7] The results of evaluating phosphorylated STAT3 (pSTAT3) in each CAR-T cell are shown. Figure 7A shows the flow cytometry results based on pSTAT3 in each CAR-T cell. Figure 7B shows the flow cytometry results based on pSTAT3 in each CAR-T cell in the presence or absence of IL-6 (IL6+) or (IL6-). Figure 7C shows the quantitative results of pSTAT3. The results are the mean values ​​from an experiment with n=4, and the error bars indicate the standard deviation. The p-values ​​shown in the figures indicate statistical significance by one-way ANOVA. [Figure 8] The results of evaluating phosphorylated STAT5 (pSTAT5) in each CAR-T cell are shown. Figure 8A shows the flow cytometry results based on pSTAT5 in the absence of IL-6 in each CAR-T cell. Figure 8B shows the quantitative results of pSTAT5 in the presence and absence of IL-6. The results are the mean values ​​from experiments with n=4, and the error bars indicate the standard deviation. The p-values ​​shown in the figures indicate statistical significance by one-way ANOVA. [Figure 9] This figure shows the cell proliferation rate of each CAR-T cell type. Each CAR-T cell type targeting CD19 was cultured together with the CD19-positive tumor cell line NALM6, and the multiplicative change in the number of CAR-T cells after 7 days compared to the start of co-culture is shown. Figure 9A shows the results for CAR-T cells introduced with the FMC63-28z CAR. The results show the mean value of an experiment with n=4, and the error bars show the standard deviation. The p-values ​​shown in the figure indicate statistical significance by one-way ANOVA. Figure 9B shows the results for CAR-T cells introduced with the FMC63-BBz CAR. The results show the mean value of an experiment with n=4, and the error bars show the standard deviation. The p-values ​​shown in the figure indicate statistical significance by Student's t-test. [Figure 10] This figure shows the results of co-culturing each CAR-T cell line with the CD19-positive tumor cell line NALM6, and then quantifying the proportion of IFN-γ-producing cells in the CD4-positive T cell fraction and the CD8-positive T cell fraction. The results show the mean values ​​from an experiment with n=4, and the error bars indicate the standard deviation. The p-values ​​shown in the figure indicate statistical significance by Student's t-test. [Figure 11] This figure shows the results of analyzing Granzyme B (GZMB) production after co-culturing each CAR-T cell line with either the K562-CD19 cell line or the Raji cell line. Figure 11A shows the results of analyzing Granzyme B production after co-culturing with the K562-CD19 cell line. Figure 11B shows the results of detecting Granzyme B production when co-culturing with different target cells. The results show the mean value of an experiment with n=3, and the error bars show the standard deviation. The P values ​​shown in the figures indicate statistical significance by Student's t-test. [Figure 12]This figure shows the survival rates of tumor cells (K562-CD19 cell line or NALM6 cell line) co-cultured with each CAR-T cell line. The results show the mean values ​​from experiments with n=3, and the error bars indicate the standard deviation. The p-values ​​shown in the figure indicate statistical significance by Student's t-test. [Figure 13] This figure shows the effects of the activity-suppressing mutations Y449F, M452L, or Y456F on the chimeric cytokine receptor GP130-IL6R-ca7R. Figure 13A shows the positions of Y449F, M452L, and Y456F on the IL-7 receptor α chain. Figure 13B shows the results of Western blotting analysis of STAT3, STAT5, and Akt phosphorylation status in CAR-T cells co-transferred with each mutation of the chimeric cytokine receptor GP130-IL6R-ca7R. [Figure 14] This figure shows the results of functional analysis of CAR-T cells co-introduced with the chimeric cytokine receptor GP130-IL6R-ca7R(M452L). Figure 14A shows the multiplicative change in the number of each CAR-T cell type after 7 days of co-culture with the CD19-positive tumor cell line NALM6. The P values ​​shown in the figure indicate statistical significance by one-way ANOVA. Figure 14B shows the results of flow cytometry analysis of the memory phenotype of each CAR-T cell type after 7 days of co-culture with NALM6. Figure 14C shows the multiplicative change in the number of CAR-T cells with undifferentiated memory phenotype (CD62L-positive and CCR7-positive). The P values ​​indicate statistical significance by one-way ANOVA. [Figure 15] This figure shows the cell proliferation rate of each CAR-T cell line. Each CAR-T cell line targeting mesothelin or GD2 was cultured together with the K562-mesothelin cell line or the NALM6-GD2 cell line, and the multiplicative change in the number of CAR-T cells after 7 days compared to the start of co-culture is shown. The results show the mean of an experiment with n=4, and the error bars show the standard deviation. The p-values ​​shown in the figure indicate statistical significance by Student's t-test. [Figure 16]This figure shows the results of co-culturing mesothelin-targeting CAR-T cells with the K562-mesothelin cell line, staining with anti-IFN-γ antibody and anti-TNF-α antibody, and then analyzing the results by flow cytometry. Figure 16A shows the flow cytometry results. Figure 16B shows the percentage of IFN-γ-producing cells. Figure 16C shows the percentage of TNF-α-producing cells. Figure 16D shows cells that produce both IFN-γ and TNF-α. The results show the mean values ​​from experiments with n=3, and the error bars show the standard deviation. The p-values ​​shown in the figures indicate statistical significance by Student's t-test. [Figure 17] This figure shows the results of co-culturing each GD2-targeting CAR-T cell line with the NALM6-GD2 cell line, staining with anti-IFN-γ antibody and anti-TNF-α antibody, and then analyzing the results by flow cytometry. Figure 17A shows the percentage of IFN-γ-producing cells. Figure 17B shows the percentage of TNF-α-producing cells. Figure 17C shows cells that produce both IFN-γ and TNF-α. The results show the mean values ​​from experiments with n=3, and the error bars show the standard deviation. The p-values ​​shown in the figures indicate statistical significance by Student's t-test. [Figure 18] This figure shows the structure of the chimeric cytokine receptor GP130-IL6R-CD28(T195P). Figure 18A shows the structure of the IL6 receptor α subunit (IL6RA). Figure 18B shows the structure of GP130-IL6R-CD28(T195P). In the figures, SP indicates the signal peptide, and T195P indicates the constitutively activating mutation of CD28. Figure 18C shows the cell proliferation rate of each CAR-T cell. Each CAR-T cell targeting CD19 was cultured together with the CD19-positive tumor cell line NALM6, and the multiplicative change in the number of CAR-T cells after 7 days compared to the start of co-culture is shown. The results show the mean of an experiment with n=4, and the error bars show the standard deviation. Statistical significance was tested by one-way ANOVA. [Figure 19]This figure shows the structures of the chimeric cytokine receptors CSF2RA-ca7R, CSF2RB-ca7R, and IL1R2-ca7R. Figure 19A shows the structure of CSF2RA-ca7R. Figure 19B shows the structure of CSF2RB-ca7R. Figure 19C shows the structure of IL1R2-ca7R. In the figures, PPCL represents the constitutively active insertion mutation, the four amino acid residue Pro-Pro-Cys-Leu (SEQ ID NO: 2). [Figure 20] This figure shows the results of functional analysis of CAR-T cells co-introduced with both CSF2RA-ca7R and CSF2RB-ca7R. Figure 20A shows the multiplicative change in the number of CAR-T cells after 7 days of co-culturing each CAR-T cell with the CD19-positive tumor cell line NALM6. Figure 20B shows the results of measuring the GM-CSF capture ability of each CAR-T cell. GM-CSF was added to each CAR-T cell, and the GM-CSF concentration was measured in the supernatant. Figures 20C and 20D show the results of measuring the GM-CSF concentration (Figure 20C) and IL-6 concentration (Figure 20D) in the culture medium after co-culturing each CAR-T cell with NALM6 cells and then adding the monocytic leukemia cell line THP1. The P values ​​shown in the figures indicate statistical significance by Student's t-test. [Figure 21] This figure shows the results of measuring the cell proliferation rate and cytokine (IL-1β) scavenging ability of each CAR-T cell group. Figure 21A shows the multiplicative change in the number of CAR-T cells after 7 days when each CAR-T cell group targeting CD19 was cultured together with the CD19-positive tumor cell line NALM6. The results show the mean values ​​from an experiment with n=4, and the error bars indicate the standard deviation. The p-values ​​shown in the figure indicate statistical significance by Student's t-test. Figure 21B shows the results of measuring the IL-1β concentration in the supernatant after adding IL-1β to each CAR-T cell group. The results show the mean values ​​from an experiment with n=3, and the error bars indicate the standard deviation. The p-values ​​shown in the figure indicate statistical significance by Student's t-test. [Figure 22]This figure shows the results of measuring blood IL-6 concentrations at 1, 2, and 4 hours after administering human IL-6 via tail vein to NSG mice treated with each type of CAR-T cell. The P values ​​shown in the figure indicate statistical significance using Student's t-test on the logarithm of the concentration. Figure 22A shows the blood IL-6 concentration at 1 hour. Figure 22B shows the blood IL-6 concentration at 2 hours. Figure 22C shows the blood IL-6 concentration at 4 hours. [Figure 23-1] This figure shows the results of measuring the cytokine (IL-6 and IL-1β) scavenging ability of CAR-T cells co-introduced with chimeric cytokine receptors and full-length IL-1 receptor type 2. Figure 23-1A shows the experimental procedure. Figure 23-1B shows the results of measuring the IL-6 concentration in the supernatant after adding recombinant IL-6 to the above CAR-T cells. "nd" indicates that IL-6 was not detected. Figure 23-1C shows the results of measuring the IL-1β concentration in the supernatant after adding recombinant IL-1β to the above CAR-T cells. The results show the mean values ​​from experiments with n=3, and the error bars show the standard deviation. The P values ​​shown in the figures indicate statistical significance by Student's t-test. [Figure 23-2] This is a continuation of Figure 23-1. Figure 23-2D shows the experimental procedure. Figure 23-2E shows the results of culturing the above CAR-T cells with the CD19-positive tumor cell line NALM6, adding the monocyte-derived cell line THP-1, and measuring the IL-6 concentration in the supernatant. "nd" indicates that IL-6 was not detected. Figure 23-2F shows the results of adding the monocyte-derived cell line THP-1 to the above CAR-T cells and measuring the IL-1β concentration in the supernatant. The results show the mean values ​​from an experiment with n=3, and the error bars show the standard deviation. The P values ​​shown in the figures indicate statistical significance by Student's t-test. [Figure 24]This figure shows the results of measuring the scavenging ability of CAR-T cells co-transmitted with GP130-IL6R-ca7R(M452L) and FMC63-28z to capture IL-6 produced from monocyte cells THP1-CD19 / EGFP-Luc2 in vivo. Figure 24A shows the experimental procedure. Figure 24B shows the plasma IL-6 concentrations 1, 5, and 8 days after CAR-T cell administration. The results show the mean values ​​from n=8 experiments, and the error bars show the standard deviation. The P values ​​shown in the figure indicate statistical significance by Student's t-test. [Figure 25-1] This figure shows the antitumor effect of CAR-T cells co-transfected with GP130-IL6R-ca7R(M452L) in an in vivo leukemia model. Figure 25-1A shows the experimental procedure. Figure 25-1B shows the percentage of human CD45-positive T cells measured by flow cytometry in peripheral blood collected 10 to 38 days after CAR-T cell administration. The results show the mean values ​​from experiments with n=2 to 7, and the error bars show the standard deviation. The P values ​​shown in the figure indicate statistical significance by Student's t-test, and "ns" indicates that no significant difference was detected. [Figure 25-2] This is a continuation of Figure 25-1. Figure 25-2C shows the results of measuring NALM6 tumor volume as luciferase luminescence by IVIS Imaging 10, 24, 38, and 60 days after CAR-T cell administration. Figure 25-2D shows the quantitative results of logarithmically transformed luciferase luminescence. The results show the mean values ​​from the n=7 experiment, and the error bars show the standard deviation. The P values ​​shown in the figures indicate statistical significance by Student's t-test. [Figure 25-3] This is a continuation of Figure 25-2. Figure 25-3E shows the results of the analysis of the progression-free survival rate based on the transplantation date of NALM6-GL cells. The p-values ​​shown in the figure indicate statistical significance using the log-rank test. [Figure 26-1]This figure shows the antitumor effect of second-generation CAR-T cells co-transferred with GP130-IL6R-ca7R(M452L) in an in vivo solid tumor model (NSG mice subcutaneously transplanted with the mesothelin-positive pancreatic cancer cell line AsPC-1). Figure 26-1A shows the experimental procedure. Figure 26-1B shows the percentage of human CD45-positive T cells measured by flow cytometry in peripheral blood collected 14 to 35 days after CAR-T cell administration. The results show the mean values ​​from an experiment with n=7, and the error bars show the standard deviation. The P values ​​shown in the figure indicate statistical significance by Student's t-test, and "ns" indicates that no significant difference was detected. [Figure 26-2] This is a continuation of Figure 26-1. Figure 26-2C shows the results of measuring the luciferase luminescence of second-generation CAR-T cells that infiltrated subcutaneous tumors 14 to 35 days after administration of second-generation CAR-T cells using IVIS Imaging. Figure 26-2D shows the quantitative results of logarithmically transformed luciferase luminescence. Note that in Figure 26-2, unlike Figure 25-2, CAR-T cells, not tumor cells, are labeled with luciferase. The results show the mean values ​​from an experiment with n=7, and the error bars show the standard deviation. The P values ​​shown in the figure indicate statistical significance by Student's t-test, and "ns" indicates that no significant difference was detected. [Figure 26-3] This is a continuation of Figure 26-2. Figure 26-3E shows the change in tumor volume over time after subcutaneous transplantation of AsPC-1 cells. The results show the mean values ​​from an experiment with n=7, and the error bars show the standard deviation. Figure 26-3F shows the results of the analysis of the progression-free survival rate based on the day of AsPC-1 cell transplantation. The p-values ​​shown in the figure indicate statistical significance by the log-rank test. [Modes for carrying out the invention]

[0016] 1. Chimeric cytokine receptors 1-1. Overview A first aspect of the present invention is a chimeric cytokine receptor. The chimeric cytokine receptor of the present invention includes a ligand-binding region at the N-terminus and a T-cell activation region at the C-terminus, the T-cell activation region including the transmembrane domain and intracellular domain of the IL-7 (interleukin-7) receptor α chain. The ligand-binding region at the N-terminus consists of the cytokine-binding region of a cytokine receptor such as the IL-6 receptor, IL-1 receptor type 2, GM-CSF receptor α chain, or GM-CSF receptor β chain. When the chimeric cytokine receptor of the present invention is introduced into immune cells such as T cells, it is possible to confer activity to capture cytokines from outside the cell to the immune cells and to activate the immune cells.

[0017] 1-2.Definition The following terms, which are frequently used in this specification, are defined below. "Adoptive immunotherapy" is a treatment method that involves collecting cells, such as immune cells, from a donor, culturing, stimulating, manipulating, and proliferating them outside the body, and then introducing them into a recipient. For example, in cancer patients, it can enhance the toxic activity of immune cells against cancer cells. Examples of adoptive immunotherapy include tumor-infiltrating lymphocyte therapy, TCR gene-modified T-cell therapy, and chimeric antigen receptor-T (CAR-T) cell therapy. Adoptive immunotherapy may also include leukocyte apheresis. Adoptive immunotherapy mainly refers to autologous transplantation, where the donor providing the cells and the recipient into whom the cells are introduced are the same individual, but it also includes allogeneic transplantation (allogeneic or xenogeneic transplantation), where the donor and recipient are different individuals.

[0018] "Tumor-infiltrating lymphocyte therapy" is a treatment method that involves collecting tumor-infiltrating lymphocytes (TILs) from a donor, processing them outside the body through activation and amplification, and then introducing them into the recipient.

[0019] "TCR gene-transferred T-cell therapy" is a treatment method that involves introducing cancer antigen-specific T cell receptor (TCR) genes into immune cells such as T cells collected from the peripheral blood of a donor, and then introducing them into a recipient.

[0020] Chimeric antigen receptor T-cell therapy (CAR-T cell therapy) is a treatment method that involves introducing chimeric antigen receptor (CAR) genes into immune cells such as T cells collected from the peripheral blood of a donor to create CAR-T cells that can recognize and attack cancer cells, and then introducing these cells into the recipient via infusion or other means. Currently, CAR-T cell therapy is approved for B-cell hematological malignancies such as leukemia and lymphoma, and is expected to be a groundbreaking cancer treatment technology following immune checkpoint inhibitor therapy.

[0021] A "chimeric antigen receptor (CAR)" refers to a fusion protein containing an extracellular domain capable of binding to antigens such as cancer antigens, a transmembrane domain, and an intracellular domain with signal transduction activity. More specifically, it refers to a fusion protein containing a variable region (V) of the light and heavy chains derived from a monoclonal antibody that recognizes cancer cell surface antigens. L and V HCARs are artificial antigen receptors that combine a single-chain antibody (scFv) conjugated with CD3ζ (also known as CD247) with a transmembrane domain and a signaling domain of a co-stimulatory molecule involved in T cell activation. CARs can be broadly classified into first to third generations based on the structure and type of signaling domain they contain. First-generation CARs consist solely of the CD3ζ (also known as CD247) signaling domain. Second-generation CARs contain one co-stimulatory molecule such as CD28 or CD137 (4-1BB) in addition to the CD3ζ signaling domain. Representative examples of second-generation CARs include the FMC63-28z CAR, which contains a portion of CD28 as a co-stimulatory molecule, and the FMC63-BBz CAR, which contains a portion of 4-1BB as a co-stimulatory molecule. Both the FMC63-28z CAR and the FMC63-BBz CAR contain anti-CD19 scFv. Third-generation CARs have a signaling domain that includes multiple co-stimulatory molecules such as CD28 and CD137(4-1BB) in addition to CD3ζ. In T cells expressing CARs, the CAR recognizes tumor cells and activates T cells, thereby efficiently killing tumor cells. T cells into which the CAR gene has been introduced are called "chimeric antigen receptor T cells (CAR-T cells)".

[0022] "Cytokine release syndrome" refers to symptoms or conditions caused by the release of cytokines, such as inflammatory cytokines. Cytokine release syndrome can occur in conjunction with the administration of antibody drugs and CAR-T cell therapy. In CAR-T cell therapy, cytokines such as IL-6 are released from monocytes and macrophages activated by CAR-T cells. These cytokines can induce fever and increased vascular permeability, which may result in hypotension and multiple organ failure. 77% of patients who received CAR-T cell therapy for B-cell acute lymphoblastic leukemia developed cytokine release syndrome (Maude SL, et al., N Engl J Med, 2018, 378(5):439-448).

[0023] "Neurotomic syndrome" refers to symptoms or conditions resulting from neurotoxicity caused by the administration of drugs or other substances. In CAR-T cell therapy, CAR-T cells activate monocytes and macrophages, releasing cytokines such as IL-1β, which can lead to neurotoxicity, resulting in aphasia, mental disorders, seizures, cerebral edema, etc. Approximately 40% of patients who received CAR-T cell therapy for B-cell acute lymphoblastic leukemia developed neurotoxic syndrome (Maude SL, et al., N Engl J Med, 2018, 378(5):439-448).

[0024] "Cytokines" are a general term for proteins with relatively small molecular weights that are secreted by cells. Cytokines are mainly secreted by immune cells and are responsible for intercellular communication. Examples of cytokines include interleukins, interferons, chemokines, hematopoietic factors, cell growth factors, and tumor necrosis factors. Specific examples of interleukins include IL-1, IL-6, and IL-7, which will be discussed later. Specific examples of hematopoietic factors include GM-CSF, which will be discussed later.

[0025] IL-1 (interleukin-1) is known as an inflammatory cytokine. IL-1 includes IL-1α and IL-1β. In this specification, when simply referred to as "IL-1," it includes IL-1 derived from any biological species. Specific examples of IL-1 include human IL-1α and human IL-1β. In neurotoxic syndromes, IL-1β can be released from monocytes, macrophages, and other cells.

[0026] IL-6 (interleukin-6) is known as an inflammatory cytokine. IL-6 is produced by T cells, macrophages, and other cells. In this specification, when simply referred to as "IL-6," it includes IL-6 derived from any species. Human IL-6 is a specific example of IL-6. In cytokine release syndrome, IL-6 can be released from monocytes, macrophages, and other cells.

[0027] "IL-7 (interleukin-7)" is known as a hematopoietic growth factor. IL-7 is secreted from stromal cells, etc. In this specification, when "IL-7" is used, it includes IL-7 derived from any biological species. Human IL-7 is a specific example of IL-7.

[0028] Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as CSF2, is a type of cytokine that functions as a hematopoietic growth factor and an immunomodulator. It is secreted by T cells, macrophages, NK cells, etc. In this specification, when "GM-CSF" is used, it includes GM-CSF derived from any biological species. Human GM-CSF is a specific example of GM-CSF.

[0029] A "cytokine receptor" is a receptor to which cytokines bind as ligands. Specific examples of cytokine receptors include interleukin receptors, interferon receptors, chemokine receptors, hematopoietic factor receptors, cell growth factor receptors, and tumor necrosis factor receptors. Specific examples of interleukin receptors include the IL-1 receptor, IL-6 receptor, IL-7 receptor α chain, gp130, and common γ chain described later, as well as the IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-9 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-15 receptor, IL-21 receptor, IL-23 receptor, IL-27 receptor, IL-10 receptor, IL-20 receptor, IL-22 receptor, and IL-28 receptor. Specific examples of hematopoietic factor receptors include the GM-CSF receptor described later. Specific examples of cell growth factor receptors include the TGF-β receptor. Specific examples of tumor necrosis factor receptors include TNF-α receptors (e.g., type 1 and type 2) and FAS receptors.

[0030] The "IL-1 receptor (interleukin-1 receptor)" is a receptor to which IL-1 binds as a ligand, and two types of IL-1 receptors are known: IL-1 receptor type 1 (also referred to as IL1R1 in this specification) and IL-1 receptor type 2 (also referred to as IL1R2 in this specification).

[0031] "IL-1 receptor type 1" includes wild-type and mutant IL-1 receptor type 1 derived from any species. Examples include human IL-1 receptor type 1, consisting of the amino acid sequence shown in SEQ ID NO: 7, and its orthologs. Examples of mutant IL-1 receptor type 1 or IL-1 receptor type 1 orthologs include amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 7, or IL-1 receptor type 1 consisting of an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 7.

[0032] An "IL-1 receptor type 1 gene" is a gene that codes for IL-1 receptor type 1. Specific examples of IL-1 receptor type 1 genes include the IL-1 receptor type 1 gene that codes for IL-1 receptor type 1 consisting of the amino acid sequence shown in Sequence ID No. 7, for example, the human IL-1 receptor type 1 gene consisting of the nucleotide sequence shown in Sequence ID No. 8. Furthermore, IL-1 receptor type 1 genes consisting of nucleotide sequences in which one or more bases are deleted, substituted, or added to the nucleotide sequence shown in Sequence ID No. 8, or nucleotide sequences having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of nucleotide identity with respect to the nucleotide sequence shown in Sequence ID No. 8, are also included.

[0033] "IL-1 receptor type 2" includes wild-type and mutant IL-1 receptor type 2 derived from any species. For example, human IL-1 receptor type 2, consisting of the amino acid sequence shown in SEQ ID NO: 9, and its orthologs are included. Examples of mutant IL-1 receptor type 2 or IL-1 receptor type 2 orthologs include amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 9, or IL-1 receptor type 2 consisting of an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 9. In human IL-1 receptor type 2, the extracellular domain is the region located N-terminal to the transmembrane domain after signal peptide cleavage, for example, positions 14 to 343 in the amino acid sequence shown in SEQ ID NO: 9. In the human IL-1 receptor type 2, the cytokine-binding region is a region included in the extracellular domain that is capable of binding to cytokines, and is, for example, located at positions 14-343 or 14-296 in the amino acid sequence shown in SEQ ID NO: 9.

[0034] An "IL-1 receptor type 2 gene" is a gene that codes for IL-1 receptor type 2. Specific examples of IL-1 receptor type 2 genes include the IL-1 receptor type 2 gene that codes for IL-1 receptor type 2 consisting of the amino acid sequence shown in SEQ ID NO: 9, and the human IL-1 receptor type 2 gene consisting of the nucleotide sequence shown in SEQ ID NO: 10. Furthermore, IL-1 receptor type 2 genes consisting of nucleotide sequences in which one or more bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 10, or IL-1 receptor type 2 genes consisting of nucleotide sequences having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more nucleotide identity with respect to the nucleotide sequence shown in SEQ ID NO: 10.

[0035] The IL-6 receptor (interleukin-6 receptor) is a receptor to which IL-6 binds as a ligand. The IL-6 receptor is also known as the IL-6 receptor α subunit or CD126, and is sometimes denoted as IL6R or IL6RA. The IL-6 receptor, together with gp130 (glycoprotein 130), which will be described later, binds to IL-6, forming a heterotrimer consisting of IL-6, the IL-6 receptor, and gp130. IL-6 receptors include wild-type and mutant IL-6 receptors derived from any species. For example, the human IL-6 receptor, consisting of the amino acid sequence shown in Sequence ID No. 11, and its orthologs are examples. Examples of mutant IL-6 receptors or IL-6 receptor orthologues include amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 11, or IL-6 receptors consisting of amino acid sequences that have 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 11. In the human IL-6 receptor, the extracellular domain is the region located N-terminal to the transmembrane domain after signal peptide cleavage, for example, positions 21 to 365 in the amino acid sequence shown in SEQ ID NO: 11. In the human IL-6 receptor, the cytokine-binding region is a region included in the extracellular domain that is capable of binding to cytokines, for example, positions 21 to 365 or 110 to 338 in the amino acid sequence shown in SEQ ID NO: 11.

[0036] An "IL-6 receptor gene" is a gene that codes for the IL-6 receptor. Specific examples of IL-6 receptor genes include the IL-6 receptor gene that codes for the IL-6 receptor consisting of the amino acid sequence shown in SEQ ID NO: 11, and the human IL-6 receptor gene consisting of the nucleotide sequence shown in SEQ ID NO: 12. Furthermore, IL-6 receptor genes consisting of nucleotide sequences in which one or more bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 12, or nucleotide sequences having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the nucleotide identity with respect to the nucleotide sequence shown in SEQ ID NO: 12, are also included.

[0037] "gp130 (glycoprotein 130)" is also known as IL6ST or CD130. gp130 includes wild-type and mutant gp130 derived from any species. For example, human gp130 consisting of the amino acid sequence shown in SEQ ID NO: 13, and its orthologs. Examples of mutant gp130 or gp130 orthologs include amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 13, or gp130 consisting of an amino acid sequence that has 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 13. In human gp130, the extracellular domain is located in the region located N-terminal to the transmembrane domain after signal peptide cleavage, for example, in the amino acid sequence shown in SEQ ID NO: 13, from position 23 to 619. In human gp130, the cytokine-binding region is a region included in the extracellular domain that is capable of binding to cytokines, and for example, it consists of positions 23 to 326 in the amino acid sequence shown in SEQ ID NO: 13.

[0038] The "gp130 gene" is a gene that codes for gp130. Specific examples of the gp130 gene include the gp130 gene that codes for gp130 consisting of the amino acid sequence shown in SEQ ID NO: 13, for example, the human gp130 gene consisting of the nucleotide sequence shown in SEQ ID NO: 14. Furthermore, gp130 genes consisting of nucleotide sequences in which one or more bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 14, or nucleotide sequences having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of nucleotide identity with respect to the nucleotide sequence shown in SEQ ID NO: 14, are also included.

[0039] The IL-7 receptor (interleukin-7 receptor) is a receptor to which IL-7 binds as a ligand. It is a heterodimer consisting of an IL-7 receptor α chain and a common γ chain (also known as the γc chain or CD132). The main signaling pathways activated by IL-7 binding to the IL-7 receptor include the Jak-Stat pathway and the PI3K-Akt pathway. When IL-7 binds to the IL-7 receptor, Jak kinase is phosphorylated, followed by phosphorylation of tyrosine residues (Y401, Y449, Y456, etc.) in the IL-7 receptor α chain. Of these, the phosphorylated tyrosine residue at position 449 (pY449) is known to be particularly important (Jiang Q, et al., Mol Cell Biol., 2004, 24(14):6501-13). pY449 functions as a docking site for SH2 domain proteins containing Stat family transcription factors activated by phosphorylation by Jak. SOCS family proteins are also known to be involved in IL-7 receptor signaling. IL-7 receptor signaling in lymphocytes is known to lead to survival, proliferation, and differentiation of lymphocytes, depending on the stage of lymphocyte development.

[0040] The "IL-7 receptor α chain" is also known as CD127 and will be referred to as IL7RA or IL7Rα in this specification. The IL-7 receptor α chain includes wild-type and mutant IL-7 receptor α chains derived from any species. Examples include the human IL-7 receptor α chain consisting of the amino acid sequence shown in SEQ ID NO: 1, and its orthologs. Examples of mutant IL-7 receptor α chains or IL-7 receptor α chain orthologs include amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 1, and IL-7 receptor α chains consisting of amino acid sequences that have 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1. In the human IL-7 receptor α chain, the extracellular domain may be located in the region located N-terminal to the transmembrane domain after signal peptide cleavage, for example, in positions 21 to 239 of the amino acid sequence shown in SEQ ID NO: 1. In the human IL-7 receptor α chain, the transmembrane domain may be located at positions 240-264 in the amino acid sequence shown in SEQ ID NO: 1. In the human IL-7 receptor α chain, the intracellular domain may be located at positions 265-459 in the amino acid sequence shown in SEQ ID NO: 1. The intracellular domain of the human IL-7 receptor α chain contains several functional motifs, including the JAK binding motif (also known as the "BOX1 motif"), the STAT3 association motif, and the STAT5 / PI3K association motif (also known as the "SH2 domain binding motif") (Figure 1). In the human IL-7 receptor α chain, which consists of the amino acid sequence shown in SEQ ID NO: 1, the JAK binding motif is located at positions 272-280 (Val-Trp-Pro-Ser-Leu-Pro-Asp-His-Lys sequence, SEQ ID NO: 15), the STAT3-related motif is located at positions 456-459 (Tyr-Gln-Asn-Gln sequence, SEQ ID NO: 16), and the STAT5 / PI3K-related motif is located at positions 449-452 (Tyr-Val-Thr-Met sequence, SEQ ID NO: 17).

[0041] Constitutively active mutations are known in the IL-7 receptor α chain. A "constitutively active IL-7 receptor α chain" or "IL-7 receptor α chain with a constitutively active mutation" is a variant of the IL-7 receptor α chain that can be activated even when IL-7 is not bound as a ligand. In cells expressing a constitutively active IL-7 receptor α chain, at least a portion of the downstream signaling pathways of the IL-7 receptor α chain can be activated even in the absence of IL-7. Known constitutively active insertion mutations include the insertion of the amino acid sequence Pro-Pro-Cys-Leu (SEQ ID NO: 2) between positions 243 and 244 in the amino acid sequence shown in SEQ ID NO: 1, the insertion of the amino acid sequence Phe-Ser-Cys-Gly-Pro (SEQ ID NO: 3) between positions 241 and 242 in the amino acid sequence shown in SEQ ID NO: 1, the insertion of the amino acid sequence Cys-His-Leu (SEQ ID NO: 4) between positions 244 and 245 in the amino acid sequence shown in SEQ ID NO: 1, the insertion of the amino acid sequence Pro-Pro-Val-Cys-Ser-Val-Thr (SEQ ID NO: 5) between positions 244 and 245 in the amino acid sequence shown in SEQ ID NO: 1, and the insertion of the amino acid sequence Lys-Cys-His (SEQ ID NO: 6) between positions 246 and 247 in the amino acid sequence shown in SEQ ID NO: 1.

[0042] Furthermore, mutations that suppress the activity of the aforementioned functional motifs in the intracellular domain of the IL-7 receptor α chain are known (Figure 13A). Examples of mutations that suppress the activity of the STAT5 / PI3K-related motif include the Y449F mutation, which replaces the Tyr residue at position 449 with a Phe residue in the amino acid sequence shown in SEQ ID NO: 1, and the M452L mutation, which replaces the Met residue at position 452 with a Leu residue in the amino acid sequence shown in SEQ ID NO: 1. An example of a mutation that suppresses the activity of the STAT3-related motif is the Y456F mutation, which replaces the Tyr residue at position 456 with a Phe residue in the amino acid sequence shown in SEQ ID NO: 1.

[0043] An "IL-7 receptor α chain gene" is a gene that codes for the IL-7 receptor α chain. Specific examples of IL-7 receptor α chain genes include the IL-7 receptor α chain gene that codes for the IL-7 receptor α chain consisting of the amino acid sequence shown in SEQ ID NO: 1, for example, the human IL-7 receptor α chain gene consisting of the nucleotide sequence shown in SEQ ID NO: 19. Furthermore, IL-7 receptor α chain genes consisting of nucleotide sequences in which one or more bases are deleted, substituted, or added to the nucleotide sequence shown in SEQ ID NO: 19, or nucleotide sequences having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the nucleotide identity with respect to the nucleotide sequence shown in SEQ ID NO: 19 are also included.

[0044] The granulocyte-macrophage colony-stimulating factor receptor (GM-CSF receptor) is a receptor to which GM-CSF binds as a ligand, and it is a heterodimer composed of a GM-CSF receptor α chain and a GM-CSF receptor β chain.

[0045] The "GM-CSF receptor α chain" is also known as CD116 and will be referred to as CSF2RA in this specification. The GM-CSF receptor α chain includes wild-type and mutant GM-CSF receptor α chains derived from any species. Examples include the human GM-CSF receptor α chain consisting of the amino acid sequence shown in Sequence ID No. 20, and its ortholog. Examples of mutant GM-CSF receptor α chains or GM-CSF receptor α chain orthologs include amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in Sequence ID No. 20, and GM-CSF receptor α chains consisting of amino acid sequences that have 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in Sequence ID No. 20. In the human GM-CSF receptor α chain, the extracellular domain is the region located N-terminal to the transmembrane domain after signal peptide cleavage, for example, the region from positions 23 to 320 in the amino acid sequence shown in SEQ ID NO: 20. In the human GM-CSF receptor α chain, the cytokine binding region is a region included in the extracellular domain that is capable of binding to cytokines, for example, the region consisting of positions 25 to 320 in the amino acid sequence shown in SEQ ID NO: 20, or the entire extracellular domain consisting of positions 23 to 320.

[0046] A "GM-CSF receptor α chain gene" is a gene that codes for the GM-CSF receptor α chain. Specific examples of GM-CSF receptor α chain genes include the GM-CSF receptor α chain gene that codes for the GM-CSF receptor α chain consisting of the amino acid sequence shown in SEQ ID NO: 20, and the human GM-CSF receptor α chain gene consisting of the nucleotide sequence shown in SEQ ID NO: 21. Furthermore, GM-CSF receptor α chain genes with nucleotide sequences in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 21, or GM-CSF receptor α chain genes having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more nucleotide identity with respect to the nucleotide sequence shown in SEQ ID NO: 21, are also included.

[0047] The term "GM-CSF receptor β-chain" includes wild-type and mutant GM-CSF receptor β-chains derived from any species. Examples include the human GM-CSF receptor β-chain consisting of the amino acid sequence shown in SEQ ID NO: 22, and its ortholog. Examples of mutant GM-CSF receptor β-chains or GM-CSF receptor β-chain orthologs include amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 22, and GM-CSF receptor β-chains consisting of amino acid sequences that have 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 22. In the human GM-CSF receptor β-chain, the extracellular domain is the region located N-terminal to the transmembrane domain after signal peptide cleavage, for example, positions 17 to 443 in the amino acid sequence shown in SEQ ID NO: 22. In the human GM-CSF receptor β chain, the cytokine-binding region is a region included in the extracellular domain that is capable of binding to cytokines, and is, for example, located at positions 17-132 and / or 339-436 in the amino acid sequence shown in SEQ ID NO: 22.

[0048] A "GM-CSF receptor β-chain gene" is a gene that codes for the GM-CSF receptor β-chain. Specific examples of GM-CSF receptor β-chain genes include the GM-CSF receptor β-chain gene that codes for the GM-CSF receptor β-chain consisting of the amino acid sequence shown in SEQ ID NO: 22, and the human GM-CSF receptor β-chain gene consisting of the nucleotide sequence shown in SEQ ID NO: 23. Furthermore, GM-CSF receptor β-chain genes with nucleotide sequences in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 23, or GM-CSF receptor β-chain genes having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more nucleotide identity with respect to the nucleotide sequence shown in SEQ ID NO: 23, are also included.

[0049] In this specification, "chimeric cytokine receptor" means a fusion protein that includes a portion derived from two or more cytokine receptors.

[0050] In this specification, “immune cells” include cell types that can function as part of the immune system, as well as undifferentiated cells and progenitor cells (e.g., immune progenitor cells) that can differentiate into such cell types. Specific examples of immune cells include lymphocytes, granulocytes, dendritic cells, macrophages, and monocytes. Examples of lymphocytes include T cells, B cells, and natural killer cells (NK cells). Lymphocytes may also be tumor-infiltrating lymphocytes. Examples of T cells include killer T cells (cytotoxic T cells), helper T cells, and regulatory T cells. T cells may be either CD8-positive T cells or CD4-positive T cells. T cells can also be classified as naive T cells, memory T cells, and effector T cells, or any of these. Naive T cells and memory T cells are known to be abundant in peripheral blood, while effector T cells are rarely found in peripheral blood. Examples of granulocytes include neutrophils, eosinophils, and basophils. While the term "immune cells" may not strictly include stem cells, in this specification it is to include stem cells that can differentiate into lymphocytes (e.g., hematopoietic stem cells).

[0051] A "signal peptide" is an extracellular translocation signal necessary for the secretion of proteins biosynthesized by gene expression into the extracellular space. The signal sequence may include a region composed of hydrophobic amino acids. After translation, the signal peptide is cleaved and removed by a signal peptidase before translocation to the extracellular space. Signal peptide sequences are present at the N-terminus of many secretory and membrane proteins, typically ranging from 15 to 30 amino acids in length. Signal peptides may originate from any species, including humans or non-humans, such as insect cells or viruses, but are preferably human-derived. A specific example of a signal peptide is the signal peptide derived from human oncostatin M.

[0052] A "linker peptide" is a peptide that can be inserted between the fused parts of a fusion protein, such as a chimeric cytokine receptor, in order for each part to perform its intended function. The length of the linker peptide is not limited, but typically it is 3 to 100 amino acids long, preferably 5 to 50 amino acids long. Peptides containing a large amount of amino acids with relatively small side chains, such as serine or glycine, are often used.

[0053] A "tag peptide" is a short peptide consisting of tens to several tens of amino acids that can label proteins and is used for protein detection and purification. Typically, the base sequence encoding the tag peptide is ligated to the 5' or 3' end of the gene encoding the protein to be labeled, and the protein is expressed as a fusion protein with the tag peptide. Various types of tag peptides have been developed in this field, and any tag peptide may be used. Specific examples of tag peptides include FLAG, HA, His, PA, and myc.

[0054] In this specification, "capture" refers to the binding of ligands, such as cytokines, to ligand-binding regions contained in chimeric cytokine receptors or their fragments. When cytokines are captured by chimeric cytokine receptors, their function, such as signal transduction mediated by endogenous cytokine receptors, may be suppressed or inhibited. Cytokines captured by chimeric cytokine receptors on the cell membrane can be absorbed into the cell.

[0055] In this specification, "multiple" means two or more integers, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3 integers.

[0056] In this specification, "amino acid identity (amino acid sequence identity)" refers to the percentage of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides being compared are aligned by inserting gaps as needed into one or both of them to maximize the number of matching amino acid residues. "Base identity (base sequence identity)" can be determined in the same manner.

[0057] In this specification, "amino acid substitution" refers to substitutions between the 20 amino acids that make up natural proteins. Amino acid substitutions are preferably within a group of conserved amino acids that have similar properties such as charge, side chain, polarity, and aromaticity. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp).

[0058] 1-3. Structure The chimeric cytokine receptor of the present invention includes a ligand-binding region at the N-terminus and a T-cell activation region at the C-terminus.

[0059] In this specification, "ligand-binding region" means a region in the extracellular domain of the chimeric cytokine receptor of the present invention that can bind to a ligand. In the chimeric cytokine receptor of the present invention, the ligand-binding region consists of the cytokine-binding region of the cytokine receptor. The cytokine receptor from which the cytokine-binding region originates is, in principle, a cytokine receptor other than the IL-7 receptor α chain.

[0060] In this specification, "cytokine-binding region" refers to a region capable of binding to a cytokine. The cytokines to which the cytokine-binding region binds are not limited and include, for example, IL-6, IL-1, or GM-CSF. As the cytokine-binding region constituting the ligand-binding region of the chimeric cytokine receptor of the present invention, cytokine-binding regions derived from cytokine receptors other than the IL-7 receptor α chain can be used. For example, it may be the cytokine-binding region of the IL-6 receptor, the cytokine-binding region of the IL-1 receptor type 2, the cytokine-binding region of the GM-CSF receptor α chain, and / or the cytokine-binding region of the GM-CSF receptor β chain.

[0061] The cytokine-binding region of the IL-6 receptor is a region within the extracellular domain of the IL-6 receptor that is capable of binding to cytokines such as IL-6. More specifically, it is sufficient if the region includes the D2 domain and / or D3 domain, which are known to be important for IL-6 binding in the IL-6 receptor (Schwantner A., ​​et al., J Biol Chem., 2004, 279(1):571-6; Yawata H., et al., EMBO J, 1993, 12(4):1705-1712). Specific examples of the cytokine-binding region of the IL-6 receptor include the region consisting of positions 110-365 or 110-338 in the amino acid sequence shown in Sequence ID No. 11, as well as the entire extracellular domain consisting of positions 21-365.

[0062] The cytokine-binding region of the IL-1 receptor type 2 is a region included in the extracellular domain of the IL-1 receptor type 2 that is capable of binding to cytokines such as IL-1. More specifically, it can be any region in the IL-1 receptor type 2 that contains three IgC2 domains and a protease cleavage region, and an example of such a region is the extracellular domain of the IL-1 receptor type 2 (Liu C, et al., J Biol Chem, 1996, 271(34):20965-20972). Specific examples of the cytokine-binding region of the IL-1 receptor type 2 include the region consisting of positions 14 to 296 in the amino acid sequence shown in Sequence ID No. 9, and the entire extracellular domain consisting of positions 14 to 343.

[0063] The cytokine-binding region of the GM-CSF receptor α chain is a region included in the extracellular domain of the GM-CSF receptor α chain that is capable of binding to cytokines such as GM-CSF. More specifically, it is sufficient if the region contains the D1 domain (e.g., the region consisting of positions 25-113 in the amino acid sequence shown in SEQ ID NO: 20) and / or the D2-D3 domain (also known as the Class I cytokine receptor homology module, exemplified by the region consisting of positions 122-320 in the amino acid sequence shown in SEQ ID NO: 20), which are known to be important for GM-CSF binding (Mirza S., et al., Biochem J, 2010, 426(3):307-17; Hansen G., Cell, 2008, 134(3):496-507). A specific example of such a region is the extracellular domain of the GM-CSF receptor α chain, for example, the extracellular domain consisting of positions 25-320 in the amino acid sequence shown in SEQ ID NO: 20.

[0064] The cytokine-binding region of the GM-CSF receptor β-chain is a region included in the extracellular domain of the GM-CSF receptor β-chain that is capable of binding to cytokines such as GM-CSF. More specifically, it is sufficient if the region includes the D1 domain and / or D4 domain, which are known to be important for GM-CSF binding in the GM-CSF receptor β-chain, and more preferably the D2 domain and / or D3 domain, which are known to be important for multimer formation between the α-chain and β-chain (example of the α-chain binding region in the GM-CSF receptor β-chain: positions 133-240 and 241-338 in the amino acid sequence shown in SEQ ID NO: 22) (Hansen G., et al., Cell, 2008, 134(3):496-507; Haman A., et al., J Biol Chem, 1999, 274(48):34155-63). Specific examples of such regions include the extracellular region containing positions 17-132 and / or 339-436 in the amino acid sequence shown in Sequence ID No. 22, for example, the entire extracellular region consisting of positions 17-443, or the region consisting of positions 17-132 and / or 339-436.

[0065] In one embodiment, the cytokine receptor may include the cytokine-binding domain of the IL-6 receptor and the ligand-binding domain of gp130 (glycoprotein 130). In this case, the position of the ligand-binding domain of gp130 may be either on the N-terminal or C-terminal side of the cytokine-binding domain of the IL-6 receptor, but it is preferably on the N-terminal side.

[0066] The ligand-binding region of gp130 is a region within the extracellular domain of gp130 that is capable of binding to cytokines such as IL-6. More specifically, it should be a region containing the D1, D2, and / or D3 domains of gp130, which are known to be important for IL-6 binding (Chow D., et al., Science, 2001, 291(5511):2150-5; Pflanz S., Biochem J, 2001, 356(Pt 2):605-12.). A specific example of such a region is the extracellular domain of gp130, for example, the extracellular region consisting of positions 23 to 326 in the amino acid sequence shown in Sequence ID No. 13.

[0067] In this specification, the "T cell activation region" refers to a region in the chimeric cytokine receptor of the present invention that can activate T cells. The T cell activation region can activate functions such as the proliferative capacity and cytotoxic activity of T cells. The T cell activation region of the chimeric cytokine receptor of the present invention includes the transmembrane domain and the intracellular domain of the IL-7 (interleukin-7) receptor α chain.

[0068] In this specification, "transmembrane domain of the IL-7 receptor α chain" refers to the portion of the IL-7 receptor α chain consisting of an amino acid sequence that spans the cell membrane. The transmembrane domain of the IL-7 receptor α chain can be determined by known methods based on the amino acid sequence of the IL-7 receptor α chain. The transmembrane domain of the human IL-7 receptor α chain may be located at positions 240 to 264 in the amino acid sequence shown in Sequence ID No. 1.

[0069] In this specification, the "intracellular domain of the IL-7 receptor α chain" refers to the portion of the IL-7 receptor α chain consisting of amino acid sequences located intracellularly. The intracellular domain of the IL-7 receptor is a region located C-terminal to the transmembrane domain of the IL-7 receptor α chain described above. For example, the region of the IL-7 receptor α chain containing the Box1 motif (e.g., positions 272-280 in the amino acid sequence shown in SEQ ID NO: 1) and / or the SH2 domain binding motif (e.g., positions 449-452 in the amino acid sequence shown in SEQ ID NO: 1), for example, the intracellular region consisting of positions 265-459 in the amino acid sequence shown in SEQ ID NO: 1.

[0070] In this specification, "containing the transmembrane domain and intracellular domain of the IL-7 receptor α chain" means including both the transmembrane domain and the intracellular domain of the IL-7 receptor α chain as described above. A specific example of a T cell activation region containing the transmembrane domain and intracellular domain of the IL-7 receptor α chain is the region including positions 240 to 459 in the amino acid sequence shown in Sequence ID No. 1.

[0071] The T cell activation region in the chimeric cytokine receptor of the present invention preferably has a constitutive activation mutation in the transmembrane domain of the IL-7 receptor. The constitutive activation mutation can be any mutation capable of activating T cells, and constitutive activation mutations known in the art can be used. The constitutive activation mutation may also be an insertion mutation. Constitutive activation insertion mutations are known (Shochat C., et al., J Exp Med, 2011, 208(5):901-8). For example, you can choose from the following group of amino acids: (a) insertion of an amino acid sequence consisting of Pro-Pro-Cys-Leu (Sequence ID 2) between positions 243 and 244 in the amino acid sequence shown in Sequence ID 1, (b) insertion of an amino acid sequence consisting of Phe-Ser-Cys-Gly-Pro (Sequence ID 3) between positions 241 and 242 in the amino acid sequence shown in Sequence ID 1, (c) insertion of an amino acid sequence consisting of Cys-His-Leu (Sequence ID 4) between positions 244 and 245 in the amino acid sequence shown in Sequence ID 1, (d) insertion of an amino acid sequence consisting of Pro-Pro-Val-Cys-Ser-Val-Thr (Sequence ID 5) between positions 244 and 245 in the amino acid sequence shown in Sequence ID 1, and (e) insertion of an amino acid sequence consisting of Lys-Cys-His (Sequence ID 6) between positions 246 and 247 in the amino acid sequence shown in Sequence ID 1.

[0072] The chimeric cytokine receptor of the present invention may have mutations that suppress the activity of one or more motifs selected from the group consisting of the JAK-binding motif, the STAT3-related motif, and the STAT5 / PI3K-related motif, which are included in the intracellular domain of the IL-7 receptor α chain. By adding mutations to the amino acid sequences constituting these motifs, it is possible to suppress only specific downstream signals, thereby enhancing the therapeutic effect of the chimeric cytokine receptor of the present invention.

[0073] In one embodiment, the chimeric cytokine receptor of the present invention may have mutations at the Y449 residue and / or the M452 residue in the STAT5 / PI3K-related motif contained in the intracellular domain of the IL-7 receptor α chain. Furthermore, the chimeric cytokine receptor of the present invention may have a mutation at the Y456 residue in the STAT3-related motif contained in the intracellular domain of the IL-7 receptor α chain. The Y449 residue is known to be an important residue involved in the recruitment of STAT3 and STAT5 proteins, and mutations at the Y449 residue may attenuate the IL-7-mediated cell proliferation signal (Lin JX, et al., Immunity 1995, 2(4):331-9; Corcoran, AE, et al., EMBO J, 1996, 15(8): 1924-1932). The M452 residue is known to be involved in the recruitment of the PI3K protein. Mutations in the M452 residue attenuate the PI3K-Akt signal, while maintaining the STAT3 / 5 signal, thus not affecting IL-7-mediated cell proliferation. Excessive PI3K signaling can promote T cell differentiation and reduce long-term viability, suggesting that mutations in the M452 residue may promote the formation of memory T cells with long-term viability (Cui G., et al., J Immunol, 2020, 204(4):844-857). Mutations in the Y456 residue are also known to attenuate the proliferation signal (Zhong J., et al., BMC Immunol, 2010, 11:5.). Specific examples of Y449, M452, and / or Y456 mutations include the Y449F mutation, the M452L mutation, and / or the Y456F mutation. In one embodiment, the chimeric cytokine receptor of the present invention has the M452L mutation in the STAT5 / PI3K-related motif contained in the intracellular domain of the IL-7 receptor α chain.

[0074] In addition to the ligand-binding region and T-cell activation region described above, the chimeric cytokine receptor of the present invention may optionally include a signal peptide, a linker peptide, and / or a tag peptide. If the ligand-binding region does not contain a signal peptide, it is preferable that the chimeric cytokine receptor of the present invention includes a signal peptide on the N-terminal side of the ligand-binding region.

[0075] In one embodiment, the ligand-binding region of the chimeric cytokine receptor of the present invention is derived from the IL-6 receptor, and the T cell-activating region is derived from the IL-7 receptor α chain. An example of such a chimeric cytokine receptor is the one having the amino acid sequence shown in SEQ ID NO: 24.

[0076] In one embodiment, the ligand-binding region of the chimeric cytokine receptor of the present invention is derived from the IL-6 receptor, the T cell activation region is derived from the IL-7 receptor α chain, and the chimeric cytokine receptor further includes a gp130 ligand-binding region at the N-terminus of the ligand-binding region derived from the IL-6 receptor. An example of such a chimeric cytokine receptor is the one having the amino acid sequence shown in SEQ ID NO: 25.

[0077] In one embodiment, the ligand-binding region of the chimeric cytokine receptor of the present invention is derived from IL-1 receptor type 2, and the T cell-activating region is derived from the IL-7 receptor α chain. An example of such a chimeric cytokine receptor is the one having the amino acid sequence shown in SEQ ID NO: 26.

[0078] In one embodiment, the ligand-binding region of the chimeric cytokine receptor of the present invention is derived from the GM-CSF receptor α chain, and the T cell-activating region is derived from the IL-7 receptor α chain. An example of such a chimeric cytokine receptor is the one consisting of the amino acid sequence shown in SEQ ID NO: 27.

[0079] In one embodiment, the ligand-binding region of the chimeric cytokine receptor of the present invention is derived from the GM-CSF receptor β chain, and the T cell activation region is derived from the IL-7 receptor α chain. An example of such a chimeric cytokine receptor is the one consisting of the amino acid sequence shown in SEQ ID NO: 28.

[0080] 1-4. Effects The chimeric cytokine receptor of the present invention can activate immune cells and capture extracellular cytokines. Based on these two functions, it is possible to achieve both improved therapeutic efficacy and reduced side effects in adoptive immunotherapy.

[0081] The ligand-binding domain in the chimeric cytokine receptor of the present invention can suppress cytokine activity by capturing extracellular cytokines such as IL-6, IL-1β, and GM-CSF. This can suppress or avoid cytokine release syndrome and neurotoxic syndrome associated with adoptive immunotherapy such as CAR-T cell therapy. More specific mechanisms of suppression / avoidance are not limited to the following but can be exemplified as follows: The ligand-binding domain derived from the IL-6 receptor can mediate the intracellular absorption of IL-6. The ligand-binding domain derived from the IL-1 receptor type 2 can be cleaved and released extracellularly as a soluble receptor, capturing IL-1β and / or mediating the intracellular absorption of IL-1β without being cleaved. The ligand-binding domain derived from the GM-CSF receptor α / β chain can reduce the activation of monocytes and / or macrophages by GM-CSF by capturing GM-CSF, and consequently suppress the secretion of cytokines such as IL-6 from monocytes / macrophages.

[0082] The chimeric cytokine receptor of the present invention can be used regardless of the type of T cell receptor or CAR that is co-introduced. Therefore, it can be widely applied to adoptive immunotherapy.

[0083] 2. Nucleic acids encoding chimeric cytokine receptors 2-1. Overview A second aspect of the present invention is a nucleic acid encoding a chimeric cytokine receptor.

[0084] 2-2. Composition The "nucleic acid encoding a chimeric cytokine receptor" can be any nucleic acid encoding any of the chimeric cytokine receptors described in the first embodiment. The base sequence of such nucleic acid is not limited. For example, a nucleic acid containing a nucleic acid encoding a ligand-binding region at the 5' end and a nucleic acid encoding a T cell activation region at the 3' end in a frame is an example. Codon-optimized base sequences and base sequences with a start codon (ATG) added to the 5' end are also examples.

[0085] In one embodiment, the nucleic acid of this embodiment is a nucleic acid encoding a chimeric cytokine receptor in which the ligand-binding region is derived from the IL-6 receptor and the T cell-activating region is derived from the IL-7 receptor α chain. Examples of such nucleic acids include those encoding a chimeric cytokine receptor consisting of the amino acid sequence shown in SEQ ID NO: 24, and, for example, those consisting of the nucleotide sequence shown in SEQ ID NO: 29.

[0086] In one embodiment, the nucleic acid of this embodiment is a nucleic acid encoding a chimeric cytokine receptor in which the ligand-binding region is derived from the IL-6 receptor, the T cell activation region is derived from the IL-7 receptor α chain, and the ligand-binding region of gp130 is further included at the N-terminus of the ligand-binding region derived from the IL-6 receptor. Examples of such nucleic acids include those encoding a chimeric cytokine receptor consisting of the amino acid sequence shown in SEQ ID NO: 25, and, for example, those consisting of the nucleotide sequence shown in SEQ ID NO: 30.

[0087] In one embodiment, the nucleic acid of this embodiment is a nucleic acid encoding a chimeric cytokine receptor in which the ligand-binding region is derived from the IL-1 receptor type 2 and the T cell-activating region is derived from the IL-7 receptor α chain. Examples of such nucleic acids include those encoding a chimeric cytokine receptor consisting of the amino acid sequence shown in SEQ ID NO: 26, and, for example, those consisting of the nucleotide sequence shown in SEQ ID NO: 31.

[0088] In one embodiment, the nucleic acid of this embodiment is a nucleic acid encoding a chimeric cytokine receptor in which the ligand-binding region is derived from the GM-CSF receptor α chain and the T cell-activating region is derived from the IL-7 receptor α chain. Examples of such nucleic acids include those encoding a chimeric cytokine receptor consisting of the amino acid sequence shown in SEQ ID NO: 27, and, for example, those consisting of the nucleotide sequence shown in SEQ ID NO: 32.

[0089] In one embodiment, the nucleic acid of this embodiment is a nucleic acid encoding a chimeric cytokine receptor in which the ligand-binding region is derived from the GM-CSF receptor β chain and the T cell-activating region is derived from the IL-7 receptor α chain. Examples of such nucleic acids include those encoding a chimeric cytokine receptor consisting of the amino acid sequence shown in SEQ ID NO: 28, and, for example, those consisting of the nucleotide sequence shown in SEQ ID NO: 33.

[0090] 3. Gene expression vector containing nucleic acids encoding chimeric cytokine receptors 3-1. Overview A third aspect of the present invention is a gene vector (hereinafter referred to as "chimeric cytokine receptor expression vector") containing nucleic acids encoding chimeric cytokine receptors in an expressible state.

[0091] 3-2. Composition The gene expression vector of this embodiment comprises the nucleic acid and promoter described in the second embodiment, and is a gene expression vector capable of expressing a chimeric cytokine receptor in a cell. In addition to the nucleic acid and promoter, which are the components, the gene expression vector may optionally include components such as a labeling gene (selection marker), enhancer, terminator, origin of replication, and poly(A) signaling. The gene expression vector of this embodiment may include a nucleic acid encoding one type of chimeric cytokine receptor as the nucleic acid described in the second embodiment, or it may include a combination of nucleic acids encoding two or more types of chimeric cytokine receptors. When including nucleic acids encoding two or more types of chimeric cytokine receptors, the combination may be any combination, and an example is a combination of a chimeric cytokine receptor containing the cytokine binding region of the GM-CSF receptor α chain and a chimeric cytokine receptor containing the cytokine binding region of the GM-CSF receptor β chain. The nucleic acids encoding two or more types of chimeric cytokine receptors may be included in the same gene expression vector.

[0092] In this specification, "gene expression vector" means a vector that contains a gene or gene fragment (hereinafter referred to as "gene etc.") in an expressible state and contains an expression unit that can control the expression of such gene etc. Gene expression vectors may also be plasmid vectors or viral vectors.

[0093] In this specification, "expression-ready state" means that the gene to be expressed is located in the downstream region of the promoter, which is under the control of the promoter. While plasmid vectors and viral vectors are known types of vectors, any type of vector can be used. Typically, a plasmid vector, which is easy to recombinate, or a viral vector, which can easily introduce genes into immune cells, is sufficient.

[0094] Plasmid vectors may include commercially available mammalian cell expression vectors such as Promega's pCI vectors and pSI vectors, or shuttle vectors that can replicate between mammalian cells and bacteria such as E. coli.

[0095] Viral vectors that can be used include, for example, retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, Simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Viral vectors that lack replication ability to prevent self-replication within infected cells may also be used.

[0096] When retroviral vectors are used, suitable packaging cells and packaging signal sequences can be selected based on the LTR sequence to produce retroviral particles. Examples of packaging cells include PG13 (ATCC® CRL-10686®), PA317 (ATCC® CRL-9078®), GP+E-86 and GP+envAm-12 (US Patent No. 5,278,056), and Psi-Crip (Proceedings of the National Academy of Sciences of the United States of America, vol. 85, pp. 6460-6464 (1988)). Retroviral particles can also be produced using 293 cells or 293T cells with high transfection efficiency. Many types of viral vectors produced based on packaging cells suitable for packaging retroviruses and retroviral vectors are commercially available from many companies.

[0097] In this specification, "promoter" refers to a gene expression regulatory region that can control the expression of a gene located downstream (at the 3' end) in a cell into which a gene expression vector has been introduced. Promoters can be classified into ubiquitous promoters (systemic promoters) and site-specific promoters based on the location in which they express a gene under expression control. A ubiquitous promoter is a promoter that controls the expression of a target gene (target gene, etc.) in all cells, i.e., the entire host organism. A site-specific promoter is a promoter that controls the expression of a target gene, etc. only in specific cells or tissues. The promoter included in the gene expression vector of the present invention may be either a ubiquitous promoter or a site-specific promoter, but it is preferable that it can induce expression in immune cells.

[0098] Furthermore, promoters are classified into constitutively active promoters, expression-inducing promoters, or time-specific active promoters based on the timing of expression. Constitutively active promoters can constitutively express target genes, etc., within cells. Expression-inducing promoters can induce the expression of target genes, etc., within cells at any desired time. Time-specific active promoters can induce the expression of target genes, etc., within cells only at specific stages of development. All of these promoters can be understood as overexpression promoters because they can lead to the overexpression of target genes within host cells. The promoter included in the gene expression vector of the present invention is preferably a constitutively active promoter, which enables long-term persistence of therapeutic effects.

[0099] In the gene expression vector of this embodiment, the promoter is a promoter capable of inducing the expression of a nucleic acid encoding a chimeric cytokine receptor within cells such as immune cells. Since the target cells into which the gene expression vector of the present invention is introduced are, in principle, mammalian cells, particularly human-derived cells, such as human-derived immune cells, any promoter capable of expressing downstream genes within those cells is acceptable. Examples include the CMV promoter (CMV-IE promoter), SV40 initial promoter, RSV promoter, EF1α promoter, Ub promoter, and 5' LTR promoter. In the case of retroviral vectors, the nucleic acid encoding a chimeric cytokine receptor can be placed downstream of the 5' LTR promoter to induce its gene expression.

[0100] In this specification, "labeled gene" refers to a gene that encodes a labeled protein, also called a selection marker or reporter protein. "Labeled protein" refers to a peptide whose activity allows for the determination of whether or not a labeled gene is expressed. Detection of activity may involve directly detecting the activity of the labeled protein itself, or it may be indirectly detected via metabolites generated by the activity of the labeled protein, such as dyes. Detection may be biological (including detection by binding of peptides or nucleic acids such as antibodies and aptamers), chemical (including enzymatic detection), physical (including behavioral analysis), or sensory detection by the detector (including detection by sight, touch, smell, hearing, and taste).

[0101] The type of labeled protein encoded by a labeled gene is not particularly limited, as long as its activity can be detected by methods known in the field. Labeled proteins that are less invasive to the transformant during detection are preferred. Examples include tag peptides, drug resistance proteins, pigment proteins, fluorescent proteins, and luminescent proteins.

[0102] "Drug-resistant proteins" are proteins that confer resistance to antibiotics and other drugs added to culture media, and most are enzymes. Examples include β-lactamase, which confers resistance to ampicillin; aminoglycoside 3'-phosphotransferase, which confers resistance to kanamycin; tetracycline efflux transporter, which confers resistance to tetracycline; and CAT (chloramphenicol acetyltransferase), which confers resistance to chloramphenicol.

[0103] "Pigment proteins" are proteins involved in the biosynthesis of pigments, or proteins that enable the chemical detection of transformants by pigment through the application of substrates; they are usually enzymes. Here, "pigments" refer to low-molecular-weight compounds or peptides that can confer pigment to transformants, regardless of their type. Examples include β-galactosidase (LacZ), β-glucuronidase (GUS), melanin-synthetic proteins, ommochrome pigments, or pteridine pigments.

[0104] A "fluorescent protein" is a protein that emits fluorescence at a specific wavelength when irradiated with excitation light of a specific wavelength. It may be either a native or unnatural type. There are no particular limitations on the excitation wavelength or fluorescence wavelength. Specifically, examples include CFP, RFP, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, and GFP (including derivatives such as EGFP and 3xP3-EGFP).

[0105] A "luminescent protein" refers to a substrate protein that can emit light without requiring excitation light, or an enzyme that catalyzes the luminescence of such a substrate protein. Examples include luciferin or aequorin as substrate proteins, and luciferase as an enzyme.

[0106] In this specification, "enhancer" is not particularly limited as long as it can enhance the expression efficiency of a gene or fragment thereof within a vector.

[0107] In this specification, "terminator" is a sequence that can terminate the transcription of a gene or the like expressed by the activity of the promoter. The type of terminator is not particularly limited. Preferably, it is a terminator derived from the same species as the promoter. In a single gene expression regulatory system, a terminator paired with the promoter on the genome is particularly preferred.

[0108] 4. Host cells containing gene expression vectors 4-1. Overview A fourth aspect of the present invention is a host cell. The host cell of this aspect includes a gene vector containing nucleic acids encoding chimeric cytokine receptors in an expressible state. The host cell of this aspect is, for example, a CAR-T cell containing a chimeric antigen receptor (CAR) expression vector.

[0109] 4-2. Composition The host cells of this embodiment include a chimeric cytokine receptor expression vector as an essential component and a CAR expression vector and / or an IL-1 receptor type 2 expression vector as a selective component.

[0110] The essential component, the chimeric cytokine receptor expression vector, is as described in the third embodiment. The host cell in this embodiment may contain one type of chimeric cytokine receptor expression vector, or it may contain a combination of two or more types of chimeric cytokine receptor expression vectors. When two or more types of chimeric cytokine receptor expression vectors are included, the combination may be any combination. For example, an example is a combination of an expression vector that expresses a chimeric cytokine receptor containing the cytokine binding region of the GM-CSF receptor α chain and an expression vector that expresses a chimeric cytokine receptor containing the cytokine binding region of the GM-CSF receptor β chain.

[0111] The type of host cell in this embodiment is not limited. Examples of host cells include immune cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells, skin keratinocytes, mesenchymal stem cells, hematopoietic stem cells, various cancer cell lines, and neural stem cells, iPS cells, and ES cells. Preferred host cells are immune cells. Immune cells may be T cells, NK cells, or macrophages. Naive T cells and memory T cells are preferred. Host cells such as immune cells may be cells derived from living organisms, immortalized cell lines, or cells differentiated from ES cells or induced pluripotent stem cells (iPS cells).

[0112] In this specification, "chimeric antigen receptor (CAR) expression vector" refers to a gene expression vector that comprises a nucleic acid encoding a chimeric antigen receptor (CAR) and a promoter, and is capable of expressing CAR in cells. In addition to the nucleic acid and promoter components, the CAR expression vector may optionally include components such as a labeling gene (selection marker), enhancer, terminator, origin of replication, and poly(A) signaling. The CAR expression vector may be a plasmid vector or a viral vector.

[0113] The type of CAR expressed by the CAR expression vector is not limited, nor is the type of antigen targeted by the CAR. Examples of antigens include viral antigens, bacterial antigens, parasitic antigens, cell surface markers on target cells associated with specific disease states, tumor antigens, and surface molecules of immune cells. It is preferable for the CAR to target antigens on the surface of tumor cells.

[0114] An "IL-1 receptor type 2 expression vector" is a gene expression vector that includes a nucleic acid encoding IL-1 receptor type 2 and a promoter, and is capable of expressing IL-1 receptor type 2 in cells. In addition to the nucleic acid and promoter components, the IL-1 receptor type 2 expression vector may optionally include components such as a labeling gene (selection marker), enhancer, terminator, origin of replication, and poly(A) signaling. The IL-1 receptor type 2 expression vector may also be a plasmid vector or a viral vector.

[0115] 4-3. Effects The host cells of the present invention, based on the ligand-binding region at the N-terminus of the chimeric cytokine receptor, can capture cytokines outside the host cell and, in some cases, absorb cytokines into the cell. Furthermore, based on the T cell activation region, the proliferative capacity and cytotoxic activity of the host cells are enhanced, and the therapeutic effect based on cytotoxic activity can be sustained over the long term.

[0116] Host cells according to this embodiment, containing a CAR expression vector, can be used as CAR-T cells in CAR-T cell therapy.

[0117] Furthermore, if the host cells of this embodiment contain a gene expression vector encoding a chimeric cytokine receptor whose ligand-binding region is derived from the IL-6 receptor, and an IL-1 receptor type 2 expression vector, the host cells of this embodiment can capture both IL-6 and IL-1β. Therefore, both cytokine release syndrome and neurotoxic syndrome can be suppressed.

[0118] 5. Cell Therapy 5-1. Overview A fifth aspect of the present invention is a cell preparation. The cell preparation of this aspect contains the host cells of the fourth aspect as an active ingredient and can be used in adoptive immunotherapy. According to the cell preparation of this aspect, cytokine release syndrome and neurotoxic syndrome can be suppressed in adoptive immunotherapy, and the therapeutic effect can be maintained over the long term.

[0119] 5-2.Definition In this specification, "subject" refers to the target of application of the cell preparation according to this embodiment. For example, it may be a tissue, organ, or individual. In the case of an individual, for example, it may be a mammal, preferably a human individual. The human individual may be a patient, such as a cancer patient.

[0120] In this specification, "subject information" refers to various information about the characteristics and condition of the subject. For example, if the subject is a human individual, this may include age, weight, sex, overall health status, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.

[0121] In this specification, “treatment” means the alleviation or elimination of symptoms associated with a disease, and / or the prevention or suppression of the progression of the disease, as well as the cure of the disease.

[0122] In this specification, "disease" is not limited. Examples of diseases include cancer, inflammatory diseases, autoimmune diseases, hepatitis, and infectious diseases. Examples of infectious diseases include viral infections such as influenza and HIV, bacterial infections, and fungal infections. The disease is preferably cancer.

[0123] In this specification, the types of cancer are not limited, but examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specific types of cancer include, for example, malignant melanoma, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, colorectal cancer (including colon and rectal cancer), small intestine cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer (including gallbladder and bile duct cancer), brain tumors, head and neck cancers, mesothelioma, osteosarcoma, soft tissue sarcoma, glioma, neuroblastoma and other pediatric tumors, hematological cancers, lymphoma, and myeloma. Examples of blood cancers include leukemia (e.g., B-cell leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., non-Hodgkin lymphoma), and myeloma (e.g., multiple myeloma). In this specification, cancer may refer to either solid tumors or blood cancers.

[0124] 5-3. Composition 5-3-1. Components The components of the cell preparation of this aspect will be described. The cell preparation of this aspect contains, as essential components, one or more active ingredients, and a solvent and / or a carrier. Hereinafter, each component will be specifically described.

[0125] (Active ingredient) The cell preparation of this aspect includes, as an essential active ingredient, the host cell described in the fourth aspect. The cell preparation of this aspect can contain one or more types of host cells.

[0126] The content of the active ingredient contained in the cell preparation of the present invention is not particularly limited. Generally, the content varies depending on the type of the active ingredient, the dosage form, and the types of the solvent and the carrier, which are other components described later. Therefore, it may be appropriately determined in consideration of each condition. It is sufficient that the cell preparation of a single application dose contains an effective amount of the active ingredient. However, when it is necessary to administer a large amount of the cell preparation to the subject in order to obtain the pharmacological effect of the active ingredient, it can also be administered in several divided doses to reduce the burden on the subject. In this case, the amount of the active ingredient only needs to include an effective amount in the total amount.

[0127] The "effective amount" refers to the amount necessary to exert the function as an active ingredient and that hardly or does not impart any harmful side effects to the subject to which it is applied. This effective amount can vary depending on various conditions such as the information of the subject, the application route, and the number of applications. Therefore, when the cell preparation of this aspect is used as a medicine, the content of the active ingredient is ultimately determined by the judgment of a doctor or a pharmacist, etc.

[0128] The amount of the host cell contained in the cell preparation of this aspect is, for example, 10 5 cells~10 10 cells or 10 6 cells~10 9 cells, preferably 10 7 cells~10 8 cells. Although not limited, when targeting children, in the case of a body weight of 50 kg or less, it is 0.2 to 5.0×10 6 cells / kg, and in the case of a body weight exceeding 50 kg, it is 0.1 to 2.5×10 8Cells are used as an example. For adults, the ratio is 0.6 to 6.0 × 10⁻⁶. 8 Cells are given as an example.

[0129] (solvent) The cell preparation of the present invention may optionally contain a pharmaceutically acceptable solvent. A "pharmaceutically acceptable solvent" refers to a solvent commonly used in the pharmaceutical technology field. Examples include water or aqueous solutions, or organic solvents. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, phosphate-buffered saline, sodium acetate buffers, glycols, or ethanol solutions. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. An example of an organic solvent is ethanol.

[0130] (carrier) The cell formulation of the present invention may optionally include a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology. Examples include excipients and human serum albumin.

[0131] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides; inorganic salts such as hydrochloride, hydrobromide, phosphate, or sulfate; salts of organic acids such as acetate, propionate, malonate, or benzoate; metal salts; citric acid, tartaric acid, glycine, polyethylene glycol, kaolin, silicic acid, or combinations thereof.

[0132] In addition to the above, if necessary, the composition may also contain solubilizers, suspending agents, diluents, dispersants, surfactants, analgesics, stabilizers, absorption enhancers, bulking agents, preservatives, antiseptics, antioxidants, buffering agents, isotonic agents, etc., which are commonly used in pharmaceutical compositions.

[0133] The carrier is used to avoid or inhibit the degradation of the active ingredient by enzymes, etc., within the subject's body, as well as to facilitate formulation and administration methods, and to maintain the dosage form and efficacy. It should be used as appropriate as needed.

[0134] 5-3-2. Dosage Form The dosage form of the cell preparation of the present invention is not particularly limited. Any form that can deliver the active ingredient to the target site in the subject's body without inactivating it is acceptable.

[0135] The specific dosage form will vary depending on the method of application, which will be described later. The methods of application can be broadly classified into parenteral administration and oral administration, but parenteral administration is preferred.

[0136] If the administration method is parenteral, the preferred dosage form is a liquid formulation that can be administered directly to the target site or systemically via the circulatory system. A good example of a liquid formulation is an injectable formulation. Injectable formulations can be formulated by mixing a solvent with excipients, suspensions, surfactants, stabilizers, pH adjusters, etc., in a unit dose form generally accepted for pharmaceutical production.

[0137] 5-3-3. Application method The method of application of the cell preparation of the present invention is not particularly limited, and the route of administration is not limited, but for example, parenteral administration is also possible. Parenteral administration can be further subdivided into systemic administration and local administration. Local administration includes, for example, intradermal administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intranasal administration, intratumoral administration, tissue administration, and organ administration. Systemic parenteral administration includes intracirculatory administration, such as intravenous administration (intravenous injection), intra-arterial administration, and intralymphatic administration. The preferred route of administration is intravenous administration, which may be infusion by drip infusion (for example, a single intravenous infusion).

[0138] 5-4. Effects The cell preparation of this embodiment provides a method for treating and / or preventing a disease, comprising the step of administering the cell preparation of the present invention to a subject. The disease may be, for example, cancer, and the subject may be a cancer patient. The treatment / prevention method is characterized by long-lasting effects such as cytotoxic activity, and reduced side effects such as cytokine release syndrome and neurotoxic syndrome. This treatment / prevention method may also be adoptive immunotherapy such as CAR-T cell therapy.

[0139] According to this embodiment of the cell preparation, a method is also provided for providing antitumor immunity to a subject, which includes the step of administering the cell preparation to the subject.

[0140] The use of the chimeric cytokine receptor, gene expression vector, host cell, or cell preparation of the present invention in the manufacture of pharmaceuticals for treating and / or preventing diseases such as cancer is also provided.

[0141] 6. Method for producing chimeric antigen receptor (CAR)-transformed cells with long-lasting cytotoxic activity 6-1. Overview A sixth aspect of the present invention is a method for producing chimeric antigen receptor (CAR)-transformed cells having long-lasting cytotoxic activity. The method of this aspect includes, as essential steps, a peripheral blood mononuclear cell isolation step and a vector introduction step. According to the method of this aspect, CAR-transformed cells having long-lasting cytotoxic activity can be produced. CAR-transformed cells produced by the method of this aspect can be used in CAR-T cell therapy in which the therapeutic effect is maintained over the long term.

[0142] In this specification, "long-lasting cytotoxic activity" means that cytotoxic activity against target cells such as cancer cells persists for a long period of time. "Long-lasting" means that it persists for a longer period than, for example, the duration of therapeutic effect of therapeutic cells in conventional adoptive immunotherapy. More specifically, it means that the duration of therapeutic effect is longer compared to CAR-introduced cells that have not been introduced with the chimeric cytokine receptor expression vector of the present invention. For example, it means that detectable cytotoxic activity persists for 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 1 week or more, 10 days or more, 2 weeks or more, 3 weeks or more, 24 days or more, 4 weeks or more, 1 month or more, 5 weeks or more, 38 days or more, 40 days or more, 6 weeks or more, 45 days or more, 7 weeks or more, 50 days or more, 55 days or more, 60 days or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, 6 months or more, or 1 year or more. Furthermore, "persistent" cytotoxic activity means that at least detectable cytotoxic activity is maintained over any of the above periods, for example, that higher cytotoxic activity is maintained compared to CAR-introduced cells that have not been introduced with the chimeric cytokine receptor expression vector of the present invention.

[0143] 6-2. Method The method for producing CAR-introduced cells according to this embodiment includes a peripheral blood mononuclear cell isolation step and a vector introduction step as essential steps, and a wide-batch culture step as a selective step. Each step will be described in detail below.

[0144] (Peripheral blood mononuclear cell isolation process) The "peripheral blood mononuclear cell isolation process" is a process for isolating peripheral blood mononuclear cells from the peripheral blood of a subject. The purpose of this process is to isolate peripheral blood mononuclear cells that contain immune cells.

[0145] In this specification, "Peripheral Blood Mononuclear Cells (PBMCs)" means cells or cell populations, including monocytes and / or lymphocytes such as T cells, isolated from the peripheral blood of a human or animal.

[0146] The method for separating peripheral blood mononuclear cells is not limited. For example, peripheral blood mononuclear cells can be separated by density gradient centrifugation or hemolysis. Density gradient centrifugation can be performed by layering a diluted whole blood sample on top of a solvent and centrifuging it. Ficoll-hypaque® or similar solvents can be used, with a solvent adjusted to a density of 1.077 g / mL being an example. As a result of density gradient centrifugation, the mononuclear cell component can be separated from red blood cells, granulocytes, and plasma as an intermediate layer. Hemolysis is a method of removing red blood cells with a hypotonic solution, but this method does not remove the granulocyte component. Therefore, this step is usually performed by density gradient centrifugation.

[0147] (Vector introduction process) The "vector introduction step" is a step in which a gene expression vector containing nucleic acid encoding the chimeric cytokine receptor described in the third embodiment (hereinafter referred to as the "chimeric cytokine receptor expression vector") and a CAR expression vector are introduced into peripheral blood mononuclear cells isolated in the peripheral blood mononuclear cell isolation step. The CAR expression vector is a gene expression vector containing nucleic acid encoding the chimeric antigen receptor (CAR) in an expressible state, and its specific composition is as described in the fourth embodiment. Cells into which the chimeric cytokine receptor expression vector and the CAR expression vector have been introduced in this step can be used, for example, as CAR-T cells in adoptive immunotherapy such as CAR-T cell therapy.

[0148] The method for introducing the chimeric cytokine receptor expression vector and the CAR expression vector into peripheral blood mononuclear cells is not particularly limited.

[0149] If each vector is a viral vector, the method of viral infection of cells is known in the art. Functional substances that improve the efficiency of viral infection may be used to introduce viral vectors, such as fibronectin or fibronectin fragments (for example, retronectin® or Vecofusin-1®, which are fibronectin fragments having a heparin-binding site).

[0150] The following is an example of a viral infection method using retronectin: After treating a cell culture plate with retronectin, the bottom of the plate is blocked with a 2% BSA / PBS solution for 30 minutes. Then, after washing with PBS, retrovirus solution derived from PG13 packaging cells is loaded onto the culture plate, and the plate is centrifuged at 32°C and 2000g for 2 hours. After centrifugation, the virus solution is removed, and cells are seeded onto the plate.

[0151] If each vector is a non-viral vector such as a plasmid, then a gene transfer method (transformation method) known in the field, as described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc., may be used. Examples include lipofection, electroporation, microinjection, calcium phosphate, DEAE-Dextran, and particle impact.

[0152] Peripheral blood mononuclear cells into which the vector has been introduced in this process can usually be used as is, but if necessary, only the cells into which the vector has been introduced may be isolated.

[0153] (Expanded culture process) The "expansion culture step" is a step in which peripheral blood mononuclear cells obtained after the peripheral blood mononuclear cell isolation step are stimulated with an anti-CD3 antibody, and can be performed as a selective step between the peripheral blood mononuclear cell isolation step and the vector introduction step described above. The anti-CD3 antibody used in this step is not limited as long as it can stimulate peripheral blood mononuclear cells, and for example, soluble anti-CD3 antibodies or cells expressing membrane-bound anti-CD3 antibodies may be used.

[0154] In this process, the proliferation of peripheral blood mononuclear cells can also be maintained by culturing peripheral blood mononuclear cells stimulated with anti-CD3 antibodies in the presence of cytokines such as IL-2. The expanded culture process can improve the efficiency of T cell proliferation and CAR introduction. [Examples]

[0155] <Example 1: Preparation of chimeric cytokine receptors IL6R-ca7R and GP130-IL6R-ca7R> (the purpose) We will create a chimeric cytokine receptor IL6R-ca7R containing a portion of the interleukin-6 receptor α subunit (hereinafter referred to as "IL6RA") and a portion of the interleukin-7 receptor α subunit (hereinafter referred to as "IL7RA"). In addition, we will create a chimeric cytokine receptor GP130-IL6R-ca7R containing a portion of glycoprotein 130 (hereinafter referred to as "gp130"), a portion of IL6RA, and a portion of IL7RA.

[0156] (Methods and Results) (1) Creation of the chimeric cytokine receptor IL6R-ca7R IL6R-ca7R was constructed as an artificial receptor by linking a signal peptide, a ligand-binding region, and a T cell activation region in that order from the N-terminus (Figure 2A). The components are as follows:

[0157] The signal peptide used was derived from silkworm fibroin L protein (SEQ ID NO: 34).

[0158] The ligand-binding region consists of the extracellular domain of IL6RA. Specifically, in the full-length human IL6RA shown in Sequence ID No. 11, it comprises positions 20 to 365.

[0159] The T cell activation region consists of the portion from the hinge region to the intracellular domain in IL7RA with a constitutively activating mutation. Specifically, it consists of the amino acid sequence from positions 232 to 459 of the full-length human IL7RA shown in SEQ ID NO: 1, with the constitutively activating insertion mutation Pro-Pro-Cys-Leu (SEQ ID NO: 2) inserted between positions 243 and 244.

[0160] The full amino acid sequence of IL6R-ca7R is shown in SEQ ID NO: 24. The nucleotide sequence of the gene encoding IL6R-ca7R (hereinafter referred to as the "IL6R-ca7R gene") is shown in SEQ ID NO: 29.

[0161] (2) Production of the chimeric cytokine receptor GP130-IL6R-ca7R GP130-IL6R-ca7R was constructed as an artificial receptor by linking the ligand-binding domain derived from gp130, the linker peptide, the ligand-binding domain derived from IL6RA, and the T cell activation domain in that order from the N-terminus (Figure 2B). The components are as follows.

[0162] The ligand-binding region derived from gp130 consists of the extracellular domain of gp130, with the signal peptide derived from gp130 contained at its N-terminus. Specifically, it occupies positions 1 to 326 in the full-length human gp130 shown in Sequence ID No. 13.

[0163] The ligand-binding region derived from IL6RA consists of the extracellular domain of IL6RA. Specifically, it comprises positions 110 to 365 in the full-length human IL6RA shown in Sequence ID No. 11.

[0164] The T cell activation region consists of an amino acid sequence in which the constitutively active insertion mutation Pro-Pro-Cys-Leu (sequence number 2) is inserted between positions 243 and 244 of the full-length human IL7RA shown in sequence number 1, as described in (1) above.

[0165] The full amino acid sequence of GP130-IL6R-ca7R is shown in SEQ ID NO: 25. The nucleotide sequence of the gene encoding GP130-IL6R-ca7R (hereinafter referred to as the "GP130-IL6R-ca7R gene") is shown in SEQ ID NO: 30.

[0166] <Example 2: Cell surface expression of chimeric cytokine receptors> (the purpose) The chimeric cytokine receptors IL6R-ca7R and GP130-IL6R-ca7R are introduced into T cells along with chimeric antigen receptors (CARs), and their cell surface expression is analyzed by flow cytometry.

[0167] (Methods and Results) RPMI 1640 medium supplemented with 10% FBS, penicillin (100 units / mL), streptomycin (100 μg / mL), and recombinant IL2 (100 IU / mL) was used as the culture medium for T cells.

[0168] On the first day of culture, peripheral blood mononuclear cells (HHU20180703 or HHU20180821) were stimulated with an anti-CD3 antibody (initial stimulation). For the initial stimulation, a K562 cell line expressing a single-chain variable region fragment (scFV) derived from an anti-CD3 antibody (clone OKT3) and the co-stimulatory molecule CD80 on its cell surface was used. After arresting cell division of this K562 cell line by mitomycin treatment, the K562 cell line and peripheral blood mononuclear cells were co-cultured in a 1:7 ratio. The initial stimulation was performed in all the following examples with the aim of improving T cell proliferation and CAR delivery efficiency.

[0169] On the second day of culture, the IL6R-ca7R gene or GP130-IL6R-ca7R gene prepared in Example 1 was introduced into T cells along with the FMC63-28z CAR gene using retrovirus. Specifically, the pMX plasmid (Kitamura T. et al., Exp Hematol., 2003, 31:1007-1014.) was introduced into Plat-E packaging cells by transient transfection using TranslT293 (Mirus Bio) as the retroviral plasmid, and the resulting ecotropic retroviral vector was stably introduced into PG13 packaging cells. The resulting viral vector derived from PG13 cells was introduced into T cells using RetroNectin (Takara Bio). The FMC63-28z CAR is a CAR created by linking a single-chain variable region fragment derived from an anti-CD19 antibody (clone FMC63; Nicholson et al., Mol Immunol. 1997, 34(16-17):1157-65.), the transmembrane domain and cytoplasmic domain of CD28, and the cytoplasmic domain of CD3z. It was constructed based on the literature (Kochenderfer JN, et al., J Immunother. 2009 Sep;32(7):689-702.).

[0170] Next, on day 5 of culture, T cells in the CD8-positive fraction were labeled with an anti-IL6RA antibody (BioLegend, 352812) to analyze the expression of chimeric cytokine receptors and / or IL6RA on the cell surface using flow cytometry. Specifically, after staining the cells with the antibody, data was acquired using BD LSRFortessa (BD Biosciences), and the data was analyzed using Flowjo software (BD Biosciences).

[0171] Figure 3 shows representative flow cytometry plots. As shown in Figure 3, in CAR-T cells into which the chimeric cytokine receptor IL6R-ca7R or GP130-IL6R-ca7R was introduced, 90% and over 80% of the cells, respectively, showed positivity. On the other hand, in control CAR-T cells into which only the FMC63-28z CAR gene was introduced, almost no positive cells were detected.

[0172] <Example 3: Dynamics of chimeric cytokine receptor internalization following IL-6 administration> (the purpose) We will administer recombinant IL-6 (interleukin-6) to CAR-T cells co-introduced with chimeric cytokine receptors and analyze the dynamics of chimeric cytokine receptor internalization.

[0173] (Methods and Results) The GP130-IL6R-ca7R gene was introduced into T cells along with the NGFR-FMC63-28z CAR gene using a retrovirus in the same manner as in Example 2 (hereinafter referred to as "GP130-IL6R-ca7R-introduced CAR-T cells"). NGFR-FMC63-28z CAR stands for tNGFR-Furin-SGSG-P2A-FMC63-28z CAR and is a polypeptide containing, in order from the N-terminus, a truncated NGFR labeling marker, a Furin protease cleavage sequence (RAKR), a flexible linker (SGSG), a P2A sequence derived from porcine scoparium virus, and the FMC63-28z CAR.

[0174] GP130-IL6R-ca7R-transformed CAR-T cells were treated with recombinant IL-6 (PEPROTECH, 200-06) at a concentration of 50 ng / mL. After standing for 30 minutes in the presence of IL-6, the culture medium was changed to one without IL-6 to remove the IL-6, and the cells were cultured for a further 3.5 hours.

[0175] At each of the following time points—before IL-6 addition (0 min), after standing for 30 minutes in the presence of IL-6 (30 min), and after culturing for 3.5 hours without IL-6 (4 hours)—the chimeric cytokine receptors on the cell surface of T cells in the CD8-positive fraction were labeled with anti-IL6RA antibody and analyzed by flow cytometry. In this example, T cells into which only the NGFR-FMC63-28z CAR gene was introduced were used as control cells.

[0176] The results of flow cytometry analysis are shown in Figure 4. In GP130-IL6R-ca7R-introduced CAR-T cells, the chimeric cytokine receptor was internalized into the cell in the presence of IL-6 (Figure 4, 30 min), and after IL-6 was removed, the chimeric cytokine receptor was recycled to the cell surface (Figure 4, 4 h). On the other hand, this dynamic of receptor internalization was not observed in control cells.

[0177] The results of flow cytometry analysis are shown in Figure 5. In GP130-IL6R-ca7R-transformed CAR-T cells, the chimeric cytokine receptor on the cell surface decreased in the presence of IL-6 (Figure 5A, 0 min vs. 30 min) and recovered after IL-6 removal (Figure 5A, 30 min vs. 4 h). Furthermore, the proportion of cells labeled with anti-IL6RA antibody decreased in the presence of IL-6 (Figure 5B, 0 min vs. 30 min) and subsequently recovered after IL-6 removal (Figure 5B, 30 min vs. 4 h). Such dynamics of receptor internalization were not observed in control cells.

[0178] These results demonstrate that in GP130-IL6R-ca7R-introduced CAR-T cells, the addition of IL-6 leads to the internalization of the chimeric cytokine receptor within the cell.

[0179] <Example 4: IL-6 scavenging ability of CAR-T cells with chimeric cytokine receptors> (the purpose) CAR-T cells co-introduced with chimeric cytokine receptors will be treated with recombinant IL-6, and the IL-6 concentration in the supernatant will be measured to evaluate IL-6 scavenging ability.

[0180] (Methods and Results) (1) Analysis of IL-6 capture by CAR-T cells with chimeric cytokine receptors. The FMC63-28z CAR gene, which encodes a CAR for CD19, and the IL6R-ca7R gene or GP130-IL6R-ca7R gene were co-introduced into T cells using retrovirus. Retroviral gene transfer was performed in the same manner as in Example 2. Hereinafter, T cells into which both the CAR gene and the chimeric cytokine receptor gene (IL6R-ca7R gene or GP130-IL6R-ca7R gene) have been introduced will be referred to as "chimeric cytokine receptor-introduced CAR-T cells" ("IL6R-ca7R-introduced CAR-T cells" or "GP130-IL6R-ca7R-introduced CAR-T cells"). Cells cultured on day 5 were 2.5 × 10⁶ 5 Cells were seeded in a 48-well plate at a rate of one cell per well, and recombinant IL-6 was added to a final concentration of 3000 pg / mL. 48 hours after IL-6 addition, the culture supernatant was collected and the IL-6 concentration in the supernatant was measured by ELISA. The experimental procedure is shown in Figure 6A. In this example, T cells into which only the NGFR-FMC63-28z CAR gene was introduced were used as control cells.

[0181] Figure 6B shows the results of measuring the IL-6 concentration in the culture supernatant. In IL6R-ca7R-transformed CAR-T cells, the IL-6 concentration in the supernatant was significantly lower compared to control cells, indicating that most of the added IL-6 was captured. In GP130-IL6R-ca7R-transformed CAR-T cells, the IL-6 concentration in the supernatant was further reduced, indicating that almost all of the IL-6 was captured.

[0182] (2) Verification of the involvement of chimeric cytokine receptors on the cell membrane surface in IL-6 capture. Next, we investigated whether IL-6 capture was mediated by receptors on the cell membrane surface or by receptors secreted into the culture supernatant. 2.5 × 10⁶ IL6R-ca7R-expressed CAR-T cells or GP130-IL6R-ca7R-expressed CAR-T cells were used. 5 Cells were seeded in a 48-well plate, and after 48 hours, only the culture supernatant was transferred to another 48-well plate. Recombinant IL-6 was then added to the supernatant to a final concentration of 3000 pg / mL. After 24 hours, the supernatant was collected and the IL-6 concentration was measured by ELISA.

[0183] The results are shown in Figure 6C. In Figure 6C, IL-6 was added to the culture supernatants of control cells, IL6R-ca7R-transformed CAR-T cells, and GP130-IL6R-ca7R-transformed CAR-T cells, and the results of measuring the IL-6 concentration in the supernatant are shown as "control_sup", "IL6R-ca7R_sup", and "GP130-IL6R-ca7R_sup". Also, using the same method as in (1) above, IL-6 was added to cells, and the results of measuring the IL-6 concentration in the supernatant are shown as "control_cell", "IL6R-ca7R_cell", and "GP130-IL6R-ca7R_cell". As shown in Figure 6C, no significant IL-6 scavenging effect was detected when IL-6 was added to the cell culture supernatant.

[0184] These results indicate that the IL-6 scavenging effect is due to chimeric cytokine receptors on the cell membrane surface, and not to chimeric cytokine receptors secreted into the culture supernatant. It is thought that IL-6 captured by chimeric cytokine receptors on the cell membrane surface is absorbed into the cell.

[0185] <Example 5: Activation of the JAK-STAT pathway based on chimeric cytokine receptors> (the purpose) We will measure the levels of phosphorylated STAT3 / STAT5 in chimeric cytokine receptor-introduced CAR-T cells and investigate the activation of the JAK-STAT pathway.

[0186] (Methods and Results) The FMC63-28z CAR gene and the IL6R-ca7R gene or GP130-IL6R-ca7R gene were co-introduced into T cells using the same method as in Example 4. After the initial stimulation on day 1 of culture and the chimeric cytokine receptor-introduced CAR-T cells on day 6 were rested in cytokine-free medium for 1 day, recombinant IL-6 was added to the medium at 10 ng / mL and allowed to stand at 37°C for 30 minutes. Subsequently, phosphorylated STAT3 (pSTAT3) and phosphorylated STAT5 (pSTAT5) were detected in the CD3-positive T cell fraction by intracellular flow cytometry.

[0187] The results of the pSTAT3 analysis are shown in Figure 7. Figure 7 shows the distribution of pSTAT3 signal intensity after 1 day resting of IL6R-ca7R-introduced CAR-T cells and GP130-IL6R-ca7R-introduced CAR-T cells in cytokine-free medium (Figure 7A), the distribution of pSTAT3 signal intensity after IL-6 addition (Figure 7B), and the results of quantifying the fluorescence intensity indicating pSTAT3 (Figure 7C).

[0188] The results of the pSTAT5 analysis are shown in Figure 8. Figure 8 shows the distribution of pSTAT5 signal intensity after a 1-day rest period (Figure 8A), and the quantification of the fluorescence intensity indicating pSTAT5 before and after IL-6 addition after a 1-day rest period (Figure 8B).

[0189] These results indicate that in IL6R-ca7R-transformed CAR-T cells and GP130-IL6R-ca7R-transformed CAR-T cells, the JAK-STAT pathway is constitutively activated regardless of cytokine addition, and that STAT3 signaling is further activated by the addition of IL-6.

[0190] <Example 6: Proliferative capacity of CD19-targeted CAR-T cells with chimeric cytokine receptors> (the purpose) This study evaluates the proliferative capacity of CD19-targeted CAR-T cells that have been transfused with chimeric cytokine receptors.

[0191] (Methods and Results) A CAR gene targeting CD19 (FMC63-28z CAR gene or FMC63-BBz CAR gene) and an IL6R-ca7R gene or GP130-IL6R-ca7R gene were co-introduced into T cells. Hereinafter, T cells into which both a CAR gene targeting CD19 (FMC63-28z CAR gene or FMC63-BBz CAR gene) and a gene encoding a chimeric cytokine receptor (IL6R-ca7R gene or GP130-IL6R-ca7R gene) have been introduced will be referred to as "chimeric cytokine receptor-introduced CD19-targeted CAR-T cells" ("IL6R-ca7R-introduced CD19-targeted CAR-T cells" or "GP130-IL6R-ca7R-introduced CD19-targeted CAR-T cells"). The FMC63-BBz CAR is a CAR containing a single-chain variable region fragment derived from an anti-CD19 antibody (clone FMC63) and a portion of 4-1BB. It was constructed based on the sequence described in the literature (Milone MC, et al., Mol Ther, 2009, 17(8):1453-64). On day 5 of culture, 2.5 × 10⁶ CD19-targeted CAR-T cells with chimeric cytokine receptor transducers and control cells with only the CAR gene transduced were transduced. 5 The cells were seeded at a rate of one cell per well in 48-well plates and co-cultured in a 1:1 ratio with the CD19-positive tumor cell line NALM6 (Tohoku University Institute of Development, Aging and Cancer, Medical Cell Resource Center, TKG 0413). Culture was performed in RPMI 1640 medium containing 10% FBS, penicillin (100 units / mL), and streptomycin (100 μg / mL) under conditions without cytokine supplementation, and the medium was changed on day 5 of culture. Seven days after the start of co-culture, the number of chimeric cytokine receptor-transformed CD19-targeted CAR-T cells and control cells was counted, and their cell proliferation capacity was evaluated.

[0192] Figure 9A shows the multiplier change in cell number from the start of co-culture when the FMC63-28z CAR gene was used as the CAR gene. In control cells, the proliferation rate during co-culture was less than 3 times, while the proliferation rate of IL6R-ca7R-transformed CD19-targeted CAR-T cells and GP130-IL6R-ca7R-transformed CD19-targeted CAR-T cells was more than 12 times. Similar results were obtained when the FMC63-BBz CAR gene was used as the CAR gene (Figure 9B).

[0193] These results demonstrate that the introduction of chimeric cytokine receptors significantly enhances the proliferative capacity of CAR-T cells, regardless of the type of CAR.

[0194] <Example 7: Functional evaluation of CD19-targeted CAR-T cells with chimeric cytokine receptors> (the purpose) We will evaluate the function of CD19-targeted CAR-T cells that have been transduced to chimeric cytokine receptors. Specifically, we will examine the proportion of IFN-γ-producing cells, granzyme B production, and tumor cell survival rate.

[0195] (Methods and Results) (1) IFN-γ producing cells GP130-IL6R-ca7R-introduced CD19-targeted CAR-T cells were co-cultured in a 1:1 ratio with the CD19-positive tumor cell line NALM6. Two hours after the start of co-culture, Brefeldin A (BioLegend, 420601) at a 1 / 1000 dilution was administered. Four hours later, T cells were stained with anti-IFN-γ antibody (BioLegend, 502528), and IFN-γ-producing cells in the CD4-positive T cell fraction and CD8-positive T cell fraction were analyzed by flow cytometry.

[0196] The results are shown in Figure 10. In GP130-IL6R-ca7R-introduced CD19-targeted CAR-T cells, the proportion of IFN-γ-producing cells was significantly increased compared to control cells.

[0197] (2) Granzyme B production GP130-IL6R-ca7R-transformed CD19-targeted CAR-T cells and CD19-positive tumor cells (K562-CD19 cell line or Raji cell line) were co-cultured in a 1:1 ratio. K562-CD19 cells were prepared by stably introducing the CD19 gene into the K562 cell line (JCRB cell bank, JCRB0019). The Raji cell line was obtained from the JCRB cell bank (JCRB cell bank, JCRB9012). Three hours after the start of co-culture, the amount of Granzyme B produced was analyzed by intracellular flow cytometry using a Granzyme B antibody (Invitrogen, GRB04).

[0198] Figure 11A shows the distribution of Granzyme B (GZMB) production after co-culture with the K562-CD19 cell line. It was shown that GP130-IL6R-ca7R-transformed CD19-targeted CAR-T cells produced increased Granzyme B compared to control cells (FMC63-28z CAR-T cells).

[0199] Figure 11B shows the quantitative results of Granzyme B production when co-cultured with different target cells. In both the K562-CD19 cell line and the Raji cell line, GP130-IL6R-ca7R-transformed CD19-targeted CAR-T cells showed increased Granzyme B production compared to control cells.

[0200] (3) Tumor cell survival rate GP130-IL6R-ca7R-introduced CD19-targeted CAR-T cells and CD19-positive tumor cells (K562-CD19 cell line or Raji cell line) were co-cultured overnight in a 1:1 ratio, and the number of viable tumor cells was measured by flow cytometry. Viable tumor cells were identified by fluorescence from the EGFP gene stably introduced into each cell line and by the LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific, L10119), which selectively stains dead cells. The survival rate of tumor cells under each condition was calculated, with the number of viable tumor cells in a well containing only tumor cells set as 100%.

[0201] Figure 12 shows the results of measuring tumor cell survival rates. In both the K562-CD19 cell line and the NALM6 cell line, tumor cell survival rates were significantly lower in GP130-IL6R-ca7R-introduced CD19-targeted CAR-T cells compared to control cells. Therefore, it was shown that GP130-IL6R-ca7R-introduced CD19-targeted CAR-T cells exhibit increased cytotoxic activity against tumor cells.

[0202] <Example 8: Effects of activity-inhibiting mutations Y449F, M452L, and Y456F> (the purpose) In the intracellular domain of the IL-7 receptor α chain, mutations that suppress the activity of each functional motif are known to be Y449F, M452L, and Y456F, as shown in Figure 13A. We will introduce these activity-suppressing mutations into the chimeric cytokine receptor GP130-IL6R-ca7R and investigate their effects.

[0203] (Methods and Results) (1) Measurement of activity of the JAK-STAT pathway and the Akt pathway CAR genes targeting CD19 and GP130-IL6R-ca7R genes were co-introduced into T cells. The GP130-IL6R-ca7R genes used included the GP130-IL6R-ca7R(WT) gene without amino acid substitution mutations, the GP130-IL6R-ca7R(Y449F) gene with the Y449F amino acid substitution mutation, the GP130-IL6R-ca7R(M452L) gene with the M452L amino acid substitution mutation, and the GP130-IL6R-ca7R(Y456F) gene with the Y456F amino acid substitution mutation. After resting chimeric cytokine receptor-introduced CAR-T cells in cytokine-free medium for one day, intracellular proteins were collected, and phosphorylated STAT3 (pSTAT3), phosphorylated STAT5 (pSTAT5), and phosphorylated Akt (pAkt) were detected by Western blotting. Simultaneously, total STAT3, total STAT5, and total Akt, as well as β-actin as an internal control, were detected.

[0204] The results are shown in Figure 13B. In CAR-T cells co-transfected with GP130-IL6R-ca7R(WT), GP130-IL6R-ca7R(Y449F), or GP130-IL6R-ca7R(Y456F), activation of the JAK-STAT and Akt pathways was observed compared to control cells. On the other hand, in CAR-T cells transfected with GP130-IL6R-ca7R(M452L), the Akt pathway was not activated, and only the JAK-STAT pathway was selectively activated.

[0205] (2) Evaluation of the proliferative capacity and undifferentiated traits of CAR-T cells introduced with GP130-IL6R-ca7R(M452L) A CAR gene targeting CD19 as an antigen, along with either the GP130-IL6R-ca7R(WT) gene or the GP130-IL6R-ca7R(M452L) gene, was co-introduced into T cells. Each CAR-T cell was divided into 2.5 × 10⁶ cells. 5 Cells were seeded at a rate of one cell per well in 48-well plates and co-cultured in a 1:1 ratio with the CD19-positive tumor cell line NALM6. Cell counts were counted 7 days after the start of co-culture, and their proliferative capacity was evaluated. Figure 14A shows the multiplicative change in cell count from the start of co-culture. Compared to control cells, CAR-T cells co-transfected with GP130-IL6R-ca7R(WT) and GP130-IL6R-ca7R(M452L) both showed significantly higher proliferative capacity.

[0206] Next, Figure 14B shows the results of flow cytometry analysis of the cell surface expression of CD62L and CCR7 in each CAR-T cell after proliferation. CD62L and CCR7 are known to be highly expressed in undifferentiated memory T cells (Gattinoni, L., et al., Nat Med., 2011, 17(10):1290-1297). In CAR-T cells co-transfected with GP130-IL6R-ca7R(WT), the proportion of CD62L-positive CCR7-positive T cells corresponding to undifferentiated memory T cells was reduced after proliferation compared to control cells, indicating accelerated differentiation (Figure 14B, 47.1% vs. 13.4%). On the other hand, in CAR-T cells co-transferred with GP130-IL6R-ca7R(M452L), differentiation was suppressed compared to GP130-IL6R-ca7R(WT) cells (Figure 14B, 19.0% vs. 13.4%).

[0207] Figure 14C shows the magnification changes from the start of co-culture of CAR-T cells with undifferentiated phenotype. In CAR-T cells co-introduced with GP130-IL6R-ca7R(M452L), the proliferation rate of undifferentiated memory T cells was significantly increased compared to both control cells and CAR-T cells co-introduced with GP130-IL6R-ca7R(M452L).

[0208] These results demonstrate that the introduction of the M452L mutation makes it possible to selectively activate only the JAK-STAT pathway without activating the Akt pathway, thereby achieving high proliferative capacity and long-term survival while inhibiting excessive T cell differentiation.

[0209] <Example 9: Proliferative capacity of chimeric cytokine receptor-transformed mesothelin / GD2-targeted CAR-T cells> (the purpose) We will evaluate the effect of introducing chimeric cytokine receptors on the cell proliferation capacity of CAR-T cells targeting antigens other than CD19, such as mesothelin or GD2.

[0210] (Methods and Results) CAR genes targeting mesothelin or ganglioside GD2 and the GP130-IL6R-ca7R gene were co-introduced into T cells. The mesothelin-targeting CAR gene was synthesized based on the amino acid sequence of antibody clone ss1, referencing the literature (Li, Q., et al., Anticancer Res., 2004, 24(3a):1327-35; Carpenito, C., et al., Proc Natl Acad Sci US A., 2009, 106(9):3360-3365). The CAR gene targeting GD2 was synthesized based on an amino acid sequence that incorporated the E101K mutation to enhance affinity to antibody clone 14g2a, referencing the following literature (Rossig, C., et al., Med Pediatr Oncol., 2000, 35(6):692-5; Horwacikm I., et al., Mol Cell Proteomics., 2015, 14(10):2577-90; Richman, SA, et al., Cancer Immunol Res., 2018, 6(1):36-46.).

[0211] Hereinafter, T cells into which both a CAR gene targeting mesothelin or GD2 and a gene encoding a chimeric cytokine receptor (GP130-IL6R-ca7R gene) have been introduced will be referred to as "chimeric cytokine receptor-introduced mesothelin-targeted CAR-T cells" ("GP130-IL6R-ca7R-introduced mesothelin-targeted CAR-T cells") or "chimeric cytokine receptor-introduced GD2-targeted CAR-T cells" ("GP130-IL6R-ca7R-introduced GD2-targeted CAR-T cells").

[0212] The above CAR-T cells and control cells were cultured in the presence of IL-2, and after removing IL-2 on day 5 of culture, 2.5 × 10⁶ cells were cultured. 5The cells were seeded at a rate of one cell per well in 48-well plates and co-cultured with cells expressing the antigen targeted by each CAR (mesothelin or GD2). GP130-IL6R-ca7R-transformed mesothelin-targeted CAR-T cells were co-cultured in a 4:1 ratio with the K562 cell line (referred to as "K562-mesothelin cell line") in which mesothelin had been stably introduced. The K562-mesothelin cell line was prepared by introducing the mesothelin gene into the K562 cell line (JCRB cell bank, JCRB0019) using a retroviral vector, and then isolating mesothelin-positive cells by flow cytometry. GP130-IL6R-ca7R-transformed GD2-targeted CAR-T cells were co-cultured in a 1:1 ratio with the NALM6 cell line (referred to as "NALM6-GD2 cell line") in which GD2 had been stably introduced. The NALM6-GD2 cell line was prepared by co-introducing the aforementioned NALM6 cell line with the B4GALNT1 gene encoding GM2 / GD2 synthase and the ST8SIA1 gene encoding GD3 synthase using retrovirus, and then obtaining clones that stably high-express GD2 on the cell surface. Co-culture was performed under conditions without cytokine addition. Cell proliferation capacity was evaluated by counting the number of GP130-IL6R-ca7R-expressed mesothelin-targeted CAR-T cells and GP130-IL6R-ca7R-expressed GD2-targeted CAR-T cells 7 days after the start of co-culture.

[0213] Figure 15 shows the multiplicative change in cell number from the start of co-culture. GP130-IL6R-ca7R-transformed mesothelin-targeted CAR-T cells and GP130-IL6R-ca7R-transformed GD2-targeted CAR-T cells showed significantly enhanced proliferative capacity compared to control cells.

[0214] <Example 10: Cytokine production capacity of chimeric cytokine receptor-modified mesothelin / GD2-targeted CAR-T cells> (the purpose) We will evaluate the cytokine production capacity of chimeric cytokine receptor-transformed mesothelin or GD2-targeted CAR-T cells.

[0215] (Methods and Results) GP130-IL6R-ca7R-expressed mesothelin-targeted CAR-T cells were co-cultured with the K562-mesothelin cell line in a 2:1 ratio. Brefeldin A was administered 2 hours after the start of co-culture. Four hours later, T cells were stained with anti-IFN-γ antibody and anti-TNF-α antibody. IFN-γ-producing cells, TNF-α-producing cells, and IFN-γ / TNF-α-producing cells in the CD8-positive T cell fraction were analyzed by flow cytometry.

[0216] The results are shown in Figure 16. Compared to control cells, GP130-IL6R-ca7R-transformed mesothelin-targeted CAR-T cells showed an increased proportion of IFN-γ-producing cells (Figure 16B), TNF-α-producing cells (Figure 16C), and IFN-γ / TNF-α-producing cells (Figure 16D).

[0217] Next, GP130-IL6R-ca7R-introduced GD2-targeted CAR-T cells were co-cultured with the NALM6-GD2 cell line in a 1:1 ratio. Cytokine production capacity in CD8-positive T cells was evaluated using the same method as above.

[0218] The results are shown in Figure 17. In GP130-IL6R-ca7R-introduced GD2-targeted CAR-T cells, the proportion of IFN-γ-producing cells (Figure 17A), TNF-α-producing cells (Figure 17B), and IFN-γ / TNF-α-producing cells (Figure 17C) increased compared to control cells.

[0219] <Example 11: Production and functional analysis of the chimeric cytokine receptor GP130-IL6R-CD28 (T195P)> (the purpose) We will create a chimeric cytokine receptor GP130-IL6R-CD28(T195P) based on the CD28 receptor with a constitutively active mutation and evaluate its function.

[0220] (Methods and Results) (1) Creation of the chimeric cytokine receptor GP130-IL6R-CD28 (T195P) GP130-IL6R-CD28(T195P) was constructed as an artificial receptor by sequentially linking a signal peptide, a ligand-binding domain derived from gp130, a linker peptide, a ligand-binding domain derived from IL6RA, and a T cell activation domain from the N-terminus (Figure 18B). The following describes each of its components.

[0221] The signal peptide, ligand-binding domain derived from gp130, linker peptide, and ligand-binding domain derived from IL6RA of GP130-IL6R-ca7R have the same configuration as those of the aforementioned GP130-IL6R-ca7R, with only the configuration of the T cell activation domain differing between the two (Figures 2B and 18B).

[0222] The T cell activation region of GP130-IL6R-CD28(T195P) consists of the portion from the hinge region to the intracellular domain of the CD28 receptor containing the constitutively activating T195P mutation (known to increase the proliferative capacity of T cells; Yoo HY, et al. Nat Genet, 2014, 46(4) 371-5; Lee SH, et al., Haematologica, 2015, 100(12): e505-e507). Specifically, the T cell activation region consists of positions 114-220 of the constitutively activating CD28 receptor in the full-length human CD28 receptor shown in Sequence ID No. 35, in which the Thr residue at position 195 is replaced with a Pro residue.

[0223] The full amino acid sequence of GP130-IL6R-CD28(T195P) is shown in Sequence ID No. 36. The nucleotide sequence of the gene encoding GP130-IL6R-CD28(T195P) (hereinafter referred to as the "GP130-IL6R-CD28(T195P) gene") is shown in Sequence ID No. 18.

[0224] (2) Functional analysis of the chimeric cytokine receptor GP130-IL6R-CD28 (T195P) The above GP130-IL6R-CD28(T195P) gene, IL-6 receptor α-chain (IL6RA) gene, IL6R-ca7R gene, or GP130-IL6R-ca7R gene was introduced into T cells together with the FMC63-28z CAR gene (hereinafter, they are respectively denoted as "GP130-IL6R-CD28(T195P)-introduced CD19-targeted CAR-T cells", "IL6R-introduced CD19-targeted CAR-T cells", "IL6R-ca7R-introduced CD19-targeted CAR-T cells", and "GP130-IL6R-ca7R-introduced CD19-targeted CAR-T cells"). After co-culturing with the CD19-positive tumor cell line NALM6 in the same manner as in Example 6, the cell number was counted, and their cell proliferation ability was evaluated.

[0225] The fold change in the cell number from the start of co-culture is shown in Fig. 18C. In the IL6R-introduced CD19-targeted CAR-T cells and the GP130-IL6R-CD28(T195P)-introduced CD19-targeted CAR-T cells, no significant difference in cell proliferation ability was detected compared with the control cells.

[0226] From this result, it was found that chimeric cytokine receptors based on the constitutively active CD28 receptor cannot obtain the effect of enhancing T cell proliferation ability. This result also showed that it is important for the T cell activation region of the chimeric cytokine receptor to be based on the constitutively active IL-7 receptor α-chain in order to obtain the advantageous effects of the present invention.

[0227] <Example 12: Preparation and functional analysis of chimeric cytokine receptors CSF2RA-ca7R and CSF2RB-ca7R> (Objective) To prepare chimeric cytokine receptors CSF2RA-ca7R and CSF2RB-ca7R, which contain a part of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (hereinafter denoted as "CSF2RA") or β-chain (hereinafter denoted as "CSF2RB") and a part of IL7RA, and evaluate their functions.

[0228] (Method and result) (1) Creation of chimeric cytokine receptors CSF2RA-ca7R and CSF2RB-ca7R CSF2RA-ca7R and CSF2RB-ca7R were constructed as artificial receptors by linking the ligand-binding region and the T-cell activation region in order from the N-terminus (Figures 19A and 19B). Their individual components are described below.

[0229] The ligand-binding domain of CSF2RA-ca7R consists of the signal peptide and extracellular domain of the GM-CSF receptor α chain (CSF2RA). Specifically, it consists of positions 1 to 320 in the full-length human CSF2RA shown in Sequence ID No. 20. The ligand-binding domain of CSF2RB-ca7R consists of the signal peptide and extracellular domain of the GM-CSF receptor β chain (CSF2RB). Specifically, it consists of positions 1 to 443 in the full-length human CSF2RB shown in Sequence ID No. 22.

[0230] The T cell activation regions of CSF2RA-ca7R and CSF2RB-ca7R consist of the portion from the hinge region to the intracellular domain of IL7RA containing a constitutively activating mutation. More specifically, it consists of an amino acid sequence in which the constitutively activating insertion mutation Pro-Pro-Cys-Leu (SEQ ID NO: 2) is inserted between positions 243 and 244 of the full-length human IL7RA shown in SEQ ID NO: 1, from position 232 to 459.

[0231] The full-length amino acid sequences of CSF2RA-ca7R and CSF2RB-ca7R are shown in Sequence IDs 27 and 28. Additionally, the nucleotide sequences of the genes encoding CSF2RA-ca7R and CSF2RB-ca7R (hereinafter referred to as "CSF2RA-ca7R gene" and "CSF2RB-ca7R gene") are shown in Sequence IDs 32 and 33.

[0232] (2) Functional analysis of chimeric cytokine receptors CSF2RA-ca7R and CSF2RB-ca7R Both the CSF2RA-ca7R and CSF2RB-ca7R genes described above were introduced into T cells along with the FMC63-28z CAR gene (hereinafter referred to as "CSF2RA-ca7R / CSF2RB-ca7R-introduced CAR-T cells"). After co-culturing with the CD19-positive tumor cell line NALM6 using the same method as in Example 6, the number of cells was counted and their cell proliferation ability was evaluated. Figure 20A shows the multiplier change in cell number from the start of co-culturing. Significant cell proliferation was observed compared to control cells into which only the CAR gene was introduced.

[0233] Next, we measured the cells from the 5th day of culture at 2.5 × 10⁶ 5 Cells were seeded in a 48-well plate at a rate of one cell per well, and recombinant GM-CSF (PEPROTECH, 300-03) was added to a final concentration of 3000 pg / mL. 48 hours after GM-CSF addition, the culture supernatant was collected and the GM-CSF concentration in the supernatant was measured by ELISA. In this example, T cells into which only the NGFR-FMC63-28z CAR gene was introduced were used as control cells. The results of measuring the GM-CSF concentration in the culture supernatant are shown in Figure 20B. In CSF2RA-ca7R / CSF2RB-ca7R-introduced CAR-T cells, the GM-CSF concentration in the supernatant decreased to below the detection limit compared to control cells, indicating that the added GM-CSF was captured.

[0234] Similarly, 2.5 × 10⁶ cells cultured on day 5 5 Seeds were sown in a 48-well plate at a rate of 1 per well, 2.5 × 10 5 The CD19-positive leukemia cell line NALM6 was co-cultured with 1.25 × 10¹⁶ cells. After 24 hours, the monocytic leukemia cell line THP1 was cultured with 1.25 × 10¹⁶ cells. 5Cells were added per well, and the culture supernatant was collected after 24 hours. The GM-CSF and IL-6 concentrations in the supernatant were measured by ELISA. The measurement results for GM-CSF and IL-6 concentrations are shown in Figures 20C and 20D, respectively. In CAR-T cells introduced with CSF2RA-ca7R / CSF2RB-ca7R, the GM-CSF concentration in the supernatant was significantly lower compared to control cells, indicating that GM-CSF secreted by the CAR-T cells themselves in response to antigen stimulation by NALM6 was captured. Furthermore, while GM-CSF secretion upon CAR-T cell activation causes the monocyte-derived cell line THP-1 to secrete IL-6, the capture of GM-CSF resulted in suppression of IL-6 secretion.

[0235] <Example 13: Production and functional analysis of the chimeric cytokine receptor IL1R2-ca7R> (the purpose) We will create a chimeric cytokine receptor IL1R2-ca7R containing a portion of the IL-1 receptor type 2 (hereinafter referred to as "IL1R2") and a portion of IL7RA, and evaluate its function.

[0236] (Methods and Results) (1) Creation of the chimeric cytokine receptor IL1R2-ca7R IL1R2-ca7R was constructed as an artificial receptor by linking the ligand-binding region and the T-cell activation region in order from the N-terminus (Figure 19C). The individual components are described below.

[0237] The ligand-binding region consists of the IL1R2 signal peptide and extracellular domain. Specifically, it comprises positions 1 to 343 in the full-length human IL1R2 shown in Sequence ID No. 9.

[0238] The T cell activation region consists of the portion of IL7RA with a constitutively activating mutation from the hinge region to the intracellular domain. Specifically, it consists of the amino acid sequence in which the constitutively activating insertion mutation Pro-Pro-Cys-Leu (SEQ ID NO: 2) is inserted between positions 243 and 244 of the full-length human IL7RA shown in SEQ ID NO: 1, from positions 232 to 459.

[0239] The full-length amino acid sequence of IL1R2-ca7R is shown in SEQ ID NO: 26. Also, the nucleotide sequence of the gene encoding IL1R2-ca7R (hereinafter referred to as the "IL1R2-ca7R gene") is shown in SEQ ID NO: 31.

[0240] (2) Functional analysis of chimeric cytokine receptor IL1R2-ca7R The above IL1R2-ca7R gene was introduced into T cells together with the FMC63-28z CAR gene (hereinafter referred to as "IL1R2-ca7R-introduced CD19-targeted CAR-T cells"). After co-culture with the CD19-positive tumor cell line NALM6 in the same manner as in Example 6, the cell number was counted and its cell proliferation ability was evaluated.

[0241] The fold change in the cell number from the start of co-culture is shown in FIG. 21A. In the control cells transfected with only the CAR gene, the growth rate was 3-fold or less, whereas the growth rate of the IL1R2-ca7R-introduced CD19-targeted CAR-T cells was 10-fold or more.

[0242] Next, the IL-1β capture activity was analyzed by the same method as in Example 4. Recombinant IL-1β (PEPROTECH, 200-01B) was added to the ILR2-ca7R-introduced CD19-targeted CAR-T cells, and the IL-1β concentration in the supernatant was measured.

[0243] The results of measuring the IL-1β concentration in the culture supernatant are shown in FIG. 21B. In the ILR2-ca7R-introduced CD19-targeted CAR-T cells, the IL-1β concentration in the supernatant was significantly lower compared to the control cells transfected with only the CAR gene, and most of the added IL-1β was captured.

[0244] [[ID=

[24] ]<Example 14: IL-6 capture / absorption effect in vivo by chimeric cytokine receptor-introduced CAR-T cells> (Objective) To examine the effect on changes in blood IL-6 concentration by administering human IL-6 via the tail vein to NSG mice administered with GP130-IL6R-ca7R-introduced CAR-T cells.

[0245] (Methods and Results) NALM6 cell line (referred to as "NALM6-GL cell line") stably introduced with EGFP-P2A-Luc2(GL) 2.5 × 10⁻¹⁴ 6 Individual doses were administered via tail vein to NSG mice (Charles River Japan, JAX Mice Stock No: 005557). After 10 days, 3 × 10⁶ control cells with only the CAR gene introduced or GP130-IL6R-ca7R-introduced CAR-T cells were administered. 6 Human IL-6 was administered intravenously to mice individually via tail vein. Seven days later, 2 μg of human IL-6 was administered intravenously, and blood samples were collected 1, 2, and 4 hours later. Plasma IL-6 concentrations were measured by ELISA. The control cells administered to NSG mice, or GP130-IL6R-ca7R-transformed CAR-T cells, were re-stimulated and proliferated in the NALM6-GL cell line on day 7 of culture, and then cultured for 11 days.

[0246] The results are shown in Figure 22. NSG mice administered with GP130-IL6R-ca7R-transformed CAR-T cells showed significantly lower plasma IL-6 concentrations compared to NSG mice administered with control cells at all three time points: 1 hour (Figure 22A), 2 hours (Figure 22B), and 4 hours (Figure 22C) after IL-6 administration. This result indicates that IL-6 is captured / absorbed in vivo upon administration of GP130-IL6R-ca7R-transformed CAR-T cells.

[0247] <Example 15: Capture / absorption effect of IL-6 and IL-1β by CAR-T cells co-transmitted with chimeric cytokine receptor and full-length IL-1 receptor type 2> (the purpose) CAR-T cells co-transmitted with the chimeric cytokine receptor GP130-IL6R-ca7R(M452L) and full-length IL-1 receptor type 2 were treated with recombinant IL-6 or IL-1β produced from the THP1 cell line, and the scavenging ability of IL-6 or IL-1β was evaluated by measuring the concentration of IL-6 or IL-1β in the supernatant.

[0248] (Methods and Results) (1) Capturing ability for recombinant IL-6 and IL-1β The GP130-IL6R-ca7R(M452L) gene was introduced into T cells together with the FMC63-28z-IL1R2 gene (hereinafter referred to as "GP130-IL-6R-ca7R (M452L) + FMC63-28z-IL1R2-introduced CAR-T cells"). FMC63-28z-IL1R2 is a polypeptide containing the full-length human IL-1 receptor type 2, consisting of FMC63-28z CAR, a P2A sequence derived from porcine tesshou virus, and the amino acid sequence shown in Sequence ID No. 9. 2.5 × 10⁶ of the above CAR-T cells cultured on day 5 were used. 5 Cells were seeded in 500 μL of medium supplemented with IL-2 (100 IU / mL) and IL-15 (10 ng / mL) in a 48-well plate at a rate of one cell per well to promote cell proliferation. Recombinant IL-6 or IL-1β was then added to a final concentration of 3000 pg / mL. 48 hours after IL-6 addition, the culture supernatant was collected and the concentration of IL-6 or IL-1β in the supernatant was measured by ELISA. The experimental procedure is shown in Figure 23-1A. In this example, T cells into which only the FMC63-28z CAR gene was introduced were used as control cells. The results of measuring the concentrations of IL-6 or IL-1β in the culture supernatant are shown in Figures 23-1B and 23-1C, respectively. In CAR-T cells introduced with GP130-IL-6R-ca7R (M452L) + FMC63-28z-IL1R2, the IL-6 concentration in the supernatant decreased to below the detection limit (Figure 23-1B), and the IL-1β concentration also decreased significantly compared to control cells (Figure 23-1C).

[0249] (2) Capturing ability of IL-6 and IL-1β produced from THP1 cell lines 2.5 × 10⁶ CAR-T cells (GP130-IL-6R-ca7R (M452L) + FMC63-28z-IL1R2) or control cells (T cells with only the FMC63-28z CAR gene introduced) cultured on day 5. 5 Seeded in 500 μL of medium per well in a 48-well plate (without adding IL-2 and IL-15), 2.5 × 10⁶5 Individual CD19-positive tumor cells were co-cultured with the NALM6 cell line. After 24 hours, 1.25 × 10⁶ cells were cultured. 5 One monocyte cell line, THP-1 (JCRB cell bank, cell number: JCRB0112), was added. After 48 hours, the culture supernatant was collected, and the concentration of IL-6 or IL-1β was measured by ELISA. The experimental procedure is shown in Figure 23-2D. The results of measuring the concentrations of IL-6 or IL-1β in the culture supernatant are shown in Figures 23-2E and 23-2F, respectively. In CAR-T cells transduced with GP130-IL-6R-ca7R (M452L) + FMC63-28z-IL1R2, the IL-6 concentration in the supernatant decreased to below the detection limit (Figure 23-2E), and the IL-1β concentration also decreased significantly compared to control cells, with almost all of it being captured (Figure 23-2F). These results demonstrate that CAR-T cells co-introduced with the chimeric cytokine receptor GP130-IL6R-ca7R(M452L) and the full-length IL-1 receptor type 2 can capture both IL-6 and IL-1β with extremely high efficiency.

[0250] <Example 16: Capture / absorption effect on IL-6 produced by monocyte cells in vivo> (the purpose) NSG mice administered the monocyte cell line THP-1 via tail vein were then given GP130-IL6R-ca7R(M452L)-transformed CAR-T cells, and the blood concentration of IL-6 produced from the THP-1 cell line was evaluated. (method) 3 × 10¹⁶ monocytic cell line THP-1 (hereinafter referred to as "THP1-CD19 / EGFP-Luc2") in which CD19 and EGFP-Luc2 have been stably introduced. 6 Individuals / mice were administered via tail vein to NSG mice. After 24-29 days, 5 × 10⁶ CAR-T cells co-transfected with the GP130-IL6R-ca7R(M452L) gene and the FMC63-28z gene (hereinafter referred to as "GP130-IL6R-ca7R(M452L)-transfected CAR-T cells"), or control cells transfected with only the FMC63-28z gene, were introduced. 6Individual mice were administered via tail vein. Peripheral blood was collected at 1, 5, and 8 days later, and plasma IL-6 concentrations were measured by ELISA (Figure 24A).

[0251] (result) The results are shown in Figure 24B. NSG mice administered with GP130-IL6R-ca7R(M452L)-transformed CAR-T cells showed significantly lower plasma IL-6 concentrations at 1 and 5 days after THP1-CD19 / EGFP-Luc2 administration compared to NSG mice administered with control cells. This result indicates that administration of GP130-IL6R-ca7R(M452L)-transformed CAR-T cells leads to the capture and absorption of IL-6 produced in vivo.

[0252] <Example 17: Evaluation of antitumor effect in an in vivo leukemia model> (the purpose) We will evaluate the antitumor effect in an in vivo leukemia model by administering GP130-IL6R-ca7R(M452L)-transformed CAR-T cells to NSG mice that have been administered the NALM6-GL cell line via tail vein. (method) NALM6 cell line (NALM6-GL cell line) stably introduced with the EGFP-P2A-Luc2(GL) gene: 2.5 × 10⁻¹⁴ 6 Individuals / mice were administered via tail vein to NSG mice. Three days later, 0.5 × 10⁶ CAR-T cells co-transfected with the GP130-IL6R-ca7R(M452L) gene and the FMC63-28z gene (hereinafter referred to as "GP130-IL6R-ca7R(M452L)-transfected CAR-T cells"), or control cells transfected with only the FMC63-28z gene, were introduced. 6Cells were administered via tail vein at a dose per mouse (Figure 25-1A). GP130-IL6R-ca7R(M452L)-transformed CAR-T cells or control cells administered to NSG mice were given on day 9 of culture. Peripheral blood was collected 10, 24, and 38 days after CAR-T cell administration, and the percentage of human CD45-positive T cells was measured by flow cytometry. Furthermore, NALM6-GL cells in vivo were detected by luciferase luminescence using the IVIS® Spectrum in vivo imaging system (PerkinElmer) at 0, 10, 24, 38, and 60 days after CAR-T cell administration. The progression-free survival rate of mice up to approximately 60 days after NALM6-GL cell transplantation was analyzed. In the progression-free survival analysis, a weight loss of 20% or more was used as the endpoint.

[0253] (result) Figure 25-1B shows the percentage of human CD45-positive T cells 10, 24, and 38 days after CAR-T cell administration. NSG mice administered with GP130-IL6R-ca7R(M452L)-transformed CAR-T cells showed a significantly higher percentage of human CD45-positive T cells in peripheral blood 10 and 24 days after CAR-T cell administration compared to mice administered with control cells, demonstrating the long-term persistence of GP130-IL6R-ca7R(M452L)-transformed CAR-T cells (Figure 25-1B). Figures 25-2C and 25-2D show the results of measuring NALM6 tumor mass as luciferase luminescence using IVIS Imaging. NSG mice administered with GP130-IL6R-ca7R(M452L)-expressed CAR-T cells showed significantly lower NALM6 tumor mass compared to mice administered with control cells at 10, 24, and 38 days after CAR-T cell administration (Figure 25-2D). Furthermore, mice administered with GP130-IL6R-ca7R(M452L)-expressed CAR-T cells showed a significantly increased progression-free survival compared to mice administered with control cells (Figure 25-3E). These results clearly demonstrate that GP130-IL6R-ca7R(M452L)-expressed CAR-T cells exhibit enhanced antitumor effects over a long period.

[0254] <Example 18: Evaluation of antitumor effect in an in vivo solid tumor model> (the purpose) We will evaluate the antitumor effect in an in vivo solid tumor model by administering GP130-IL6R-ca7R(M452L)-transformed CAR-T cells to NSG mice subcutaneously transplanted with the mesothelin-positive pancreatic cancer cell line AsPC-1 as a solid tumor model. Unlike Example 17, in this example, CAR-T cells, rather than tumor cells, are labeled with luciferase. (method) 1.5 × 10 6 Individual mesothelin-positive pancreatic cancer cell lines AsPC-1 (ATCC CRL-1682) were mixed with Matrigel to promote engraftment and then subcutaneously transplanted into NSG mice. Thirteen days after AsPC-1 transplantation, 0.5 × 10⁶ second-generation CAR-T cells (hereinafter referred to as "GP130-IL6R-ca7R(M452L)-transformed second-generation CAR-T cells") co-transformed with the GP130-IL6R-ca7R(M452L) gene and the second-generation CAR gene Luc2-ss1-28z, or control cells transfected with only the Luc2-ss1-28z gene, were transplanted. 6The drug was administered via tail vein at a dose per mouse (Figure 26-1A). Note that GP130-IL6R-ca7R(M452L)-transformed second-generation CAR-T cells or control cells administered to NSG mice were given on day 10 of culture. Luc2-ss1-28z stands for Luc2-P2A-tNGFR-Furin-SGSG-P2A-ss1 28z, and is a polypeptide containing, in order from the N-terminus, a luciferase gene, a P2A sequence, a truncated NGFR, a Furin protease cleavage sequence (RAKR), a flexible linker (SGSG), a P2A sequence, and the ss1 28z CAR. Here, the ss1 28z CAR is derived from the anti-mesothelin antibody clone ss1 and is a second-generation CAR gene possessing a CD28 signaling domain for mesothelin. The Luc2 contained in Luc2-ss1-28z allows for the time-series observation of CAR-T cell localization from outside the body based on luciferase luminescence. Peripheral blood was collected 14, 21, 28, and 35 days after CAR-T cell administration, and the percentage of human CD45-positive T cells was measured by flow cytometry. In addition, CAR-T cells infiltrating subcutaneous tumors were detected by luciferase luminescence using the IVIS® Imaging System (PerkinElmer) 14, 21, 28, and 35 days after CAR-T cell administration. Furthermore, the progression-free survival rate of mice up to approximately 80 days after AsPC-1 cell transplantation was analyzed. In the progression-free survival rate analysis, the tumor volume was continuously 200 mm². 3 The first point in time when the above conditions were met was designated as the endpoint.

[0255] (result) Figure 26-1B shows the percentage of human CD45-positive T cells 14, 21, 28, and 38 days after CAR-T cell administration. In NSG mice administered with GP130-IL6R-ca7R(M452L)-transformed second-generation CAR-T cells, the percentage of human CD45-positive T cells in peripheral blood 14, 21, and 28 days after CAR-T cell administration was significantly higher compared to mice administered with control cells, demonstrating the long-term persistence of GP130-IL6R-ca7R(M452L)-transformed second-generation CAR-T cells (Figure 26-1B). Figures 26-2C and 26-2D show the results of measuring the luciferase luminescence of CAR-T cells that infiltrated subcutaneous tumors using IVIS Imaging. In NSG mice administered with GP130-IL6R-ca7R(M452L)-transformed second-generation CAR-T cells, tumor infiltration of CAR-T cells was significantly enhanced compared to control cell administration at 14, 21, and 28 days after CAR-T cell administration (Figure 26-2D), and tumor volume was significantly reduced (Figure 26-3E). Furthermore, mice administered with GP130-IL6R-ca7R(M452L)-transformed second-generation CAR-T cells showed a significantly increased progression-free survival compared to mice administered with control cells (Figure 26-3F). These results revealed that second-generation CAR-T cells introduced with GP130-IL6R-ca7R(M452L) exhibit enhanced antitumor effects against solid tumors over the long term. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. It is a chimeric cytokine receptor, It includes a ligand-binding region at the N-terminus and a T-cell activation region at the C-terminus. The ligand-binding region consists of the cytokine-binding region of a cytokine receptor selected from the group consisting of IL-6 (interleukin-6) receptor, IL-1 (interleukin-1) receptor type 2, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α chain, and GM-CSF receptor β chain. The T cell activation region includes the transmembrane domain and intracellular domain of the IL-7 (interleukin-7) receptor α chain. The aforementioned transmembrane domain is (a) The amino acid sequence shown in SEQ ID NO: 2, between positions 243 and 244 in the amino acid sequence shown in SEQ ID NO: 1, (b) The amino acid sequence shown in SEQ ID NO: 3 between positions 241 and 242 in the amino acid sequence shown in SEQ ID NO: 1, (c) The amino acid sequence shown in SEQ ID NO: 4 between positions 244 and 245 in the amino acid sequence shown in SEQ ID NO: 1, (d) The amino acid sequence shown in SEQ ID NO: 5 between positions 244 and 245 in the amino acid sequence shown in SEQ ID NO: 1, and (e) The amino acid sequence shown in SEQ ID NO: 6 between positions 246 and 247 in the amino acid sequence shown in SEQ ID NO: 1 The chimeric cytokine receptor, wherein one selected from the group consisting of the following is inserted.

2. The chimeric cytokine receptor according to claim 1, wherein the cytokine receptor is an IL-6 receptor and further comprises a ligand-binding region for gp130 (glycoprotein 130) at its N-terminal side.

3. The chimeric cytokine receptor according to claim 1 or 2, wherein the intracellular domain has a mutation selected from the group consisting of Y449F, M452L, and Y456F in the amino acid sequence shown in SEQ ID NO:

1.

4. A nucleic acid encoding a chimeric cytokine receptor according to any one of claims 1 to 3.

5. A gene expression vector comprising the nucleic acid described in claim 4 in an expressible state.

6. A host cell comprising the gene expression vector described in claim 5.

7. The host cell according to claim 6, further comprising a CAR expression vector containing a nucleotide sequence encoding a chimeric antigen receptor (CAR) in an expressible state.

8. The host cell according to claim 6 or 7, further comprising an IL-1 receptor type 2 expression vector containing a nucleotide sequence encoding the full-length IL-1 receptor type 2 in an expressible state.

9. A host cell according to any one of claims 6 to 8, which is an immune cell.

10. The host cell according to claim 9, wherein the immune cell is a T cell, an NK cell, or a macrophage.

11. A cell preparation comprising the host cells described in any one of claims 6 to 10.

12. A method for producing chimeric antigen receptor (CAR)-transformed cells that have long-lasting cytotoxic activity, A process for isolating peripheral blood mononuclear cells from isolated peripheral blood, and A step of introducing the gene expression vector and CAR expression vector described in claim 5 into peripheral blood mononuclear cells isolated in the above step. Includes, The method wherein the CAR expression vector contains a nucleotide sequence encoding a chimeric antigen receptor (CAR) in a state capable of expression.

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