Chimeric cytokine receptors

Abstract

Some of the embodiments of the methods and compositions provided herein relate to chimeric cytokine receptors. In some embodiments, the chimeric cytokine receptor may include an IL-7 domain and / or an IL-21 domain. In some embodiments, the chimeric cytokine receptor may be a hybrid IL-7 / IL-21 cytokine receptor. In some embodiments, the chimeric cytokine receptor can induce combinatorial interleukin signaling when combined with a T cell. In some embodiments, T cells comprising the chimeric cytokine receptor can be readily activated and / or expanded in the absence of exogenous cytokines.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 362,368, entitled "CHIMERIC CYTOKINE RECEPTOR," filed April 1, 2022, the disclosure of which is expressly incorporated by reference in its entirety herein.

[0002] Sequence Listing Reference This application has been filed with a sequence listing in electronic format. The sequence listing is: It was provided as a file of approximately 46,728 bytes with the file name SCRI364WOSEQLISTXML, created on March 23, 2023. The information set forth in this electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] Some of the embodiments of the methods and compositions provided herein relate to chimeric cytokine receptors. In some embodiments, the chimeric cytokine receptor may include an IL-7 domain and / or an IL-21 domain. In some embodiments, the chimeric cytokine receptor may be a hybrid IL-7 / IL-21 cytokine receptor. In some embodiments, the chimeric cytokine receptor can induce combinatorial interleukin signaling when combined with a T cell. In some embodiments, T cells comprising the chimeric cytokine receptor can be readily activated and / or expanded in the absence of exogenous cytokines. [Background technology]

[0004] In cell-based adoptive immunotherapy, T cells isolated from a patient are modified to express synthetic proteins and the cells are then reintroduced into the patient to exert a new therapeutic function. Examples of such synthetic proteins include chimeric antigen receptors (CARs) and recombinant T cell receptors (TCRs). One example of a currently used CAR is a fusion protein consisting of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen binding to the CAR, the intracellular signaling portion of the CAR initiates an activation-associated response (e.g., release of cytolytic molecules) in immune cells, inducing tumor cell death.

[0005] Preclinical models have shown that CAR T cell therapy can be improved by supplementation with cytokines that bind to the gamma chain, soluble factors that promote T cell proliferation and survival. However, systemic administration of cytokines to patients has been shown in clinical trials to result in toxic side effects, making systemic administration of cytokines not a viable therapeutic solution. Summary of the Invention [Means for solving the problem]

[0006] Disclosed herein are chimeric cytokine receptors. In some embodiments, the chimeric cytokine receptors of the present disclosure comprise: (i) the extracellular domain; (ii) the transmembrane domain of the interleukin-7 receptor (IL-7R); and (iii) the intracellular domain of IL-7R Including, The extracellular domain may comprise a cell surface selectable polypeptide, and the cell surface selectable polypeptide may be selected from the extracellular domain of HER2, the extracellular domain of EGFR, and the extracellular domain of CD19. In some embodiments, the transmembrane domain of the IL-7R is modified to promote dimerization and / or constitutive signaling of the chimeric receptor. In some embodiments, the chimeric cytokine receptor of the present disclosure further comprises a signal sequence of GMCSF. In some embodiments, the signal sequence of GMCSF has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:7, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:7. In some embodiments, (i) the extracellular domain comprises the extracellular domain of HER2, which may comprise or consist of a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:8, e.g., may comprise or consist of a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:8; (ii) the extracellular domain comprises the extracellular domain of CD19, which extracellular domain of CD19 comprises or optionally comprises a truncated CD19 polypeptide (CD19t) consisting of a polypeptide having an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:28; or (iii) the extracellular domain comprises the extracellular domain of EGFR, which may comprise a truncated EGFR polypeptide (EGFRt). In some embodiments, the HER2 extracellular domain lacks domain I, domain II, and domain III of the HER2 extracellular protein. In some embodiments, the IL-7R transmembrane domain has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:9, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the IL-7R transmembrane domain is modified to promote dimerization of the chimeric receptor and comprises the amino acid sequence TCP. In some embodiments, the intracellular domain of the IL-7R has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 10, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the chimeric cytokine receptor of the present disclosure further comprises an interleukin-21 receptor (IL-21R) polypeptide attached to the C-terminus of the IL-7R domain via a linker. In some embodiments, the linker has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 11, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the linker is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the length of the linker is 3 amino acids to 30 amino acids.In some embodiments, the IL-21R peptide has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the intracellular domain of IL-7R is modified to remove a STAT5 signaling motif. In some embodiments, the intracellular domain of IL-7R has at least 80% identity to the amino acid sequence of SEQ ID NO: 13, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the intracellular domain of IL-7R comprises a Y449F mutation. In some embodiments, a chimeric cytokine receptor of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, e.g., an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0007] Also disclosed herein are polynucleotides. In some embodiments, the polynucleotides of the present disclosure encode a chimeric cytokine receptor according to any of the embodiments disclosed herein.

[0008] Also disclosed herein is a polynucleotide encoding a chimeric cytokine receptor according to any of the embodiments of the present disclosure, the polynucleotide comprising a nucleotide sequence having at least 80% sequence identity with SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, for example, comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. In some embodiments, the polynucleotide of the present disclosure further comprises a promoter. In some embodiments, the promoter is a constitutive promoter, for example, an EF1α promoter or an MND promoter (e.g., SEQ ID NO:21). In some embodiments, the polynucleotide of the present disclosure further comprises a transcription terminator. In some embodiments, the transcription terminator is an RBG polyA sequence (e.g., SEQ ID NO:22). In some embodiments, the polynucleotide of the present disclosure further comprises a translation factor. In some embodiments, the translation factor is EF1α. In some embodiments, the polynucleotide of the present disclosure further comprises a nucleic acid encoding a selection marker, for example, a truncated EGFR (EGFRt) polypeptide or a truncated CD19 (CD19t) polypeptide (e.g., SEQ ID NO: 30). In some embodiments, the polynucleotide of the present disclosure further comprises a selection factor and / or a resistance cassette. In some embodiments, the selection factor and / or the resistance cassette encodes a double mutant dihydrofolate reductase (DHFRdm) polypeptide. In some embodiments, the polynucleotide of the present disclosure further comprises a coding sequence for a ribosomal skipping sequence and / or a self-cleaving peptide. In some embodiments, the ribosomal skipping sequence comprises a T2A skipping sequence, and / or the self-cleaving peptide comprises a P2A self-cleaving peptide sequence.In some embodiments, the polynucleotides of the disclosure further comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), such as, for example, an anti-CD19 CAR. In some embodiments, the polynucleotides of the disclosure encode a chimeric receptor optimized for expression in human cells.

[0009] Also disclosed herein is a vector. In some embodiments, the vector of the present disclosure comprises a polynucleotide according to any of the embodiments disclosed herein. In some embodiments, the vector of the present disclosure is a viral vector. In some embodiments, the vector of the present disclosure is selected from a lentiviral vector, an adeno-associated viral vector, and an adenoviral vector. In some embodiments, the vector of the present disclosure is a lentiviral vector.

[0010] Also disclosed herein is a cell. In some embodiments, the cell of the present disclosure comprises one or more of the polypeptides or polynucleotides and / or vectors disclosed herein. In some embodiments, the cell of the present disclosure further comprises a polynucleotide encoding a chimeric antigen receptor (CAR). In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a cell derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo cell.

[0011] Also disclosed herein is a method of treating, inhibiting or alleviating a disease or disorder in a subject. In some embodiments, the method comprises administering to a subject in need of treating, inhibiting or alleviating a disease or disorder a cell according to any of the embodiments disclosed herein. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is inflammation. In some embodiments, the disease or disorder is cancer. In some embodiments, the treating or alleviating the disease or disorder does not comprise co-administering a cytokine to the subject. In some embodiments, the method further comprises co-administering to the subject a cytokine, the dose of which is reduced compared to the dose of the cytokine co-administered to a subject administered a CAR-containing cell that does not comprise a polypeptide according to any of the embodiments of the present disclosure disclosed herein, a polynucleotide according to any of the embodiments of the present disclosure disclosed herein, and / or a vector according to any of the embodiments of the present disclosure disclosed herein. In some embodiments, the cytokine is selected from IL-7 and IL-21. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-21. In some embodiments, the cell is an autologous cell obtained from the subject. In some embodiments, the cancer comprises a solid tumor, such as colorectal cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, renal cancer, pancreatic cancer, brain cancer, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, liver cancer; or a non-solid tumor, such as leukemia, multiple myeloma. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0012] Also disclosed herein are methods for preparing a cell population comprising a chimeric antigen receptor (CAR). In some embodiments, the methods include: (a) transducing a T cell with a polynucleotide according to any of the embodiments of the present disclosure; (b) transducing the T cells with a polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells in a culture medium that does not contain exogenous cytokines under conditions that stimulate activation and proliferation of the T cells. Includes. In some embodiments, step (b) is performed before step (a). In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, the cytokine is selected from IL-7 and IL-21. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-21. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a cell derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo cell.

[0013] Also disclosed herein are methods for preparing a cell population comprising a chimeric antigen receptor (CAR). In some embodiments, the methods include: (a) transducing a T cell with a polynucleotide according to any of the embodiments of the present disclosure; (b) transducing the T cells with a polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells under conditions sufficient to stimulate activation and proliferation of the T cells. Including, The medium used for the culture contains a reduced amount of exogenous cytokines compared to the amount sufficient to stimulate the activation and proliferation of T cells that do not contain a polynucleotide encoding the chimeric receptor. In some embodiments, step (b) is performed before step (a). In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, the cytokine is selected from IL-7 and IL-21. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-21. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a cell derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo cell.

[0014] Further provided herein is a method for culturing cells, comprising: (a) obtaining a cell comprising a polynucleotide according to any of the embodiments of the present disclosure; and (b) culturing the cells in a medium that does not contain exogenous cytokines or that contains reduced amounts of exogenous cytokines. Including, The method disclosed herein is characterized in that the amount of exogenous cytokine is reduced compared to the amount of exogenous cytokine in a medium used to culture cells not containing the polypeptide, and the culture results in a population of cells sufficient in number for infusion. In some embodiments, the amount of the cytokine in the medium is sufficient to activate and / or proliferate the cells. In some embodiments, the cells comprise a chimeric antigen receptor (CAR). In some embodiments, the cytokine is selected from IL-7 and IL-21. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-21. In some embodiments, the cells are T cells. In some embodiments, the cells are CD8+ T cells or CD4+ T cells. In some embodiments, the cells are cells derived from CD8+ T cells or CD4+ T cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are ex vivo cells. [Brief description of the drawings]

[0015] [Figure 1] The left panel shows a schematic diagram of a HER polypeptide with its extracellular portion shown in a dotted box; a constitutively active IL7R mutant with a transmembrane domain containing a mutation and an intracellular STAT5 signaling domain (each domain shown in a dotted box); and an IL21 signaling complex with the STAT3 signaling domain shown in a dotted box. The right panel shows a schematic diagram of an example of a chimeric receptor (CIndR) that contains the extracellular domain of HER2, the transmembrane domain of IL7R containing a mutation, the intracellular STAT5 signaling domain of IL7R, and the signaling domain of the IL21 signaling complex linked via a linker to the other portions of the chimeric receptor.

[0016] [Diagram 2]The top panel shows a schematic diagram showing various chimeric receptors and a control marker-only receptor. The bottom panel shows a polynucleotide encoding the CIndR linked to the MND promoter; the EF1α promoter linked to a polynucleotide encoding the anti-CD19 CAR, a T2A sequence, a selection marker (DHFRdm), a P2A sequence, and a cell surface marker (EGFRt) in a vector.

[0017] [Figure 3A] Schematic diagram of ClndR7 chimeric receptor. ClndR7 contains the extracellular domain of a truncated HER2 polypeptide (Her2tG) (1), the transmembrane domain of IL7R containing an insertion (2), and the intracellular domain of IL7R (3).

[0018] [Figure 3B] Another schematic diagram of the ClndR7 chimeric receptor containing the Y449 residue in the intracellular domain of IL7R (3) is shown.

[0019] [Figure 3C] A schematic diagram of the ClndR7 / 21 chimeric receptor. ClndR7 / 21 contains a flexible linker (4) and the IL21R peptide (5).

[0020] [Figure 3D] A schematic diagram of the ClndR7 / 21 chimeric receptor containing the Y449 residue in the intracellular domain of IL7R is shown.

[0021] [Figure 3E] A schematic of the ClndR21 chimeric receptor with the F449 mutation is shown. ClndR21 was generated by removing the STAT5 signaling motif from the cytoplasmic domain of the IL7 receptor (6).

[0022] [Figure 4] FIG. 1 shows a non-limiting example of a schematic of a polynucleotide construct encoding ClndR7 and a truncated CD19 polypeptide (CD19t) with a T2A self-cleaving peptide sequence in between.

[0023] [Diagram 5] FACS analysis of ClndR7-expressing T cells expressing the polynucleotide constructs of Figure 4. The analysis showed a correlation between co-expressed CD19t and Her2tG, indicating that Her2tG can be used as a cell surface marker for ClndR7 expression.

[0024] [Figure 6] FACS analysis of phosphorylated STAT3 (pSTAT3) abundance in untreated CD8+ T cells (dark grey peak) or CD8+ T cells treated with IL21 (light grey peak) is shown.

[0025] [Figure 7A] FACS analysis of phosphorylated STAT5 (pSTAT5) abundance in unmodified CD8+ T cells (dark grey peak) or CindR-expressing CD8+ T cells expressing ClndR7 (light grey peak) is shown.

[0026] [Figure 7B] FACS analysis of phosphorylated STAT3 (pSTAT3) abundance in unmodified CD8+ T cells (dark grey peak) or CindR-expressing CD8+ T cells expressing ClndR7 (light grey peak) is shown.

[0027] [Figure 7C] FACS analysis of phosphorylated STAT5 (pSTAT5) abundance in unmodified CD8+ T cells (dark grey peak) or CindR-expressing CD8+ T cells expressing ClndR7 / 21 (light grey peak) is shown.

[0028] [Figure 7D] FACS analysis of phosphorylated STAT3 (pSTAT3) abundance in unmodified CD8+ T cells (dark grey peak) or CindR-expressing CD8+ T cells expressing ClndR7 / 21 (light grey peak) is shown.

[0029] [Figure 8A] Bar graph analysis of median fluorescence intensity (MFI) of intracellular flow cytometric quantification of phosphorylated STAT3 (pSTAT3) in cells (n=3 donors). T cells were engineered to express a non-active control protein (Her2tG) or one of the three ClndRs. Control cells were pre-treated with IL-7, IL-21, or no treatment. Cells were gated on lymphocytes, singlets, or dead cell exclusion staining. Significant values ​​for markers were determined by one-way ANOVA. "*" indicates p<0.05, "**" indicates p<0.01, "***" indicates p<0.001, and "****" indicates p<0.0001.

[0030] [Figure 8B] Bar graph analysis of median fluorescence intensity (MFI) of intracellular flow cytometric quantification of phosphorylated STAT5 (pSTAT5) in cells (n=3 donors). T cells were engineered to express a non-active control protein (Her2tG) or one of the three ClndRs. Control cells were pre-treated with IL-7, IL-21, or no treatment. Cells were gated on lymphocytes, singlets, or dead cell exclusion staining. Significant values ​​for markers were determined by one-way ANOVA. "*" indicates p<0.05, "**" indicates p<0.01, "***" indicates p<0.001, and "****" indicates p<0.0001.

[0031] [Figure 9] A schematic diagram of the ClndR7 / 21 chimeric receptor with a flexible linker moiety (7) is shown. For this flexible linker moiety, various types of linker candidates were tested, and the linker candidates used were GGG (SEQ ID NO: 23), GGGGS (SEQ ID NO: 24), (GGGGS)2 (SEQ ID NO: 25), or (GGGGS)3 (SEQ ID NO: 26).

[0032] [Figure 10A] Bar graph analysis of median fluorescence intensity (MFI) of phosphorylated STAT5 (pSTAT5) quantified by intracellular flow cytometry in cells. Relative MFI values ​​were calculated relative to the MFI values ​​of mock cells. Measurements are shown for mock cells, mock cells treated with IL-21, ClndR7 cells, or ClndrR7 / 21 cells incorporating GGG, GGGGS, (GGGGS)2, or (GGGGS)3 as linker candidates.

[0033] [Figure 10B] Bar graph analysis of median fluorescence intensity (MFI) of phosphorylated STAT3 (pSTAT3) quantified by intracellular flow cytometry in cells. Relative MFI values ​​were calculated relative to the MFI values ​​of mock cells. Measurements are shown for mock cells, mock cells treated with IL-21, ClndR7 cells, or ClndrR7 / 21 cells incorporating GGG, GGGGS, (GGGGS)2, or (GGGGS)3 as linker candidates.

[0034] [Figure 11A] A schematic diagram of an example of a construct for introducing Her2tG into T cells is shown. This construct contains a Her2tG sequence, an MND sequence, an EF1α sequence, a CD19 CAR sequence, a T2A sequence, a DHFRdm sequence, a P2A sequence, and an EGFRt sequence in a PBNP construct.

[0035] [Figure 11B] A schematic diagram of an example of a construct for introducing ClndR7 into T cells is shown, which contains ClndR7, MND, EF1α, CD19 CAR, T2A, DHFRdm, P2A and EGFRt sequences in a PBNP construct.

[0036] [Figure 11C]A schematic diagram of an example of a construct for introducing ClndR7 / 21 into T cells is shown. This construct contains ClndR7 / 21 sequence, MND sequence, EF1α sequence, CD19 CAR sequence, T2A sequence, DHFRdm sequence, P2A sequence and EGFRt sequence in a PBNP construct.

[0037] [Figure 11D] A schematic diagram of an example of a construct for introducing ClndR21 into T cells is shown, which contains ClndR21, MND, EF1α, CD19 CAR, T2A, DHFRdm, P2A and EGFRt sequences within a PBNP construct.

[0038] [Figure 12A] Schematic of the assay in which effector anti-CD19 CAR T cells containing various chimeric receptors were co-cultured with target Raji cells (target cells). Raji cells were added to the co-culture on days 3, 6, 9 and 12. Tumor cell abundance was monitored throughout the experimental period.

[0039] [Figure 12B] A graph showing the amount of red fluorescent signal in T cell populations from donor A. CAR T cells were repeatedly exposed to Raji tumor cells in vitro every 3 days (black arrows). Raji cells were previously engineered to express the red fluorescent protein mCherry, and tumor cell abundance was monitored over a 3-week period using an Incucyte live cell fluorescence imager.

[0040] [Figure 12C] A graph showing the amount of red fluorescent signal in T cell populations from donor B. CAR T cells were repeatedly exposed to Raji tumor cells in vitro every 3 days (black arrows). Raji cells were previously engineered to express the red fluorescent protein mCherry, and tumor cell abundance was monitored over a 3-week period using an Incucyte live cell fluorescence imager.

[0041] [Figure 12D] A graph showing the amount of red fluorescent signal in T cell populations from donor C. CAR T cells were repeatedly exposed to Raji tumor cells in vitro every 3 days (black arrows). Raji cells were previously engineered to express the red fluorescent protein mCherry, and tumor cell abundance was monitored over a 3-week period using an Incucyte live cell fluorescence imager.

[0042] [Figure 12E] Graph showing time course of tumor signal in co-cultures of anti-CD19 CAR T cells with various chimeric receptors and Raji cells.

[0043] [Figure 12F] Graph showing time course of tumor signal in co-cultures of Raji cells with anti-CD19 CAR T cells containing receptor with control marker only treated with IL-2 and / or IL-21.

[0044] [Figure 13A] Schematic of the assay in which effector anti-CD19 CAR T cells containing various chimeric receptors were co-cultured with target Raji cells (target cells). Co-cultures were replated on day 7. T cells were quantified on days 7 and 14.

[0045] [Figure 13B] Graph showing the relative fold expansion of CAR T cells containing various chimeric receptors versus the number of expansions when CAR T cells containing the marker were treated with IL7 and IL21. From top to bottom, the graph shows lines CIndR21, CIndR7 / 21, CIndR21, and the marker alone.

[0046] [Figure 13C]Bar graph showing the mean relative fold proliferation in cells from three donors at day 14, with asterisks indicating statistical significance. Statistical analysis was performed using one-way ANOVA, with p values ​​indicated as p<0.05*, 0.0001****.

[0047] [Figure 14A] Schematic diagram of the assay in which effector anti-CD19 CAR T cells containing various chimeric receptors were co-cultured with target Raji cells (target cells). On day 7, exogenous cytokines were removed from the culture medium. Annexin V analysis was performed on day 10.

[0048] [Figure 14B] Bar graph showing the percentage of cells that are 7-AAD+ (dead cells), 7-AAD-Annexin V- (normal cells), or 7-AAD-Annexin V+ (pre-apoptotic cells) at day 10. Statistical analysis was performed using one-way ANOVA, and p values ​​were expressed as p<0.05*, 0.01**.

[0049] [Figure 14C] Graph showing relative fold change of live CAR T cells containing various chimeric receptors over time.

[0050] [Figure 15A] Schematic of the assay in which effector anti-CD19 CAR T cells containing various chimeric receptors were co-cultured with target Raji cells (target cells). Co-cultures were replated on day 3. Cell surface markers were analyzed by flow cytometry on days 3 and 6.

[0051] [Figure 15B] Graphs showing the percentage of CD45RA+, CD62L+ and CCR7+ CD8+ cells on day 3 (left panel) or day 6 (right panel) are shown.

[0052] [Figure 16]Graph showing the amount of light flux in mice administered CD19+ tumor cells containing a fluorescent marker on day 0. Mice were administered anti-CD19 CAR cells containing various chimeric receptors on day 7. The graph shows, from top to bottom, mock (on day 15); MND_Her2tG_CD19CAR; MND_CIndR7_CD19CAR; MND_CIndR7 / 21-CD19CAR; and MND-CIndR21_CD19CAR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Successful adoptive T cell therapy requires robust proliferation and persistence of administered T cells, a behavior that is highly dependent on the signals that T cells receive from the surrounding environment. Preclinical models have shown that chimeric antigen receptor (CAR) T cell therapy can be improved by supplementation with cytokines (soluble factors that promote T cell proliferation and survival) that bind to the gamma chain. However, systemic administration of such cytokines is not a therapeutically effective solution, as clinical trials have shown that systemic administration of cytokines to patients leads to toxic side effects (Jeught V.et al.(2014)Oncotarget 6:1359-81). In light of this, to obtain the benefits of combining CAR T cell therapy with cytokine supplementation without causing systemic toxicity, various chimeric cytokine receptors were constructed to provide constitutive interleukin signaling endogenous to T cells. These chimeric cytokine receptors recapitulated the signaling events of specific gamma chain cytokines. Moreover, unexpectedly, the activity of CAR T cells containing specific chimeric cytokine receptors was enhanced.

[0054] CAR T cells expressing chimeric cytokine receptors were used in vitro to test the survival, proliferation, and tumor cell elimination capabilities of the T cell populations following a series of tumor challenges. CAR T cells expressing chimeric cytokine receptors were found to exhibit enhanced proliferation and survival in the absence of exogenous cytokines.

[0055] Three signals are required for optimal T cell activation and proliferation: T cell receptor activation, costimulatory cytokines, and stimulatory cytokines. CAR T cells can provide the first two signals by the CAR, but the third signal is dependent on environmental or exogenous cytokines, which may be scarce in the tumor microenvironment. Cytokine supplementation may improve the efficacy of CAR T cell therapy, but systemic administration of cytokines has been shown to cause toxicity in clinical trials. Developing a method to selectively provide this third signal to CAR T cells without or with minimal systemic toxicity would be clinically beneficial.

[0056] Disclosed herein are chimeric cytokine receptors comprising interleukin 7 and / or interleukin 21 domains for combinatorial interleukin signaling. In some embodiments, the cytokine receptor of the disclosure is ClndR7. In some embodiments, the cytokine receptor of the disclosure is ClndR7 / 21. In some embodiments, the cytokine receptor of the disclosure is ClndR21. In some embodiments, the hybrid cytokine receptor of the disclosure features signaling outputs of different types of endogenous receptors.

[0057] As disclosed herein, a cytokine-independent receptor for interleukin-7 and interleukin-21 signaling (CIndR7 / 21) was engineered. CIndR7 / 21 is composed of a fusion of a portion derived from the human interleukin-7 receptor (IL7R) and a portion derived from the human interleukin-21 receptor (IL21R). Briefly, CIndR7 / 21 contains (1) a truncated HER2 polypeptide (Her2tG), the extracellular domain of a cell surface marker protein; (2) the IL7R transmembrane domain with an insertion that allows receptor homodimerization and constitutive signaling; (3) the intracellular domain of IL7R; and (4) an IL21R-derived peptide appended to the C-terminus of IL7R via a flexible linker sequence (Figure 3A).

[0058] Preclinical studies have shown that cytokine supplementation can improve CAR T cell therapy. However, systemic administration of cytokines is not a clinically viable option due to dose-limiting toxicity in patients administered in clinical trials. The ambitious goal of the present invention is to confer the therapeutic benefits of cytokine supplementation of CAR T cells without causing systemic toxicity. Furthermore, the disclosed CIndR7 / 21 provides a novel approach to further optimize signaling to induce CAR T cell proliferation, persistence, and antitumor activity by combining signaling output from two cytokines that may exert complementary effects on T cells. IL7 enhances T cell survival and proliferation activity, whereas IL21 enhances T cell cytotoxicity and exhaustion resistance. It is desirable to enhance the therapeutic promoting effects of both cytokines in CAR T cells with a single transgenic receptor in the form of CIndR7 / 21. Commercial use of CIndR7 / 21 is useful as an adjunct to CAR T cell therapy or other types of adoptive T cell therapy.

[0059] As disclosed herein, CIndR7 / 21 provides the first constitutively active receptor to induce signaling output from IL7 and IL21, which can be used to engineer CAR T cells to induce constitutive signaling from IL7 and IL21 that T cells inherently possess, thereby promoting T cell activity and thus further enhancing the efficacy of CAR T cell therapy. Such effects included enhanced CAR T cell proliferation, persistence, cytotoxicity, and resistance to T cell exhaustion. Preclinical studies in mice also showed that CIndR7 / 21 and CIndR21 enhanced CAR T cell-mediated tumor suppression.

[0060] Definition of Terms The terms used in this disclosure have their common and ordinary meaning according to this specification. Those skilled in the art will understand each term used by reading the entire specification.

[0061] As used herein, the term "comprising" is synonymous with the terms "including," "containing," or "characterized by" and is open-ended and inclusive and does not exclude additional elements or steps not described herein.

[0062] As used herein, "a" or "an" may mean one or more than one.

[0063] As used herein, "about" indicates that the particular numerical value includes the degree of error inherent in the method employed to determine the numerical value or experimental variation.

[0064] As used herein, a "chimeric receptor" includes a synthetically engineered receptor that includes a ligand-binding domain of an antibody or other protein sequence that binds to a molecule associated with a disease or disorder, linked, preferably via a spacer domain, to one or more intracellular signaling domains (e.g., costimulatory domains) derived from a T cell receptor or other receptor. Chimeric receptors may also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, or chimeric antigen receptors (CARs).

[0065] As used herein, a "chimeric cytokine receptor" includes a synthetically engineered receptor that includes a cytokine tethered to an extracellular domain of a cytokine receptor polypeptide, a transmembrane domain, and an intracellular domain of a cytokine receptor linked to the extracellular domain of the cytokine receptor polypeptide via the transmembrane domain. In some embodiments, the cytokine can be selected from cytokines that bind to type I cytokine receptors, such as IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21, etc. In some embodiments, the extracellular domain of the cytokine receptor polypeptide can be derived from a type I cytokine receptor, such as IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor, IL-15 receptor, IL-21 receptor, etc. In some embodiments, the transmembrane domain can be derived from a type I cytokine receptor, such as IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor, IL-15 receptor, IL-21 receptor, etc. In some embodiments, the intracellular domain of the cytokine receptor polypeptide may be derived from a type I cytokine receptor, such as the IL-2 receptor, IL-4 receptor, IL-7 receptor, IL-9 receptor, IL-13 receptor, IL-15 receptor, or IL-21 receptor.

[0066] As used herein, "chimeric antigen receptor (CAR)" may refer to an artificial T cell receptor, also known as a chimeric T cell receptor, which is a genetically engineered receptor, capable of grafting any specificity onto an effector immune cell. Chimeric antigen receptors can be used to graft, for example, the specificity of a monoclonal antibody onto a T cell, and the coding sequence of the monoclonal antibody can be easily transferred, for example, by using a retroviral vector or other suitable gene delivery system. The structure of a CAR may include a single chain variable fragment (scFv) from a monoclonal antibody, which is fused to the transmembrane and intracellular domains of CD3 zeta. These molecules induce zeta signaling in response to the scFv being recognized by its target. In some embodiments, a gene delivery vector with a self-inactivating transposase system is utilized. In some embodiments, the gene delivery vector further comprises a sequence encoding at least one protein. In some embodiments, the protein is a chimeric antigen receptor. Chimeric receptors can also be referred to as artificial T cell receptors, chimeric T cell receptors, chimeric immune receptors, and / or chimeric antigen receptors (CARs). CAR is a genetically engineered receptor that can be grafted with any specificity into cells expressing immune receptors. CAR may include an antibody or antibody fragment, a spacer, a signaling domain and / or a transmembrane domain. However, CAR has been modified in various components or domains (e.g., epitope binding regions (e.g., antibody fragments, scFvs or parts thereof), spacers, transmembrane domains and / or signaling domains, etc.), which has resulted in surprising effects, and in some contexts herein, the components of CAR are described as including characteristics as independent individual components. The various components included in CAR have various variations, which, for example, enhance the binding affinity to a particular epitope.

[0067] Artificial T cell receptors, or CARs, can be used as a treatment for cancer or viral infections using a technique called adoptive cell transfer. T cells are taken from a subject and modified to express a receptor specific for a molecule presented by cancer cells, viruses, or virus-infected cells. The genetically modified T cells can recognize and kill cancer cells or virus-infected cells or promote viral clearance, and the genetically modified T cells thus produced are reintroduced into the subject. In some embodiments, the gene delivery vector may include a sequence encoding a chimeric antigen receptor. In some embodiments, a method is provided for producing recombinant multiplex T cells for adoptive T cell immunotherapy. In the broadest sense, the method may include providing a gene delivery vector according to any of the embodiments described herein, introducing the gene delivery vector into T cells, and selecting the cells containing the gene delivery vector, the selection including isolating the T cells expressing a particular phenotype under selective pressure.

[0068] To generate an effective immune response, it is desirable to costimulate T cells, which occurs when lymphocytes are activated. Costimulatory signals are antigen non-specific and are generated by interaction of T cells with costimulatory molecules expressed on the cell membrane of antigen-bearing cells. Costimulatory molecules include, but are not limited to, CD28, CD80, and CD86. In some embodiments, a method is provided for generating recombinant multiplex T cells for adoptive T cell immunotherapy. In some embodiments, the T cells are T cells bearing a chimeric antigen receptor. In some embodiments, the T cells bearing a chimeric antigen receptor are genetically modified to express a costimulatory ligand. In some embodiments, a method is provided for treating, inhibiting, or alleviating cancer or a viral infection in a subject. In the broadest sense, the method may include administering to the subject T cells according to any of the embodiments described herein. In some such embodiments, the subject is an animal, such as a livestock or pet animal, and in other embodiments, the subject is a human. In some such embodiments, the T cells bearing a chimeric antigen are genetically modified to express a costimulatory molecule. In some embodiments, the gene delivery vector comprises a sequence encoding at least one costimulatory molecule, hi some embodiments, the gene delivery vector is at least 1 kb to 20 kb.

[0069] As used herein, "genetically modifying" and "genetic recombination" may include methods of modifying organisms and cells, such as bacteria, T cells, bacterial cells, eukaryotic cells, insects, plants, and mammals, using genetic material (such as nucleic acids) modified using genetic engineering techniques. For example, nucleic acids such as DNA can be inserted into a host genome by first isolating the genetic material of interest using molecular cloning techniques, replicating it to create a DNA sequence, or synthesizing the DNA, and then inserting such resulting constructs into the host organism. Nucleases can be used to delete or "knock out" genes. Gene targeting is another technique for modifying endogenous genes by homologous recombination and can be used for gene deletion, exon removal, gene addition, or point mutation introduction.

[0070] Herein, genetic modification by transduction is described. "Transduction" refers to the method of transferring genetic material, such as DNA or RNA, into cells using vectors. Common techniques use viral vectors, electroporation, or chemical reagents that increase cell permeability. DNA can be transferred by viruses or viral vectors. Herein, methods of modifying immune cells, CD4+ T cells and / or CD8+ T cells, are described. T cells are transduced with genetic material, for example, encoding a specific protein or chimeric antigen receptor, to achieve high expression of a therapeutic gene and / or to increase the amount of chimeric antigen receptor expressed on the cell surface. T cells can be genetically modified, for example, using viruses. Viruses commonly used in gene therapy are, for example, adenoviruses, adeno-associated viruses (AAV), retroviruses, or lentiviruses.

[0071] A variety of transduction techniques have been developed that utilize recombinant infectious viral particles to deliver nucleic acids encoding chimeric antigen receptors. These transduction techniques are currently the preferred approach for transducing T lymphocytes. As described herein, viral vectors used for transduction can include viral vectors derived from Simian Virus 40, adenovirus, adeno-associated virus (AAV), lentiviral vectors, or retroviruses. Thus, there are a variety of gene transfer and expression methods, the essential function of which is to introduce and express genetic material in mammalian cells. Some of the transduction techniques mentioned above can be used to transduce hematopoietic cells or lymphocytes, including calcium phosphate transfection; protoplast fusion; electroporation; or infection with recombinant adenovirus, adeno-associated virus, retrovirus, lentivirus, or retrovirus vectors. Primary T lymphocytes have been successfully transduced by electroporation or infection with retroviruses or lentiviruses. Thus, retroviral or lentiviral vectors can be used to transfer genes into eukaryotic cells (e.g., T cells) very efficiently. Furthermore, retroviral or lentiviral integration can be controlled, and one or several copies of new genetic information can be stably integrated into each cell. Also, as described herein, cells can be transduced in situ.

[0072] As used herein, a "vector" or "construct" refers to a nucleic acid used to introduce a heterologous nucleic acid into a cell, which may contain various regulatory elements, thereby allowing the expression of the heterologous nucleic acid in the cell. Vectors include, but are not limited to, plasmids, minicircles, yeast, and viral genomes. In some embodiments, the vector is a plasmid, minicircle, viral vector, DNA, or mRNA. In some embodiments, the vector is a lentiviral vector or a retroviral vector. In some embodiments, the vector is a lentiviral vector. As used herein, "Vpx" refers to a virion-associated protein encoded by HIV type 2 and some simian immunodeficiency virus strains. Vpx can enhance the replication of HIV-2 in humans. Lentiviral vectors packaged with Vpx protein can be used for transfection to increase the infection rate of myeloid cells. In some embodiments, the lentiviral vector is packaged with Vpx protein. As used herein, "Vpr" protein may refer to Viral Protein R. Vpr is a 14 kDa protein that plays a key role in controlling the nuclear translocation of HIV-1 preintegration complexes and is required for viral replication in non-dividing cells. Non-dividing cells include, for example, macrophages. In some embodiments, the lentiviral vector can be packaged with the Vpr protein or a portion thereof. In some embodiments, the lentiviral vector is packaged with a viral accessory protein. In some embodiments, the viral accessory protein is selected from the group consisting of Vif, Vpx, Vpu, Nef, and Vpr. Such accessory proteins, such as vif, Vpx, vpu, and nef, function as adapter molecules by interacting with intracellular ligands, allowing the virus to exploit the normal functions of host factors for its own purposes.HIV accessory proteins have been reported by Strebel et al. (HIV Accessory Proteins versus Host Restriction Factors, Curr Opin Virol. 2013 Dec;3(6):10.1016 / j.coviro.2013.08.004; which is expressly incorporated herein by reference in its entirety).

[0073] As used herein, the term "promoter" refers to a nucleotide sequence that induces transcription of a structural gene. In some embodiments, the promoter is located in the non-coding region at the 5' end of the gene, near the transcription start site of the structural gene. Sequence elements within the promoter that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Such promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation specific elements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993); expressly incorporated herein by reference in its entirety), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman et al., Seminars in Cancer Biol. 1:47 (1990); expressly incorporated herein by reference in its entirety), glucocorticoid response elements (GREs), and other transcription factor binding sites, such as CRE / ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP2 (Ye et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP3 (Ye et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP4 (Ye et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP5 (Ye et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP6 (Ye et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP7 (Ye et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP8 (Ye et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP9 (Ye et al., J al., J.Biol.Chem.269:25728(1994); expressly incorporated herein in its entirety by reference), SP1, cAMP response element binding protein (CREB; Loeken et al., Gene Expr.3:253(1993); expressly incorporated herein in its entirety by reference), and octamer factor (for a review, see Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin / Cummings Publishing Company, Inc.1987; expressly incorporated herein in its entirety by reference) and Lemaigre and Rousseau, Biochem.J.303:1(1994) (expressly incorporated herein in its entirety by reference)). In the present specification, the promoter may be a constitutively active promoter, a repressible promoter, or an inducible promoter.If a promoter is an inducible promoter, the rate of transcription increases in response to an inducing agent. In contrast, if a promoter is a constitutive promoter, the rate of transcription is not regulated by an inducing agent. Repressible promoters are also known.

[0074] As used herein, "treat," "treating," "treated," or "treatment" can refer to both therapeutic and prophylactic treatment, depending on the context.

[0075] As used herein, the terms "alleviate", "alleviating", "alleviation" or "alleviated" in relation to a disorder may refer to the alleviation of symptoms of the disorder, the stabilization of the disease, or the prevention of the progression of the disorder. In the case of a disorder such as cancer, these terms may include the reduction in tumor size, the inhibition of the growth or proliferation of cancer cells or the reduction in the number of cancer cells, the complete or partial elimination of the tumor (e.g., complete or partial response), the stabilization of the disease, the prevention of the progression of the cancer (e.g., progression-free survival), or other effects on the cancer that are considered by the physician to be therapeutic effects.

[0076] As used herein, "administer," "administering," or "administered" may refer to a method for introducing a compound or a pharma- ceutically acceptable salt thereof, or a recombinant cell composition into a patient or subject, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, or transdermal administration.

[0077] As used herein, a "subject" or "patient" may refer to any organism to which an embodiment described herein may be used or administered, for example, for experimental, diagnostic, prophylactic and / or therapeutic purposes. A subject or patient may include, for example, an animal. In some embodiments, the subject is a mouse, a rat, a rabbit, a non-human primate, or a human. In some embodiments, the subject is a cow, a sheep, a pig, a horse, a dog, a cat, a primate, or a human.

[0078] As used herein, "co-administration" may refer to the administration of two or more therapeutic agents in combination with another therapeutic agent. Each therapeutic agent may be administered sequentially or simultaneously such that they are present in the bloodstream of an organism at the same time.

[0079] Certain cytokine receptors Some embodiments provided herein include a chimeric cytokine receptor polypeptide. In some embodiments, the chimeric cytokine receptor of the present disclosure comprises: (i) an extracellular domain, e.g., a polypeptide capable of selecting the cell surface, e.g., the extracellular domain of HER2, the extracellular domain of EGFR, or the extracellular domain of CD19; (ii) the transmembrane domain of the interleukin-7 receptor (IL-7R); and (iii) the intracellular domain of IL-7R Includes. In some embodiments, the extracellular domain is a domain selected to allow for easy identification and / or selection of cells expressing this extracellular domain.

[0080] In some embodiments, the transmembrane domain of the IL-7R is modified to promote dimerization and / or constitutive signaling of the chimeric receptor. In some embodiments, the chimeric cytokine receptor of the present disclosure further comprises a signal sequence of GMCSF. In some embodiments, the signal sequence of GMCSF has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:7, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:7. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is a percentage within a range between any two of the above percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0081] In some embodiments, the extracellular domain comprises the extracellular domain of HER2. In some embodiments, the extracellular domain comprises the extracellular domain of CD19, including a truncated CD19 polypeptide (CD19t). In some embodiments, the CD19t comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the extracellular domain comprises the extracellular domain of EGFR, for example, a truncated EGFR polypeptide (EGFRt).

[0082] In some embodiments, the extracellular domain of HER2 comprises a polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:8, for example, a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:8. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or is 100%, or is a percentage within a range of any two of the percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0083] In some embodiments, the HER2 extracellular domain lacks domain I, domain II, and domain III of the HER2 extracellular protein. In some embodiments, the IL-7R transmembrane domain has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 9, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9. In some of such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is within a range of any two of the above percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0084] In some embodiments, the transmembrane domain of the IL-7R is modified to promote dimerization of the chimeric receptor and comprises the amino acid sequence TCP. In some embodiments, the transmembrane domain of the IL-7R has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 10, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10. In some of such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is within a range of any two of the above percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0085] In some embodiments, a chimeric cytokine receptor of the present disclosure further comprises an interleukin 21 receptor (IL-21R) polypeptide attached to the C-terminus of the IL-7R domain via a linker. In some embodiments, the linker has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:11, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:11. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, 100%, or within any two of the above percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%. In some embodiments, the linker is selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. In some such embodiments, the linker is selected from a sequence having greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, 100%, or within any two of the above percentages. In some such embodiments, the identity is greater than about 95% identity to any of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. In some such embodiments, the identity is greater than 95% identity to any of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. In some embodiments, the linker is about 3 amino acids to about 30 amino acids in length. In some embodiments, the linker is about 3 amino acids in length. In some embodiments, the linker is about 4 amino acids in length.In some embodiments, the linker is about 5 amino acids in length. In some embodiments, the linker is about 10 amino acids in length. In some embodiments, the linker is about 15 amino acids in length. In some embodiments, the linker is about 20 amino acids in length. In some embodiments, the linker is about 30 amino acids in length.

[0086] In some embodiments, the IL-21R peptide has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 12, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 12. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is a percentage within a range of any two of the percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0087] In some embodiments, the intracellular domain of IL-7R is modified to remove STAT5 signaling motifs. In some embodiments, the intracellular domain of IL-7R has at least 80% identity with the amino acid sequence of SEQ ID NO: 13, for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 13. In some such embodiments, the identity is more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is within a range of any two of the percentages. In some such embodiments, the identity is more than about 95%. In some such embodiments, the identity is greater than 95%.

[0088] In some embodiments, the intracellular domain of IL-7R comprises a Y449F mutation. In some embodiments, a chimeric cytokine receptor of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, e.g., an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, 100% identity, or a percentage identity between any two of the above percentages. In some such embodiments, the identity is greater than about 95% identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In some such embodiments, the identity is greater than 95% identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0089] Specific Polynucleotides Some embodiments provided herein include a polynucleotide encoding a chimeric cytokine receptor polypeptide. In some embodiments, the polynucleotide encodes a chimeric cytokine receptor according to any of the embodiments disclosed herein. In some embodiments, the polynucleotide preferably comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, e.g., preferably comprises a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, 100% identity, or a percentage identity between any two of the above percentages. In some such embodiments, the identity is greater than about 95% identity to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. In some such embodiments, the identity is greater than 95% identity to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0090] In some embodiments, the polynucleotide further comprises a promoter. In some embodiments, the promoter is an inducible promoter or a constitutive promoter. In some embodiments, the promoter is an EF1α promoter or an MND promoter (e.g., a promoter shown in SEQ ID NO:21; or a promoter having at least 80% sequence identity with SEQ ID NO:21; e.g., a promoter having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:21). In some such embodiments, the identity to SEQ ID NO:21 is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is a percentage within a range of any two of said percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0091] In some embodiments, the polynucleotide further comprises a transcription terminator. In some embodiments, the transcription terminator is a RBG polyA sequence (e.g., a transcription terminator as set forth in SEQ ID NO:22; or a transcription terminator having at least 80% sequence identity to SEQ ID NO:22; e.g., a transcription terminator having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:22). In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is a percentage within a range between any two of the above percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0092] In some embodiments, the polynucleotide further comprises a translation factor, hi some embodiments, the translation factor is EF1α.

[0093] In some embodiments, the polynucleotide further comprises a nucleic acid encoding a selectable marker, e.g., a truncated CD19 (CD19t) polypeptide or a truncated EGFR (EGFRt) polypeptide. In some embodiments, the polynucleotide encodes a truncated CD19 (CD19t) as set forth in SEQ ID NO:30, or encodes a CD19t having at least 80% sequence identity to SEQ ID NO:30, e.g., a CD19t having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:30. In some such embodiments, the identity is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or is 100%, or is a percentage within a range between any two of the above percentages. In some such embodiments, the identity is greater than about 95%. In some such embodiments, the identity is greater than 95%.

[0094] In some embodiments, the polynucleotide further comprises a selection agent and / or a resistance cassette. In some embodiments, the selection agent and / or the resistance cassette encodes a double mutant dihydrofolate reductase (DHFRdm) polypeptide.

[0095] In some embodiments, the polynucleotide further comprises a coding sequence for a ribosomal skipping sequence and / or a self-cleaving peptide. In some embodiments, the ribosomal skipping sequence comprises a T2A skipping sequence and / or a self-cleaving peptide comprising a P2A self-cleaving peptide sequence.

[0096] In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a chimeric antigen receptor, such as, for example, an anti-CD19 CAR.

[0097] vector Some embodiments disclosed herein relate to vectors. In some embodiments, the vectors of the present disclosure comprise a polynucleotide as described herein. In some embodiments, the vectors are suitable for or configured to transduce cells.

[0098] In some embodiments, the vector comprises a viral vector. In some embodiments, the vector is selected from a lentiviral vector, an adeno-associated viral vector, and an adenoviral vector. In some embodiments, the vector is a lentiviral vector.

[0099] In some embodiments, a vector of the present disclosure may comprise a polynucleotide encoding a chimeric cytokine receptor provided herein and / or a polynucleotide encoding a CAR.

[0100] cell Some embodiments disclosed herein relate to a cell. In some embodiments, the cell comprises one or more of the polypeptides described in any of the embodiments of the disclosure disclosed herein, the polynucleotides described in any of the embodiments of the disclosure disclosed herein, and / or the vectors described in any of the embodiments of the disclosure disclosed herein. In some embodiments, the cell comprises a polypeptide described herein. In some embodiments, the cell comprises a polynucleotide described herein. In some embodiments, the cell comprises a vector described herein. In some embodiments, the cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR) and / or a CAR protein. In some embodiments, the cell is a white blood cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a CD8+ T cell. In some embodiments, the cell is a CD4+ T cell. In some embodiments, the cell is a cell derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a cell derived from a CD8+ T cell. In some embodiments, the cells are derived from CD4+ T cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are cell lines. In some embodiments, the cells are produced from cell culture. In some embodiments, the cells are obtained directly from a tissue of a subject. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are ex vivo cells.

[0101] Treatment method Some embodiments relate to therapeutic methods. In some embodiments, the therapeutic methods disclosed herein relate to the treatment, suppression or alleviation of a disease or disorder (e.g., cancer) in a subject. Some such methods include administering a cell provided herein to a subject in need of treatment, suppression or alleviation of a disease or disorder. In some embodiments, the method includes administering a cell according to any of the embodiments disclosed herein to a subject in need of treatment, suppression or alleviation of a disease or disorder. In some embodiments, the cell includes a chimeric cytokine receptor or includes a polynucleotide encoding a chimeric cytokine receptor. In some embodiments, the cell further includes a CAR or includes a polynucleotide encoding a CAR.

[0102] In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is inflammation. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer can include solid tumors such as colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, pancreatic cancer, brain tumor, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, liver cancer; or non-solid tumors such as leukemia, multiple myeloma. In some embodiments, the cancer includes brain tumors.

[0103] In some embodiments, treating or alleviating the disease or disorder does not include co-administering a cytokine to the subject. In some embodiments, the method further comprises co-administering a cytokine to the subject, the dose of the cytokine being reduced compared to the dose of the cytokine co-administered to a subject administered a CAR-containing cell that does not comprise a polypeptide described in any of the embodiments of the present disclosure disclosed herein, a polynucleotide described in any of the embodiments of the present disclosure disclosed herein, and / or a vector described in any of the embodiments of the present disclosure disclosed herein.

[0104] Utilizing the chimeric cytokine receptors provided herein reduces or eliminates the need to supplement a subject's treatment with co-administration of cytokines. For example, when CAR T cells that do not comprise a chimeric cytokine receptor are administered to a subject, exogenous cytokines can be co-administered to further stimulate or activate the CAR T cells administered to the subject. As described herein, T cells comprising a chimeric cytokine receptor provided herein can be provided in a fully stimulated and / or activated state in the absence of exogenous cytokines, or can be provided in a fully stimulated and / or activated state in the presence of a dose of exogenous cytokines that is significantly reduced compared to the dose of T cells that do not comprise a chimeric cytokine receptor provided herein. In some embodiments, the treating or alleviating cancer does not include co-administering a cytokine to the subject. Some embodiments further include co-administering a cytokine to the subject, where the dose of the cytokine is reduced compared to the dose of the cytokine co-administered to a subject administered CAR-containing cells that do not comprise a chimeric cytokine receptor provided herein. In some such embodiments, the dose of cytokine co-administered to a subject administered cells comprising a CAR and a chimeric cytokine receptor can be reduced by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, or by more than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%, or by a percentage within a range of any two of the above percentages, compared to the dose of cytokine co-administered to a subject administered cells comprising a CAR that do not comprise a chimeric cytokine receptor.

[0105] In some embodiments, administration of CAR T cells that comprise a chimeric cytokine receptor provided herein improves tumor elimination from a subject compared to a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein.

[0106] In some such embodiments, administration of CAR T cells comprising a chimeric cytokine receptor provided herein reduces tumor volume in a subject compared to tumor volume in a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein. In some such embodiments, tumor volume in a subject administered CAR T cells comprising a chimeric cytokine receptor provided herein can be reduced compared to tumor volume in a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein, and the difference in tumor volume can be greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, greater than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100%, or can be a percentage within a range bounded by any two of the percentages.

[0107] In some such embodiments, administering CAR T cells that comprise a chimeric cytokine receptor provided herein reduces tumor volume in a subject at a rate that is faster than the rate of reduction in tumor volume in a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein.

[0108] In some embodiments, administering CAR T cells that comprise a chimeric cytokine receptor provided herein extends the overall survival of a subject compared to the overall survival of a subject administered CAR T cells that do not comprise a chimeric cytokine receptor provided herein.

[0109] In some embodiments, the cytokine is selected from IL-7 and IL-21. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-21. In some embodiments, the cells are autologous cells obtained from the subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0110] Method for preparing cell populations Some embodiments provided herein relate to a method for preparing a cell population comprising a chimeric antigen receptor (CAR), in some embodiments, the method comprising: (a) transducing a T cell with a polynucleotide according to any of the embodiments of the present disclosure; (b) transducing the T cells with a polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells in a culture medium that does not contain exogenous cytokines under conditions that stimulate activation and proliferation of the T cells. Includes. In some embodiments, step (b) is performed before step (a). In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, the cytokine is selected from IL-7 and IL-21. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-21. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a cell derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo cell.

[0111] In some embodiments, the method further comprises: (a) transducing a T cell with a polynucleotide according to any of the embodiments of the present disclosure; (b) transducing the T cells with a polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells under conditions sufficient to stimulate activation and proliferation of the T cells. Including, The medium used for the culture contains a reduced amount of exogenous cytokines compared to the amount sufficient to stimulate the activation and proliferation of T cells that do not contain a polynucleotide encoding the chimeric receptor. In some embodiments, step (b) is performed before step (a). In some embodiments, steps (a) and (b) are performed simultaneously. In some embodiments, the cytokine is selected from IL-7 and IL-21. In some embodiments, the cytokine is IL-7. In some embodiments, the cytokine is IL-21. In some embodiments, the cell is a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a cell derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an ex vivo cell.

[0112] In some embodiments, the amount of exogenous cytokine sufficient to stimulate activation and proliferation of T cells comprising a chimeric cytokine receptor and a CAR provided herein is reduced compared to the amount of exogenous cytokine sufficient to stimulate activation and proliferation of CAR-containing T cells not comprising a chimeric cytokine receptor provided herein, the difference being greater than, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about, greater than about In some embodiments, the cell is a cell derived from a CD8+ T cell or a CD4+ T cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a mammalian cell, such as a human cell. In some embodiments, the cell is an ex vivo cell.

[0113] Particular sequences useful in embodiments of the methods and compositions provided herein are listed in Table 1 below. [Table 1] TIFF2025512886000003.tif246170TIFF2025512886000004.tif250170TIFF2025512886 000005.tif248170TIFF2025512886000006.tif250170TIFF2025512886000007.tif74170 EXAMPLES

[0114] Adoptive T cell therapy depends on the persistence and functional durability of administered T cells. T cell persistence and functional durability are heavily influenced by environmental signals, such as cytokine inputs, that T cells receive. As described herein, IL-7 stimulation of CAR T cells resulted in longer persistence, while IL-21 stimulation enhanced cytotoxicity and functional durability. We hypothesized that combining the signaling inputs of these two interleukins could exert complementary effects on CAR T cell functionality. Herein, we provide the design of a novel chimeric cytokine receptor that can provide both IL-7 and IL-21 signaling outputs. In this study, we investigated the survival, proliferation, cytotoxicity, and antitumor potential of CAR T cells in long-term coculture with tumor cells. CAR T cells with a hybrid IL-7 / IL-21 signaling cytokine receptor were found to exhibit improved persistence and functional durability. These properties make this hybrid cytokine receptor a promising candidate for adjunctive means of CAR T cell therapy, particularly in the solid tumor space where the microenvironment contributes to T cell inactivation. In parallel with this investigation, we also investigated a transcriptional regulatory system that allows spatial and temporal control of the expression of the hybrid cytokine receptor of the present disclosure, so that CAR T cell therapy using the hybrid cytokine receptor of the present disclosure can be carried out in a clinically practical manner.

[0115] Example 1 - Design of cytokine-independent receptors for signal transduction In this example, a hybrid IL7 / IL21 cytokine receptor for combinatorial interleukin signaling is disclosed as a non-limiting example. This cytokine-independent receptor for IL7 signaling (CIndR7) was designed based on a naturally occurring mutant IL7R with an insertion in the transmembrane domain commonly found in leukemia. The IL7R homodimer obtained by dimerization of this receptor transmits signals independent of cytokines and gamma chains.

[0116] FIG. 1 (left panel) shows a schematic diagram of a HER polypeptide with an extracellular portion shown in a dotted box; a constitutively active IL7R mutant with a transmembrane domain containing a mutation and an intracellular STAT5 signaling domain (each domain shown in a dotted box); and an IL21 signaling complex with a STAT3 signaling domain shown in a dotted box. FIG. 1 (right panel) shows a schematic diagram of an example of a chimeric receptor (CIndR) that includes an extracellular domain of HER2; a transmembrane domain of IL7R containing a mutation; an intracellular STAT5 signaling domain of IL7R; and a signaling domain of an IL21 signaling complex linked to other portions of the chimeric receptor via a linker. This cytokine-independent receptor (CIndR) incorporates, from the N-terminus to the C-terminus, (1) a Her2 extracellular domain for cell surface detection, (2) a mutant IL7R transmembrane domain for constitutive signaling, (3) an intracellular domain of IL7R, and (4) an IL21R peptide tethered via a flexible linker. The intracellular domain of IL7R contains a STAT5 activation motif, and the IL21R peptide contains a STAT3 activation motif.

[0117] Figure 2 (top panel) shows a schematic diagram depicting the various chimeric receptors and the control marker-only receptor. Three types of CIndRs with different intracellular domain designs were generated: 1) CIndR7 (incorporating only the IL7R intracellular domain), 2) CIndR7 / 21 (incorporating the IL21R peptide and the IL7R intracellular domain), and 3) CIndR21 (incorporating the IL7R intracellular domain with the STAT5-activating tyrosine residue mutated to phenylalanine to ablate STAT5 signaling, and the IL21R peptide). Each polynucleotide encoding these various CIndRs was cloned into a dual promoter construct, which also contained polynucleotides encoding the anti-CD19 CAR, a selection marker (DHFRdm), and a cell surface marker (EGFRt). FIG. 2 (bottom panel) shows, in a vector, a polynucleotide encoding CIndR linked to the MND promoter; and the EF1α promoter linked to a polynucleotide encoding an anti-CD19 CAR, a T2A sequence, a selection marker (DHFRdm), a P2A sequence, and a cell surface marker (EGFRt).

[0118] Examples of chimeric receptors are shown in Figures 3A-3D. Figure 3A shows a polypeptide containing (1) the extracellular domain of Her2tG, (2) the transmembrane domain of IL7R with an insertion, and (3) the intracellular domain of IL7R. Figure 3B shows CindR7, which further contains a Y449 mutation in the IL7R domain, which can promote the formation of disulfide bridges between IL7R monomers. We designed the CIndR7 / 21 chimeric receptor by adding an IL21 receptor-derived peptide (5) to the C-terminus of CIndR7 via a flexible linker (4) (Figure 3C) and introducing the Y449 mutation in the IL7R domain (Figure 3D). We further improved the design by removing the STAT5 signaling motif from the cytoplasmic domain of the IL7 receptor, generating CIndR21 (Figure 3E).

[0119] Example 2 - Proof of concept of Her2tG as a cell surface marker for CIndR7 To determine whether a truncated HER2 (Her2tG) polypeptide could be used as a cell surface marker for testing CIndR7, we transfected T cells with a plasmid containing a truncated CD19 polypeptide (CD19t), ClndR7, and T2A (Figure 4). The incorporation of T2A ensured that CD19t and CIndR7 were translated into two separate proteins. Her2 staining was directly proportional to CD19 staining in these cells as measured by FACS (Figure 5). The flow cytometry scatter plots showed a correlation between the abundance of the co-expressed truncated CD19 marker protein and Her2tG, indicating that Her2tG can be used as a cell surface marker for CIndR7 expression.

[0120] Example 3 - Generation and enrichment of CIndR-expressing CAR T cells Each CIndR sequence was introduced into a PiggyBac nanoplasmid construct as a self-contained cistron whose expression was driven by the MND promoter and terminated by the RBG polyadenylation sequence. These constructs further contained a human anti-CD19 CAR and another cistron encoding a drug resistance gene, a double mutant of dihydrofolate reductase (DHFRdm), and a cell surface marker protein in the form of a truncated epidermal growth factor polypeptide (EGFRt) (Figures 11A-11D). As an inactive control, the Her2tG cargo was used.

[0121] CIndR-encoding constructs were co-transfected with mRNA encoding a PiggyBac transposon into CD4+ and CD8+ T cells by electroporation, allowing stable integration into the genome. T cells with stable integration of the transgenic sequence were enriched for by specific activation of the CAR in combination with methotrexate selection, to which resistance is conferred by expression of the DHFRdm protein.

[0122] Example 4 - Flow cytometry for detection of phosphorylated STAT proteins T cells were harvested from culture, washed twice with PBS to remove added cytokines, and cultured overnight without added cytokines. The next day, cells were treated with IL-7 (5 ng / mL) or IL-21 (5 ng / mL) for 20 min or no treatment was added. Cells were then fixed with BD CytoFix reagent, permeabilized with BD Perm Buffer III, stained with antibodies specific for phosphorylated STAT5 (pSTAT5) and phosphorylated STAT3 (pSTAT3), and finally analyzed by flow cytometry.

[0123] Example 4 - Hybrid cytokine receptors recapitulate IL-7 and IL-21 signaling in human T cells CD19 CAR T cells expressing each CIndR variant were assessed by flow cytometry using an antibody specific for phosphorylated STAT3. Controls included CAR T cells expressing the marker alone and treated with exogenous IL-7 or exogenous IL-21.

[0124] Control and CIndR and IL21R peptide-expressing T cell populations exposed to IL-21 showed STAT3 activation (Fig. 6, Fig. 8A), indicating that the IL21R signaling domains integrated into CIndR7 / 21 and CIndR21 were functional and showed the same pattern of STAT3 activation as cells exposed to exogenous IL-21.

[0125] Furthermore, control and T cell populations expressing CIndR and the unmutated IL7R intracellular domain exposed to IL-7 displayed STAT5 activation (Fig. 8B), indicating that the IL7R signaling domains integrated into CIndR7 and CIndR7 / 21 were functional and displayed the same pattern of STAT5 activation as cells exposed to exogenous IL-7.

[0126] In contrast, expression of CIndR7 in human CD8+ T cells increased phosphorylated STAT5 (pSTAT5) (Figure 7A), but only weakly phosphorylated STAT3 (Figure 7B). On the other hand, expression of CIndR7 / 21 resulted in robust phosphorylation of STAT3 (Figure 7D), while pSTAT5 remained at high levels (Figure 7C). Overall, CIndR7-expressing T cells showed significantly higher pSTAT5 levels, CIndR21-expressing T cells showed significantly higher pSTAT3 levels, and CIndR7 / 21-expressing T cells showed significantly higher pSTAT3 levels and significantly higher pSTAT5 levels. The results indicate that a range of CIndR proteins, alone or in combination, conferred STAT3 signaling activity and STAT5 signaling activity.

[0127] Example 5 - Linker length of IL21R peptide Linkers between the cytoplasmic domain of IL7R and the IL21R peptide (Figure 7) were tested to determine which linker would maximize STAT3 and STAT5 signaling (Figure 9). Polypeptides containing various types of flexible linker candidates were tested for signaling ability. Linker candidates were GGG (SEQ ID NO: 23), GGGGS (SEQ ID NO: 24), (GGGGS)2 (SEQ ID NO: 25), or (GGGGS)3 (SEQ ID NO: 26). Flow cytometry analysis showed that the linker with four glycines plus two serines ((GGGGS)2 (SEQ ID NO: 25)) maximized STAT3 and STAT5 activation, so this length of linker was incorporated into all constructs for further evaluation (Figures 10A-B).

[0128] Example 6 - In vitro antitumor activity assay A 3-week repeated tumor challenge assay was used to investigate the long-term antitumor activity of T cells containing CindR and anti-CD19 CAR. The experimental method is outlined in Figure 12A. Briefly, CD4 + Cells and CD8 +The cells (effector cells, i.e., anti-CD19 CAR T cells) were mixed at a 1:1 ratio and co-cultured with human lymphoma Raji cells (CD19+ target cells) at an effector:target ratio of 2:1. Raji cells were pre-modified to express the red fluorescent protein mCherry, and tumor abundance was tracked over time by live cell fluorescence imaging using an Incucyte S3 instrument. Raji cells were added to the co-cultures at the same dose every 3 days until differences in tumor abundance were observed between each T-cell experimental group. Live imaging was used to quantitate the ability of CAR T cells to suppress tumor growth over a 15-day period. The amount of red fluorescent signal in each T-cell group was quantified for three different donors.

[0129] As shown in Figures 12B-12D, the arrows indicate the timing of the addition of Raji tumor cells to the co-cultures. Overall, CIndR-containing CAR T cells maintained antitumor activity for a longer period compared to CIndR-free CAR T cells. In all three donors, CIndR7 / 21 showed the most potent tumor growth suppression.

[0130] Figure 12E and Figure 12F show the results of additional experiments showing that CAR T cells expressing CIndRs that induce IL-21 signaling (CIndR7 / 21 and CIndR21) significantly suppressed tumor growth compared to other groups (Figure 12E). Notably, only control CAR T cells treated with IL-21 were able to suppress tumor growth, and the same pattern of tumor suppression was observed when this marker-control CAR T cells were exposed to this exogenous cytokine (Figure 12F). These findings further demonstrated that hybrid cytokine receptors that induce IL-21 signaling could improve the antitumor activity of CAR T cells under long-term tumor exposure, an effect that recapitulated the benefits obtained by exposure to exogenous IL-21.

[0131] Example 7 - Hybrid cytokine receptors increase CAR T cell proliferation The experimental method outlined in Figure 13A was used to evaluate the proliferation-enhancing effect of each CIndR on CAR T cells. Briefly, on day 0, each CAR T cell population was co-cultured with Raji target cells. On day 7, T cells were quantified and re-seeded with a fresh batch of Raji target cells. On day 14, T cells were quantified again and T cell proliferation numbers over 2 weeks were calculated. As controls, marker-only CAR T cells were cultured with exogenous IL-7, IL-21, or a combination of IL-7 and IL-21.

[0132] The fold expansion of each CAR T cell group relative to the maximum expansion achieved by the control group exposed to IL-7 and IL-21 was calculated for each donor. The relative fold expansion values ​​over time are shown in Figure 13B, and the final relative fold expansion values ​​are shown in Figure 13C. The relative fold expansion showed that the IL-21 signaling receptor significantly enhanced CAR T cell proliferation, with the enhancement of proliferation being most pronounced after the second exposure to Raji tumor cells. Furthermore, these findings demonstrated that CIndR, which induces IL-21 signaling, increased CAR T cell proliferation in response to antigen stimulation to a statistically significant extent.

[0133] Example 8 - Inhibition of apoptosis by hybrid cytokine receptors Using the experimental method outlined in Figure 14A, each CIndR was assessed for its ability to suppress apoptosis and induce cytokine-independent proliferation in CAR T cells. Briefly, on day 0, each CAR T cell population was co-cultured with irradiated Raji target cells. On day 7, cultures were washed to remove cytokines from the culture medium, T cells were re-seeded, and subsequent proliferation in cytokine-free conditions was observed. On day 10, T cells were harvested and T cell viability was assessed by Annexin V binding flow cytometry analysis. T cell viability was measured by performing a flow cytometry experiment on day 10 using Annexin V binding and uptake of 7-AAD viability dye by T cell populations. Annexin V binds to pre-apoptotic cells, and 7-AAD is taken up by dead cells, thus inhibiting the proliferation of normal cells (Annexin V). - 7-AAD- ), pre-apoptotic cells (Annexin V + 7-AAD - ) and dead cells (7-AAD + ) can be quantified.

[0134] Statistical analysis showed that CIndR induced IL-21 signaling significantly reduced T cell apoptosis compared to the marker control group (Figure 14B). T cell proliferation was observed after cytokine removal on day 7. As shown in Figure 14C, CIndR extended CAR T cell survival and did not induce cytokine-independent T cell proliferation in the absence of antigen.

[0135] Example 9 - Maintenance of a subset of stem cell memory CAR T cells by IL-21 signaling receptor The effect of each CIndR on the expression of CAR T cell differentiation markers was evaluated using the experimental method outlined in Figure 15A. Briefly, on day 0, each CAR T group was co-cultured with Raji target cells. On day 3, the expression of T cell differentiation markers was evaluated by flow cytometry, and T cells were quantified and re-seeded with a fresh batch of Raji target cells. On day 6, the expression of T cell differentiation markers was again evaluated by flow cytometry.

[0136] As shown in Figure 15B, CAR T cells with CIndRs that induce IL-21 signaling showed a higher proportion of T cells expressing stem cell memory T cell markers. This effect was most statistically significant in the CIndR21-expressing CAR T cell group, and the difference between this group and the control T cell group increased between the first and second target cell exposures. A similar pattern was observed in the control marker-only CAR T cell group exposed to exogenous IL-21. These findings revealed that the IL-21 signaling receptor maintained a subpopulation of stem cell memory T cells.

[0137] Example 10 - Enhancement of tumor suppression by IL-21 signaling receptor in leukemia-bearing mice A systemic leukemia model in immunodeficient (NSG) mice was used to evaluate each CIndR for its enhancing effect on tumor suppression by CAR T cells in vivo. Briefly, on day 0, 11-13 week old female mice were systemically injected with CD19+ human Nalm-6 leukemia cells. Nalm-6 cells were pre-modified to express a firefly luciferase bioluminescence reporter gene to observe tumor abundance in mice by luminescence imaging. On day 7, mice were systemically injected with human T cells from five experimental treatment groups: (1) unmodified T cells; (2) CD19 CAR T cells co-expressing markers only; (3) CD19 CAR T cells co-expressing CIndR7; (4) CD19 CAR T cells co-expressing CIndR7 / 21; or (5) CD19 CAR T cells co-expressing CIndR21.

[0138] Tumor burden quantification by bioluminescence showed that mice treated with either CD19 CAR T cell population had reduced tumor burden by day 15 compared to mice treated with unmodified T cells (Figure 16). In contrast, mice treated with CD19 CAR T cells that further contained CIndR to induce IL-21 signaling showed a significant reduction in tumor burden at day 15. Furthermore, some of these mice had almost complete elimination of tumors. These results suggest that the IL-21 signaling function conferred by the hybrid cytokine receptor technology described herein enhances CAR T cells, making them more effective anti-cancer therapeutics.

[0139] The foregoing description discloses some methods and materials of the present invention. The methods and materials of the present invention may be modified, as may the manufacturing methods and equipment. Such modifications will be readily apparent to those of skill in the art in light of this disclosure or practice of the invention disclosed herein. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but will encompass all modifications and other aspects that fall within the true scope and spirit of the invention.

[0140] All references cited herein, including but not limited to published patent applications, unpublished patent applications, patents, and journal articles, are incorporated herein by reference in their entirety and made a part of this specification. In the event that any reference, patent, or patent application incorporated by reference conflicts with the disclosure of this specification, the disclosure of this specification will govern and / or take precedence over such conflicting matter. [Sequence List]

Claims

1. 1. A chimeric cytokine receptor, comprising: (i) the extracellular domain; (ii) the transmembrane domain of the interleukin-7 receptor (IL-7R); and (iii) the intracellular domain of IL-7R Including, The extracellular domain may comprise a cell surface selectable polypeptide, and the cell surface selectable polypeptide may be selected from the extracellular domain of HER2, the extracellular domain of EGFR, and the extracellular domain of CD19. Chimeric cytokine receptors.

2. The chimeric cytokine receptor of claim 1, wherein the transmembrane domain of the IL-7R is modified to promote dimerization and / or constitutive signaling of the chimeric receptor.

3. The chimeric cytokine receptor of claim 1 or 2, further comprising a GMCSF signal sequence.

4. The chimeric cytokine receptor of claim 3, wherein the GMCSF signal sequence has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:7, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:

7.

5. (i) the extracellular domain is the extracellular domain of HER2, which may comprise or consist of a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:8, e.g., may comprise or consist of a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:8; (ii) the extracellular domain is the extracellular domain of CD19, and the extracellular domain of CD19 comprises or may comprise a truncated CD19 polypeptide (CD19t) consisting of a polypeptide having an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28; or (iii) the extracellular domain is an extracellular domain of EGFR, and the extracellular domain of EGFR may comprise a truncated EGFR polypeptide (EGFRt); A chimeric cytokine receptor according to any one of claims 1 to 4.

6. The chimeric cytokine receptor of any one of claims 1 to 5, wherein the extracellular domain of HER2 lacks domain I, domain II and domain III of the HER2 extracellular protein.

7. 7. The chimeric cytokine receptor of any one of claims 1 to 6, wherein the transmembrane domain of IL-7R has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:9, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:

9.

8. The chimeric cytokine receptor of claim 6, wherein the transmembrane domain of the IL-7R has been modified to promote dimerization of the chimeric receptor and comprises the amino acid sequence TCP.

9. The chimeric cytokine receptor of any one of claims 1 to 8, wherein the intracellular domain of IL-7R has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 10, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

10.

10. The chimeric cytokine receptor of any one of claims 1 to 9, further comprising an interleukin 21 receptor (IL-21R) polypeptide attached via a linker to the C-terminus of the intracellular domain of the IL-7R.

11. The chimeric cytokine receptor of claim 10, wherein the linker has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:11, for example at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:

11.

12. The chimeric cytokine receptor of claim 10 or 11, wherein the linker comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:

26.

13. The chimeric cytokine receptor according to any one of claims 10 to 12, wherein the linker is 3 to 30 amino acids in length.

14. 14. The chimeric cytokine receptor of any one of claims 10 to 13, wherein the IL-21R polypeptide has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:12, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

12.

15. The chimeric cytokine receptor of any one of claims 1 to 14, wherein the intracellular domain of the IL-7R has been modified to remove the STAT5 signaling motif.

16. The chimeric cytokine receptor of any one of claims 1 to 15, wherein the intracellular domain of IL-7R has at least 80% identity to the amino acid sequence of SEQ ID NO: 13, e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

13.

17. The chimeric cytokine receptor of claim 15 or 16, wherein the intracellular domain of the IL-7R comprises a Y449F mutation.

18. The chimeric cytokine receptor of claim 1, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, for example, an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

19. A polynucleotide encoding the chimeric cytokine receptor of any one of claims 1 to 18.

20. 20. The polynucleotide of claim 19, comprising a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, e.g., comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

21. 21. The polynucleotide of claim 19 or 20, further comprising a promoter.

22. 22. The polynucleotide of claim 21, wherein the promoter is a constitutive promoter, such as an EF1α promoter or an MND promoter (e.g., SEQ ID NO: 21).

23. The polynucleotide of any one of claims 19 to 22, further comprising a transcription terminator.

24. 24. The polynucleotide of claim 23, wherein the transcription terminator is a RBG polyA sequence (e.g., SEQ ID NO: 22).

25. 25. The polynucleotide of any one of claims 19 to 24, further comprising a nucleic acid encoding a selection marker, the selection marker being, for example, a truncated EGFR (EGFRt) polypeptide or a truncated CD19 (CD19t) (e.g., SEQ ID NO: 30).

26. The polynucleotide of any one of claims 19 to 25, further comprising a selection factor and / or a resistance cassette.

27. 27. The polynucleotide of claim 26, wherein the selection agent and / or the resistance cassette encodes a double mutant dihydrofolate reductase (DHFRdm) polypeptide.

28. The polynucleotide of any one of claims 19 to 27, further comprising a coding sequence for a ribosomal skipping sequence and / or a self-cleaving peptide.

29. 29. The polynucleotide of claim 28, wherein the ribosomal skipping sequence comprises a T2A skipping sequence and / or the self-cleaving peptide comprises a P2A self-cleaving peptide sequence.

30. The polynucleotide of any one of claims 19 to 29, further comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

31. 31. The polynucleotide of claim 30, wherein the CAR is an anti-CD19 CAR.

32. The polynucleotide of any one of claims 19 to 31, wherein the chimeric receptor is optimized for expression in a human cell.

33. A vector comprising the polynucleotide according to any one of claims 19 to 32.

34. The vector of claim 33 which is a viral vector.

35. 35. The vector of claim 33 or 34, which is selected from a lentiviral vector, an adeno-associated viral vector and an adenoviral vector.

36. The vector of claim 35 which is a lentiviral vector.

37. A cell comprising a polypeptide according to any one of claims 1 to 18, a polynucleotide according to any one of claims 19 to 32, and / or a vector according to any one of claims 33 to 36.

38. 38. The cell of claim 37, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR), e.g., further comprising a polynucleotide encoding an anti-CD19 CAR.

39. 39. The cell of claim 37 or 38, which is a T cell.

40. The cell according to any one of claims 37 to 39, which is a CD8+ T cell or a CD4+ T cell.

41. The cell according to any one of claims 37 to 40, which is derived from a CD8+ T cell or a CD4+ T cell.

42. The cell according to any one of claims 37 to 41, which is a primary cell.

43. The cell according to any one of claims 37 to 42, which is a mammalian cell.

44. The cell according to any one of claims 37 to 43, which is a human cell.

45. The cell according to any one of claims 37 to 44, which is an ex vivo cell.

46. 1. A method of treating, inhibiting or ameliorating a disease or disorder in a subject, comprising: A method comprising the step of administering a cell according to any one of claims 37 to 45 to a subject in need of treatment, suppression or alleviation of a disease or disorder.

47. 47. The method of claim 46, wherein the disease or disorder is an autoimmune disease.

48. 47. The method of claim 46, wherein the disease or disorder is inflammation.

49. 47. The method of claim 46, wherein the disease or disorder is cancer.

50. 50. The method of any one of claims 46 to 49, wherein treating or alleviating the disease or disorder does not comprise co-administering a cytokine to the subject.

51. The method of claim 50, further comprising the step of co-administering a cytokine to the subject, wherein the dose of the cytokine is reduced compared to the dose of the cytokine co-administered to a subject administered a CAR-containing cell that does not comprise the polypeptide of any one of claims 1 to 18, the polynucleotide of any one of claims 19 to 32, and / or the vector of any one of claims 33 to 36.

52. 52. The method of claim 50 or 51, wherein the cytokine is selected from IL-7 and IL-21.

53. The method of any one of claims 50 to 52, wherein the cytokine is IL-7.

54. The method of any one of claims 50 to 53, wherein the cytokine is IL-21.

55. 55. The method of any one of claims 46 to 54, wherein the cells are autologous cells obtained from the subject.

56. The method of any one of claims 49 to 55, wherein the cancer comprises a solid tumor such as colorectal cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, pancreatic cancer, brain tumor, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer, liver cancer; or a non-solid tumor such as leukemia, multiple myeloma.

57. The method of any one of claims 46 to 56, wherein the subject is a mammal.

58. The method of any one of claims 46 to 57, wherein the subject is a human.

59. 1. A method for preparing a cell population comprising a chimeric antigen receptor (CAR), comprising: (a) transducing a T cell with a polynucleotide according to any one of claims 19 to 32; (b) transducing the T cells with a polynucleotide encoding a chimeric antigen receptor (CAR) (e.g., an anti-CD19 CAR); and (c) culturing the transduced T cells in a culture medium that does not contain exogenous cytokines under conditions that stimulate activation and proliferation of the T cells. A method comprising:

60. 1. A method for preparing a cell population comprising a chimeric antigen receptor (CAR), comprising: (a) transducing a T cell with a polynucleotide according to any one of claims 19 to 32; (b) transducing the T cells with a polynucleotide encoding a chimeric antigen receptor (CAR); and (c) culturing the transduced T cells under conditions sufficient to stimulate activation and proliferation of the T cells. Including, the medium used for the culture contains a reduced amount of exogenous cytokines compared to the amount sufficient to stimulate activation and proliferation of T cells that do not contain a polynucleotide encoding the chimeric receptor; method.

61. 61. The method of claim 59 or 60, wherein step (b) is carried out before step (a).

62. 61. The method of claim 59 or 60, wherein steps (a) and (b) are carried out simultaneously.

63. 63. The method of any one of claims 59 to 62, wherein the cytokine is selected from IL-7 and IL-21.

64. The method of any one of claims 59 to 63, wherein the cytokine is IL-7.

65. The method of any one of claims 59 to 64, wherein the cytokine is IL-21.

66. 66. The method of any one of claims 59 to 65, wherein the cell is a CD8+ T cell or a CD4+ T cell.

67. The method of any one of claims 59 to 65, wherein the cell is a cell derived from a CD8+ T cell or a CD4+ T cell.

68. 68. The method of any one of claims 59 to 67, wherein the cells are primary cells.

69. The method of any one of claims 59 to 68, wherein the cell is a mammalian cell.

70. The method of any one of claims 59 to 69, wherein the cell is a human cell.

71. The method of any one of claims 61 to 72, wherein the cells are ex vivo cells.

72. Use of a chimeric cytokine receptor according to any one of claims 1 to 18, a polynucleotide according to any one of claims 19 to 32, a vector according to any one of claims 33 to 36 or a cell according to any one of claims 37 to 45 as a medicament, for example for the treatment or suppression of an autoimmune disease, an inflammatory condition or cancer, comprising The use, wherein the cancer is, for example, colon cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, malignant melanoma, kidney cancer, pancreatic cancer, brain tumor, glioblastoma, neuroblastoma, medulloblastoma, sarcoma, bone cancer or liver cancer, or a non-solid tumor, for example, leukemia or multiple myeloma.

73. 1. A method for culturing cells, comprising: (a) obtaining a cell comprising a polypeptide according to any one of claims 1 to 18; and (b) culturing the cells in a medium that does not contain exogenous cytokines or that contains reduced amounts of exogenous cytokines. Including, the amount of said exogenous cytokine is reduced compared to the amount of said exogenous cytokine in a medium used to culture said polypeptide-free cells, and said culture results in a population of cells sufficient in number for infusion; method.

74. 74. The method of claim 73, wherein the amount of said cytokine in said medium is sufficient for activation and / or proliferation of said cells.

75. 75. The method of claim 73 or 74, wherein the cell comprises a chimeric antigen receptor.

76. 76. The method of any one of claims 73 to 75, wherein the cytokine is selected from IL-7 and IL-21.

77. 77. The method of any one of claims 73 to 76, wherein the cytokine is IL-7.

78. The method of any one of claims 73 to 77, wherein the cytokine is IL-21.

79. The method of any one of claims 73 to 78, wherein the cell is a T cell.

80. 80. The method of any one of claims 73 to 79, wherein the cell is a CD8+ T cell or a CD4+ T cell.

81. The method of any one of claims 73 to 80, wherein the cell is a cell derived from a CD8+ T cell or a CD4+ T cell.

82. The method of any one of claims 73 to 81, wherein the cells are primary cells.

83. The method of any one of claims 73 to 82, wherein the cell is a mammalian cell.

84. The method of any one of claims 73 to 83, wherein the cell is a human cell.

85. The method of any one of claims 73 to 84, wherein the cells are ex vivo cells.

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