Use and production of engineered immune cells

Abstract

Disclosed are immune cells engineered to reduce or eliminate the expression and / or function of NR4A, TOX, NR4A + TOX, or immune cells engineered to reduce or eliminate the expression and / or function of NR4A + TOX and increase the expression of IL-21. The invention also provides cells engineered to inhibit the expression and / or function of the NFAT / AP-1 pathway. The cells disclosed in the invention are T cells and NK cells. It can be expanded to produce a homogeneous or heterogeneous population of cells and / or in combination with a pharmaceutically acceptable carrier. It may be a CAR cell. Also disclosed are methods of inducing an immune response and treating a condition in need of selective immunotherapy, comprising contacting a target cell with a cell or a composition of the invention. The contacting may be performed in vitro or in vivo to provide immunotherapy to the subject. In addition, the invention provides methods of producing such engineered cells. The invention also provides kits containing materials for making and using the cells.

Description

[0001] This application is a divisional application of the invention patent application which entered the Chinese national phase on July 15, 2020, with application number 201880086647.7, application date November 21, 2018, and invention name “Application and Production of Engineered Immune Cells”. Cross-references to related patent applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 589,562, filed on November 22, 2017, the entire contents of which are incorporated herein by reference. Government support statement

[0002] This invention was made with government support from the National Institutes of Health (AI109842 and AI040127). The government has certain rights in this invention. Technical Field

[0003] Embodiments of the present invention relate to improving adoptive immune cell therapy, particularly T cell therapy for the treatment of cancer or infection, by depletion of Nr4a and / or Tox family transcription factors. Background Art

[0004] Adoptive cell therapy for cancer is an increasingly common strategy for infusing T cells to recognize and eliminate tumor cells. T cells expressing chimeric antigen receptor (CAR T) targeting human CD19 (huCD19) antigen 1,2 B-cell leukemia and lymphoma 3,4 CAR T cells have shown impressive clinical efficacy. However, they are less effective against solid tumors. 5,6 , in part because they enter low-reactivity 7 (“Exhaustion” 8-11 or "dysfunctional" 12,13 ) state, which is triggered by chronic antigenic stimulation and is characterized by upregulation of several inhibitory receptors and loss of effector function 14,15 Therefore, there is a need in the art to provide immunotherapies that target low-responsive tumors. The present invention provides compositions and methods that meet this unmet need. SUMMARY OF THE INVENTION

[0005] The present invention provides cells engineered to reduce or eliminate the expression and / or function of NR4A transcription factors in cells. The present invention also provides cells engineered to reduce or eliminate the expression and / or function of TOX transcription factors in cells. In one aspect, the present invention also provides cells engineered to reduce or eliminate the expression and / or function of NR4A and TOX transcription factors in cells. In another aspect, cells are engineered to reduce or eliminate the expression and / or function of NR4A and TOX transcription factors in cells, and to increase the expression of IL-21. In another aspect, the present invention provides cells engineered to inhibit the expression and / or function of NFAT / AP-1 pathways in cells. In one aspect, for the disclosed cells, the cells are immune cells, such as T cells and NK cells.

[0006] The cells can be expanded to produce a homogenous or heterogenous cell population and / or a homogenous or heterogenous cell population combined with a carrier (eg, a pharmaceutically acceptable carrier).

[0007] The present invention also provides methods of inducing an immune response and treating a condition requiring selective immunotherapy, the method comprising contacting a target cell with a cell or composition as described herein, or consisting essentially of the step, or consisting of the step. The contacting can be performed in vitro or in vivo, thereby providing immunotherapy to a subject, such as a human patient.

[0008] The present invention also provides a method for producing an engineered cell, the method comprising, consisting essentially of, or consisting of reducing or eliminating the expression and / or function of the NR4A transcription factor in the cell.

[0009] The present invention also provides a method for producing engineered cells, the method comprising reducing or eliminating the expression and / or function of TOX transcription factors in cells. On the other hand, a method for producing engineered cells, the method comprising reducing or eliminating the expression and / or function of NR4A transcription factors and TOX transcription factors in cells, or consisting essentially of this step, or consisting of this step. On the other hand, the present invention also provides a method for producing engineered cells, the method comprising reducing or eliminating the expression and / or function of NR4A transcription factors and TOX transcription factors in cells and increasing the expression of IL-21, or consisting essentially of this step, or consisting of this step. On the other hand, the present invention provides a method for producing engineered cells, the method comprising inhibiting the expression and / or function of the NFAT / AP-1 pathway in cells, or consisting essentially of this step, or consisting of this step.

[0010] In one aspect, for the disclosed methods, the cells are immune cells, such as T cells and NK cells.

[0011] A kit containing materials for making and using the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings illustrate embodiments of the present technology, but are not limiting. For clarity and ease of illustration, the accompanying drawings are not drawn to scale, and in some cases various aspects may be shown in an exaggerated or enlarged manner to facilitate understanding of specific embodiments.

[0013] Figure 1A -1F: Provides a non-limiting example of adoptive transfer of chimeric antigen receptor (CAR) expressing mouse CD8+ T cells, which exhibit an exhaustion phenotype similar to endogenous CD8+ T cells in a shorter period of time. Figure 1A A schematic diagram of a tumor experiment is provided. FIG. 1B provides a flow cytometry graph showing a CAR CD8+T cell population and an endogenous CD8+T cell population on day 8 after adoptive transfer. FIG. 1C provides a bar graph showing the percentage of total CD8+T cells for both populations. FIG. 1D provides a flow cytometry graph showing the expression of PD-1 and Tim3 by CAR CD8+T cells or endogenous CD8+T cells. FIG. 1E provides a flow cytometry graph showing four populations ( Figure 1A Figure 1F provides a flow cytometry graph of cytokine re-stimulation of CAR CD8+T cells compared with endogenous CD8+T cells. Cells were stimulated or not stimulated with PMA / ionomycin.

[0014] Figure 2A-2G: Provided are non-limiting examples of mice bearing tumors adoptively transferred with CAR CD8+T cells lacking all three Nr4a family members exhibiting increased tumor regression rates and prolonged survival compared to mice transferred with CAR CD8+T cells lacking only Nr4a3. Figure 2A provides a schematic diagram of the tumor experiment. Figure 2B provides a time course of average tumor growth, n=13 for Nr4a3- / - at d0; n=14 for Nr4aTKO at d0. At d21, p values ​​were calculated using a t-test (assuming equal variances); equal variances were determined by an f-test. The experiment calculated using a one-sided Mann-Whitney-Wilcoxon test had an efficacy of 91%. Figure 2C provides tumor growth curves for the corresponding individual mice. Figure 2D shows the survival curves, and the p values ​​were calculated using the log-rank (Mantel-Cox) test. Figure 2E provides a schematic diagram of the tumor experiment. Figure 2F provides a bar graph showing the surface expression of PD-1, Tim3, and Lag3 of CAR NGFR+CD8+T cells on day 8 after adoptive transfer. The data are the average of the data points with two biological replicates. Figure 2G provides a bar graph showing TNF and IFNγ after restimulation of CAR NGFR+CD8+T cells with PMA / ionomycin on day 8 after adoptive transfer. IL-2 was not detected above background in both groups (not shown). The data show the average of the data points with two biological replicates.

[0015] Figures 3A-3G : B16-OVA-huCD19 tumors infiltrated with adoptively transferred CAR-expressing mouse CD8+ T cells and OT-1 TCR transgenic T cells show a phenotype similar to the endogenous CD8+ TIL phenotype. (Figure 3A) Experimental design for evaluating the properties of CD45.1+CD8+ CAR-expressing TILs and endogenous CD45.2+ TILs isolated 21 days after tumor injection and 8 days after adoptive transfer of 1.5 million CAR T cells. (Figure 3B) Left: CAR CD8 +Flow cytometry of TIL colonies and endogenous CD8+TIL colonies; CAR TIL is expressed by CD45.1+ and Thy1.1 expression encoded in CAR retroviral vectors. On the right, flow cytometry of surface expression of PD-1 and TIM3 on CAR CD8+TIL and endogenous CD8+TIL is shown. (Fig. 3C-3D) Experimental design for evaluating CD45.1+OT-1TCR transgenic TIL characteristics; other details are as (Fig. 3B). (Fig. 3E) CAR TIL and OT-1TIL percentages in total CD8+TIL are shown in histograms. The histogram shows the average values ​​of data points for 6, 5 and 11 biological replicates for CARTIL, OT-I TIL and endogenous TIL, respectively. Flow cytometry graphs represent all biological replicates. (Fig. 3F, Fig. 3G) Quantification of cytokine production after restimulation with CAR, OT-1, and endogenous CD8+ TILs compared to CD8+ T cells retrotransduced with CAR and restimulated in vitro. Cells were stimulated with PMA / ionomycin or not. (Fig. 3F) The bar graphs shown are the mean of data points with three biological replicates. All p values ​​were calculated using an unpaired t-test with Welch correction. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. (Fig. 3G) Representative flow cytometry plots of cytokine production after restimulation (data from (Fig. 3F)).

[0016] Figure 4A-4F: Gene expression and chromatin accessibility profiles of PD-1hiTIM3hi CAR CD8+TILs are similar to those of PD-1hiTIM3hi endogenous CD8+TILs. (Figure 4A) Principal component analysis (PCA) of RNA sequencing (RNA-seq) data from various populations of TILs expressing CAR (CARTIL PD-1hiTIM3hi (Figure 4A) and PD-1hiTIM3lo (Figure 4B)) and endogenous TIL PD-1hiTIM3hi (Figure 4C), PD-1hiTIM3lo (Figure 4D), PD-1loTIM3lo (Figure 4E) populations. The variance percentages of PC1 and PC2 are shown. (Figure 4B) Top, heat map of mouse CD8+T cell ATAC-seq data, showing log2-fold changes from the row means of 9 clusters determined by k-means clustering. Bottom, heat map of motif enrichment analysis. Data for a representative member of a transcription factor family that is enriched in at least one cluster compared to all accessible regions is shown. (Fig. 4C) Representative flow cytometry graphs of protein level expression of Nr4a1, Nr4a2, and Nr4a3 compared between CAR TIL PD-1hiTIM3hi (Fig. 4A) and PD-1hiTIM3lo (Fig. 4B) populations, and between endogenous TIL PD-1hi-TIM3hi (Fig. 4C), PD-1hiTIM3lo (Fig. 4D), and PD-1loTIM3lo (Fig. 4E); (Fig. 4D) Quantification of Nr4a expression levels; data show averages and individual values ​​from three independent biological assays. (Fig. 4E) Human CD8+TIL 20 Figure 4F. Single-cell RNA-seq scatter plots plotting the expression of PDCD1 and HAVCR2 in single cells (x and y axes, respectively), as well as the expression of different NR4A genes (color scale). Each dot represents a single cell. (Figure 4F) Top, PD-1hi TILs (two samples from human melanoma and one sample from non-small cell lung cancer (NSCLC) tumors) 19 ) and human CD8+ T cell ATAC-seq data from antigen-specific CD8+ T cells from HIV-infected individuals, showing log2-fold changes from row averages of 9 clusters identified by k-means clustering. Bottom, heatmap of motif enrichment analysis.

[0017] Figures 5A-5F: Tumor-bearing mice adoptively transferred with Nr4a TKO CAR CD8+ T cells lacking all three Nr4a family members exhibited higher tumor regression rates and prolonged survival compared to mice transferred with wild-type CAR CD8+ T cells. (Figure 5A) Experimental design for monitoring tumor growth; 3 million CAR T cells were adoptively transferred on day 7 after tumor inoculation. (Figure 5B) Top three panels, time course of tumor growth in individual mice, with 30 or more biological replicates per condition. At d7, n=21 for PBS, n=35 for WT, and n=39 for Nr4aTKO. Bottom, at day 21, n=14 for PBS, n=25 for WT, and n=36 for Nr4aTKO. Data show mean ± sd and individual values; p values ​​were calculated using ordinary one-way ANOVA with Tukey's multiple comparison test; *p = 0.0331, ****p < 0.0001. ( Figure 5C ) p-values ​​for survival curves were calculated using the log-rank (Mantel-Cox) test; ****p<0.0001. Number of surviving mice at day 90, n=0 for PBS; n=1 for WT; n=27 for Nr4aTKO. (Fig. 5D) Experimental design for evaluating CD8+ TIL characteristics; 1.5 million CAR T cells were adoptively transferred on day 13 after tumor inoculation. (Fig. 5E) Expression of PD-1 and TIM3 in WT and Nr4aTKO TILs 8 days after adoptive transfer. Both samples expressed CAR on cells at a set level in the CAR+NGFR+ population (10 3 -10 4 ) Gate. Top figure, representative flow cytometry graphs of PD-1 and TIM3 surface expression; middle and lower left, representative flow cytometry bar graphs of PD-1 and TIM3 expression. Means and individual values ​​of six independent experiments are shown, and each point represents TILs from 3-8 mice. Two-sided p values ​​were calculated using paired t-tests with Welch correction. ( Fig. 5F ) Cytokine production of WT and Nr4aTKO TILs on day 8 after adoptive transfer. Top, flow cytometry graphs showing TNF and IFNγ production by representative CAR+NGFR+CD8+ TILs that were not stimulated or stimulated with PMA / ionomycin. Bottom, bar graphs showing cytokine production by CARNGFR+CD8+ TILs stimulated with PMA / ionomycin on day 8 after adoptive transfer. +The output of TNF and IFNγ alone and together after T cell restimulation. No IL-2 above background was detected in both groups (not shown). The mean and individual values ​​of five independent experiments are shown, where each point represents TIL from 3-8 mice. For WT and Nr4aTKO, a paired t-test was used to calculate the bilateral p value between unstimulated samples and between stimulated samples. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

[0018] Figure 6A-6F: Gene expression and chromatin accessibility profiles of Nr4a TKO CAR CD8+ TILs show that they are similar to WT CD8 + TILs, the effector function is enhanced. (Fig. 6A) Average (MA) plot of differentially expressed genes in Nr4a TKO relative to WT CAR TILs. Differentially expressed genes (adjusted p-value <0.1 and log2-fold change ≥1 or ≤-1) are highlighted in different colors (as indicated by the color key). The selected genes are marked. (Fig. 6B) Heat map of genes with opposite expression changes between Nr4a deletion and Nr4a overexpression. The fold change values ​​(log2 ratio) of all genes differentially expressed in Nr4aTKO relative to WT CAR TILs were compared with the corresponding gene values ​​in cells ectopically expressing Nr4a1, Nr4a2, and Nr4a3. Different clusters were identified by the k-means method (k=7). Highlighted are genes that are downregulated after Nr4a loss and upregulated after Nr4a overexpression (e.g., Pdcd1, Havcr2, and Tox), as well as genes that are upregulated after Nr4a loss and downregulated after Nr4a overexpression (e.g., Tnf and Il21). (FIG. 6C) Scatter plot of pairwise comparison of ATAC-seq density (Tn5 insertions / kb). Differentially accessible regions and associated de novo identified motifs between Nr4a TKO and WT CAR TILs are shown. (FIG. 6D) On the left, a genome browser view of the Pdcd1 locus from cells transduced with CA-RIT-NFAT1, integrating all previously mentioned ATAC-seq samples. The gray bar shows the depletion-specific enhancer located approximately ~23 kb 5' to the Pdcd1 transcription start site. The upper right bar graph shows the expression of Nr4a in cells ectopically expressing HA-tagged versions of Nr4a1, Nr4a2, and Nr4a3. The lower right bar graph shows the expression of Nr4a in cells ectopically expressing HA-tagged versions of Nr4a1, Nr4a2, and Nr4a3. Enrichment of HA-tagged Nr4a by chromatin immunoprecipitation above background at the depletion-specific enhancer at 23 kb. Fig. 6E) Comparison of ATAC-seq data from Nr4a TKO and WT CAR TILs with ATAC-seq data from cells ectopically expressing CA-RIT-NFAT1, Nr4a1, Nr4a2, or Nr4a3 (top to bottom). Bottom graph is a comparison of ATAC-seq data from Nr4a TKO and WT CAR TILs with ATAC-seq data from cells stimulated with PMA / ionomycin. ( Fig. 6F ) Schematic diagram showing the proposed role of Nr4a in T cells undergoing chronic antigen stimulation.

[0019] Figures 7A-7H: Surface expression and functional evaluation of chimeric antigen receptors (CARs) reacting with human CD19 in mouse CD8+T cells. (Fig. 7A) Cell lines expressing huCD19. Left, EL4 cells; gray = parental EL4, black = EL4 expressing huCD19. Middle left, MC38 cells; gray = parental MC38, black = MC38 expressing huCD19. Middle right, B16-OVA cells; gray = parental B16-OVA, black = B16-OVA expressing huCD19. Right, B16-OVA-huCD19 cells in vivo; gray = isotype control, black = B16-OVA cells expressing huCD19, which were isolated from C57BL / 6J tumor-bearing mice and cultured for 7 days after isolation. (FIG. 7B) Left, tumor growth curves showing comparison of inoculation with 250K B16-OVA parental tumor cells or 250K B16-OVA-huCD19 tumor cells; n=15 for both groups. Ordinary two-way ANOVA showed no significant differences between the two groups at any time point. Right, tumor growth curves showing comparison of inoculation with 250K or 500K B16-OVA-huCD19 tumor cells; n=5 for 250K; n=6 for 500K. Ordinary two-way ANOVA showed significant differences between the two groups at day 21; *p=0.0146. (FIG. 7C) Schematic diagram of CAR constructs. LS=leader sequence; SS=signal sequence; myc=myc-tag; scFV=single chain variable fragment against human CD19; followed by mouse CD28, mouse CD3ζ signaling domain; and 2A self-cleaving peptide and mouse Thy1.1 reporter gene. (Fig. 7D) Surface expression of CAR monitored by expression of Thy1.1 reporter gene or myc tag. CD8+T cells transduced by simulation were used as controls. (Fig. 7E) Flow cytometry shows the cytokine production (TNF and IFNγ) of CD8+T cells expressing CAR after restimulation with EL4 cells expressing huCD19 or PMA / ionomycin. The data represent three independent biological replicates. (Fig. 7F) Bar graph of the data shown in (e); data are from three independent biological replicates; p values ​​are calculated using a two-sided unpaired t test. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. (Fig. 7G) In vitro killing assay of CD8+CAR T cells compared with CD8+T cells transduced by simulation; p values ​​are calculated using a common two-way ANOVA. (Fig. 7H) Expression of inhibitory surface receptors on CD8+T cells transduced with CAR or simulated transduced and cultured in vitro for 5 days; data represent three independent biological replicates. Grey shading = isotype control, black line = mock or CAR.

[0020] Figures 8A-8C: Adoptively transferred CAR-expressing mouse CD8+ T cells and OT-I TCR transgenic T cells infiltrating B16-OVA-huCD19 tumors exhibit a phenotype similar to that of endogenous CD8+ TILs. (Fig. 8A, Fig. 8B) Flow cytometry gating scheme for CAR (Fig. 8A) and OT-I CD8+ TILs (Fig. 8B). (Fig. 8C) Top, tumor growth curves of tumor-bearing mice adoptively transferred with CAR or OT-I CD8+ T cells; the figure is a summary of 3 independent experiments. At d=7, CARn=24, OT-In=21; at d=21, CARn=17, OT-In=20. Bottom, tumor growth curves of tumor-bearing mice adoptively transferred with CAR or PBS; the figure is a summary of 3 independent experiments. At d=7, CARn=35, PBSn=8. At d=21, CARn=35, PBSn=6. Tumor size at day 21 after tumor inoculation shows a p-value of 0.3527 for CAR compared to OT-I (top); p-value = 0.6240 for PBS compared to CAR (bottom); these values ​​were calculated using a two-sided unpaired t-test with Welch's correction.

[0021] Fig. 9 : Comparison of gene expression profiles of CAR-expressing TILs and endogenous CD8+TILs. Average plots (MA) of differentially expressed genes in the comparisons shown; differentially expressed genes (adjusted p-value < 0.1, and log2 fold change ≥ 1 or ≤ -1) are highlighted in different colors as shown in the legend. Selected genes are labeled. Top row, comparison of CAR TIL populations among themselves and with endogenous PD-1lo TIM3lo TILs; middle row, comparison within endogenous TIL populations; bottom row, comparison of CAR and endogenous PD-1hi TIM3hi TILs (left), and comparison of CAR and endogenous PD-1hi TIM3lo TILs (right).

[0022] Figures 10A-10C :Comparison of chromatin accessibility profiles of CAR CD8+TIL, endogenous CD8+TIL, and OT-I CD8+TIL. Fig. 10A ) Pairwise Euclidean distance comparison of log2-transformed ATAC-seq density (per kilobase of Tn5 inserted) across all replicates at all peaks accessible in at least one replicate. ( Fig. 10B ) Scatter plot of pairwise comparison of ATAC-seq density (per kb of Tn5 insertion) between the indicated samples. ( Fig. 10C) Genome browser view of sample loci Pdcd1 (left), Itgav (right); the scale range of all tracks is 0-1000, and the data are the average of all replicates. CD8+TIL populations are shown and defined in Figure 1B and Figure 1D: (A) PD-1hi TIM3hi CAR, (B) PD-1hi TIM3lo CAR, (C) endogenous PD-1hi TIM3hi, (D) endogenous PD-1hi TIM3hi, (E) endogenous PD-1lo TIM3hi.

[0023] Figures 11A-11B : Endogenous mouse CD8+T cells and adoptively transferred CAR-expressing mouse CD8+T cells infiltrating B16-OVA-huCD19 tumors exhibit elevated levels of Nr4a. (FIG. 11A) Flow cytometry gating scheme for CAR (left) and endogenous (right) CD8+TILs. (FIG. 11B) Representative flow cytometry bar graphs of Nr4a protein in PD-1hi TIM3hi TILs, PD-1hi TIM3lo TILs, and PD-1loTIM3lo TILs and their corresponding fluorescent-1 controls (off-white).

[0024] Figures 12A-12C: Scatter plots of single cell RNA-seq of human CD8+ TILs. Plotting the expression of PDCD1 and HAVCR2 in single cells (x and y axes, respectively), and (shown in color scale) the expression of genes differentially upregulated in PD-1hiTIM3hi TILs relative to PD-1lo TIM3lo TILs (Figure 12A). (Figure 12B) Genes differentially downregulated in PD-1hi TIM3hi TILs relative to PD-1lo TIM3lo TILs. ( Fig. 12C ) Genes encoding selected transcription factors showed differential expression in the comparison of PD-1hi TIM3hi TILs relative to PD-1hi TIM3lo TILs. Each dot represents a single cell. Human CD8+ TIL data from 20 .

[0025] Figures 13A-13C : Generate strong dual transduction efficiency of WT and Nr4a TKO CAR T cells for adoptive transfer. (FIG. 13A) CD8a-only stained CAR T cell control (previously tested to be identical to Fluo-1 control in CAR+ expression and NGFR+ expression) before adoptive transfer. (FIG. 13B) CAR and NGFR expression of CD8+WT CAR T cells before adoptive transfer. (FIG. 13C) CAR and NGFR expression of CD8+Nr4a TKO CAR T cells before adoptive transfer.

[0026] Figures 14A-14D: Tumor-bearing mice adoptively transferred with CAR CD8+T cells lacking three Nr4a family members exhibited longer survival compared to mice transferred with wild-type CAR CD8+T cells or CAR CD8+T cells lacking only one of the three Nr4a family members. (Figure 14A) Experimental design for monitoring tumor growth; 3 million CAR T cells were adoptively transferred on day 7 after tumor inoculation. (Figure 14B) Time course of tumor growth in individual mice bearing B16-OVA-huCD19 tumors, each consisting of 17 or more biological replicates (these data include WT and Nr4a TKO data from Figure 3). At d7, n=31 for PBS mice; n=35 for WT; n=17 for Nr4a1 KO; n=17 for Nr4a2 KO; n=32 for Nr4a3- / -; n=39 for Nr4a TKO. At day 21, the number of mice was n=14 for PBS, n=25 for WT, n=12 for Nr4a1 KO, n=15 for Nr4a2 KO, n=22 for Nr4a3- / -, and n=36 for Nr4a TKO. (FIG. 14A) The mean ± sd and individual values ​​of B16-OVA-huCD19 tumor size at day 21 after inoculation are shown. The p value was calculated using ordinary one-way ANOVA with Tukey's multiple comparison test; PBS vs WT, *p=0.0395; WT vs Nr4a1 KO, p=ns=0.0511; WT vs Nr4a2 KO, **p=0.002, WT vs Nr4a3- / -, *p=0.0161; WT vs Nr4a TKO, ****p<0.0001. (d) Survival curve of mice. p values ​​for survival curves were calculated using the log-rank (Mantel-Cox) test; ****p<0.0001. Number of mice at d90 for PBS n=0; for WT, n=1; for Nr4a1 KO, n=0; for Nr4a2 KO, n=1; for Nr4a3- / -, n=11; for Nr4a TKO, n=27.

[0027] Figures 15A-15D : Ectopic expression of Nr4a1, Nr4a2, and Nr4a3 in mouse CD8+ T cells results in reduced cytokine production and increased expression of inhibitory surface markers. Mouse CD8+ T cells were isolated, activated, and transduced with empty vector or HA-tagged Nr4a1, Nr4a2, or Nr4a3 vectors with a human NGFR reporter gene. Cells were assayed on day 5 after activation. Fig.15A) Flow cytometry gating was performed on CD8+NGFR+ empty vector control cells, cells expressing Nr4a1, Nr4a2, and Nr4a3 based on the constant expression level of the NGFR reporter gene. ( Fig. 15B ) Quantification of surface receptor expression (data from three independent biological replicates). Fig. 15C ) Top, representative flow cytometry plots of cytokine production upon restimulation with PMA / ionomycin; bottom, quantification of cytokine production after restimulation (data from three independent biological replicates). All p values ​​were calculated using ordinary one-way ANOVA with Dunnett's multiple comparison test; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. ( Fig.15D ) Quantification of transcription factors (data from three independent biological replicates).

[0028] Figures 16A-16B : Comparison of gene expression profiles of mouse CD8+T cells expressing Nr4a1, Nr4a2, and Nr4a3 ectopically in vitro. (Figure 16A) Principal component analysis (PCA) of RNA sequencing (RNA-seq) data from mouse CD8+T cells expressing Nr4a1, Nr4a2, Nr4a3 and empty vector controls in vitro. The percentage of variance of PC1 and PC2 is shown. (Figure 16B) Average mean (MA) plots of differentially expressed genes in comparisons of Nr4a1, Nr4a2 or Nr4a3 ectopic expression with empty vectors (top row), and paired comparisons between ectopic expression of various Nr4a family members (bottom row). As shown in the PCA plot in (a), different colors are used to highlight differentially expressed genes (adjusted p-value <0.1 and log2 fold change ≥1 or ≤-1). Selected genes are marked.

[0029] Fig.17 : Comparison of chromatin accessibility profiles of mouse CD8+ T cells ectopically expressing Nr4a1, Nr4a2, and Nr4a3 in vitro. Scatter plots of pairwise comparisons of ATAC-seq density (per kb Tn5 insertion) between the samples shown.

[0030] Figures 18A-18D : From adoptively transferred Nr4a TKO CAR CD8 cells lacking all three Nr4a family members + T cells or WT CAR CD8 +Phenotype of TIL isolated from tumor-bearing mice with T cells. (Figure 18A) On day 13 after tumor inoculation, delayed tumor growth of 1.5 million CAR T cells adoptively transferred; TILs for phenotypic analysis were isolated from these tumors. Tumor growth curves of Rag1- / - mice adoptively transferred with WT and Nr4aTKO CAR CD8+T cells; at d=7, WT=47 and Nr4aTKO=41; at d=21, WT=35 and Nr4aTKO=32. The p value was calculated using an ordinary two-way ANOVA with Tukey's multiple comparison test; for WT vs Nr4aTKO, p value=ns=0.6908. (Figure 18B) Flow cytometry gating scheme for the determination of surface markers, cytokines, and transcription factors expressed by WT (top) and Nr4aTKO (bottom) TILs. CAR expression (10 3 -10 4 ) was gated. (FIG. 18C) Histogram of TIL counts and MFI of Ki67 for WT and Nr4aTKO CAR TILs. (FIG. 18D) Top, histogram of transcription factor expression for WT and Nr4aTKO CAR TILs. Bottom, representative flow cytometry graphs of TIM3 and TCF1 expression in WT and Nr4aTKO CAR TILs. Two-sided p values ​​were calculated using paired t-tests. *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

[0031] Figures 19A-19C: Gene expression of Nr4a TKO CAR CD8+TILs indicates enhanced effector function compared to WT CAR CD8+TILs. (Figure 19A) Principal component analysis (PCA) of RNA sequencing (RNA-seq) data from Nr4a TKO CAR TILs or WT CAR TILs. Percent variance of PC1 and PC2 is shown. (Figure 19B) Normalized enrichment scores (NES) of gene sets defined by effector, memory, and exhausted CD8+T cells from LCMV-infected mice 17 . ( Fig.19C ) Gene set enrichment analysis of RNA-seq data of Nr4a TKO CAR TILs and WT CAR TILs, with genes ranked by log2 fold change between these conditions.

[0032] Figures 20A-20B: Nr4a family members bind to the predicted Nr4a binding motif, which is more accessible in WT CAR TILs than Nr4a TKO CAR TILs. (Figure 20A) Top right, representative bar graphs show Nr4a expression in cells expressing ectopically HA-tagged versions of Nr4a1, Nr4a2, and Nr4a3. In the middle, genome browser views of the Ccr7, Ccr6, and Ifng loci of WT CAR TILs compared to Nr4aTKO CAR TILs, including binding motifs for NFAT, Nr4a (Nur77), bZIP, NFκB, and the positions of the qPCR primers used. The scale range for all channels is 0-1000, and the data are the averages of all replicates. On the right, the bar graph shows the enrichment of Nr4a in the detected area. (FIG. 20B) Genome browser view of the Il21 (left), Tnf (right) loci binding to WT CAR TILs compared to Nr4a TKO CAR TILs, including binding motifs for NFAT, Nr4a (Nur77), bZIP, and NFκB. The scale for all tracks ranges from 0-1000, and the data are the average of all replicates.

[0033] Fig.21 : Nr4a family members showed moderate reduction in mRNA expression in antigen-specific cells from LCMV-infected mice treated with anti-PDL1 or IgG control. The mean (MA) plot of differentially expressed genes in cells treated with anti-PDL1 compared to cells treated with IgG control highlights two different types of differentially expressed genes: adjusted p-value <0.1 and log2 fold change ≥0.5 or ≤-0.5 (light color); and adjusted p-value <0.1 and log2 fold change ≥1 or ≤-1 (darker color). The selected genes are labeled. Shown is the number of genes in each category. The sequencing data in this analysis were obtained from 29 Obtained.

[0034] Figures 22A-22D: Immunocompetent tumor-bearing mice adoptively transferred Nr4a TKO CAR CD8+T cells lacking all three Nr4a family members showed prolonged survival compared to mice transferred WT CAR CD8+T cells. (Figure 22A) Experimental design for monitoring tumor growth; 6 million CAR T cells were adoptively transferred on day 7 after tumor inoculation. (Figure 22B) The three figures on the left are the time course of tumor growth in individual mice bearing B16-OVA-huCD19 tumors, each containing 13-15 biological replicates. On day 7, the number of mice was n=13 for PBS, n=15 for WT, and n=14 for Nr4a TKO. On day 21, the number of mice was n=11 for PBS, n=11 for WT, and n=13 for Nr4a TKO. The three right panels are time courses of tumor growth for individual mice bearing MC38-huCD19 tumors, each condition containing 10 biological replicates. At d7, the number of mice was n=10 for PBS, n=10 for WT, and n=10 for Nr4a TKO. At day 19, the number of mice was n=9 for PBS, n=7 for WT, and n=10 for Nr4a TKO. (FIG. 22C) Left, the mean ± sd and individual values ​​of B16-OVA-huCD19 tumor size at day 21 post-inoculation are shown; p values ​​were calculated using ordinary one-way ANOVA with Tukey's multiple comparison test, which did not show any significant differences. Right, the mean ± sd and individual values ​​of MC38-huCD19 tumor size at day 19 post-inoculation are shown; p values ​​were calculated using ordinary one-way ANOVA with Tukey's multiple comparison test; *p=0.012, ***p=0.0001. (FIG. 22D) Survival curves of mice bearing B16-OVA-huCD19 tumors (left) and MC38-huCD19 tumors (right). The p-values ​​for the survival curves were calculated using the log-rank (Mantel-Cox) test, ****p<0.0001. For mice bearing B16-OVA-huCD19 tumors at 90 days, for PBS, number of mice n=0; for WT, n=0; for Nr4a TKO n=2; *p=0.0026. For mice bearing MC38-huCD19 tumors, all mice died at d23; *p=0.0138.

[0035] Figures 23A-23C: In solid tumors, Tox and PD-1 are highly expressed in CAR-T cells. Experimental protocol: 12 days after injection of B16-huCD19 tumors, CAR-T cells were adoptively transferred, and TIL expressing CD45.1+CD8+CAR was isolated at 16, 20 and 25 days. PD-1 and TOX expression levels were analyzed by flow cytometry from CAR-T cells (day 12: before transfer) and TIL expressing CAR (days 16, 20 and 24) (Figure 23A-C).

[0036] Fig.24 : C57BL / 6N mice bearing B16-huCD19 tumors adoptively transferred with TOX / TOX2-deficient CAR-T cells had suppressed tumor growth and prolonged survival compared with mice transferred with WT CAR-T cells. Three million CAR-T cells were adoptively transferred on day 7 after tumor inoculation. Tumor size was measured by caliper every 2 days.

[0037] Fig.25 : Time course of tumor growth and survival curves in individual mice.

[0038] Fig.26 : Experimental protocol: CAR-T cells lacking TOX and TOX2 or WT CAR T cells were adoptively transferred on day 12 after injection of B16-huCD19 tumors, and TILs expressing CD45.1+CD8+CAR were isolated on day 24, and PD-1, Tim-3, Lag-3, and CD160 expression levels were analyzed by flow cytometry from CAR-expressing TILs.

[0039] Fig. 27 : Experimental protocol: CAR-T cells lacking TOX and TOX 2 or WT CAR T cells were adoptively transferred 12 days after injection of B16-huCD19 tumors, and TILs expressing CD45.1+CD8+CAR were isolated on day 24, and IFN-γ and TNF-α expression levels were analyzed by flow cytometry from CAR-expressing TILs after PMA / ionomycin stimulation (4 hours).

[0040] Fig.28 : Experimental protocol: CAR-T cells lacking TOX and TOX 2 or WT CAR T cells were adoptively transferred on day 12 after injection of B16-huCD19 tumors, and TILs expressing CD45.1+CD8+CAR were isolated on day 24, and T-bet and Eomes expression levels were analyzed by flow cytometry from CAR-expressing TILs.

[0041] Fig.29: RAG1- / - mice bearing B16-huCD19 tumors adoptively transferred with TOX / TOX2-deficient CAR-T cells had suppressed tumor growth and prolonged survival compared with mice transferred with WT CAR-T cells. Three million CAR-T cells were adoptively transferred on day 7 after tumor inoculation. Time course of tumor growth and survival curves in individual mice. Tumor size was measured by caliper every 2 days.

[0042] Fig.30 : Plasmid map of plasmid JC31 carrying the CAR-2A-Thy 1.1 construct.

[0043] Fig.31 : Plasmid sequence of plasmid JC31 carrying the CAR-2A-Thy 1.1 construct, with the following elements: MESV between 579 and 920, gag (truncated) between 987 and 1403, Kozak sequence between 1416 and 1425, Myc between 1485 and 1514, CAR between 1515 and 2900, P2A between 2901 and 2957, mouse Thy 1.1 between 2958 and 3446, 3'LTR between 3496 and 3010, M13 between 4179 and 4195 rev, the lac operator between 4203 and 4219, the lac promoter between 4220 and 4271, the CAP binding site between 4272 and 4293, the ori between 4581 and 5169, the AmpR between 5340 and 6200, and the AmpR promoter between 6201 and 6305. Detailed description

[0044] It should be understood that the present invention is not limited to the specific aspects described, as they may, of course, vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0045] A number of embodiments of the present invention have been described. However, it should be understood that various modifications may be made without departing from the spirit and scope of the present invention. Therefore, the following examples are intended to illustrate rather than limit the scope of the disclosure described in the claims.

[0046] In the absence of explicit recitation it should be inferred, and unless otherwise intended, that when the technology relates to polypeptides, proteins, polynucleotides or antibodies, equivalents or biological equivalents thereof are intended to be included within the scope of the technology.

[0047] Throughout the invention, various publications, patents, and published patent specifications are referenced by explicit reference. Complete bibliographic information for the references can be found immediately preceding the claims. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, just as if each document were individually incorporated by reference. In the event of a conflict, the present specification (including definitions) controls.

[0048] Each patent, patent application, publication or any other reference or document cited herein is hereby incorporated by reference in its entirety. In the event of conflict, the specification, including definitions, will control.

[0049] The citation of any patent, patent application, publication or any other document does not constitute an admission that any of the above is relevant prior art nor does it constitute an admission of the contents or date of such publication or document.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0051] All features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature having the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, the disclosed features (e.g., antibodies) are an example of a genus of equivalent or similar features.

[0052] As used herein, unless the context clearly indicates otherwise, all numerical values ​​or numerical ranges include integers within such ranges and fractions of numerical values ​​or integers within such ranges. In addition, when a list of numerical values ​​is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%), the list includes all intermediate values ​​and fractional values ​​thereof (e.g., 54%, 85.4%). Thus, for example, reference to 80% or more identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., and 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc., and so on.

[0053] Reference to an integer greater than (more than) or less than includes any number greater than or less than the referenced number, respectively. Thus, for example, reference to less than 100 includes 99, 98, 97, etc., up to the number one (1); and less than 10 includes 9, 8, 7, etc., up to the number one (1).

[0054] As used herein, all values ​​or ranges include values ​​and fractions of integers and fractions of integers within such ranges, unless the context clearly indicates otherwise. Thus, for illustration, reference to a numerical range, such as 1-10, includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 1.1, 1.2, 1.3, 1.4, 1.5, etc., and so on. Thus, reference to a range of 1-50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to and including 50, and 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., and so on.

[0055] Reference to a series of ranges includes ranges that constitute the boundaries of the different ranges within the series. Thus, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,000, 3,000-4,000, 5,000-6,000, 6,000-7,000, 7,000-8,000, 8,000-9,000, 9,000-10,000-11,000, 9,000-12,000, 9,000-13,000, 9,000-14,000, 9,000-15,000, 9,000-1 Ranges of 500, 2,500-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000 include ranges of 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000, and the like.

[0056] The foregoing may be modified without departing from the basic aspects of the present technology. Although the present technology has been described in basic detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and that such modifications and improvements are within the scope and spirit of the present technology.

[0057] Affirmative language is generally used herein to disclose the present invention to describe a large number of embodiments and aspects. The present invention also specifically includes embodiments in which a particular subject matter is excluded in whole or in part, such as substances or materials, method steps and conditions, schemes or procedures. For example, in certain embodiments or aspects of the present invention, materials and / or method steps are excluded. Therefore, even if the present invention is generally not expressed in this article in a way that the present invention does not include which aspects, aspects that are not explicitly excluded in the present invention are still disclosed herein.

[0058] The technology described illustratively herein can be suitably put into practice in the absence of any element not specifically disclosed herein.Therefore, for example, in each case herein, any one of the terms "comprising", "consisting essentially of ... " and "consisting of ... " can be replaced by any one of the other two terms.The terms and expressions adopted are used as descriptive terms, rather than restrictive, and the use of such terms and expressions does not exclude any equivalent form of the features shown and described or its fragments, and various modifications can be made within the scope of the present technology.The term "one" or "one" can refer to one or more (for example, "a reagent" can represent one or more reagents) in the element modified by it, unless the context clearly describes one or more than one in the element.As used herein, the term "about" refers to a value within 10% of the basic parameter (that is, plus or minus 10%), and the term "about" is used at the beginning of a string of values ​​to modify each of these values ​​(that is, "about 1, 2 and 3" refers to about 1, about 2 and about 3).For example, the weight of "about 100 grams" can include the weight between 90 grams and 110 grams. As used herein, the term "substantially" refers to a value modifier, which means "at least 95%, "at least 96%, "at least 97%, "at least 98%", or "at least 99%", and may include 100%. For example, a composition that is substantially free of X may contain less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of X, and / or X may not be present or cannot be detected in the composition.

[0059] Therefore, it should be understood that although the present technology has been specifically disclosed through representative embodiments and optional features, modifications and variations of the concepts disclosed herein may be made by those skilled in the art and such modifications and variations may be considered within the scope of the technology. definition

[0060] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an NR4A transcription factor" or "an TOX transcription factor" includes a plurality of such NR4A or TOX transcription factors.

[0061] As used herein, the term "comprising" is intended to mean that the composition or method includes the recited steps or elements, but not excluding others. "Consisting essentially of" shall be meant to make the claim open-ended only to those steps or elements that do not materially affect the basic and novel characteristics of the claimed composition and method. "Consisting of" shall be meant to exclude any element or step not specified in the claim. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0062] As used herein, the term "about" is used to indicate a value that includes the standard deviation of error for the device or method used to determine the value. When used before a numerical designation that includes a range (e.g., temperature, time, amount, and concentration), the term "about" indicates that the approximate value may vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1%.

[0063] "Eukaryotic cells" include all kingdoms of life except protozoa. They can be easily distinguished by the membrane-bound nucleus. Animals, plants, fungi and protists are eukaryotic organisms or organisms whose cells are organized into complex structures by endomembranes and cytoskeleton. The most typical membrane-bound structure is the nucleus. Unless specifically stated, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plants, insects and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include monkeys, cattle, pigs, mice, rats, birds, reptiles and humans.

[0064] As used herein, "population of cells" means a collection of more than one cell that is phenotypically and / or genotypically identical (clonal) or different.

[0065] As used herein, a "substantially homogeneous" cell population is one that has at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% of the same phenotype as determined by a preselected marker, phenotype, or genomic trait. In one aspect, the colony is a clonal colony.

[0066] As used herein, a "heterogeneous" population of cells is a population having up to 69%, or up to 60%, or up to 50%, or up to 40%, or up to 30%, or up to 30%, or up to 20%, or up to 10%, or up to 5%, or up to 4%, or up to 3%, or up to 2%, or up to 61%, or up to 60%, or up to 0.5% of the same phenotype as determined by a pre-selected marker, phenotype or genomic trait.

[0067] As used herein, "treating" a disease in a subject refers to (1) preventing symptoms or disease from occurring in a subject susceptible to or not yet showing symptoms of the disease; (2) inhibiting a disease or preventing its development; (3) ameliorating or causing regression of a disease or a symptom of a disease. As understood in the art, "treatment" is a method for obtaining a beneficial or desired result (including a clinical result). For the purposes of the present technology, whether detectable or undetectable, a beneficial or desired result may include (but is not limited to) one or more of the following: alleviating or ameliorating one or more symptoms, reducing the extent of a condition (including a disease), stabilizing (i.e., not worsening) the state of a condition (including a disease), delaying or slowing the condition (including a disease), progression, improving and slowing the state of a condition (including a disease), and alleviating (whether partially or completely). Therapies containing the disclosed compositions and methods may be first-line, second-line, third-line, fourth-line, fifth-line therapies, and are intended to be used as a single therapy or in combination with other suitable therapies. In one aspect, treatment excludes prevention.

[0068] The phrases "first-line" or "second-line" or "third-line" refer to the order in which a patient receives treatment. A first-line treatment regimen is the first treatment given, while a second-line or third-line treatment is given after the first-line treatment or after the second-line treatment, respectively. The National Cancer Institute defines first-line therapy as "the first treatment for a disease or condition. In patients with cancer, the primary treatment may be surgery, chemotherapy, radiation therapy, or a combination of these therapies. For those skilled in the art, first-line therapy is also referred to as "primary treatment and primary therapy." See the National Cancer Institute's website at www.cancer.gov, last visited on May 1, 2008. Typically, a subsequent chemotherapy regimen is given to a patient because the patient has not shown a positive clinical or subclinical response to the first-line treatment or the first-line treatment has been stopped.

[0069] As used herein, "anti-tumor immunity" in a subject refers to the reduction or prevention of symptoms or development of cancer in a subject susceptible to or not yet manifesting cancer.

[0070] As used herein, providing "immunity to pathogen infection" in a subject refers to preventing the onset of symptoms or pathogen infection in a subject susceptible to or not yet showing symptoms of pathogen infection. In one aspect, immunity reduces or decreases the course or severity of symptoms and the duration of infection.

[0071] In some embodiments, the subject is in need of a treatment, cell, or composition described herein. In certain embodiments, the subject has or is suspected of having a neoplastic condition, a neoplasia, a tumor, a malignancy, or a cancer. In certain embodiments, the subject is in need of a treatment, cell, or composition described herein has or is suspected of having a neoplastic condition, a neoplasia, a tumor, a malignancy, or a cancer. In certain embodiments, the engineered T cells described herein are used to treat a subject having or suspected of having a neoplastic condition, a neoplasia, a tumor, a malignancy, or a cancer.

[0072] In some embodiments, provided herein are methods of treating a subject having or suspected of having a neoplasia, a neoplastic condition, a tumor, a cancer, or a malignant tumor. In certain embodiments, a method of treating a subject comprises administering to the subject a therapeutically effective amount of an engineered T cell. In certain embodiments, the method comprises reducing or inhibiting the proliferation of a neoplastic cell, a tumor, a cancer, or a malignant tumor cell, comprising contacting the cell, tumor, cancer, or malignant tumor cell with an engineered T cell in an amount sufficient to reduce or inhibit the proliferation of the neoplastic cell, a tumor, a cancer, or a malignant tumor cell.

[0073] In some embodiments, a method of reducing or inhibiting the metastasis of a neoplasia, tumor, cancer or malignancy to other sites or the formation or establishment of a metastatic neoplasia, tumor, cancer or malignancy at other sites distant from a primary neoplasia, tumor, cancer or malignancy comprises administering to a subject an amount of engineered T cells sufficient to reduce or inhibit the metastasis of a neoplasia, tumor, cancer or malignancy to other sites or the formation or establishment of a metastatic neoplasia, tumor, cancer or malignancy at other sites distant from a primary neoplasia, tumor, cancer or malignancy.

[0074] Non-limiting examples of neoplasia, neoplastic conditions, tumors, cancers or malignancies include carcinoma, sarcoma, neuroblastoma, cervical cancer, hepatocellular carcinoma, mesothelioma, glioblastoma, myeloma, lymphoma, leukemia, adenoma, adenocarcinoma, glioma, glioblastoma, retinoblastoma, astrocytoma, oligodendroglioma, meningioma or melanoma. Neoplasia, neoplastic conditions, tumors, cancers or malignancies may include or involve hematopoietic cells. Non-limiting examples of sarcomas include lymphosarcoma, liposarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma or fibrosarcoma. In some embodiments, the neoplasia, neoplastic condition, tumor, cancer or malignancy is a myeloma, lymphoma or leukemia. In some embodiments, the neoplasia, neoplastic condition, tumor, cancer, or malignancy comprises a neoplasia, tumor, or cancer of the lung, thyroid, head or neck, nasopharynx, larynx, nose or sinuses, brain, spine, chest, adrenal gland, pituitary gland, thyroid gland, lymphatic, gastrointestinal tract (mouth, esophagus, stomach, duodenum, ileum, jejunum (small intestine), colon, rectum), genitourinary tract (uterus, ovary, cervix, endometrium, bladder, testis, penis, prostate), kidney, pancreas, liver, bone, bone marrow, lymphatic, blood, muscle, or skin. In some embodiments, the neoplasia, neoplastic condition, tumor, cancer, or malignancy comprises small cell lung cancer or non-small cell lung cancer. In some embodiments, the neoplasia, neoplastic condition, tumor, cancer, or malignancy comprises a stem cell neoplasia, tumor, cancer, or malignancy. In some embodiments, the neoplasia, neoplastic condition, tumor, cancer, or malignancy.

[0075] In some embodiments, a method inhibits or reduces the recurrence or progression of neoplasia, neoplastic conditions, tumors, cancers or malignancies. In some embodiments, the method comprises administering an anti-cell proliferation, anti-neoplastic, anti-tumor, anti-cancer or immune enhancing treatment or therapy. In some embodiments, the treatment method results in partial or complete destruction of neoplasia, tumor, cancer or malignant cell masses; reduction in cell volume, size or number of neoplasia, neoplastic conditions, tumors, cancers or malignant tumor masses; stimulation, induction or increase in necrosis, dissolution or apoptosis of neoplasia, tumor, cancer or malignant tumor cells; reduction of neoplasia, tumor, cancer or malignant tumor cell masses; inhibition or prevention of progression or increase in volume, mass, size or cell number of neoplasia, tumor, cancer or malignant tumors; or extension of life span. In some embodiments, the treatment method results in a reduction or decrease in the severity, duration or frequency of adverse symptoms or complications associated with or caused by neoplasia, tumor, cancer or malignancy. In some embodiments, the treatment method results in a reduction or decrease in pain, discomfort, nausea, weakness or drowsiness. In some embodiments, the treatment method results in increased energy, appetite, improved mobility or mental health.

[0076] The term "contacting" means direct or indirect binding or interaction between two or more entities (e.g., between a target cell population and a T cell engineered to reduce or eliminate the expression and / or function of the NR4A transcription factor in the cell). A specific example of a direct interaction is binding. A specific example of an indirect interaction is one entity acting on an intermediate molecule, which in turn acts on a second mentioned entity. Contacting as used herein includes in solution, in a solid phase, in vitro, ex vivo, in a cell, and in vivo. In vivo contacting can be referred to as administration or dosing.

[0077] As used herein, the term "binding" or "antibody binding" or "specific binding" means that the antigen binding domain of an antibody, antibody fragment, CAR, TCR, engineered TCR, BCR, MHC, immunoglobulin-like molecule, scFv, CDR or other antigen presenting molecule binds to an antigen, epitope or peptide with a specific binding affinity of less than 10. -5 The binding affinity (K D ) contact. In some aspects, the antigen binding domain binds to the complex of the antigen and the MHC molecule. In some aspects, the antigen binding domain is about less than 10 -6 M, 10 -6 M, and preferably 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M. In certain aspects, specific binding refers to the binding of an antigen to an MHC molecule, or the binding of an antigen binding domain of an engineered T cell receptor to an antigen or an antigen-MHC complex.

[0078] As used herein, the term "exhausted T cells" or "exhausted CAR T cells" refers to low-responsiveness triggered by chronic antigen stimulation. 7 (“Exhaustion” 8-11 or "dysfunctional" 12,13 ) state, characterized by upregulation of several inhibitory receptors and loss of effector function 14,15 .

[0079] As used herein, the term "administering" or "administering" is used to refer to the introduction of a therapeutic agent (e.g., a polynucleotide, a vector, a cell, a modified cell, a colony) into a subject. The therapeutic administration of the substance is used to alleviate any symptoms, or prevent the occurrence of other symptoms. When the administration is for the purpose of preventing or reducing the possibility of the development of an autoimmune disease or condition, the substance may be provided before any visible or detectable symptoms occur. Routes of administration include, but are not limited to, oral (e.g., tablets, capsules, or suspensions), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural, and intrathecal.

[0080] As used herein, the term "expression" refers to the process in which a polynucleotide is transcribed into mRNA and / or the process in which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. On the one hand, the expression level of a gene from a sample can be directly compared with the expression level of the gene from a control or reference sample. On the other hand, after applying the compound, the expression level of a gene from a sample can be directly compared with the expression level of the gene from the same sample. When used in the context of gene expression, the terms "up-regulation" and "down-regulation" and their deformations are respectively generally referred to as normal or expected threshold values ​​relative to cells (especially subtypes of cells) to increase and reduce gene expression.

[0081] As used herein, the term "gene expression profiling" refers to measuring the expression levels of multiple genes to create an expression profile of a particular sample.

[0082] As used herein, the term "reducing or eliminating ... expression and / or function" refers to reducing or eliminating the transcription of the polynucleotide to mRNA, or reducing or eliminating the translation of the mRNA to a peptide, polypeptide or protein, or reducing or eliminating the function of the peptide, polypeptide or protein. In a non-limiting example, the transcription of the polynucleotide to mRNA is reduced to at least half of its normal level found in wild-type cells.

[0083] As used herein, the term "increase the expression of..." refers to increasing the transcription of the polynucleotide to mRNA, or increasing the translation of the mRNA to a peptide, polypeptide or protein, or increasing the function of the peptide, polypeptide or protein. In a non-limiting example, the transcription of the polynucleotide to mRNA is increased to at least twice its normal level found in wild-type cells.

[0084] As used herein, the term "transcription factor" refers to a polypeptide capable of sequence-specific interaction with a portion of a gene or gene regulatory region. The interaction can be direct sequence-specific binding, in which the transcription factor directly contacts the nucleic acid; or indirect sequence-specific binding mediated or promoted by other auxiliary proteins, in which the transcription factor is bound to the nucleic acid by direct nucleic acid binding proteins. In addition, some transcription factors exhibit inductive or synergistic binding. Transcription factors affect the level of gene transcription. On the one hand, transcription factors upregulate or increase gene expression. On the other hand, transcription factors downregulate or reduce gene expression.

[0085] As used herein, the term "NR4A transcription factor" refers to a member of the NR4A subfamily of nuclear hormone receptors that bind to DNA and regulate gene expression. Non-limiting examples of members of the NR4A transcription factor family are human NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (NOR1), encoded by the sequences provided by SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

[0086] As used herein, the term "TOX transcription factor" refers to a member of the TOX subfamily of nuclear hormone receptors that bind to DNA and regulate gene expression. Non-limiting examples of TOX transcription factor family members are human TOX1, TOX2, TOX3 and TOX4 encoded by the sequences provided by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0087] As used herein, the term "IL-21" (interleukin 21) refers to a member of the common gamma chain family of cytokines with immunomodulatory activity. A non-limiting example is human IL-21 encoded by the sequence provided by SEQ ID NO: 10.

[0088] As used herein, the term "inhibiting the expression and / or function of the NFAT / AP-1 pathway" refers to reducing or eliminating the transcription of genes in the pathway, or reducing or eliminating the translation of the mRNA into a pathway peptide, polypeptide, or protein, or reducing or eliminating the function of the peptide, polypeptide, or protein of the pathway. Non-limiting examples of inhibiting the expression and / or function of the NFAT / AP-1 pathway include: inhibiting the expression and / or function of NR4A transcription factor or TOX transcription factor, or increasing the expression of IL-21.

[0089] An "effective amount" is an amount sufficient to achieve the intended purpose (non-limiting examples of which include: inducing an immune response, regulating an immune response, inhibiting an inflammatory response, and regulating T cell activity or T cell populations). In one aspect, an effective amount is an amount that acts to achieve the therapeutic purpose, such as a therapeutically effective amount. As described in detail herein, the effective amount or dosage depends on the purpose and composition, and can be determined according to the present invention.

[0090] As used herein, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes (such as B cells) by the presence of T cell receptors on the cell surface. T cells can be isolated or obtained from commercial channels. "T cells" include all types of immune cells expressing CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg) and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells and neutrophils, which are capable of mediating cytotoxic reactions. Non-limiting examples of commercially available T cell lines include: BCL2 (AAA) Jurkat ( CRL-2902 TM )、BCL2(S70A)Jurkat( CRL-2900 TM )、BCL2(S87A)Jurkat( CRL-2901 TM )、BCL2 Jurkat( CRL-2899 TM )、Neo Jurkat( CRL-2898 TM), TALL-104 cytotoxic human T cell line (ATCC #CRL-11386). Additional examples include, but are not limited to, mature T cell lines, such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T cell lines, such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13. MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to T14, TALL-1, TALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3 and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4; 11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and skin T-cell lymphoma cell lines, such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162). Leukemia-free cell lines, including but not limited to REH, NALL-1, KM-3, L92-221, are another commercially available source of immune cells, as well as cell lines derived from other leukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (or ATCC) (http: / / www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).

[0091] As used herein, the term "engineered T cell receptor" refers to a molecule comprising the following elements: (a) an extracellular antigen binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some aspects, the engineered T cell receptor is a genetically modified TCR, a modified TCR, a recombinant TCR, a transgenic TCR, a partial TCR, a chimeric fusion protein, a CAR, a first generation CAR, a second generation CAR, a third generation CAR, or a fourth generation TRUCK. In some aspects, the engineered T cell receptor comprises an antibody or an antibody fragment. In a specific aspect, the engineered T cell receptor is a genetically modified TCR or CAR.

[0092] As used herein, the term "receptor" or "T cell receptor" or "TCR" refers to a cell surface molecule found on T cells, whose function is to recognize and bind to antigens presented by antigen presenting molecules. Typically, TCR is a heterodimer of an alpha chain (TRA) and a beta chain (TRB). Some TCRs consist of alternative gamma (TRG) chains and delta (TRD) chains. T cells expressing this version of TCR are called gamma delta T cells. TCR is part of the immunoglobulin superfamily. Therefore, like antibodies, each chain of TCR contains three hypervariable CDR regions. There is also a hypervariable region (HV4) on the beta chain. TCR heterodimers are typically present in an octamer complex, which also contains three dimer signaling modules CD3γ / ε, CD3δ / ε, and CD247ζ / ζ or ζ / η. A non-limiting exemplary amino acid sequence of a human TCR-alpha chain: METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCAPVLSGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS. Non-limiting exemplary amino acid sequence of a human TCR-beta chain: DSAVYLCASSLLRVYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPPEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQP.

[0093] The term "modified TCR" refers to a genetically engineered TCR, and / or a transgenic TCR, and / or a recombinant TCR. Non-limiting examples of modified TCRs include single-chain VαVβTCRs (scTv), full-length TCRs produced using a T cell display system, and TCRs in which the CDR regions have been engineered to recognize specific antigens, peptides, fragments, and / or MHC molecules. Methods for developing and engineering modified TCRs are known in the art. See, for example, Stone, J.D. et al. Methods in Enzymology 503: 189-222 (2012), PCT application WO2014018863A1.

[0094] Type 1 T regulation (T R 1) Cells are a subset of CD4+ T cells that have regulatory properties and are able to suppress antigen-specific immune responses in vitro and in vivo. R 1 cells are defined by their unique cytokine profile and produce high levels of IL-10 and TGF-β, but not IL-4 or IL-2. IL-10 and TGF-β produced by these cells mediate suppression of primitive naive T cells in vitro. There is also evidence that T R Cells exist in vivo, and it has been documented that CD4(+) T cells producing high levels of IL-10 are present in patients with severe combined immunodeficiency who have received allogeneic stem cell transplantation. R 1 cells are involved in the regulation of peripheral tolerance and they may be used as cell therapy to regulate immune responses in vivo. See, for example, Levings, M. et al. J. Allergy Clin. Immunol. 106(1Pt2):S109-12 (2000).

[0095] T R Tr1 cells are defined by their ability to produce high levels of IL-10 and TGF-β. Tr1 cells specific for various antigens are generated in vivo but may also be differentiated from natural CD4+ T cells in vitro in the presence of IL-10. R The proliferation capacity of T1 cells is very low, which can be overcome by IL-15. R 1 cells suppress naive and memory T helper cell type 1 or type 2 responses by producing IL-10 and TGF-β. R Further characterization of T cells at the molecular level will define their mechanism of action and elucidate their relationship to other subsets of TR cells. R1 cells to identify new targets for the development of new therapeutic agents and as cell therapies to regulate peripheral tolerance. See, for example, Roncarolo, M. et al. Immunol. Rev. 182: 68-79 (2001).

[0096] The terms "subject", "host", "individual" and "patient" are used interchangeably herein to refer to an animal, typically a mammal. Any suitable mammal can be treated by the methods, cells or compositions described herein. Non-limiting examples of mammals include: humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, adolescent, child, infant, or intrauterine mammal). The mammal can be a male animal or a female animal. The mammal can be a pregnant female animal. In some embodiments, the subject is a human. In some embodiments, the human suffers from or is suspected of suffering from cancer or a neoplastic disease.

[0097] "Composition" generally means an active agent (e.g., engineered immune cells (such as T cells, modified T cells, NK cells, chimeric antigen cells), cells containing engineered immune cells (e.g., T cells, NK cells, CAR T cells or CARNK cells), antibodies, cytokines IL-12, compounds or compositions) and naturally occurring or non-naturally occurring inert (e.g., detectable agents or markers) or active carriers in combination, such as adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. and include pharmaceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetrasaccharides and oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, and which alone or in combination account for 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, etc. Representative amino acid / antibody components that can also function in a buffering capacity include: alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartic acid, etc. Carbohydrate excipients should also be included in the technology, examples of which include, but are not limited to: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and inositol.

[0098] Compositions used according to the present invention include cells, treatments, therapies, medicaments, drugs and pharmaceutical preparations, which can be packaged in dosage unit form to facilitate drug administration and dosage uniformity. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in an object, each unit containing a composition calculated to produce a predetermined amount of a desired response associated with its administration (i.e., appropriate approach and scheme). Depending on the number of treatments and the unit dose, the dosage depends on the desired result and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is unique to each person. Factors affecting dosage include the physical and clinical state of the object, the route of administration, the expected therapeutic goal (symptom relief and cure) and the efficacy, stability and toxicity of a particular composition. After preparation, the solution will be administered in a manner compatible with the dosage formulation and in an amount for the treatment or prevention of an effective amount. The preparation is easy to administer in various dosage forms (e.g., the type of injectable solution described herein).

[0099] As used herein, the term "isolated" refers to a material that is substantially free of molecules or organisms or cells of other materials. On the one hand, the term "isolated" refers to nucleic acids (e.g., DNA or DNA), or proteins or polypeptides (e.g., antibodies or derivatives thereof), or cells or organelles, or tissues or organs that are separated from other DNA or RNA, proteins or polypeptides, cells or organelles, or tissues or organs present in natural sources. The term "isolated" also refers to nucleic acids or polypeptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or that are substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acids" are intended to include nucleic acid fragments that are not present as fragments in natural situations and are not found in natural states. The term "isolated" is also used herein to refer to polypeptides separated from other cell proteins, and is intended to include purified and recombinant polypeptides. The term "isolated" is also used herein to refer to cells or tissues separated from other cells or tissues, and is intended to include cultured and engineered cells or tissues.

[0100] As used herein, the term "isolated cell" generally refers to a cell that is substantially separated from other cells of a tissue.

[0101] As used herein, the term "animal" refers to a living multicellular vertebrate organism, a category that includes, for example, mammals and birds. The term "mammal" includes humans and non-human mammals, such as cows, dogs, cats, rats, rodents, monkeys, horses, and humans. Other examples include adults, juveniles, and infants.

[0102] As used herein, the term "antibody" ("Ab") generally refers to immunoglobulins (or "Ig") or immunoglobulin-like molecules, including but not limited to antibodies of the following isotypes: IgM, IgA, IgD, IgE, IgG, and combinations thereof. Immunoglobulin-like molecules include but are not limited to similar molecules produced during the immune response of vertebrates (e.g., mammals such as humans, rats, goats, rabbits, and mice), as well as non-mammalian species such as shark immunoglobulins (see Feige, M. et al. Proc. Nat. Ac. Sci. 41 (22): 8155-60 (2014)). Unless otherwise specifically stated, the term "antibody" includes intact immunoglobulins and "antibody fragments" or "antigen-binding fragments" that specifically bind to a target molecule (or a group highly similar to a target molecule) to the exclusion of binding to other molecules (e.g., antibodies and antibody fragments have a binding constant for the target molecule that is at least 10 greater than the binding constant for other molecules in the biological sample). 3 M -1 , at least 10 4 M -1 or at least 10 5 M -1). The term "antibody" also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0103] As used herein, the term "monoclonal antibody" refers to an antibody produced by a cell, wherein the light chain and heavy chain genes of a single antibody are transfected into the cell or more traditionally by a single clone transfection of a B lymphocyte. Monoclonal antibodies generally have affinity for a single epitope (i.e., they are monovalent), but can be engineered to have specificity (e.g., bispecific) for two or more epitopes. Methods for producing monoclonal antibodies are well known to those skilled in the art, such as by creating hybridomas by fusing myeloma cells with immune spleen cells, phage display, amplifying single cells from B cell populations, single plasma cell interrogation technologies, and single B cell culture. Monoclonal antibodies include recombinant antibodies, chimeric antibodies, humanized antibodies, and human antibodies.

[0104] The general structure of an antibody consists of a heavy (H) chain and a light (L) chain connected by disulfide bonds. The structure may also contain glycans attached at conserved amino acid residues. Each heavy and light chain contains a constant region and a variable region (also called a "domain"). There are two types of light chains, λ and κ. There are five main types of heavy chains that determine the isotype (or class) of the antibody molecule: γ, δ, α, μ, and ε. The constant region of the heavy chain also contributes to the effector function of the antibody molecule. Antibodies containing heavy chains μ, δ, γ3, γ1, α1, γ2, γ4, ε, and α2 generate the following isotypes: IgM, IgD, IgG3, IgG1, IgA1, IgG2, IgG4, IgE, and IgA2, respectively. The IgY isotype, which is related to mammalian IgG, is found in reptiles and birds. The IgW isotype, which is related to mammalian IgD, is found in cartilaginous fish. The conversion of classes is the process of replacing the constant region of the immunoglobulin heavy chain with different immunoglobulin heavy chains to produce antibodies of different isotypes by the reorganization of the heavy chain locus of the B cell. Antibodies can exist as monomers (e.g., IgG), dimers (e.g., IgA), tetramers (e.g., fish IgM), pentamers (e.g., mammalian IgM) and / or in complexes formed with other molecules. In some embodiments, antibodies can be bound to the cell surface or secreted by the cell.

[0105] The variable region of immunoglobulin heavy chain and light chain specifically binds antigen. "Framework" region is a part of Fab, which plays the role of the support of three hypervariable regions known as "complementarity determining region" (CDR). The collection of CDR is called paratope. The framework region of different light chains or heavy chains is relatively conservative within a species. The combined framework region of the antibody (comprising the region from the light chain and the heavy chain) mainly adopts the β-fold conformation, and CDR forms a loop, which connects the β-fold and forms a part of the β-fold structure in some cases. Therefore, the framework region positions the CDR in the correct direction by the non-covalent interaction between the chains. The framework region and CDR of many antibodies have been defined, and can be obtained from the database maintained online (Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991).

[0106] The heavy and light chain variable regions (V H and V L ) is responsible for binding to the antigen epitope. A limited number of amino acid positions in the CDR are directly involved in antigen binding. These positions in the CDR are called specificity determining residues (SDRs). The CDRs of the heavy or light chain are numbered sequentially starting from the N-terminus (i.e., CDR1, CDR2, and CDR3). For example, V L CDR3 is the middle CDR located in the variable domain of the antibody light chain. H CDR1 is the first CDR in the variable region of the antibody heavy chain. H and V L The region sequence, and therefore the specific CDR sequence. Antibodies with different specificities (ie different binding sites for different antigens) have different CDRs.

[0107] When used for antibodies, the term "humanization" means that the amino acid sequence of the antibody has one or more non-human amino acid residues (e.g., mouse, rat, goat, rabbit, etc.) of the complementary determining region (CDR), which specifically binds to the desired antigen in the recipient human immunoglobulin molecule; and one or more human amino acid residues in the Fv framework region (FR), which are amino acid residues located on the side of the CDR. Such antibodies generally have reduced immunogenicity compared to the non-human parent antibody from which the one or more CDRs are obtained, and therefore have a longer half-life in the human body.

[0108] "Fragment antigen binding" (Fab) refers to the region of an antibody that corresponds to two of the three fragments produced by papain digestion. The Fab fragment contains the region that binds to the antigen and is composed of one variable region and one constant region from each heavy and light chain. F(ab') 2 A fragment refers to a fragment of an antibody digested by pepsin or the enzyme IdeS (immunoglobulin degrading enzyme from Streptococcus pyogenes) that contains two Fab regions linked by a disulfide bond. A single-chain variable fragment ("scFv") refers to a fusion protein that contains at least one V and V regions connected by a linker between 5 and 30 amino acids. H and at least one V L Region. Methods and techniques for producing scFvs that bind to specific antigens are known in the art (see, for example, Ahmad, ZA et al., Clinical and Developmental Immunology, 2012: 980250 (2012)).

[0109] As used herein, the term "antigen" refers to a compound, composition or substance that can be specifically bound and / or recognized by a specific humoral immune product or a cellular immune product and an antigen recognition molecule (including but not limited to antibody molecules, single-chain variable fragments (scFv), cell surface immunoglobulin receptors, B cell receptors (BCR), T cell receptors (TCR), engineered TCR, modified TCR or CAR). The term "epitope" refers to an antigen and an antigen fragment, region, site or domain recognized by an antigen recognition molecule. Antigens can be any type of molecule, including but not limited to peptides, proteins, lipids, phospholipid haptens, simple intermediate metabolites, sugars (eg, monosaccharides or oligosaccharides), hormones and macromolecules such as complex carbohydrates (eg, polysaccharides). Some non-limiting examples of antigens include antigens (including self-antigens), tumor antigens, toxins and other antigens involved in autoimmune diseases, allergies and transplant rejection. Non-limiting examples of tumor antigens include mesothelin, ROR1 and EGFRvIII, ephrin type A receptor 2 (EphA2), interleukin (IL) -13rα2, EGFR VIII, PSMA, EpCAM, GD3, fucosyl GM1, PSCA, PLAC1, sarcoma breakpoints, Wilms tumor 1, hematological differentiation antigens, surface glycoproteins, gangliosides (GM2), growth factor receptors, matrix antigens, vascular antigens or combinations thereof. Antigens expressed by pathogens include but are not limited to microbial antigens, such as viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens.

[0110] As used herein, the term "target cell population" refers to a population of cells presenting an antigen that can be targeted by engineered T cells. Non-limiting examples of target cell populations include tumor cells, cancer cells, and cells infected by pathogens. Non-limiting examples of pathogens include viral pathogens and bacterial pathogens.

[0111] As used herein, the term "antigen binding domain" refers to any protein or polypeptide domain that can specifically bind to an antigen target (including a target complex of an antigen and an MHC molecule).

[0112] As used herein, the term "autologous" with respect to cells, tissues and / or transplants refers to cells, tissues and / or transplants that are isolated from and subsequently administered back to the same subject, patient, recipient and / or host. "Allogeneic" refers to non-autologous cells, tissues and / or transplants.

[0113] As used herein, the term "B cell" refers to a type of lymphocyte in the humoral immunity of the adaptive immune system. After activation by antigen interaction, the main function of B cells is to make antibodies, act as antigen presenting cells, release cytokines and develop memory B cells. B cells are distinguished from other lymphocytes (such as T cells) by the presence of B cell receptors on the cell surface. B cells can be isolated or obtained from commercially available sources. Non-limiting examples of commercially available B cell lines include the cell line AHH-1 ( CRL-8146 TM )、BC-1( CRL-2230 TM )、BC-2( CRL-2231 TM )、BC-3( CRL-2277 TM )、CA46( CRL-1648 TM )、DG-75[DG-75]( CRL-2625 TM )、DS-1( CRL-11102 TM ), EB-3[EB3]( CCL-85 TM), Z-138(ATCC#CRL-3001), DB(ATCCCRL-2289), Toledo(ATCC CRL-2631), Pfiffer(ATCC CRL-2632), SR(ATCC CRL-2262), JM-1(ATCC CRL-10421), NFS-5C-1(ATCC CRL-1693), NFS-70C10 (ATCCCRL-1694), NFS-25C-3 (ATCC CRL-1695) and SUP-B15 (ATCC CRL-1929). Additional examples include, but are not limited to, cell lines derived from anaplastic and large cell lymphomas, such as DEL, DL-40, FE-PD, JB6, Karpas 299, Ki-JK, Mac-2A Ply1, SR-786, SU-DHL-1, -2, -4, -5, -6, -7, -8, -9, -10 and -16, DOHH-2, NU-DHL-1, U-937, Granda 519, USC-DHL-1, RL; cell lines of Hodgkin lymphoma, such as DEV, HD-70, HDLM-2, HD-MyZ, HKB-1, KM-H2, L428, L 540, L1236, SBH-1, SUP-HD1, SU / RH-HD-1. Non-limiting exemplary sources of such commercially available cell lines include: the American Type Culture Collection (or ATCC) (www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).

[0114] As used herein, a "target cell" is any cell that expresses an antigenic target that can be bound by an engineered T cell.

[0115] As used herein, "cancer" is a disease state characterized by the presence of cells that exhibit abnormal, uncontrolled replication in a subject, and can be used interchangeably with the term "tumor". In some embodiments, the cancer is a leukemia or lymphoma. "Cells associated with cancer" refer to those subject cells that exhibit abnormal, uncontrolled replication. In certain embodiments, the cancer is acute myeloid leukemia or acute lymphocytic leukemia. As used herein, "leukemia" is a blood or bone marrow cancer characterized by an abnormal increase in immature white blood cells. The specific condition of acute myeloid leukemia (AML) (also known as acute myeloid leukemia or acute granulocytic leukemia) is a cancer of myeloid-derived blood cells, characterized by the rapid growth of abnormal myeloid cells that accumulate in the bone marrow and interfere with the production of bone marrow and normal blood cells. The specific condition of acute lymphoblastic leukemia (ALL) (also known as acute lymphoblastic leukemia or acute lymphoid leukemia) is a cancer of the white blood cells characterized by the overproduction and accumulation of malignant, immature white blood cells (lymphocytes), resulting in a lack of normal, healthy blood cells. As used herein, "lymphoma" is a cancer of the blood characterized by the development of tumors of the blood cells and symptoms of enlarged lymph nodes, fever, clammy sweats, unintentional weight loss, itching, and frequent tiredness.

[0116] As used herein, "pathogen-infected cell population" or "pathogen-infected cells" refers to a cell population or cell infected by a pathogen. Examples of pathogenic infections include, but are not limited to, bacterial infections such as group A Streptococcus, Mycobacterium tuberculosis, Shigella flexneri, Salmonella enterica, Listeria monocytogenes, Francisella tularensis, and viral infections such as herpes simplex virus.

[0117] Those skilled in the art can use methods such as RNA sequencing, DNA microarray, real-time PCR or chromatin immunoprecipitation (ChIP) to monitor the expression of transcription factors. Methods such as flow cytometry, immunoblotting, 2D gel electrophoresis or immunoassay can be used to monitor the expression of proteins.

[0118] Those skilled in the art can use methods such as RNA interference (RNAi), CRISPR, TALEN, ZFN or other methods targeting specific sequences to reduce or eliminate the expression and / or function of NR4A or TOX transcription factors. CRISPR, TALEN, ZFN or other genome editing tools can also be used to increase the expression and / or function of IL-21.

[0119] As used herein, "RNAi" (RNA interference) refers to a method of reducing or eliminating gene expression in cells by targeting specific mRNA sequences for degradation by introducing short double-stranded RNA (dsRNA) and small interfering RNA (e.g., siRNA, shRNA or miRNA, etc.) (Agrawal, N. et al.; Microbiol Mol Biol Rev. 2003; 67: 657-685, Arenz, C. et al.; Naturwissenschaften. 2003; 90: 345-359, Hannon GJ.; Nature. 2002; 418: 244-251).

[0120] As used herein, the term "CRISPR" refers to a technique for sequence-specific gene manipulation that relies on clustered regularly spaced short palindromic repeat pathways. CRISPR can be used to perform gene editing and / or gene regulation, and only target proteins to specific genomic locations. "Gene editing" refers to a type of genetic engineering, in which the nucleotide sequence of a target polynucleotide is changed by introducing deletions, insertions, single-strand or double-strand breaks or base substitutions into a polynucleotide sequence. In some aspects, CRISPR-mediated gene editing utilizes the pathway of non-homologous end joining (NHEJ) or homologous recombination to perform editing. Gene regulation refers to increasing or decreasing the production of a specific gene product (eg, protein or RNA).

[0121] As used herein, the term "gRNA" or "guide RNA" refers to a guide RNA sequence used to target a specific polynucleotide sequence for gene editing using CRISPR technology. Techniques for designing gRNAs and donor therapeutic polynucleotides for target specificity are known in the art. For example, Doench, J., et al. Nature biotechnology 2014; 32 (12): 1262-7, Mohr, S. et al. (2016) FEBS Journal 283: 3232-38 and Graham, D., et al. Genome Biol. 2015; 16: 260. The gRNA comprises a fusion polynucleotide of CRISPR RNA (crRNA) and a trans-activating CRIPSPR RNA (tracrRNA), or consists essentially of the fusion polynucleotide, or consists of the fusion polynucleotide, or the gRNA comprises a polynucleotide having CRISPR RNA (crRNA) and a trans-activating CRIPSPR RNA (tracrRNA). In some aspects, the gRNA is synthetic (Kelley, M. et al. (2016) J of Biotechnology 233 (2016) 74-83).

[0122] The term "Cas9" refers to the CRISPR-associated nuclease referred to by the name. Non-limiting exemplary Cas9s include Staphylococcus aureus Cas9, nuclease-inactivated Cas9, and each of its orthologs and bioequivalents. Orthologs include, but are not limited to, Streptococcus pyogenes Cas9 ("spCas9"); Cas9s from Streptococcus thermophilus, Legionella pneumophila, Neisseria lactis, Neisseria meningitidis, Francisella novicida; and Cpf1s from various bacteria including Acidaminococcus spp. and Francisella novicida U112 (performing a cleavage function similar to Cas9).

[0123] As used herein, "TALEN" (transcription activator-like effector nuclease) refers to an engineered nuclease comprising a non-specific DNA-cutting nuclease fused to a TALE DNA binding domain that can target a DNA sequence and be used for genome editing. Boch (2011) Nature Biotech. 29: 135-6; Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501. TALE is a protein secreted by bacteria of the genus Xanthomonas. The DNA binding domain contains a repetitive, highly conserved sequence of 33-34 amino acids (except the 12th and 13th amino acids). These two positions are highly variable and show a strong correlation with specific nucleotide recognition. Therefore, they can be engineered to bind to the desired DNA sequence. To produce TALEN, the TALE protein is fused to a nuclease (N), which is a wild-type or mutant Fok1 endonuclease. Several mutations of Fok1 have been made for use in TALENs, which, for example, improve cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39:e82; Miller et al. (2011) Nature Biotech. 29:143-8; Hockemeyer et al. (2011) Nature Biotech. 29:731-734; Wood et al. (2011) Science 333:307; Doyon et al. (2010) Nature Methods 8:74-79; Szczepek et al. (2007) Nature Biotech. 25:786-793; and Guo et al. (2010) J. Mol. Bio. 200:96. The Fok1 domain functions as a dimer, requiring two constructs with unique DNA binding domains to form sites in the target genome with appropriate orientation and spacing. The number of amino acid residues between the TALE DNA binding domain and the Fok1 cleavage domain and the number of bases between the two separate TALEN binding sites appear to be important parameters for obtaining high levels of activity. Miller et al. (2011) Nature Biotech. 29: 143-8. TALENs specific for sequences in immune cells can be constructed using any method known in the art, including various schemes using modular components.Zhang et al. (2011) Nature Biotech. 29:149-53; Geibler et al. (2011) PLoS ONE 6:e19509.

[0124] As used herein, "ZFN" (zinc finger nuclease) refers to an engineered nuclease comprising a non-specific DNA-cutting nuclease fused to a zinc finger DNA binding domain that can target a DNA sequence and be used for genome editing. Like TALEN, ZFN comprises a Fok1 nuclease domain (or a derivative thereof) fused to a DNA binding domain. In the case of ZFN, the DNA binding domain comprises one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160. Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. Zinc fingers can comprise, for example, Cys2His2, and can recognize a sequence of about 3 bp. Various zinc fingers of known specificity can be combined to produce a multi-finger polypeptide that recognizes a sequence of about 6, 9, 12, 15 or 18 bp. Various selection and modular assembly technologies can be used to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid systems, and two-hybrid systems, as well as mammalian cells. Like TALEN, ZFN must dimerize to cut DNA. Therefore, a pair of ZFNs is needed to target non-palindromic DNA sites. The two individual ZFNs must bind to opposite strands of DNA, and their nucleases are appropriately spaced. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5. ZFNs that are specific for sequences in immune cells can be constructed using any method known in the art. See, e.g., Provasi (2011) Nature Med. 18:807-815; Torikai (2013) Blood 122:1341-1349; Cathomen et al. (2008) Mol. Ther. 16:1200-7; Guo et al. (2010) J. Mol. Bioi. 400:96; U.S. Patent Publication 201110158957; and U.S. Patent Publication 2012 / 0060230.

[0125] "Cytotoxic cells" refer to cells that are capable of killing other cells or microorganisms. Examples of cytotoxic cells include, but are not limited to, CD8+ T cells, natural killer (NK) cells, NKT cells, and neutrophils, which are capable of mediating cytotoxic responses.

[0126] As used herein, the term "detectable label" refers to at least one label that is capable of directly or indirectly generating a detectable signal. A non-exhaustive list of such labels includes: enzymes that generate a detectable signal (e.g., by colorimetry, fluorescence, luminescence), such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent, luminescent dyes, groups whose electron density is detected by electron microscopy or by their electrical properties (e.g., conductivity, amperometry, voltammetry, impedance); detectable groups, such as detectable groups whose molecular size is sufficient to induce a detectable modification in their physical and / or chemical properties, such that detection can be performed by optical methods (e.g., diffraction, surface plasmon resonance, surface changes, contact angle changes) or physical methods (e.g., atomic force spectroscopy, tunneling) or radioactive molecules (e.g., 32 P. 35 S or 125 I) Completed.

[0127] As used herein, "disease-associated antigen" may refer to an antigen, epitope, or fragment thereof that is associated with a disease process or mechanism. For example, an antigen associated with inflammation is an antigen or fragment thereof that produces an immune response when presented. Inflammation-associated antigens that produce such an effect are selected to treat inflammation. Similarly, autoimmune-associated antigens are antigens associated with autoimmune diseases and would not be selected to treat diseases or diseases other than autoimmunity, such as cancer. Non-limiting exemplary disease-associated antigens are disclosed herein, and further, such antigens can be determined for specific diseases based on the epitope screening techniques, mechanisms, and methods described herein.

[0128] When applied to nucleic acid sequences, the term "encoding" means that if the nucleic acid can be transcribed and / or translated to produce a functional RNA (e.g., miRNA, siRNA, RNAi, tRNA, rRNA, snRNA, etc.), mRNA or polypeptide and / or its fragments in its native state or when manipulated by methods known to those skilled in the art, the polynucleic acid is said to "encode" RNA or polypeptide. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0129] As used herein, the term "enhancer" refers to a regulatory sequence element that increases, improves or reduces the transcription of a nucleic acid sequence, regardless of its position and orientation relative to the nucleic acid sequence to be expressed. An enhancer can enhance transcription from a single promoter or enhance transcription from multiple promoters simultaneously. Any truncated, mutated or otherwise modified variant of a wild-type enhancer sequence is also within the above definition as long as the function of improving transcription is retained or substantially retained (e.g., at least 70%, at least 80%, at least 90% or at least 95% of the wild-type activity (i.e., full-length sequence activity)).

[0130] In the context of nucleic acid or amino acid sequences, the term "chimeric" means that the sequence contains or consists of at least one substitution unit (e.g., fragment, region, part, domain, polynucleotide or polypeptide) derived from, obtained from, or separated from, or based on other different physical or chemical entities. For example, a chimera of two or more different proteins may contain a sequence from a variable region domain of an antibody fused to a transmembrane domain of a cell signaling molecule. In some aspects, a chimera refers to a sequence consisting of sequences from at least two different species.

[0131] As used herein, the term "chimeric antigen receptor" (CAR) refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide (different from the polypeptide from which the extracellular domain is derived), and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is sometimes also referred to as a "chimeric receptor", "T body" or "chimeric immune receptor (CIR)". "Extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide that can bind to a certain antigen. "Intracellular domain" or "intracellular signaling domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals in a cell to cause activation or inhibition of a biological process. In certain embodiments, in addition to the primary signaling domain, the intracellular domain may also include one or more costimulatory signaling domains, or may consist essentially of one or more costimulatory signaling domains, or may further include one or more costimulatory signaling domains. "Transmembrane domain" refers to any oligopeptide or polypeptide known to span the cell membrane that can function to connect the extracellular domain and the signaling domain. The chimeric antigen receptor may optionally include a "hinge domain" that acts as a linker between the extracellular domain and the transmembrane domain. Non-limiting exemplary polynucleotide sequences encoding the components of each domain are disclosed herein, for example:

[0132] Hinge domain: The polynucleotide sequence of the IgG1 heavy chain hinge: CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG, and alternatively its equivalents.

[0133] Transmembrane domain: The polynucleotide sequence of the CD28 transmembrane region: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA CAGTGGCCTTTATTATTTTCTGGGTG, and alternatively its equivalents.

[0134] Intracellular domain: The polynucleotide sequence of the 4-1BB co-stimulatory signal region: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAACTG, and optional equivalents thereof.

[0135] Intracellular domain: The polynucleotide sequence of the CD28 co-stimulatory signal region: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC CGCCCCGGGGCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCG CAGCCTATCGCTCC, and optionally its equivalents.

[0136] Intracellular domain: The polynucleotide sequence of the CD3ζ signaling region: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA, and alternatively equivalents thereof.

[0137] A non-limiting example of a CAR extracellular domain capable of binding to an antigen is an anti-CD19 binding domain sequence that specifically binds to the CD19 antigen as disclosed in the US20140271635 application.

[0138] Other embodiments of each exemplary domain component include other proteins with similar biological functions, which have at least 70% or at least 80% amino acid sequence identity with the protein encoded by the nucleic acid sequence disclosed above, preferably 90% sequence identity, more preferably at least 95% sequence identity. In addition, non-limiting examples of such domains are provided herein.

[0139] As used herein, the term "CD8α hinge domain" refers to the specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity, preferably 90% sequence identity, and more preferably at least 95% sequence identity with the CD8α hinge domain as shown herein. Pinto, RDet al. (2006) Vet. Immunol. Immunopathol. 110: 169-177 provides exemplary sequences of CD8α hinge domains of humans, mice, and other species. Pinto, RDet al. (2006) Vet. Immunol. Immunopathol. 110: 169-177 provides sequences related to the CD8α hinge domain. Non-limiting examples of such sequences include:

[0140] Human CD8 alpha hinge domain amino acid sequence: PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY, and alternatively its equivalents.

[0141] Mouse CD8α hinge domain amino acid sequence: KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY, and alternative equivalents thereof.

[0142] Cat CD8 alpha hinge domain amino acid sequence: PVKPTTTPAPRPPTQAPITTSQRVSLRPGTCQPSAGSTVEASGLDLSCDIY, and alternative equivalents thereof.

[0143] As used herein, the term "CD8α transmembrane domain" refers to a specific protein fragment associated with this name and any other molecule with a similar biological function, which has at least 70% or at least 80% amino acid sequence identity, preferably 90% sequence identity, and more preferably at least 95% sequence identity with the CD8α transmembrane domain as shown herein. The fragment sequence associated with amino acids 183 to 203 of the human T cell surface glycoprotein CD8α chain (GenBank Accession No.: NP_001759.3), or the fragment sequence associated with amino acids 197 to 217 of the mouse T cell surface glycoprotein CD8α chain (GenBank Accession No.: NP_001074579.1), and the fragment sequence associated with amino acids 190 to 210 of the rat T cell surface glycoprotein CD8α chain (GenBank Accession No.: NP_113726.1) provide other exemplary sequences of the CD8α transmembrane domain. The sequences associated with each of the listed accession numbers are provided as follows:

[0144] Amino acid sequence of human CD8α transmembrane domain: IYIWAPLAGTCGVLLLSLVIT, and alternative equivalents thereof.

[0145] Amino acid sequence of mouse CD8α transmembrane domain: IWAPLAGICVALLLSLIITLI, and alternative equivalents thereof.

[0146] Amino acid sequence of rat CD8α transmembrane domain: IWAPLAGICAVLLLSLVITLI, and alternative equivalents thereof.

[0147] As used herein, the term "CD28 transmembrane domain" refers to a specific protein fragment associated with this name and any other molecule with a similar biological function, which has at least 70% or at least 80% amino acid sequence identity, or at least 90% sequence identity, or at least 95% sequence identity with the CD28 transmembrane domain sequence as shown herein. The fragment sequences associated with GenBank Accession Nos. XM_006712862.2 and XM_009444056.1 provide other non-limiting example sequences of the CD28 transmembrane domain.

[0148] As used herein, the term "4-1BB co-stimulatory signaling region" refers to a specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity with the 4-1BB co-stimulatory signaling region sequence as shown herein, preferably 90% sequence identity, more preferably at least 95% sequence identity. Non-limiting exemplary sequences of the 4-1BB co-stimulatory signaling region are provided in U.S. Publication No. 20130266551A1 (filed as U.S. Patent Application No. 13 / 826,258), such as the exemplary sequences provided below and sequences encoded by the 4-1BB co-stimulatory signaling region amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, and optional equivalents thereof.

[0149] As used herein, the term "ICOS costimulatory signaling region" refers to a specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity, preferably 90% sequence identity, and more preferably at least 95% sequence identity with the ICOS costimulatory signaling region sequence as shown herein. Sequences of non-limiting examples of ICOS costimulatory signaling regions are provided in U.S. Patent Application Publication No. 2015 / 0017141A1. Exemplary polynucleotide sequences are provided below.

[0150] The polynucleotide sequence of the ICOS co-stimulatory signaling region: ACAAAAAAGA AGTATTCATC CAGTGTGCACGACCCTAACG GTGAATACAT GTTCATGAGA GCAGTGAACA CAGCCAAAAA ATCCAGACTC ACAGATGTGACCCTA, and alternatively its equivalents.

[0151] As used herein, the term "OX40 costimulatory signaling region" refers to the specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity, or 90% sequence identity, or at least 95% sequence identity with the OX40 costimulatory signaling region sequence as shown herein. Non-limiting exemplary sequences of the OX40 costimulatory signaling region are disclosed in U.S. Patent Application Publication No. 2012 / 20148552A1, and include the exemplary sequences provided below.

[0152] The polynucleotide sequence of the OX40 co-stimulatory signaling region: AGGGACCAG AGGCTGCCCC CCGATGCCCACAAGCCCCCT GGGGGAGGCA GTTTCCGGAC CCCCATCCAA GAGGAGCAGG CCGACGCCCA CTCCACCCTGGCCAAGATC, and alternatively equivalents thereof.

[0153] As used herein, the term "CD28 costimulatory signaling region" refers to the specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity, or 90% sequence identity, or at least 95% sequence identity with the CD28 costimulatory signaling region sequence as shown herein. Example sequences CD28 costimulatory signaling regions are provided in U.S. Patent No. 5,686,281. Geiger, TLet al. (2001) Blood 98: 2364-2371; Hombach, A. et al. (2001) J Immunol 167: 6123-6131; Maher, J. et al. (2002) Nat Biotechnol 20: 70-75; Haynes, N Met al. (2002) J Immunol.169:5780-5786(2002);Haynes,NMet al.(2002)Blood 100:3155-3163.(2001)Blood 98:2364-2371;Hombach,A.et al.(2001)J Immunol167:6123-6131;Maher,J.et al.(2002)Nat Biotechnol 20:70-75; Haynes, NM et al. (2002) J Immunol. 169:5780-5786 (2002); Haynes, NM et al. (2002) Blood 100:3155-3163. Non-limiting examples include sequences encoded by the following sequence: CD28 amino acid sequence: MLRLLLALNL FPSIQVTGNKILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLDSAVEVCVVYG NYSQQLQVYSKTGFNCDGKLGNESVTFYLQ NLYVNQTDIY FCKIEVMYPPPYLDNEKSNG TIIHVKGKHLCPSPLFPGPSKPFWVLVVVG GVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS, and equivalents thereof.

[0154] As used herein, the term "CD3 zeta signaling domain" refers to the specific protein fragment associated with this name and any other molecules with similar biological functions, which have at least 70% or at least 80% amino acid sequence identity, or 90% sequence identity, or at least 95% sequence identity with the CD3 zeta signaling domain sequence as shown herein. Non-limiting example sequences of amino acid sequences of CD3 zeta signaling domains are provided in U.S. Application No. 13 / 826,258, for example: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0155] As used herein, "first generation CAR" refers to a CAR comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide derived from the extracellular domain, and at least one intracellular domain. "Second generation CAR" refers to a first generation CAR that also includes a co-stimulatory domain (eg, 4-1BB or CD28). "Third generation CAR" refers to a first generation CAR that also includes two co-stimulatory domains (eg, CD27, CD28, ICOS, 4-1BB or OX40). "Fourth generation CAR" (also referred to as "TRUCK") refers to a CART cell that is further engineered to secrete additional factors (eg, proinflammatory cytokine IL-12). A review of these CAR technologies and cell therapies can be found in Maus, M. et al. Clin. Cancer Res. 22 (3): 1875-84 (2016).

[0156] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is typically present at the N-terminus of a newly synthesized secretory or membrane polypeptide or protein. It serves to direct the polypeptide across or into the cell membrane and then is removed. Such examples are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381 and 5,958,736.

[0157] As used herein with respect to regulatory polynucleotides, the term "operably linked" refers to the binding between a regulatory polynucleotide and a polynucleotide sequence to which it is linked, such that when a specific protein binds to the regulatory polynucleotide, the linked polynucleotide is transcribed.

[0158] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA with any sequence, isolated RNA with any sequence, nucleic acid probes and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, nucleotide structural modifications can be imparted before or after polynucleotide assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any aspect of the polynucleotides of the present technology includes a double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0159] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0160] As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence (e.g., a gene). For example, a promoter can be constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence, a region of a polynucleotide sequence, that controls the initiation and frequency of transcription. It may contain genetic elements to which regulatory proteins and molecules (e.g., RNA polymerase and other transcription factors) can bind.

[0161] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense refer to compounds having two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. On the other hand, the subunits may be linked by other bonds (e.g., esters, ethers, etc.). A protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that can constitute the sequence of a protein or peptide. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and D and L optical isomers, amino acid analogs and peptidomimetics.

[0162] As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid, peptide, protein, biocomplex or other active compound is completely or partially separated from proteins or other contaminants. Typically, a substantially purified peptide, protein, biocomplex or other active compound used in the present disclosure contains more than 80% of all macromolecular species relative to the preparation before the peptide, protein, biocomplex or other active compound is mixed with a pharmaceutical carrier, excipient, buffer, absorption enhancer, stabilizer, preservative, adjuvant or other admixture or formulated into a complete pharmaceutical preparation for therapeutic administration. More typically, the peptide, protein, biocomplex or other active compound is purified to represent greater than 90%, typically greater than 95%, of all macromolecular species present in the purified preparation before mixing with other formulation ingredients. In other cases, the purified preparation may be substantially homogeneous, wherein other macromolecular species cannot be detected by conventional techniques.

[0163] As used herein, the term "purification tag" refers to at least one tag that can be used for purification or identification. A non-exhaustive list of such tags includes: His, lacZ, GST, maltose binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry red, thioredoxin, poly (NANP), V5, Snap, HA, chitin binding protein, Softag 1, Softag 3, Streptococcus or S protein. Suitable direct or indirect fluorescent labels include: FLAG, GFP, YFP, RFP, dTomato, cherry red, Cy3, Cy5, Cy5.5, Cy 7, DNP, AMCA, biotin, digitoxin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.

[0164] As used herein, the term "suicide gene" is a gene capable of inducing apoptosis; non-limiting examples include: HSV-TK (herpes simplex virus thymidine kinase), cytosine deaminase, nitroreductase, carboxylesterase, cytochrome P450 or PNP (purine nucleoside phosphorylase), truncated EGFR or inducible caspase ("iCasp"). Suicide genes may act along a variety of pathways, and in some cases, may be induced by inducers such as small molecules. For example, the iCasp suicide gene comprises a portion of a caspase protein operably linked to a protein optimized to bind an inducer; the inducer is introduced into a cell comprising the suicide gene, resulting in activation of caspase and subsequent apoptosis of the cell.

[0165] When applied to the production of chimeric antigen receptor cells, the term "transduction" refers to the process of introducing exogenous nucleotide sequences into cells. In some embodiments, the transduction is accomplished by a vector.

[0166] As used herein, the term "vector" refers to a nucleic acid construct designed to be transferred between different hosts, including but not limited to plasmids, viruses, cosmids, phages, BACs, YACs, etc. "Vector" is defined as a recombinant virus or viral particle that contains a polynucleotide to be delivered to a host cell in vivo, in vitro, or in vitro. In some embodiments, a plasmid vector can be prepared from a commercially available vector. In other embodiments, a viral vector can be produced with baculovirus, retrovirus, adenovirus, AAV, etc. according to techniques known in the art. In one embodiment, the viral vector is a lentiviral vector. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alpha virus vectors, etc. Vectors based on infectious tobacco mosaic virus (TMV) can be used to make proteins, and have been reported to express Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat. Acad. Sci. USA 106 (15): 6099-6104). Alphavirus vectors (e.g., vectors based on Semliki Forest virus and vectors based on Sindbis virus) have also been developed for gene therapy and immunotherapy. See Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying et al. (1999) Nat. Med. 5 (7): 823-827. In terms of retroviral vector-mediated gene transfer, a vector construct refers to a polynucleotide comprising a retroviral genome or a portion thereof and a target gene (e.g., a polynucleotide encoding a CAR). Further details of modern methods for vectors for gene transfer can be found in, for example, Kotterman et al. (2015) Viral Vectors for Gene Therapy: Translational and Clinical Outlook Annual Review of Biomedical Engineering 17. A vector comprising a promoter and a cloning site to which a polynucleotide can be operably linked is well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.).

[0167] As used herein, the terms "T2A" and "2A peptide" are used interchangeably and refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or an artificial peptide comprising essential amino acids having a relatively short (about 20 amino acids in length, depending on the virus of origin) peptide sequence of the consensus polypeptide motif DV / IEXNPGP, wherein X refers to any amino acid that is generally considered to be self-cleaved.

[0168] As used herein, the term "recombinant protein" refers to a polypeptide produced by recombinant DNA technology, wherein typically DNA encoding the polypeptide is inserted into an appropriate expression vector, which in turn is used to transform host cells to produce the heterologous protein.

[0169] As used herein, the term "detectable label" refers to at least one label that is capable of directly or indirectly generating a detectable signal. A non-exhaustive list of such labels includes: enzymes that generate a detectable signal (e.g., by colorimetry, fluorescence, luminescence), such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent, luminescent dyes; groups with electron density detectable by electron microscopy or by their electrical properties (e.g., conductivity, amperometry, voltammetry, impedance), such as groups whose molecular size is sufficient to induce detectable modifications in their physical and / or chemical properties, detection of which can be accomplished by optical methods (e.g., diffraction, surface plasmon resonance, surface changes, contact angle changes) or physical methods (e.g., atomic force spectroscopy, tunneling); or radioactive molecules (e.g., 32 P. 35 S or 125 I) In one aspect, the detectable label excludes naturally fluorescent polynucleotides.

[0170] On the one hand, the term "equivalent" or "bioequivalent" of an antibody refers to the ability of an antibody to selectively bind to its epitope protein or fragment thereof as measured by ELISA or other suitable methods. Bioequivalent antibodies include, but are not limited to, antibodies, peptides, antibody fragments, antibody variants, antibody derivatives, and antibody mimetics that bind to the same epitope as a reference antibody.

[0171] In the absence of explicit narration, it should be inferred that, and unless otherwise intended, when the present disclosure relates to polypeptides, proteins, polynucleotides or antibodies, their equivalents or biological equivalents are within the scope of the present disclosure. As used herein, the term "biological equivalents thereof" is intended to be synonymous with "equivalents thereof" when referring to a reference protein, antibody, polypeptide or nucleic acid, meaning those proteins, antibodies, polypeptides or nucleic acids with minimal homology while still maintaining the desired structure or function. Unless specifically described herein, it is contemplated that any polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof. For example, an equivalent means at least about 70% homology or identity, or at least 80% homology or identity, or at least about 85%, or at least about 90%, or at least about 95%, or 98% homology or identity; and exhibits biological activity substantially equal to that of a reference protein, polypeptide or nucleic acid. Alternatively, when referring to a polynucleotide, an equivalent thereof is a polynucleotide that hybridizes with a reference polynucleotide or its complement under stringent conditions.

[0172] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence means that, when compared, the percentage of bases in the two sequences being compared is the same. Comparisons and sequence homology or identity percentages can be determined using software programs known in the art, such as "A Guide to Experimental Molecular Biology" ((Ausubel et al., eds. 1987) Appendix 30, Section 7.7.18, Table 7.7.1.1987). Preferably, comparisons are performed using default parameters. A preferred comparison program is BLAST using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard, filter = none, strand = double strand; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at the following website: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0173] As used herein, "homology" or "identity", "percent identity" or "similarity" when used in the context of two or more nucleic acid or polypeptide sequences refers to two or more identical or identical sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues, such as at least 60% identity over a specified region, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity (e.g., nucleotide sequences encoding antibodies described herein or amino acid sequences of antibodies described herein). Homology can be determined by comparing positions in each sequence, which can be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. Alignments and percent homology or sequence identity can be determined using software programs known in the art, for example in Appendix 30, Section 7.7.18, Table 7.7.1 of Molecular Biology (Ausubel et al., eds. 1987). Preferably, default parameters are used for the alignment. A preferred alignment program is BLAST using default parameters. In particular, preferred programs are BLASTN and BLASTP using the following default parameters: genetic code = standard, filter = none, strand = double strand, threshold = 60, expectation = 10, matrix = BLOSUM62, description = 50 sequences, sorting = high score, database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at the following website: ncbi.nlm.nih.gov / cgi-bin / BLAST. The term "homology" or "identity", "identity" or "similarity" percentage also refers to or can be used for the complementary sequence of the sequence being tested. The term also includes sequences with deletions and / or additions and sequences with substitutions. As described herein, the preferred algorithm can resolve gaps and other issues. Preferably, the identity exists in a region of at least about 25 amino acids or nucleotides in length, or more preferably in a region of at least 50-100 amino acids or nucleotides in length. "Unrelated" or "non-homologous" sequences have less than 40% identity, or less than 25% identity with one of the sequences disclosed herein.

[0174] "Hybridization" refers to the reaction of one or more polynucleotides to form a complex stabilized by hydrogen bonds between the bases of the nucleotide residues. Hydrogen bonds can exist through Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can contain two chains forming a duplex structure, three or more chains forming a multi-chain complex, a single self-hybridizing chain, or any combination thereof. The hybridization reaction can constitute a step in a broader process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0175] Examples of stringent hybridization conditions include: an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6x SSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a washing solution of about 4xSSC to about 8xSSC. Examples of moderate hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9x SSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a washing solution of about 5x SSc to about 2x SSC. Examples of high stringency conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1x SSC to about 0.1x SSC; a formamide concentration of about 55% to about 75%; and a washing solution of about 1x SSC, 0.1x SSC or deionized water. Typically, the hybridization incubation time is 5 minutes to 24 hours, with 1, 2 or more washing steps, and the washing incubation time is about 1, 2 or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It will be appreciated that the equivalent of SSC using other buffer systems may be employed.

[0176] "Normal cells corresponding to the cancer tissue type" refers to normal cells from the same tissue type as the cancer tissue. A non-limiting example is normal white blood cells from a patient (eg, a leukemia patient).

[0177] As used herein, the term "NK cell", also known as natural killer cell, refers to a class of lymphocytes that originate from the bone marrow and play a key role in the innate immune system. NK cells can provide a rapid immune response to virus-infected cells, tumor cells or other stressed cells even in the absence of antibodies and major histocompatibility complexes on the cell surface. NK cells can be isolated or obtained from commercial channels. Non-limiting examples of commercial NK cell lines include the cell line NK-92 ( CRL-2407 TM )、NK-92MI( CRL-2408 TM). Other examples include, but are not limited to, the NK cell lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (or ATCC) (http: / / www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).

[0178] As used herein, the term "overexpression" refers to a cell, tissue or organ expressing a protein in an amount greater than that produced in a control cell, control tissue or organ. The overexpressed protein may be endogenous or exogenous to the host cell.

[0179] As used herein, the term "recombinant protein" refers to a polypeptide produced by recombinant DNA techniques, wherein typically DNA encoding the polypeptide is inserted into an appropriate expression vector, which in turn is used to transform a host cell to produce the heterologous protein.

[0180] As used herein, the term "specific binding" refers to a binding affinity between an antibody and an antigen of at least 10 -6 M. In certain aspects, the antibody is at least about 10 -7 M affinity binding, preferably 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 M.

[0181] A "solid tumor" is an abnormal mass of tissue that usually does not contain cysts or areas of fluid. Solid tumors can be benign or malignant, metastatic or non-metastatic. Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas.

[0182] The sequences associated with each of the GenBank Accession Numbers, UniProt Reference Numbers and references listed above are incorporated herein by reference.

[0183] As used herein, the term "major histocompatibility complex" (MHC) refers to antigen presenting molecules that act as part of the immune system to bind antigens and other peptide fragments and display them on the cell surface for recognition by antigen recognition molecules (e.g., TCRs). When used in reference to human MHC, MHC can be used interchangeably with the term "human leukocyte antigen" (HLA); thus, MHC refers to all HLA subtypes, including but not limited to the classical MHC genes disclosed herein: HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR, in addition to all variants, isoforms, isotypes, and other biological equivalents thereof. MHC class I (MHC-I) and MHC class II (MHC-II) molecules utilize different antigen processing pathways. Typically, peptides derived from intracellular antigens are presented to CD8+T cells through class I MHC molecules expressed on almost all cells, while peptides derived from extracellular antigens are presented to CD4+T cells through MHC-II molecules. However, several exceptions to this dichotomy have been found. In certain embodiments disclosed herein, specific antigens, peptides and / or epitopes are recognized and presented in antigen-MHC complexes in the context of suitable class I or class II MHC proteins. The genetic composition of the object can be assessed to determine which MHC allele is suitable for a specific patient, disease or condition with a specific set of antigens. In mice, MHC genes are referred to as histocompatibility 2 (H-2) genes. Classical MHC class I subtypes of mice include H-2D, H-2K and H-2L. Non-classical MHC class I subtypes of mice include H-2Q, H-2M and H-2T. Classical MHC class II subtypes of mice include H-2A (IA) and H-2E (1-E). Nonclassical murine MHC class II subtypes include H-2M and H-2O. Canine MHC molecules are referred to as canine leukocyte antigens (DLA). Feline MHC molecules are referred to as feline leukocyte antigens (FLA). In some embodiments, orthologous or homologous MHC molecules are selected to transition a therapy or treatment involving a specific antigen-MHC complex from one species to a different species.

[0184] As used herein, the phrase "immune response" or its equivalent "immunological response" refers to the development of a cell-mediated response (e.g., mediated by antigen-specific T cells or their secretory products). Presentation of polypeptide epitopes bound to class I or class II MHC molecules can elicit a cellular immune response to treat or prevent viral infection, expand antigen-specific Breg cells, TC1, CD4 +T helper cells and / or CD8+ cytotoxic T cells and / or self-regulatory T cells and B cell "memory" cells produced by the disease. The response may also involve the activation of other components. In some aspects, the term "immune response" can be used for the formation of a regulatory network comprising immune cells. Therefore, the term "regulatory network formation" can refer to the immune response caused, whereby immune cells (preferably T cells, more preferably T regulatory cells) trigger further differentiation of other immune cells, such as, but not limited to, B cells or antigen presenting cells (non-limiting examples include dendritic cells, monocytes and macrophages). In some embodiments, the formation of the regulatory network involves the differentiation of B cells into regulatory B cells. In some embodiments, the formation of the regulatory network involves the formation of tolerogenic antigen presenting cells.

[0185] "Immune cells" include all cells generated by hematopoietic stem cells (HSCs), including but not limited to HSCs, white blood cells (leukocytes), lymphocytes (including T cells, B cells and natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "Leukocytes" include but are not limited to lymphocytes, granulocytes, monocytes and macrophages.

[0186] The terms "inflammatory response" and "inflammation" used herein represent the complex biological response of immune cells, humoral factors and vascular tissue of individuals or objects to exogenous or endogenous stimuli such as pathogens, damaged cells or stimulants and / or inflammatory signals such as proinflammatory cytokines. Inflammatory response includes the secretion of cytokines, and more particularly the secretion of proinflammatory cytokines, i.e., cytokines mainly produced by activated immune cells and participating in the amplification of inflammatory response. Exemplary proinflammatory cytokines and chemokines include but are not limited to IL-1β, TNF-α, IFN-γ, IL-8, IL-6, IL-12, IL-15, IL-16, IL-17 (including family members IL17A, IL17B, IL-17C, IL-17D, IL-17E, IL-17F), IL-18, GM-CSF, IL-21, IL-23, IL-27 and TGF-β. Exemplary anti-inflammatory cytokines include, but are not limited to, TGF-β, IL-1Rα, IL-4, IL-6, IL-10, IL-11, IL-13, IL-35, INF-α. Cytokines may have pro-inflammatory or anti-inflammatory properties according to a specific biological environment (Cavaillon, JM (2001) Cell Mol.Biol 47 (4): 695-702). Exemplary inflammation includes acute inflammation and chronic inflammation. Acute inflammation refers to a short-term process characterized by signs of typical inflammation (swelling, redness, pain, fever and loss of function) caused by infiltration of plasma and leukocytes into tissues. As long as there is a noxious stimulus, acute inflammation usually occurs, and once the stimulus is removed, destroyed or isolated by scar (fibrosis), the stimulus stops. Chronic inflammation refers to a condition characterized by concurrent active inflammation, tissue destruction and repair attempts. Chronic inflammation is not characterized by the typical symptoms of acute inflammation listed above. In contrast, chronically inflamed tissues are characterized by infiltration of mononuclear immune cells (monocytes, macrophages, lymphocytes and plasma cells), tissue destruction and healing attempts, including angiogenesis and fibrosis. In the sense of the present invention, inflammation can be inhibited by influencing and in particular inhibiting any event in an individual that forms a complex biological response associated with inflammation.

[0187] "Autoimmune diseases or conditions" include diseases or conditions caused by or directed against an individual's own tissues or organs, or manifestations thereof, or conditions resulting therefrom. In one embodiment, it refers to conditions caused by or aggravated by the production of T cells that react with normal body tissues and antigens. Examples of autoimmune diseases or conditions include, but are not limited to, arthritis (rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondylosteoarthritis and juvenile rheumatoid arthritis, osteoarthritis, chronic arthritis (arthritis chronica progrediente), deforming arthritis, chronic polyarthritis (polyarthritis chronica progrediente) primaria), primary arthritis, reactive arthritis, and ankylosing spondylitis); inflammatory hyperproliferative skin diseases, psoriasis (e.g., plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis); atopic diseases, including atopic diseases such as hay fever and Job's syndrome; dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, numerical dermatitis , seborrheic dermatitis, nonspecific dermatitis, primary contact dermatitis, and atopic dermatitis; X-linked hyper-IgM syndrome; allergic intraocular inflammatory diseases; urticaria, such as chronic allergic urticaria and chronic idiopathic urticaria including chronic autoimmune urticaria; myositis, polymyositis / dermatomyositis, juvenile dermatomyositis; toxic epidermal necrolysis; scleroderma (including systemic scleroderma), such as systemic sclerosis, such as multiple sclerosis (MS), Primary progressive MS (PPMS) and relapsing relapsing MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, diffuse sclerosis, ataxia, neuromyelitis optica spectrum disorder (NMO, also known as Devic's disease or Devic's syndrome); inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, colitis, polyposis colitis, necrotizing enterocolitis, and transmural colitis, as well as autoimmune inflammatory bowel disease), enteritis; pyoderma pyogenes; erythema nodosum; primary sclerosing cholangitis; respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS); meningitis; inflammation of the entire or partial uvea; iritis; choroiditis; autoimmune blood diseases; rheumatoid spondylitis, rheumatoid synovitis; hereditary angioedema; cranial nerve injury,Such as meningitis; herpetic gestationis, pemphigus gestationis, scrotitis, autoimmune premature ovarian failure; sudden hearing loss due to autoimmune disease; IgE-mediated diseases, such as anaphylaxis, allergic and atopic rhinitis; encephalitis, such as Rasmussen's encephalitis and limbic and / or brainstem encephalitis; uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phagocytic antigen uveitis, posterior uveitis or autoimmune uveitis; patients with or not with nephrotic syndrome (GN) such as chronic or acute glomerulonephritis, such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, including types I and II into membranous or membranous proliferative GN (MPGN), and rapidly progressive GN, proliferative glomerulonephritis, autoimmune polyglandular endocrine failure; balanitis; balanitis; erythema annulare; multiple erythema, erythema, annular granuloma, lichen needles, Scleroderma atrophicans, lichen atrophicans, chronic lichen planus, lichen planus, lichen planus, lamellar ichthyosis, epidermal lytic hyperkeratosis, malignant prekeratosis, pyoderma gangrenosum, allergic diseases and reactions, allergic allergies, eczema, eczema, eczematous eczema, muscular dystrophy eczema and vesicular palmoplantar eczema, asthma (such as bronchial asthma, bronchial asthma and autoimmune asthma), diseases involving T cell infiltration and chronic inflammatory reactions, directed against foreign antigens (such as immune responses of the fetal ABO blood type during pregnancy), immune response), chronic pulmonary disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus encephalitis, pediatric lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, lupus erythematosus, alopecia, systemic lupus erythematosus (LE) cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and lupus erythematosus, type I diabetes, type II diabetes, latent autoimmune diabetes mellitus in adults (or type 1.5 diabetes). Also considered are acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes, sarcoidosis, granulomatous diseases (including lymphomatous granulomatosis), Wegener's granulomatosis, agranulocytosis, vasculitis including vasculitis, large vessel vasculitis (including polymyalgia), medium vessel vasculitis (including Kawasaki disease and polynodular arteritis / nodular arteritis), microscopic polyinflammation, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, Churg-Strauss vasculitis or syndrome (CSS) and small vessel vasculitis associated with ANCA, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, Diamond's disease Blackfan anemia, hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia,Leukopenia, diseases involving leukocyte dialysis, CNS inflammatory diseases, Alzheimer's disease, Parkinson's disease, multiple organ damage syndromes, such as those secondary to sepsis, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, antiphospholipid syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid, pemphigoid, (including pemphigus vulgaris, pemphigus erythematosus, pemphigus erythematosus sores, mucous membranes, pemphigus and pemphigus erythematosus), autoimmune polyendocrinopathy, Rett's disease or syndrome, thermal injury, preeclampsia, immune complex diseases such as immune complex nephritis, immune complex nephritis, antibody-mediated nephritis, IgM polyneuropathy and other neuropathy asthma or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenia, such as idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP, acquired thrombocytopenic purpura, scleritis, such as idiopathic kerato-scleritis, scleritis, autoimmune diseases (including testicular and ovarian ) orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyglandular syndrome, etc. (or polyglandular endocrine disease syndrome), paraneoplastic syndromes, including paraneoplastic syndromes of the nervous system, such as Lambert-Eaton myasthenia gravis syndrome or Eaton-Lambert syndrome, Stiff man or stiff man syndrome, encephalomyelitis, such as allergic encephalomyelitis or encephalomyelitis allergic and experimental allergic encephalomyelitis (EAE), myasthenia gravis, such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, myoclonus or myoclonic-myoclonic syndrome (OMS), and sensory neuropathy, multifocal motor neuritis, autoimmune hepatitis, Sheehan's syndrome, lipoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barré syndrome, Bergey's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acanthosis dermatosis, cirrhosis (such as primary biliary cirrhosis and pneumohepatitis), autoimmune enteropathy syndrome, celiac or celiac disease, peritoneal perfusion sinusitis (gluten enteropathy), refractory enteropathy, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), autoimmune ear disease,Examples include autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis (e.g., refractory or relapsing or relapsing polychondritis), pulmonary alveolar proteinosis, Cogan's syndrome / nonsyphilitic interstitial keratitis, Bell's palsy, diabetic syndrome / syndrome, autoimmune rosacea, herpes zoster-related pain, amyloidosis, noncancerous lymphocytosis, primary lymphocytosis, including monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndromes, epilepsy, migraine, cardiac arrhythmias, muscle disorders, deafness, blindness, blindness, periodic paralysis, and channelopathies of the central nervous system , autism, inflammatory myopathies, channelopathies such as focal or segmental glomerulosclerosis (FSGS), endocrine eye diseases, uveoretinitis, chorioretinitis, autoimmune hepatitis diseases, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, progeria dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler syndrome, alopecia macrocephala, alopecia totalis, CREST syndrome (calcifications, esophageal dysmotility, sclerosis and telangiectasia), male and female autoimmune infertility, such as due to antispermia antibodies, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, polymorphism Erythema, postcardiotomy syndrome, Cushing's syndrome, bird lover's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport's syndrome, pneumonia such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosy, malaria, parasitic diseases such as schistosomiasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue fever, endocarditis, endocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema hyperplasia, mitochondrialization, fetal erythrocytosis Felty's syndrome, filariasis (flariasis), such as chronic cyclitis, metachronous cyclitis, iridocyclitis (acute or chronic) and other cyclitis or Fuch's cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echo virus infection, sepsis, endotoxemia, pancreatitis, hyperthyroidism, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune glandular failure, Sydenham's chorea, streptococcal nephritis, thromboangiitis, flooding, thyrotoxicosis, dorsal enteritis, chronic choroiditis, gianT cell polymorphism keratoconjunctivitis sicca, epidemic keratoconjunctivitis,Idiopathic nephrotic syndrome, minimal change disease, benign familial and ischemia-reperfusion injury, reperfusion of transplanted organs, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / lung disease, silicosis, aphthous ulcers, oral ulcers, arteriosclerosis, azoospermia, autoimmune hemolysis, Box's disease, cryoglobulinemia, Dupuyt's contracture, intraocular phagocytic eye, enteritis allergens, erythema nodosum leprosy, idiopathic facial palsy, chronic fatigue syndrome, rheumatic fever, Hamman-Rich's disease, sensorineural hearing loss, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, kidney disease, sympathetic eye, orchitis granuloma, pancreas inflammation, multiple radiation neuritis, non-nodular pustular muscular dystrophy, non-acquired muscular dystrophy, acquired body degeneration, flushing toxic shock syndrome, food poisoning, diseases involving T-cell infiltration, leukocyte adhesion deficiency, immune reactions mediated by cytokines and T lymphocytes related to acute and delayed hypersensitivity reactions, diseases involving leukocyte dialysis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, folliculitis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulin insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult Human episodic idiopathic hypoparathyroidism (AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermal bullous edema (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, frontal lobe, maxillary or sphenoid sinusitis, eosinophil-related diseases such as eosinophilia, pulmonary infiltration with eosinophilia, eosinophilic myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonia with Aspergillus eosinophilia, Aspergillus-containing, seronegative spondylitis, polyendocrine autoimmune disease, sclerosing cholangitis, sclerosing, episclerosing membranes, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypoglobulinemia in infancy, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, vasodilation, with autoimmune rheumatic diseases, autoimmune diseases, lymphadenitis, hypotension, vascular dysfunction, tissue damage, hyperalgesia, renal ischemia, cerebral ischemia and diseases with concomitant angiogenesis, allergic hypersensitivity, glomerular phospholipids, reperfusion injury, lymphomatous tracheobronchitis, inflammatory skin diseases, skin diseases with an acute inflammatory component, multiorgan failure, bullous diseases, renal cortical necrosis, acute purulent meningitis or other inflammatory diseases of the central nervous system, inflammatory diseases of the eye and orbit, granulocyte transfusion-related syndrome,Cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, nephritis, intimal hyperplasia, peptic ulcer, valvulitis, emphysema, alopecia areata, type II adipose tissue inflammation / diabetes, obesity-related adipose tissue inflammation / insulin resistance, and endometriosis.

[0188] The term "introduction" applied to the method of producing modified cells (e.g., chimeric antigen receptor cells) refers to the process of introducing an exogenous (i.e., exogenous or extracellular) agent into a host cell to produce a cell containing an exogenous agent. The method of introducing nucleic acid includes, but is not limited to, transduction, retroviral gene transfer, transfection, electroporation, transformation, viral infection, and other recombinant DNA techniques known in the art. In some embodiments, transduction is accomplished by a vector (e.g., a viral vector). In some embodiments, transfection is accomplished by a chemical vector, a DNA / liposome complex, or a micelle (e.g., Lipofectamine, Invitrogen). In some embodiments, viral infection is accomplished by infecting cells with viral particles (e.g., AAV) containing a polynucleotide of interest. In some embodiments, the introduction also includes CRISPR-mediated gene editing or TALEN-mediated gene editing. Methods for introducing non-nucleic acid exogenous agents (e.g., soluble factors, cytokines, proteins, peptides, enzymes, growth factors, signaling molecules, small molecule inhibitors) include, but are not limited to, culturing cells in the presence of the exogenous agent, contacting the cells with the agent, contacting the cells with a composition comprising the agent and an excipient, and contacting the cells with a vesicle or viral particle comprising the agent.

[0189] The term "culture" refers to growing T cells in a culture medium under conditions that are conducive to cell expansion and proliferation. The term "culture medium" is well-known in the art and generally refers to any substance or preparation used to culture living cells. When used for cell culture, the term "culture medium" includes components of the cell's surrounding environment. The culture medium can be a mixture of solid, liquid, gas, or various phases and materials. Culture medium includes liquid growth medium and liquid culture medium that does not maintain cell growth. Culture medium also includes gel-like culture medium, such as agar, agarose, gelatin, and collagen matrix. Exemplary gaseous media include gas phases, and cells grown on culture dishes or other solid or semi-solid supports are exposed to the gas phase. The term "culture medium" also refers to materials intended for cell culture, even if the substance is not in contact with the cells. In other words, a nutrient-rich liquid prepared for culture is a culture medium. Similarly, a powdered mixture suitable for cell culture after mixing with water or other liquids can be referred to as a "powdered culture medium." A "defined culture medium" refers to a medium made of chemically determined (usually purified) components. "Defined medium" does not contain biological extracts that are not fully characterized, such as yeast extract and beef broth. "Rich medium" includes medium designed to support the growth of most or all viable forms of a particular species. "Rich medium" generally contains complex biological extracts. "Medium suitable for high-density culture growth" is any medium in which other conditions (such as temperature and oxygen transfer rate) allow the cell culture to reach an OD600 of 3 or higher. The term "basal medium" refers to a medium that promotes the growth of many types of microorganisms and does not require any special nutritional supplements. Most basal media generally contain four basic chemical groups: amino acids, carbohydrates, inorganic salts and vitamins. Basal media are generally used as the basis for more complex culture media, to which supplements such as serum, buffer, growth factors, lipids, etc. are added. On the one hand, the growth medium can be a complex medium with essential growth factors to support the growth and expansion of the cells of the present invention while maintaining their self-renewal ability. Examples of basal media include, but are not limited to, Eagles basal medium, minimum essential medium, Dulbecco's modified Eagle's medium, Medium 199, Nutrient mixture Ham's F-10 and Ham's F-12, McCoy's 5A, Dulbecco's MEM / FI 2, RPMI 1640, and Iscove's modified Dulbecco's medium (IMDM).

[0190] "Cryoprotectants" are known in the art and include, but are not limited to, for example, sucrose, trehalose, and glycerol. Cryoprotectants that exhibit low toxicity in biological systems are generally used. Mode for carrying out the invention

[0191] To identify transcriptional and other regulatory factors that contribute to the attenuation of CAR T cell function in solid tumors, a mouse model was developed in which recipient mice bearing murine melanoma expressing the huCD19 antigen were adoptively transferred with huCD19-reactive CAR T cells. CD8+ CAR tumor-infiltrating T cells (CAR TILs) with low effector function and high expression of the inhibitory surface receptors PD-1 and TIM3 exhibited similar gene expression and chromatin accessibility profiles as endogenous TILs, with activation of the initiating transcription factor NFAT (nuclear factor of activated T cells). 16-18 , which is associated with secondary activation of three Nr4a (nuclear receptor subfamily group 4) transcription factors Nr4a1 (Nur77), Nr4a2 (Nurr1) and Nr4a3 (Nor1). 19,20 and viral antigen-specific CD8+ T cells from humans with chronic infection 21 Similar comparisons were made to data from , where high expression of Nr4a transcription factors and enrichment of Nr4a binding motifs in unique chromatin regions were observed. Treatment of tumor-bearing mice with CAR T cells lacking all three Nr4a transcription factors (Nr4a TKO) resulted in tumor regression and prolonged survival compared to mice adoptively transferred with Nr4a-sufficient (WT) CAR T cells. Nr4a TKO CAR TILs exhibited a phenotype and gene expression profile characteristic of CD8+ effector T cells, and uniquely accessible chromatin regions in Nr4a TKO CAR TILs were enriched with binding motifs of transcription factors classically involved in T cell activation compared to wild-type. As described herein, Nr4a transcription factors were identified as major players in the cell-intrinsic program of T cell hyporesponsiveness, and Nr4a inhibition is indicated as a promising strategy for cancer immunotherapy. Engineered immune cells

[0192] To this end, the present invention provides cells engineered to reduce or eliminate the expression and / or function of NR4A transcription factors in cells. On the one hand, cells are engineered to reduce or eliminate the expression and / or function of NR4A transcription factors in cells, wherein the NR4A transcription factor comprises one, two or all three of NR4A1 (Nur77), NR4A2 (Nurr1) or NR4A3 (NOR1), or substantially its composition, or consisting of it. Compared with wild-type cells, expression can be reduced by at least 10% or more, or 20% or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 85%, or 90%, or 95%, or 99%, or 100%. Non-limiting examples of cells are immune cells, such as T cells and NK cells.

[0193] In another aspect, the cell is engineered to reduce or eliminate the expression and / or function of the NR4A transcription factor in the cell, comprising, consisting essentially of, or consisting of the gene expression profile shown in Table 1 and / or Table 2. Expression may be reduced by at least 10% or more, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 85%, or 90%, or 95%, 99% or 100% compared to wild-type cells. Non-limiting examples of cells are immune cells, such as T cells and NK cells.

[0194] The present invention also provides cells engineered to reduce or eliminate the expression and / or function of TOX transcription factors in cells. In one aspect, cells are engineered to reduce or eliminate the expression and / or function of TOX transcription factors, wherein the TOX transcription factors comprise TOX1, TOX2, TOX3 or TOX4, or are substantially composed of, or are composed of. In another aspect, cells are engineered to reduce or eliminate the expression and / or function of two or more of TOX1, TOX2, TOX3 or TOX4. In another aspect, cells are engineered to reduce or eliminate the expression and / or function of four of TOX1, TOX2, TOX3 or TOX4. Compared with wild-type cells, expression can be reduced by at least 10% or more, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 85%, or 90%, or 95%, 99% or 100%. Non-limiting examples of cells are immune cells, such as T cells and NK cells.

[0195] The present invention also provides immune cells engineered to reduce or eliminate the expression and / or function of NR4A and TOX transcription factors in the immune cells. In one aspect, immune cells are engineered to reduce or eliminate the expression and / or function of NR4A and TOX transcription factors in the immune cells; wherein the NR4A transcription factor comprises NR4A (Nur77), NR4A2 (Nurr1) or NR4A3 (NOR1), or is essentially composed of it, or is composed of it; wherein the TOX transcription factor comprises TOX1, TOX2, TOX3 or TOX4, or is essentially composed of it, or is composed of it. In another aspect, wherein the immune cells are engineered to reduce or eliminate the expression and / or function of two or more, or three or more of NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (NOR1). Alternatively, the cell can be engineered to reduce one, two, three or all four TOX factors, and one, two, or three of all four NR4A transcription factors. Expression may be reduced by at least 10% or more, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 85%, or 90%, or 95%, or 99%, or 100% compared to wild-type cells.

[0196] The present invention also provides immune cells engineered to reduce or eliminate the expression and / or function of NR4A and TOX transcription factors (as described above and incorporated herein by reference) and increase the expression of IL-21 in the T cells. As used herein, the term "IL-21" (interleukin 21) refers to a member of the common γ chain family of cytokines with immunomodulatory activity. A non-limiting example is human IL-21 encoded by the sequence provided by SEQ ID NO: 10. Compared to the wild type, the expression increase includes, for example, at least about 2%, or about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 100% or more, or 150%, or 200%, or 250%, or 300%, or 350%, or 400%, or 450%, or 500%, or 550%, or 600%, or 650%, or 700%.

[0197] Also provided herein are immune cells engineered to inhibit expression and / or function of the NFAT / AP-1 pathway in immune cells. Expression may be reduced by at least 10% or more, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 85%, or 90%, or 95%, or 99%, or 100% compared to wild-type cells.

[0198] As used herein, the term "inhibiting the expression and / or function of the NFAT / AP-1 pathway" refers to reducing or eliminating the transcription of genes in the pathway, or reducing or eliminating the translation of the mRNA into a pathway peptide, polypeptide or protein, or reducing or eliminating the function of the peptide, polypeptide or protein of the pathway. Non-limiting examples of inhibiting the expression and / or function of the NFAT / AP-1 pathway include inhibiting the expression and / or function of NR4A transcription factor and / or TOX transcription factor, or increasing the expression of IL-21. Compared to wild-type cells, increased expression includes, for example, at least about 2%, or about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 100% or more, or 150%, or 200%, or 250%, or 300%, or 350%, or 400%, or 450%, or 500%, or 550%, or 600%, or 650%, or 700% or more.

[0199] Those skilled in the art can use methods such as RNA sequencing, DNA microarray, real-time PCR or chromatin immunoprecipitation (ChIP) to monitor the expression of transcription factors. Methods such as flow cytometry, immunoblotting, two-dimensional gel electrophoresis or immunoassay can be used to monitor the expression of proteins.

[0200] Those skilled in the art can use, for example, RNA interference (RNAi), CRISPR, TALEN, ZFN or other methods targeting specific sequences to reduce or eliminate the expression and / or function of NR4A or TOX transcription factors. CRISPR, TALEN, ZFN or other genome editing tools can also be used to increase the expression and / or function of IL-21.

[0201] Cells can be isolated from host or cultured immune cells. Such non-limiting samples include mammals and human cells as defined herein. On the one hand, immune cells are NK cells or T cells. When used for treatment, they can be autologous or allogeneic to the object being treated. For the purposes of the present invention, "T cells" include all types of immune cells expressing CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg) and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells and neutrophils, which are capable of mediating cytotoxic reactions. Non-limiting examples of commercially available T cell lines include: BCL2 (AAA) Jurkat ( CRL-2902 TM )、BCL2(S70A)Jurkat( CRL-2900 TM )、BCL2(S87A)Jurkat( CRL)-2901 TM )、BCL2Jurkat( CRL-2899 TM )、Neo Jurkat( CRL-2898 TM), TALL-104 cytotoxic human T cell line (ATCC #CRL-11386). Other examples include, but are not limited to, mature T cell lines such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T cell lines such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13,MOLT-16,MT-1,MT-ALL,P12 / Ichikawa,Peer,PER0117,PER-255,PF-382,PFI-285,RPMI-8402,ST-4,SUP-T1 to T14,TALL-1,TALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3 and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4; 11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and skin T-cell lymphoma cell lines, such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162). Leukemia-free cell lines, including but not limited to REH, NALL-1, KM-3, L92-221, are another commercially available source of immune cells derived from other leukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (or ATCC) (http: / / www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).These cells can be from any multicellular vertebrate, such as mammals and birds. The term "mammal" includes humans and non-human mammals, such as cows, dogs, cats, rats, mice, monkeys, horses and humans. Other examples include adults, teenagers and infants.

[0202] In some embodiments, the engineered immune cells of the present invention are CD8 T cells. In other embodiments, the engineered immune cells of the present invention are CD3 cells, T helper cells (CD4+ cells), natural killer (NK) T cells, T regulatory cells (Treg), γ-δ T cells or neutrophils.

[0203] In some embodiments, the engineered immune cells express receptors that bind to tumor antigens or antigens expressed by pathogens. In other embodiments, the engineered T cells express receptors that bind to tumor antigens, wherein the tumor antigen comprises mesothelin, ROR1 or EGFRvIII, ephrin receptor type A 2 (EphA2), interleukin (IL) -13rα2, EGFRVIII, PSMA, EpCAM, GD3, fucosyl GM1, PSCA, PLAC1, sarcoma breakpoints, Wilms tumor 1, hematological differentiation antigens, surface glycoproteins, gangliosides (GM2), growth factor receptors, matrix antigens, vascular antigens, or a combination thereof, or consists essentially of, or consists of.

[0204] In a specific embodiment, the engineered immune cells of the present invention further comprise, or consist essentially of, or consist of suicide genes. As used herein, the term "suicide gene" is a gene capable of inducing apoptosis; non-limiting examples include HSV-TK (herpes simplex virus thymidine kinase), cytosine deaminase, nitroreductase, carboxylesterase, cytochrome P450 or PNP (purine nucleoside phosphorylase), truncated EGFR or inducible caspase ("iCasp"). Suicide genes may act along a variety of pathways and, in some cases, can be induced by, for example, small molecule inducers.

[0205] On the one hand, the engineered immune cell of the present invention includes a chimeric antigen receptor (CAR), or is substantially composed of a chimeric antigen receptor (CAR), or is composed of a chimeric antigen receptor (CAR), therefore, the engineered cell is a CAR cell. Therefore, the engineered immune cell of the present invention further includes a chimeric antigen receptor (CAR), or is substantially composed of a chimeric antigen receptor (CAR), or is composed of a chimeric antigen receptor (CAR), wherein CAR also includes the following components, or is substantially composed of the following components, or is composed of the following components: (a) antigen binding domains; (b) hinge domains; (c) transmembrane domains; and (d) intracellular domains.

[0206] In one aspect, the engineered immune cells of the present invention comprise, or consist essentially of, or consist of a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor (CAR) comprises, or consists essentially of, or consists of: (a) an anti-CD19 binding domain; (b) a hinge domain; (c) a CD28 or CD8α transmembrane domain; (d) one or more co-stimulatory regions selected from a CD28 co-stimulatory signaling region, a 4-1BB co-stimulatory signaling region, an ICOS co-stimulatory signaling region, and an OX40 co-stimulatory region; and (e) a CD3ζ signaling domain. The following sequences are merely exemplary sequences that can be used to prepare cells as described herein:

[0207] Hinge domain: IgG1 heavy chain hinge polynucleotide sequence: CTCGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCG, and alternatively its equivalents.

[0208] Transmembrane domain: CD28 transmembrane region polynucleotide sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA CAGTGGCCTTTATTATTTTCTGGGTG, and alternative equivalents.

[0209] Intracellular domain: 4-1BB co-stimulatory signaling region nucleotide sequence: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGAGGATGTGAACTG, and alternative equivalents.

[0210] Intracellular domain: CD28 co-stimulatory signaling region nucleotide sequence: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC CGCCCCGGGGCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCG CAGCCTATCGCTCC, and alternative equivalents.

[0211] Intracellular domain: CD3ζ signaling region polynucleotide sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA, and alternative equivalents.

[0212] Other embodiments of each exemplary domain component include: other proteins with similar biological functions, which have at least 70% or at least 80% amino acid sequence identity with the protein encoded by the nucleic acid sequence disclosed above, preferably 90% sequence identity, more preferably at least 95% sequence identity. In addition, non-limiting examples of such domains are provided herein.

[0213] A non-limiting example of a CAR extracellular domain capable of binding to an antigen is an anti-CD19 binding domain sequence that specifically binds to the CD19 antigen as disclosed in the US20140271635 application. Thus, the polynucleotide will encode the binding domain.

[0214] As used herein, the term "CD8α hinge domain" refers to a specific protein fragment associated with this name and any other molecule with a similar biological function, which has at least 70% or at least 80% amino acid sequence identity, preferably 90% sequence identity, and more preferably at least 95% sequence identity with the CD8α hinge domain sequence as shown herein. Pinto, RDet al. (2006) Vet. Immunol. Immunopathol. 110: 169-177 provides example sequences of CD8α hinge domains of humans, mice, and other species. Pinto, RDet al. (2006) Vet. Immunol. Immunopathol. 110: 169-177 provides sequences related to the CD8α hinge domain. Such non-limiting examples include:

[0215] Human CD8 alpha hinge domain amino acid sequence: PAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY, and alternatively its equivalents.

[0216] Mouse CD8α hinge domain amino acid sequence: KVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY, and alternative equivalents thereof.

[0217] Cat CD8 alpha hinge domain amino acid sequence: PVKPTTTPAPRPPTQAPITTSQRVSLRPGTCQPSAGSTVEASGLDLSCDIY and alternative equivalents thereof.

[0218] Therefore, the polynucleotides encoding these peptides are contained within the polynucleotide encoding CAR.

[0219] As used herein, the term "CD8α transmembrane domain" refers to a specific protein fragment associated with this name and any other molecule with a similar biological function, which has at least 70% or at least 80% amino acid sequence identity with the CD8α transmembrane domain sequence as shown herein, preferably 90% sequence identity, and more preferably at least 95% sequence identity. The fragment sequence associated with amino acid positions 183 to 203 of the human T cell surface glycoprotein CD8α chain (GenBank Accession No.: NP_001759.3), or the fragment sequence associated with amino acids 197 to 217 of the mouse T cell surface glycoprotein CD8α chain (GenBank Accession No.: NP_001074579.1), and the fragment sequence associated with amino acids 190 to 210 of the rat T cell surface glycoprotein CD8α chain (GenBank Accession No.: NP_113726.1) provide other exemplary sequences of the CD8α transmembrane domain. The sequences associated with each accession number listed are as follows:

[0220] Human CD8α transmembrane domain amino acid sequence: IYIWAPLAGTCGVLLLSLVIT, and alternatively its equivalents.

[0221] Mouse CD8α transmembrane domain amino acid sequence: IWAPLAGICVALLLSLIITLI, and alternative equivalents thereof.

[0222] Rat CD8α transmembrane domain amino acid sequence: IWAPLAGICAVLLLSLVITLI, and alternative equivalents thereof.

[0223] Therefore, polynucleotides encoding these peptides are included within the polypeptides.

[0224] As used herein, the term "CD28 transmembrane domain" refers to a specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity, at least 90% sequence identity, or at least 95% sequence identity with the CD28 transmembrane domain sequence as shown herein. The fragment sequences associated with GenBank Accession Nos. XM_006712862.2 and XM_009444056.1 provide other non-limiting example sequences of CD28 transmembrane domain.

[0225] As used herein, the term "4-1BB costimulatory signaling region" refers to a specific protein fragment associated with this name and any other molecule with a similar biological function, which has at least 70% or at least 80% amino acid sequence identity, preferably 90% sequence identity, and more preferably at least 95% sequence identity with the 4-1BB costimulatory signaling region sequence as shown herein. Non-limiting example sequences of the 4-1BB costimulatory signaling region are provided in U.S. Publication No. 20130266551A1 (submitted as U.S. Patent Application No. 13 / 826,258), such as the exemplary sequences provided below.

[0226] 4-1BB costimulatory signaling region amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, and optional equivalents thereof. Therefore, the polynucleotide encoding the sequence is encoded within the polynucleotide.

[0227] As used herein, the term "ICOS costimulatory signaling region" refers to the specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity with the ICOS costimulatory signaling region sequence as shown herein, preferably 90% sequence identity, more preferably at least 95% sequence identity. Non-limiting example sequences of ICOS costimulatory signaling regions are provided in U.S. Patent Application Publication No. 2015 / 0017141A1, and exemplary polynucleotide sequences are provided below.

[0228] The polynucleotide sequence of the ICOS co-stimulatory signaling region: ACAAAAAAGA AGTATTCATC CAGTGTGCACGACCCTAACG GTGAATACAT GTTCATGAGA GCAGTGAACA CAGCCAAAAA ATCCAGACTC ACAGATGTGACCCTA, and alternatively its equivalents.

[0229] As used herein, the term "OX40 costimulatory signaling region" refers to the specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity, or 90% sequence identity, or at least 95% sequence identity with the OX40 costimulatory signaling region sequence as shown herein. Non-limiting example sequences of the OX40 costimulatory signaling region are disclosed in U.S. Patent Application Publication No. 2012 / 20148552A1, and include the exemplary sequences provided below.

[0230] The polynucleotide sequence of the OX40 co-stimulatory signaling region: AGGGACCAG AGGCTGCCCC CCGATGCCCACAAGCCCCCT GGGGGAGGCA GTTTCCGGAC CCCCATCCAA GAGGAGCAGG CCGACGCCCA CTCCACCCTGGCCAAGATC, and alternatively equivalents thereof.

[0231] As used herein, the term "CD28 co-stimulatory signaling region" refers to the specific protein fragment associated with this name and any other molecules with similar biological functions, which have at least 70% or at least 80% amino acid sequence identity, or 90% sequence identity, or at least 95% sequence identity with the CD28 co-stimulatory signaling region sequence shown herein. Exemplary sequences of CD28 costimulatory signaling domains are provided in U.S. Pat. No. 5,686,281; Geiger, TL et al. (2001) Blood 98:2364-2371; Hombach, A. et al. (2001) J Immunol 167:6123-6131; Maher, J. et al. (2002) Nat Biotechnol 20:70-75; Haynes, NM et al. (2002) J Immunol 169:5780-5786 (2002); Haynes, NM et al. (2002) Blood 100:3155-3163. Non-limiting examples include the following sequence: CD28 amino acid sequence: MLRLLLALNL FPSIQVTGNKILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLDSAVEVCVVYG NYSQQLQVYSKTGFNCDGKLGNESVTFYLQ NLYVNQTDIY FCKIEVMYPPPYLDNEKSNG TIIHVKGKHLCPSPLFPGPSKPFWVLVVVG GVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS and optionally equivalents thereof. Therefore, polynucleotides encoding these polypeptides are included in the polypeptide encoding CAR.

[0232] As used herein, the term "CD3 zeta signaling domain" refers to the specific protein fragment associated with this name and any other molecule with similar biological function, which has at least 70% or at least 80% amino acid sequence identity, or 90% sequence identity, or at least 95% sequence identity with the CD3 zeta signaling domain sequence as shown herein. Non-limiting example sequences of amino acid sequences of CD3 zeta signaling domains are provided in U.S. Application No. 13 / 826,258, for example: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR. Therefore, polynucleotides encoding these polypeptides are included in polynucleotides encoding CAR.

[0233] On the one hand, the engineered cells of the present invention include chimeric antigen receptors (CAR), or are substantially composed of chimeric antigen receptors (CAR), or are composed of chimeric antigen receptors (CAR); wherein the anti-CD19 binding domain of the CAR includes a single-chain variable fragment (scFv) that specifically recognizes humanized anti-CD19 binding domains, or is substantially composed of it, or is further composed of it. On the other hand, the engineered cells of the present invention include chimeric antigen receptors (CAR), or are substantially composed of chimeric antigen receptors (CAR), or are further composed of chimeric antigen receptors (CAR); wherein the anti-CD19 binding domain scFv of the CAR includes a heavy chain variable region and a light chain variable region, or is substantially composed of it, or is composed of it. A non-limiting example of a CAR extracellular domain capable of binding to an antigen is an anti-CD19 binding domain sequence that specifically binds to the CD19 antigen as disclosed in the US20140271635 application.

[0234] In a specific embodiment, the engineered cell of the present invention comprises a chimeric antigen receptor (CAR), or is essentially composed of a chimeric antigen receptor (CAR), or is composed of a chimeric antigen receptor (CAR), wherein the anti-CD19 binding domain scFv of CAR includes a linker polypeptide located between the anti-CD19 binding domain scFv heavy chain variable region and the anti-CD19 binding domain scFv light chain variable region, or is essentially composed of it, or is composed of it. On the other hand, the engineered T cell of the present invention comprises a chimeric antigen receptor (CAR), or is essentially composed of a chimeric antigen receptor (CAR), or is further composed of a chimeric antigen receptor (CAR), wherein the linker polypeptide of the CAR comprises a sequence of (GGGGS)n where n is an integer from 1 to 6, or is essentially composed of it, or is composed of it. Alternatively, a "linker sequence" refers to any amino acid sequence comprising 1 to 10 amino acids, or 8 amino acids, or 6 amino acids, or 5 amino acids, which can be repeated 1 to 10 times, or 1 to about 8 times, or 1 to about 6 times, or about 5 times, or 4 times, or 3 times, or 2 times. For example, a linker can comprise up to 15 amino acid residues consisting of a pentapeptide repeated three times. In one aspect, a linker sequence is a (glycine 4 serine) 3 flexible polypeptide linker comprising three copies of gly-gly-gly-gly-ser-, represented by the single letter sequence symbol GGGGS.

[0235] In a specific embodiment, the engineered cells of the invention comprise, consist essentially of, or consist of a chimeric antigen receptor (CAR), wherein the CAR further comprises, consists essentially of, or consists of a detectable marker or purification marker attached to or expressed by the CAR.

[0236] Non-limiting examples of detectable labels include enzymes that generate a detectable signal (e.g., by colorimetry, fluorescence, luminescence), such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase; chromophores, such as fluorescent, luminescent dyes; groups whose electron density is detected by electron microscopy or by their electrical properties (e.g., conductivity, amperometry, voltammetry, impedance); detectable groups, such as detectable groups whose molecular size is sufficient to induce a detectable modification in their physical and / or chemical properties, such that detection can be by optical methods (e.g., diffraction, surface plasmon resonance, surface changes, contact angle changes) or physical methods (e.g., atomic force spectroscopy, tunneling) or radioactive molecules (e.g., 32 P. 35 S or 125 I) Completed.

[0237] Non-limiting examples of purification tags include His, lacZ, GST, maltose binding protein, NusA, BCCP, c-myc, CaM, FLAG, GFP, YFP, cherry red, thioredoxin, poly (NANP), V5, Snap, HA, chitin binding protein, Softag 1, Softag 3, Streptococcus or S protein. Suitable direct or indirect fluorescent labels include: FLAG, GFP, YFP, RFP, dTomato, cherry red, Cy3, Cy5, Cy5.5, Cy 7, DNP, AMCA, biotin, digoxigenin, Tamra, Texas Red, rhodamine, Alexa fluors, FITC, TRITC or any other fluorescent dye or hapten.

[0238] In one aspect, the engineered cells of the invention comprise, consist essentially of, or consist of a polynucleotide encoding a CAR, and optionally, wherein the polynucleotide further encodes an anti-CD19 binding domain.

[0239] The CAR cells of the present invention can be produced by inserting a polynucleotide encoding CAR into an engineered immune cell and then expressing CAR in the cell. Therefore, on the one hand, the engineered T cells of the present invention include a polynucleotide encoding CAR, or consist essentially of it, or consist of it, wherein the polynucleotide also includes a promoter operably connected to the polynucleotide to express the polynucleotide in the cell, or consists essentially of it, or consists of it. Non-limiting examples of promoters include constitutive, inducible, repressible or tissue-specific promoters. The promoter transcribes the connected polynucleotide in an "operably connected" manner.

[0240] In one aspect, the polynucleotide further comprises, or consists essentially of, or further consists of a sequence encoding a 2A self-cleaving peptide (T2A), which is optionally located upstream of a polynucleotide encoding an antigen binding domain (e.g., an anti-CD19 binding domain). "T2A" and "2A peptide" are used interchangeably to refer to any 2A peptide or fragment thereof, any 2A-like peptide or fragment thereof, or an artificial peptide comprising essential amino acids, wherein the essential amino acids are relatively short (depending on the virus from which they are derived) peptide sequences containing the consensus polypeptide motif DV / IEXNPGP, wherein X refers to any amino acid that is generally considered to be self-cleaving.

[0241] In some embodiments, the engineered cells of the present invention include polynucleotides encoding CAR, or consist essentially of, or consist of, wherein the polynucleotides further include a signal peptide upstream of a polynucleotide encoding an anti-CD19 binding domain, or consist essentially of, or consist of. In a specific embodiment, the engineered cells of the present invention include polynucleotides encoding CAR, or consist essentially of, or consist of, wherein the polynucleotides further include a mouse Thy1.1 reporter gene signal polypeptide, or consist essentially of, or consist of.

[0242] In some embodiments, the engineered cells of the present invention comprise, consist essentially of, or consist of a polynucleotide encoding a CAR, wherein the polynucleotide further comprises, consists essentially of, or consists of a sequence of SEQ ID NO: 1.

[0243] In some embodiments, the engineered T cells of the present invention comprise a polynucleotide encoding CAR, or consist essentially of a polynucleotide encoding CAR, or consist of a polynucleotide encoding CAR, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 2.

[0244] The polynucleotide encoding the CAR may be contained within a vector such as a plasmid. In a separate aspect, the vector is a viral vector selected from a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0245] In some embodiments, the cells of the invention have been isolated from a subject. In a specific embodiment, the cells of the invention have been isolated from a subject, wherein the subject has cancer.

[0246] In a specific embodiment, the cells of the invention have been isolated from a subject, wherein the subject has a cancer, and the tumor antigen is expressed by cells associated with the cancer. Production cells

[0247] The present invention also provides a method for producing engineered immune cells, the method comprising reducing or eliminating the expression and / or function of the NR4A transcription factor in the cell, or consisting essentially of it, or consisting of it. In one aspect, the method for producing engineered immune cells also includes, or consists essentially of the following, or further consists of the following: isolating cells from a subject, reducing or eliminating the expression and / or function of the NR4A transcription factor in the cell, and culturing the cells under conditions that are conducive to cell expansion and proliferation.

[0248] The present invention also provides a method for producing engineered immune cells, the method comprising reducing or eliminating the expression and / or function of the TOX transcription factor in the cell. In one aspect, the method for producing engineered immune cells also comprises, or essentially consists of, or consists of: isolating immune cells from a subject, reducing or eliminating the expression and / or function of the TOX transcription factor in the cell, and culturing the immune cells under conditions conducive to cell expansion and proliferation.

[0249] Immune cells include, but are not limited to, NK cells and T cells. As used herein, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes (e.g., B cells) by the presence of T cell receptors on the cell surface. T cells can be isolated or obtained from commercial channels. "T cells" include all types of immune cells expressing CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg), and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic reactions. Non-limiting examples of commercially available T cell lines include: BCL2 (AAA) Jurkat ( CRL-2902 TM )、BCL2(S70A)Jurkat( CRL-2900 TM )、BCL2(S87A)Jurkat( CRL-2901 TM )、BCL2Jurkat( CRL-2899 TM )、Neo Jurkat( CRL-2898 TM), ALL-104 cytotoxic human T cell line (ATCC #CRL-11386). Other examples include, but are not limited to, mature T cell lines, such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T cell lines, such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13,MOLT-16,MT-1,MT-ALL,P12 / Ichikawa,Peer,PER0117,PER-255,PF-382,PFI-285,RPMI-8402,ST-4,SUP-T1 to T14,TALL-1,TALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3 and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4; 11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and skin T-cell lymphoma cell lines, such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162). Leukemia-free cell lines, including but not limited to REH, NALL-1, KM-3, L92-221, are another commercially available source of immune cells, as well as cell lines derived from other leukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (or ATCC) (http: / / www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).

[0250] The term "reduce or eliminate ... expression and / or function" is intended to reduce or eliminate the transcription of the polynucleotide to mRNA, or reduce or eliminate the translation of the mRNA to a peptide, polypeptide or protein, or reduce or eliminate the function of the peptide, polypeptide or protein. In a non-limiting example, the transcription of the polynucleotide to mRNA is reduced to at least half of its normal level found in wild-type cells.

[0251] In one aspect, the present invention provides a method for producing engineered immune cells, the method comprising the following steps, or consisting essentially of the following steps, or consisting of the following steps: reducing or eliminating the expression and / or function of NR4A and TOX transcription factors in cells. On the one hand, the method for producing engineered immune cells also comprises the following steps, or consisting essentially of the following steps, or consisting of the following steps: isolating immune cells from a subject, reducing or eliminating the expression and / or function of NR4A transcription factors in the cells, and culturing the cells under conditions that are conducive to cell expansion and proliferation. On the other hand, the method for producing engineered immune cells also comprises the following steps, or consisting essentially of the following steps, or consisting of the following steps: isolating immune cells from a subject, reducing or eliminating the expression and / or function of TOX transcription factors in the cells, and culturing the cells under conditions that are conducive to cell expansion and proliferation.

[0252] The transduced cells can be cultured under conditions that allow the cells to grow and expand.

[0253] For the purpose of this method, the term "NR4A transcription factor" refers to a member of the NR4A subfamily of nuclear hormone receptors that bind to DNA and regulate gene expression. Non-limiting examples of NR4A transcription factor family members are human NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (NOR1) encoded by the sequences provided by SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively. Also for the purpose of this disclosure, the term "TOX transcription factor" refers to a member of the TOX subfamily of nuclear hormone receptors that bind to DNA and regulate gene expression. Non-limiting examples of TOX transcription factor family members are human TOX1, TOX2, TOX3, and TOX4 encoded by the sequences provided by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. Further for the purpose of this disclosure, the term "IL-21" (interleukin 21) refers to a member of the common γ chain family of cytokines with immunomodulatory activity. A non-limiting example is human IL-21 encoded by the sequence provided by SEQ ID NO:10.

[0254] Immune cells include, but are not limited to, NK cells and T cells. As used herein, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes (e.g., B cells) by the presence of T cell receptors on the cell surface. T cells can be isolated or obtained from commercial channels. "T cells" include all types of immune cells expressing CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg), and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic reactions. Non-limiting examples of commercially available T cell lines include: BCL2 (AAA) Jurkat ( CRL-2902 TM )、BCL2(S70A)Jurkat( CRL-2900 TM )、BCL2(S87A)Jurkat( CRL-2901 TM )、BCL2Jurkat( CRL-2899 TM )、Neo Jurkat( CRL-2898 TM), ALL-104 cytotoxic human T cell line (ATCC #CRL-11386). Other examples include, but are not limited to, mature T cell lines, such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T cell lines, such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13,MOLT-16,MT-1,MT-ALL,P12 / Ichikawa,Peer,PER0117,PER-255,PF-382,PFI-285,RPMI-8402,ST-4,SUP-T1 to T14,TALL-1,TALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3 and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4; 11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and skin T-cell lymphoma cell lines, such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162). Leukemia-free cell lines, including but not limited to REH, NALL-1, KM-3, L92-221, are another commercially available source of immune cells, as well as cell lines derived from other leukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (or ATCC) (http: / / www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).

[0255] On the other hand, the present disclosure provides a method for generating engineered immune cells, the method comprising the following steps, or consisting essentially of the following steps, or consisting of the following steps: reducing or eliminating the expression and / or function of NR4A and TOX transcription factors, and increasing the expression of IL-21 in cells. On the one hand, the method for producing engineered cells also comprises the following steps, or consisting essentially of the following steps, or consisting of the following steps: isolating immune cells from a subject, reducing or eliminating the expression and / or function of NR4A transcription factors in the cells, and culturing the cells under conditions that are conducive to cell expansion and proliferation. On the other hand, the method for producing engineered immune cells further comprises the following steps, or consisting essentially of the following steps, or consisting of the following steps: isolating immune cells from a subject, reducing or eliminating the expression and / or function of TOX transcription factors in the cells, and culturing the cells under conditions that are conducive to cell expansion and proliferation. On the other hand, the method for producing engineered immune cells further comprises the following steps, or consisting essentially of the following steps, or consisting of the following steps: isolating immune cells from a subject, increasing the expression and / or function of IL-21 in the cells, and culturing the cells under conditions that are conducive to cell expansion and proliferation.

[0256] Those skilled in the art can use methods such as RNA sequencing, DNA microarray, real-time PCR or chromatin immunoprecipitation (ChIP) to monitor the expression of transcription factors. Methods such as flow cytometry, immunoblotting, 2D gel electrophoresis or immunoassay can be used to monitor the expression of proteins.

[0257] Those skilled in the art can use methods such as RNA interference (RNAi), CRISPR, TALEN, ZFN or other methods targeting specific sequences to reduce or eliminate the expression and / or function of NR4A or TOX transcription factors. CRISPR, TALEN, ZFN or other genome editing tools can also be used to increase the expression and / or function of IL-21.

[0258] In another aspect, a method of producing an engineered immune cell comprises, consists essentially of, or consists of the following steps: inhibiting expression and / or function of the NFAT / AP-1 pathway in a cell. In one aspect, a method of producing an engineered immune cell also comprises, consists essentially of, or consists of the following steps: isolating an immune cell from a subject, inhibiting expression and / or function of the NFAT / AP-1 pathway in the cell, and culturing the cell under conditions conducive to cell expansion and proliferation.

[0259] As used herein, the term "inhibiting the expression and / or function of the NFAT / AP-1 pathway" refers to reducing or eliminating the transcription of genes in the pathway, or reducing or eliminating the translation of the mRNA into a pathway peptide, polypeptide, or protein, or reducing or eliminating the function of the peptide, polypeptide, or protein of the pathway. Non-limiting examples of inhibiting the expression and / or function of the NFAT / AP-1 pathway include: inhibiting the expression and / or function of NR4A transcription factor or TOX transcription factor, or increasing the expression of IL-21.

[0260] The term "reducing or eliminating ... expression and / or function" refers to reducing or eliminating the transcription of the polynucleotide to mRNA, or reducing or eliminating the translation of the mRNA to a peptide, polypeptide or protein, or reducing or eliminating the function of the peptide, polypeptide or protein. In a non-limiting example, the transcription of the polynucleotide to mRNA is reduced to at least half of its normal level found in wild-type cells.

[0261] Immune cells include, but are not limited to, NK cells and T cells. As used herein, the term "T cell" refers to a class of lymphocytes that mature in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes (e.g., B cells) by the presence of T cell receptors on the cell surface. T cells can be isolated or obtained from commercial channels. "T cells" include all types of immune cells expressing CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg), and γ-δ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic reactions. Non-limiting examples of commercially available T cell lines include BCL2 (AAA) Jurkat ( CRL-2902 TM ), BCL2(S70A)Jurkat( CRL-2900 TM ), BCL2(S87A)Jurkat( CRL)-2901 TM ), BCL2Jurkat( CRL-2899 TM ), Neo Jurkat( CRL-2898 TM ), TALL-104 cytotoxic human T cell line (ATCC #CRL-11386). Non-limiting examples of commercially available T cell lines include: BCL2 (AAA) Jurkat ( CRL-2902 TM)、BCL2(S70A)Jurkat( CRL-2900 TM )、BCL2(S87A)Jurkat( CRL-2901 TM )、BCL2Jurkat( CRL-2899 TM )、Neo Jurkat( CRL-2898 TM), ALL-104 cytotoxic human T cell line (ATCC #CRL-11386). Other examples include, but are not limited to, mature T cell lines, such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T cell lines, such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13,MOLT-16,MT-1,MT-ALL,P12 / Ichikawa,Peer,PER0117,PER-255,PF-382,PFI-285,RPMI-8402,ST-4,SUP-T1 to T14,TALL-1,TALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3 and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4; 11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and skin T-cell lymphoma cell lines, such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162). Leukemia-free cell lines, including but not limited to REH, NALL-1, KM-3, L92-221, are another commercially available source of immune cells, as well as cell lines derived from other leukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection (ATCC) (http: / / www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (https: / / www.dsmz.de / ).

[0262] On the other hand, the method for producing engineered immune cells as described above further comprises the following steps, or consists essentially of the following steps, or consists of the following steps: isolating immune cells from a subject, wherein the cells isolated from the subject bind to a target antigen. On the one hand, the target antigen is a tumor antigen or an antigen expressed by a pathogen. On the other hand, the tumor antigen comprises, or consists essentially of, or consists of: mesothelin, ROR1 or EGFRvIII, ephrin receptor type A 2 (EphA2), interleukin (IL) -13rα2, EGFR VIII, PSMA, EpCAM, GD3, fucosyl GM1, PSCA, PLAC1, sarcoma breakpoints, Wilms tumor 1, hematological differentiation antigens, surface glycoproteins, gangliosides (GM2), growth factor receptors, matrix antigens, vascular antigens, or a combination thereof.

[0263] The term "receptor" or "T cell receptor" or "TCR" refers to a cell surface molecule found on T cells whose function is to recognize and bind to antigens presented by antigen presenting molecules. Typically, TCRs are heterodimers of an alpha chain (TRA) and a beta chain (TRB). Some TCRs consist of alternative gamma (TRG) and delta (TRD) chains. T cells expressing this version of TCR are called gamma delta T cells. TCRs are part of the immunoglobulin superfamily. Therefore, like antibodies, each TCR chain contains three hypervariable CDR regions. There is also a hypervariable region (HV4) on the beta chain. TCR heterodimers are typically present in an octamer complex, which further contains three dimeric signaling modules CD3γ / ε, CD3δ / ε, and CD247ζ / ζ or ζ / η.

[00136] A non-limiting exemplary amino acid sequence of a human TCR-alpha chain: METLLGVSLVILWLQLARVNSQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASRAADTASYFCAPVLSGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPED TFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS. Non-limiting exemplary amino acid sequence of a human TCR-beta chain: DSAVYLCASSLLRVYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPPEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQP.

[0264] On the one hand, the method for producing the engineered immune cell of the present disclosure also includes the following steps, or is substantially composed of this step, or is composed of this step: the polynucleotide (polynucleotide CAR) encoding the chimeric antigen receptor is introduced into the cell. In one embodiment, the polynucleotide CAR includes the following components, or is substantially composed of the following components, or is composed of the following components: (a) antigen binding domain, (b) hinge domain, (c) transmembrane domain, and (d) intracellular domain.

[0265] On the one hand, the method for producing the engineered immune cells of the present invention also includes introducing a polynucleotide CAR, or is essentially composed of this step, or is composed of this step; wherein the polynucleotide CAR also comprises the following components, or is essentially composed of the following components, or is composed of the following components: (a) an anti-CD19 binding domain; (b) a hinge domain; (c) a CD28 or CD8α transmembrane domain; (d) one or more co-stimulatory regions selected from the CD28 co-stimulatory signaling region, the 4-1BB co-stimulatory signaling region, the ICOS co-stimulatory signaling region, and the OX40 co-stimulatory signaling region; and (e) a CD3ζ signaling domain.

[0266] The chimeric antigen receptor may optionally comprise a "hinge domain" that acts as a linker between the extracellular domain and the transmembrane domain. Non-limiting exemplary polynucleotide sequences encoding these components are described herein.

[0267] A non-limiting example of a CAR extracellular domain capable of binding to an antigen is an anti-CD19 binding domain sequence that specifically binds to the CD19 antigen as disclosed in the US20140271635 application.

[0268] In a specific aspect, the method for producing the engineered immune cell of the present disclosure also includes introducing a polynucleotide CAR, or consisting essentially of this step, or consisting of this step, wherein the anti-CD19 binding domain of the polynucleotide CAR is a single-chain variable fragment (scFv) that specifically recognizes the humanized anti-CD19 binding domain. On the other hand, the anti-CD19 binding domain scFv of the polynucleotide CAR includes a heavy chain variable region and a light chain variable region, or is essentially composed of a heavy chain variable region and a light chain variable region, or is composed of a heavy chain variable region and a light chain variable region.

[0269] A chimeric antigen receptor may optionally comprise a "hinge domain" which acts as a linker between the extracellular and transmembrane domains.

[0270] A non-limiting example of a CAR extracellular domain capable of binding to an antigen is an anti-CD19 binding domain sequence that specifically binds to the CD19 antigen, as disclosed in the US20140271635 application.

[0271] In a specific aspect, the method for producing the engineered T cells of the present invention further comprises the following steps, or is substantially composed of the following steps, or is composed of the following steps: introducing a polynucleotide CAR, wherein the anti-CD19 binding domain scFv of the polynucleotide CAR comprises a joint polypeptide located between the anti-CD19 binding domain scFv heavy chain variable region and the anti-CD19 binding domain scFv light chain variable region, or is substantially composed of it, or is composed of it. On the other hand, the method for producing the engineered T cells of the present invention further comprises the following steps, or is substantially composed of the following steps, or is composed of the following steps: introducing a polynucleotide CAR with a joint, wherein the polynucleotide CAR joint polypeptide comprises a sequence of (GGGGS)n where n is an integer of 1 to 6, or is substantially composed of it, or is composed of it.

[0272] On the one hand, the method for producing engineered T cells of the present invention further comprises, or is essentially composed of, or is composed of the following steps: introducing a polynucleotide CAR, wherein the polynucleotide further comprises a detectable marker and / or a purification marker.

[0273] On the other hand, the method for producing the engineered immune cells of the present invention further comprises, or consists essentially of, or consists of the following steps: introducing a polynucleotide CAR, wherein the polynucleotide further comprises a promoter operably linked to the polynucleotide to express the polynucleotide in the immune cells.

[0274] On the other hand, the method for producing the engineered immune cells of the present invention further comprises, or consists essentially of, or consists of the following steps: introducing a polynucleotide CAR, wherein the polynucleotide further comprises, or consists essentially of, or consists of the following components: a polynucleotide sequence encoding a 2A self-cleaving peptide (T2A), which is optionally located upstream of a polynucleotide encoding an anti-CD19 binding domain.

[0275] In some embodiments, the method for producing engineered immune cells of the present invention comprises, or consists essentially of, or consists of the following steps: introducing a polynucleotide CAR, wherein the polynucleotide sequence comprises, or consists essentially of, or consists of SEQ ID NO: 1.

[0276] In one embodiment, the method for producing the engineered immune cells of the present invention comprises, consists essentially of, or consists of the following steps: introducing a polynucleotide CAR, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 2.

[0277] In another embodiment, the method for producing the engineered immune cells of the present invention comprises the following steps, or consists essentially of the following steps, or consists of the following steps: introducing a polynucleotide CAR, wherein the polynucleotide further comprises a vector, or consists essentially of a vector, or consists of a vector. On the one hand, the vector also comprises an isolated nucleic acid sequence containing SEQ ID NO: 1, or consists essentially of the nucleic acid sequence, or consists of the nucleic acid sequence. On the other hand, the vector is a plasmid. In a separate aspect, the vector is a viral vector selected from a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0278] The present invention also provides immune cells prepared by any of the above disclosed methods for producing engineered immune cells.

[0279] The present invention also provides a substantially homogeneous cell population of any of the engineered immune cells of the present invention.

[0280] The present invention also provides a heterogeneous cell population of any engineered immune cell of the present invention. Composition

[0281] In one aspect, compositions are provided herein, comprising a carrier and one or more engineered immune cells of the invention or a cell population of any of the engineered immune cells of the invention, or consisting essentially of these, or consisting of these. In another aspect, the carrier is a pharmaceutically acceptable carrier.

[0282] "Composition" generally means a combination of an active agent and a naturally occurring or non-naturally occurring inert (e.g., detectable agent or marker) or active carrier, wherein the active agent is, for example, an engineered T cell receptor, a modified T cell receptor, a chimeric antigen receptor, a cell comprising an engineered T cell receptor, a CAR T cell or a CAR NK cell, an antibody, a compound or a composition, wherein the carrier is, for example, an adjuvant, a diluent, an adhesive, a stabilizer, a buffer, a salt, a lipophilic solvent, a preservative, an adjuvant, etc. and includes a pharmaceutically acceptable carrier. The carrier also includes pharmaceutical excipients and additives, peptides, amino acids, lipids and carbohydrates (e.g., including monosaccharides, di-, tri-, tetra-oligosaccharides and oligosaccharides; such as sugar alcohols, aldonic acids, esterified sugars, etc. derived sugars; and polysaccharides or sugar polymers), which may exist alone or in combination, and they alone or in combination account for 1-99.99% of weight or volume. Exemplary protein excipients include, for example, serum albumin, gelatin, casein, etc. of human serum albumin (HSA), recombinant human albumin (rHA), etc. Representative amino acids / antibody components that can also function in a buffering capacity include: alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartic acid, etc. Carbohydrate excipients should also be included in the technology, and examples include, but are not limited to: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and inositol.

[0283] Compositions used according to the present invention include cells, treatments, therapies, medicaments, drugs and pharmaceutical preparations, which can be packaged in dosage unit form for easy administration and uniform dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in an object, each unit comprising a composition calculated to produce a predetermined amount of a desired response associated with its administration (i.e., appropriate approach and scheme). Depending on the number of treatments and the unit dose, the dosage depends on the desired result and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is unique to each person. Factors affecting dosage include the physical and clinical state of the object, the route of administration, the expected therapeutic goal (symptom relief and cure) and the efficacy, stability and toxicity of a particular composition. After preparation, the solution will be administered in a manner compatible with the dosage formulation and in an amount for the treatment or prevention of an effective amount. The preparation is easy to administer in various dosage forms (e.g., the type of injectable solution described herein).

[0284] On the other hand, provided herein is a composition comprising a vector and one or more of any engineered immune cells of the present invention or a cell population of any engineered immune cells of the present invention, or consisting essentially of these, or also consisting of these; wherein the composition further comprises a cryoprotectant, or optionally consists essentially of a cryoprotectant, or consists of a cryoprotectant.

[0285] Compositions used according to the present invention include cells, treatments, therapies, medicaments, drugs and pharmaceutical preparations, which can be packaged in dosage unit form for easy administration and uniform dosage. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in an object, each of which contains a composition calculated to produce a predetermined amount of a desired response associated with its administration (i.e., appropriate approach and scheme). Depending on the number of treatments and the unit dose, the dosage depends on the desired result and / or protection. The precise amount of the composition also depends on the judgment of the practitioner and is unique to each person. Factors affecting dosage include the physical and clinical state of the object, the route of administration, the expected therapeutic goal (symptom relief and cure) and the efficacy, stability and toxicity of a particular composition. After preparation, the solution will be administered in a manner compatible with the dosage formulation and in an amount for the treatment or prevention of an effective amount. The preparation is easy to administer in various dosage forms (e.g., the type of injectable solution described herein).

[0286] "Cryoprotectants" are known in the art and include, but are not limited to, for example, sucrose, trehalose, and glycerol. Cryoprotectants that exhibit low toxicity in biological systems are generally used.

[0287] The present invention provides an immune cell that binds to a target cell, wherein the immune cell is any one of the engineered immune cells of the present invention. Reagent test kit

[0288] Also provided is a kit comprising, consisting essentially of, or consisting of vectors and instructions for preparing any of the engineered immune cells of the invention, and optionally instructions for diagnostic or therapeutic use thereof. How to use

[0289] Also provided herein is a method for stimulating a cell-mediated immune response to a target cell population, the method comprising contacting the target cell population with an engineered cell of the present invention or a cell population of the present invention, or consisting essentially of the step, or consisting of the step. On the other hand, a method for stimulating a cell-mediated immune response to a target cell population is provided, the method comprising the following steps, or consisting essentially of the following steps, or consisting of the steps: contacting a target cell population with a cell of the present invention or a cell population of the present invention, wherein the contact is performed in vitro or in vivo. Contact can be a direct or indirect binding or interaction between two or more entities (e.g., a target cell population and a T cell engineered to reduce or eliminate the expression and / or function of the NR4A transcription factor in the cell). A specific example of a direct interaction is binding. A specific example of an indirect interaction is that an entity acts on an intermediate molecule, which in turn acts on a second mentioned entity. The contact used herein includes in solution, in a solid phase, in vitro, in vitro, in a cell, and in vivo. In vivo contact can be referred to as administration or administration. The target cell can be a cell infected by a pathogen or a cancer cell or a tumor cell. On the other hand, cancer is characterized by being low in response. On the one hand, cells are selected to specifically bind to target cells. Cells can be from any species, such as mammalian or human cells. They can be separated from objects (e.g., from biopsy tissue) or cultured cells.

[0290] In another aspect, a method of stimulating a cell-mediated immune response to a target cell population is provided, the method comprising, consisting essentially of, or consisting of contacting the target cell population with a cell of the invention or a cell population of the invention, wherein the contacting is in vivo and the target cell population is a cancer cell population in a subject. In another aspect, the cancer is characterized by being hyporesponsive.

[0291] Cancer cells targeted by this approach include blood cancers, such as acute myeloid leukemia or acute lymphoblastic leukemia, and solid tumors, such as carcinoma, sarcoma, neuroblastoma, cervical cancer, hepatocellular carcinoma, mesothelioma, glioblastoma, myeloma, lymphoma, leukemia, adenoma, adenocarcinoma, glioma, glioblastoma, retinoblastoma, astrocytoma, oligodendroglioma, meningioma, or melanoma.

[0292] The method can be used to treat humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). The mammal can be of any age or in any developmental stage (e.g., an adult, adolescent, child, infant, or mammal in utero). The mammal can be male or female. In some embodiments, humans suffer from or are suspected of having cancer or a neoplastic disease. The method can be used as a first-line, second-line, third-line, fourth-line, or fifth-line therapy, and can be combined with other suitable therapies (e.g., surgical resection). On the other hand, cancer is characterized by being low-responsive.

[0293] On the other hand, disclosed herein is a method for stimulating a cell-mediated immune response to pathogen-infected cells in an object, the method comprising, or consisting essentially of, or consisting of the following steps: administering to the object a cell of the disclosure or a cell population of the disclosure in an amount effective to stimulate a cell-mediated immune response. On the one hand, the cell or population specifically binds to a cell population infected with a pathogen. The pathogen-infected cell population or pathogen-infected cells treated by the method include, but are not limited to, bacterial infections caused by group A streptococci, mycobacterium tuberculosis, shigella flexneri, salmonella enterica, listeria monocytogenes, francisella tularensis, and viral infections caused by, for example, herpes simplex virus. The method can be used to treat animals, typically mammals. Any suitable mammal can be treated by the methods, cells or compositions described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, adolescent, child, infant, or in utero mammal). The mammal can be male or female. The mammal can be a pregnant female. In some embodiments, the subject is a human.

[0294] In another aspect, a method for stimulating a cell-mediated immune response to a cancer target cell population, the method comprising, consisting essentially of, or consisting of the following steps: administering to a subject a cell or cell population of the invention in an amount effective to stimulate a cell-mediated immune response. In one aspect, the subject has cancer, has had cancer, or is in need of treatment for cancer. In another aspect, the cancer is characterized by being hyporesponsive.

[0295] The present invention also provides a method for providing anti-tumor immunity in a subject, the method comprising, consisting essentially of, or consisting of the following steps: administering to the subject a cell or cell population of the present invention in an amount effective to provide immunity to the subject. In one aspect, the subject is a mammal. In another aspect, the subject is a human. The cell or population is provided to prevent the onset of symptoms or cancer in a subject susceptible to or not yet showing symptoms of cancer.

[0296] One aspect of the invention herein is a method of treating a subject having a disease, disorder or condition associated with elevated expression of a tumor antigen, the method comprising, consisting essentially of, or consisting of administering to the subject a cell or cell population of the invention in an amount effective to treat the subject. In another aspect, the tumor is characterized by low responsiveness.

[0297] Cancer cells and cancer-related antigens targeted by these methods include hematological cancers, such as acute myeloid leukemia or acute lymphoblastic leukemia, and solid tumors, such as cancer, sarcoma, neuroblastoma, cervical cancer, hepatocellular carcinoma, mesothelioma, glioblastoma, myeloma, lymphoma, leukemia, adenoma, adenocarcinoma, glioma, glioblastoma, retinoblastoma, astrocytoma, oligodendroglioma, meningioma or melanoma. On the one hand, cell or colony specificity binds to cancer target cell colony. On the other hand, cancer is characterized by low reactivity.

[0298] The method can be used to treat humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). The mammal can be of any age or at any stage of development (e.g., an adult, adolescent, child, infant, or in utero mammal). The mammal can be male or female. In some embodiments, the human has or is suspected of having cancer or a neoplastic disease. The method can be used as a first-line, second-line, third-line, fourth-line, or fifth-line therapy and can be combined with other suitable therapies (e.g., surgical resection).

[0299] In certain embodiments, the subject has or is suspected of having a neoplastic disease, neoplasia, tumor, malignancy, or cancer. In some embodiments, the subject in need of the treatment, cells, or compositions described herein has or is suspected of having a neoplastic disease, neoplasia, tumor, malignancy, or cancer.

[0300] In one aspect, a method for stimulating a cell-mediated immune response to a target cell population infected with a pathogen in a subject is provided, the method comprising, or consisting essentially of, or consisting of the following steps: administering a cell of the present invention or a cell population of the present invention to the subject. In one aspect, the subject suffers from a pathogen infection, has suffered from a pathogen infection, or needs to be treated for a pathogen infection. The pathogen-infected cell population or pathogen-infected cells treated in this manner include, but are not limited to, bacterial infections caused by group A streptococci, mycobacterium tuberculosis, shigella flexneri, salmonella enterica, listeria monocytogenes, Francisella tularensis, and viral infections caused by, for example, herpes simplex virus. The subject treated in this manner includes animals, typically mammals. Any suitable mammal can be treated by the methods, cells, or compositions described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, adolescent, child, infant, or in utero mammal). The mammal can be male or female. The mammal can be a pregnant female. In some embodiments, the subject is a human. The method can be combined with other suitable therapies or treatments.

[0301] In some embodiments, the subject is in need of a treatment, cell, or composition as described herein. In certain embodiments, the subject has or is suspected of having a pathogen infection. In some embodiments, the subject is in need of a treatment, cell, or composition as described herein has or is suspected of having a pathogen infection.

[0302] For the above methods, an effective amount is administered, and the administration of the cell or cell colony is used to alleviate any symptom or prevent the occurrence of other symptoms. When administration is for the purpose of preventing or reducing the possibility of cancer recurrence or metastasis or pathogen infection, the cell or composition can be administered before any visible or detectable symptoms occur. Routes of administration include, but are not limited to, oral (e.g., tablets, capsules, or suspensions), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural, and intrathecal.

[0303] These methods provide one or more of the following effects: (1) preventing symptoms or disease from occurring in a subject susceptible to or not yet showing symptoms of the disease; (2) inhibiting the disease or preventing its development; (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is a method for obtaining a beneficial or desired result (including a clinical result). For the purposes of the present technology, whether detectable or undetectable, a beneficial or desired result may include (but is not limited to) one or more of the following: alleviating or ameliorating one or more symptoms, reducing the extent of a condition (including a disease), stabilizing (i.e., not worsening) the state of a condition (including a disease), delaying or slowing the condition (including a disease), the process, improving and slowing the state of a condition (including a disease), and alleviating (whether partially or completely). The treatment containing the disclosed compositions and methods may be first-line, second-line, third-line, fourth-line, fifth-line therapy, and is intended to be used as a single therapy or in combination with other suitable therapies. In one aspect, treatment excludes prevention.

[0304] Also provided herein is a method for providing immunity to pathogen infection in an object, the method comprising the following steps, or consisting essentially of the following steps, or consisting of the following steps: administering any cell or cell colony of the present invention to an object in an amount that provides immunity. On the one hand, the method prevents symptoms or pathogen infection from occurring in an object that is susceptible or has not yet shown symptoms of pathogen infection. The method can be used to treat animals, typically mammals. Any suitable mammal can be treated by the methods, cells or compositions described herein. Non-restrictive humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.) of mammals, domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs) and experimental animals (e.g., mice, rats, rabbits, guinea pigs) of mammals. In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, teenager, child, infant, or mammal in the womb). The mammal can be male or female. The mammal can be a pregnant female. The method can be combined with other suitable therapies and can be used to treat viruses, bacteria, fungi and protozoa. Examples of pathogenic infections include, but are not limited to, bacterial infections caused by Group A Streptococcus, Mycobacterium tuberculosis, Shigella flexneri, Salmonella enterica, Listeria monocytogenes, Francisella tularensis, and viral infections caused by, for example, herpes simplex virus. discuss

[0305] Many existing methods for engineering adoptive T cell therapy focus on enabling T cells to recognize tumor antigens by expressing specific T cell receptors or chimeric antigen receptors. After infusion, the function of the cells is affected after entering the tumor environment. The inventors found from in vitro models that conventional T cells and CAR-T cells rapidly acquire a low-responsive, "exhausted" state and poorly clear implanted melanoma tumors. The present invention provides CAR-T cells that are genetically engineered to lack the Nr4a transcription factor, thereby maintaining function within the tumor and promoting tumor clearance.

[0306] This "exhausted" state is characterized by a unique gene expression and epigenetic profile, including increased activity and altered expression of Nr4a family transcription factors (Nr4a1 aka Nur77, Nr4a2 aka Nurr1, Nr4a3 aka Nor1). Using a mouse model of implantable melanoma, the inventors found similar "exhaustion" profiles between endogenous, polyclonal tumor-infiltrating T cells, adoptively transferred monoclonal transgenic T cells, and T (CAR-T) cells expressing chimeric antigen receptors. CAR-T cells engineered to lack Nr4a1, Nr4a2, and Nr4a3 transcription factors can clear implanted melanoma, while unmodified CAR-T cells do not limit tumor growth. Upon restimulation, CAR-T cells lacking Nr4a produce more IFN-γ and TNF than CAR-T cells expressing Nr4a. Therefore, the loss of Nr4a is a potential strategy to improve the prognosis of adoptive cell therapy, especially for CAR-T cells targeting solid tumors.

[0307] To identify changes in gene expression and associated regulatory elements in CAR T cell-infiltrating solid tumors and to compare the properties of three different classes of CD8+ tumor-infiltrating lymphocytes (TILs): CAR TILs recognizing human CD19 (huCD19), CAR TILs recognizing H-2K b OT-I T cell receptor (TCR)-transgenic TILs (OT-I TILs) and endogenous TILs from recipient syngeneic mice expressing SIINFEKL peptide of chicken ovalbumin (OVA) were expressed ( FIG. 7 ). As targets for CAR T cells, the B16-OVA mouse melanoma cell line, the EL4 mouse thymoma cell line, and the MC38 colon adenocarcinoma cell line were engineered to express human CD19 (huCD19) ( Figure 1A, left figure); The generated B16-OVA-huCD19 cells are recognized by OVA-reactive OT-I T cells and huCD19-reactive expression CAR CD8+T cells. It has been confirmed that after 18 days of subcutaneous growth in syngeneic C57BL / 6J mice and subsequent 7 days of ex vivo culture, the B16-OVA-huCD19 cell line stably maintains huCD19 expression (Fig. 7A, right figure). The growth rates of Bf16-OVA and B16-OVA-huCD19 cells in vivo are the same, indicating that the addition of huCD19 antigen does not cause rejection of tumor cells (Fig. 1B, left). According to the growth rate of tumors in mice, 500,000 B16-OVA-huCD19 tumor cells were inoculated into mice (Fig. 7B, right).

[0308] CAR T cells express a second-generation CAR in which a single-chain variable fragment specific for huCD19 and tagged with a myc epitope is expressed 22,23 Compatible with the transmembrane domain of murine CD28 and mouse CD28 and CD3ζ 1 The intracellular signaling part of the CAR was fused to the retroviral construct, followed by a 2A self-cleaving peptide and a mouse Thy1.1 reporter gene (Figure 7C). 95.5% of CAR retroviral expression was achieved in mouse CD8+ T cells. + 4.0% transduction efficiency (Figure 1D). CAR T cells are functional because they produce cytokines TNF and IFNγ after restimulation with EL4-huCD19 target cells in culture (Figure 7E, 7F) and show dose-dependent target cell lysis against B16-OVA-huCD19 cells in vitro (Figure 7G). Under resting conditions, CAR T cells are similar to mock-transduced cells in terms of surface expression of PD-1, TIM3, and LAG3 (Figure 7H).

[0309] To evaluate CAR T cell function, CD45.1+CD8+ T cells were transduced with CAR retrovirus and adoptively transferred into C57BL / 6J mice bearing B16-OVA-huCD19 tumors 13 days after tumor inoculation (Figure 7A). Eight days after adoptive transfer, CD8+CD45.1+Thy1.1 +CAR TILs and endogenous CD45.2+ host T cells (Figure 7B, left). For comparison, the same B16-OVA-huCD19 tumors infiltrated with adoptively transferred CD45.1+CD8+OT-I TCR transgenic cells were analyzed in parallel (1C, 1D; see Figures 8A, 8B for gating scheme). Tumor growth rates were comparable in mice receiving CAR or OT-I CD8+ T cells (Figure 8C, top panel), allowing direct comparison of the two TIL populations; the number of transferred CAR T cells was kept low to minimize tumor rejection (Figure 8C, bottom panel), allowing the effects of genetic manipulation to be more easily observed. On average, CAR and OT-I T cells accounted for 50% of the CD8+ TILs in the tumor. 18% and 9% (Figure 7E).

[0310] On day 8 after transfer, all three TIL populations produced low levels of cytokines TNF and IFNγ after restimulation (Figure 7F, 7G), confirming that their function had been reduced. All three TIL populations also contained PD-1, which is considered to be highly depleted. 高 TIM3 高 Cells (populations A, C, and F in Figures 7B and 7D, upper right), and PD-1, which are considered antigen-specific memory precursors that proliferate after treatment with anti-PD-1 / PD-L1 高 TIM3 低 Cells (populations B and D). Endogenous TILs also contain PD-1 低 TIM3 低 The colony of T cells (Fig. 7B, 7D, lower right) is similar to that of naive T cells (see ATAC-seq data below). Therefore, all three TIL colonies (CAR, OT-I and endogenous) develop similar phenotypes with reduced cytokine production and increased expression of inhibitory receptors.

[0311] RNA sequencing 24 (RNA-seq) and ATAC-seq 25 (Transposase Accessible Chromatin Assay followed by Sequencing) was used to compare gene expression and chromatin accessibility profiles of CD8+ tumor-infiltrating lymphocytes from different populations (AF) in a CAR T-cell system 26-28 (Figure 7B, 7D). Principal component analysis of RNA-seq data showed that PD-1 高 TIM3 高 Transcriptional profile of CAR TIL population (A) and endogenous PD-1 高 TIM3 高 The transcriptional profile of the TIL population (C) is similar but differs from that of CAR and endogenous PD-1 高 TIM3低 TIL population (B, D) and endogenous PD-1 低 TIM3 低 The transcriptional profiles of cells (E) were different ( Figure 8A , Fig. 9 ). Similarly, ATAC-seq analysis showed that endogenous OT-I and CAR PD-1 高 TIM3 高 and PD-1 高 TIM3 低 Genome-wide chromatin accessibility profiles of TIL subsets were similar to each other but not to PD-1 低 TIM3 低 The chromatin accessibility profiles of endogenous TILs were different ( Figure 8B , Figure 10 ).

[0312] With PD-1 低 TIM3 低 Tumor non-reactive P14 TIL 18 In contrast, OT-ITIL (PD-1 高 TIM3 高 ) showed increased expression of genes encoding the transcription factors Nr4a2 and Tox, various inhibitory surface receptors, and effector genes including granzymes and cytokines. Consistent with these findings, endogenous PD-1 低 TIM3 低 Compared with TILs (group E), genes encoding transcription factors (Nr4a2, Tox, Tbx21), inhibitory receptors (Pdcd1, Ctla4, Havrc2, Tigit), several granzymes, cytokines (Il21, Ifng, Tnf), and cytokine receptors (Il2ra, Il10ra) were significantly upregulated in PD-1 TILs. 高 Upregulated in CAR TIL and endogenous TIL (population AD) Fig. 9 , comparing groups A vs E, B vs E, C vs E, and D vs E). 高 TIM3 低 Compared with (groups B and D), depleted PD-1 高 TIM3 高 There was no significant difference in Nr4a mRNA expression between CAR and endogenous TIL (groups A and C). Fig. 9 , comparing groups A vs B, C vs D); however, Nr4a protein expression was significantly increased in the former TIL groups (A, C) compared with the latter (B, D) (see below). 9,17,18 Consistent with depletion of PD-1 高 TIM3高 Compared with TIL, PD-1 高 TIM3 低 Antigen-specific memory precursors resembled naive CD8+ T cells in expressing higher levels of Tcf7, Il7r, Ccr7, and Sell (encoding CD62L (L-selectin)) mRNA (Figure 3, compare graphs of populations A vs E, B vs E, C vs E, and D vs E).

[0313] Analysis of ATAC-seq data showed that endogenous PD-1 低 TIM3 低 TILs (population E) are similar to naive CD8+ T cells in chromatin accessibility and different from PD-1 高 TIM3 高 and PD-1 高 TIM3 低 TIL (group AD) (Figure 8B, top panel; Figure 10). 高 The regions selectively accessible in CAR and endogenous TILs were enriched for common Nr4a binding motifs, as well as common binding motifs for NFAT, NFκB, bZIP, and IRF-bZIP motifs (Figure 8B, clusters 8 and 9; discussed below). Again consistent with published literature 9,17,18 Consistent, memory precursor PD-1 高 TIM3 低 The chromatin accessibility profile of TILs (populations B and D) was similar to that of naive CD8+ T cells, showing a significant enrichment of Tcf7 binding motifs (Figure 8B, cluster 6).

[0314] As mentioned above, PD-1 高 TIM3 高 and PD-1 高 TIM3 低 TIL populations showed no significant differences in the expression of Nr4a family members at the mRNA level ( Fig. 9 , compare groups A vs B, C vs D). However, flow cytometry analysis clearly showed that 高 TIM3 低 Compared with the TIL group, the expression of all three Nr4a transcription factors was higher at the protein level. 高 TIM3 低 Compared with the TIL population, depleted PD-1 高 TIM3 高The increased expression of Nr4a family members and the enrichment of Nr4a binding motifs in regions differentially accessible to these cells strongly indicate that Nr4a family members are potential transcriptional effectors of CD8+ T cell responses to chronic antigen stimulation.

[0315] Data from human TILs and T cells from patients chronically infected with HIV provide further rationale for our focus on the Nr4a family. Single-cell RNA-seq data derived from CD8+ T cells infiltrating human melanoma 20 Revealed: NR4A1 and NR4A2 expression showed strong positive correlation with PDCD1 (encoding PD-1) and HAVCR2 (encoding TIM3) expression, while NR4A3 showed a moderate positive correlation. Consistent with our mouse RNA-seq data, PDCD1 and HAVCR2 mRNA expression was observed to be positively correlated with the expression of mRNA encoding surface receptors CD38, TIGIT, and CTLA4 (Figure 12A); and negatively correlated with the expression of mRNA encoding transcription factor TCF1, cell surface receptor SELL (L-selectin or CD62L), and chemokine receptor CCR7 (Figure 12B). The expression of mRNA encoding other target transcription factors TOX, TOX2, and IRF4 was also positively correlated with the expression of PDCD1 and HAVCR2 mRNA ( Fig. 12C ). Human ATAC-seq data 19,21 Analysis showed that in CD8 + PD-1 高 Nr4a nuclear receptor, NFAT, bZIP and IRF:bZIP motifs are enriched in the only accessible regions in TILs and HIV antigen-specific CD8+ T cells from infected humans. In summary, Figure 12 shows that Nr4a family members are upregulated and Nr4a nuclear receptor binding motifs are enriched in PD-1hi T cell accessible regions in human and mouse CD8+ T cells exposed to chronic antigen stimulation 17,18,21,29 .

[0316] All three members of the Nr4a family are essential for the development of regulatory T cells 30 , suggesting that they may play redundant roles in other biological contexts. Given this expected redundancy, Nr4a-sufficient (WT) CAR TILs were compared with CAR TILs triple-deficient for all three Nr4a transcription factors (Nr4a TKO) ( Fig. 9 ). Because the Nr4a gene-disrupted mice were originally derived from 129 / SvJ ES cells 31, and despite strict backcrossing, their genetic background may not be fully compatible with that of inbred C57BL / 6J mice, so mice deficient in Rag1 should be used as tumor vaccination recipients to avoid variable rejection of CAR T cells. Naive CD8+ T cells from Nr4a1 fl / fl Nr4a2 fl / flNr4a3- / - mice were simultaneously transduced with two retroviruses, the first encoding CAR-2A-Thy1.1 (Figure 7C) and the second encoding Cre followed by an IRES-NGFR cassette to generate Nr4a triple knockout (Nr4a TKO) CAR T cells (Figure 7A). As a control, naive CD8+T cells from Nr4a1 fl / fl Nr4a2 fl / fl Nr4a3+ / + mice were transduced with CAR-2A-Thy1.1 retrovirus and empty retrovirus with only IRES-NGFR to generate Nr4a WT CAR T cells (WT) (Figure 13A). CAR T cells were adoptively transferred into mice that had been injected with B16-OVA-huCD19 melanoma cells 7 days ago, and tumor growth was monitored for an additional 83 days. Compared with control mice adoptively transferred with WT CD8+CAR T cells, mice adoptively transferred with Nr4a TKO CD8+CAR T cells lacking all three Nr4a transcription factors showed significant tumor regression and improved survival ( Fig. 9 ), the difference in tumor size among the three groups (PBS, WT, and Nr4a TKO) appeared as early as day 21 after tumor inoculation (i.e., 14 days after adoptive transfer) ( Fig. 9 ). Thus, the Nr4a transcription factor inhibits tumor rejection in CAR T cell models.

[0317] To explore the redundancy of Nr4a family members in CD8+T cell function in vivo, the anti-tumor effects of CD8+CAR T cells lacking a single Nr4a protein were compared with those of WT and Nr4a TKO CAR T cells (Figure 14). CAR T cells were adoptively transferred into Rag1-deficient mice that had been injected with B16-OVA-huCD19 melanoma cells 7 days ago, and tumor growth was monitored for an additional 83 days (Figure 14A). Tumor growth curves and survival curves showed that Nr4a TKO CAR T cells exhibited superior anti-tumor activity compared with CAR T cells from mice lacking any single Nr4a protein (Figures 14B, 14C). Again, differences in tumor size between WT and various Nr4a knockouts were observed as early as day 21 after tumor inoculation.

[0318] To further confirm the redundant functions of these three Nr4a family members in CD8+ T cell function, retroviruses expressing Nr4a1, Nr4a2, and Nr4a3 in mouse CD8+ T cells were used in vitro (Figures 15-17). Ectopic expression of any of the three Nr4a transcription factors resulted in increased expression of inhibitory surface receptors PD-1, TIM3, 2B4, and GITR, and reduced cytokine TNF and IFN upon restimulation. γ production (Figure 15). Principal component analysis of RNA-seq data from cells expressing any given Nr4a transcription factor or empty vector control showed that most of the differences between these groups were genes with similar profiles in cells expressing Nr4a family members compared to empty vector (Figure 16A). In RNA-seq and ATAC-seq, pairwise comparisons showed that there were almost no differences between Nr4a family members (Figures 16B, 17A). Therefore, all three Nr4a proteins induce overlapping changes in gene expression profiles and regulatory element accessibility profiles of CD8+T cells in vitro and in vivo.

[0319] To evaluate phenotypic and genome-wide changes associated with antitumor function in Nr4a TKO and WT, experimental conditions were altered to delay tumor regression ( Fig. 9 ) and ensured that tumor size was similar between the two groups (Figure 18A), see Figure 18B for the gating scheme for TIL. The number of Nr4a TKO TILs recovered per gram of tumor was not significantly different from that recovered by WT TILs (Figure 18C). At day 21 after tumor inoculation (8 days after adoptive transfer), PD-1 expression was slightly but statistically significantly reduced when comparing WT and Nr4aTKO TILs, and total PD-1 高 The population was significantly tilted towards low TIM3 expression ( Fig. 9 ); in Nr4a TKO TILs, TIM3 低 The population was skewed toward low TCF1 expression (Figure 18D, bottom). In the effector function test, the percentage of cells expressing TNF as well as IFNγ and TNF was significantly higher in Nr4a TKO compared to WT TIL ( Fig. 9 ). Overall, there were no significant differences in the MFI of TCF1, Tbet, or Eomes (Figure 18D, top).

[0320] Genome-wide changes associated with effector function in Nr4a TKO and WT TILs were analyzed by RNA-seq and ATAC-seq. RNA-seq identified 1,076 differentially expressed genes, of which 536 genes were more highly expressed in Nr4a TKO TILs and 540 genes were more highly expressed in WT TILs ( Fig. 10A, Figure 19A). Using mice infected with LCMV 17 Separated effector, memory, and exhaustion (PD-1 高 TIM3 高 Gene set enrichment analysis (GSEA) of the gene sets of the 247 groups 32 The results showed that Nr4aTKO TILs were extensively enriched for genes associated with effector functions ( Fig. 10A , Figures 19B, 19C). Specifically, mRNAs encoding IL-2Rα, TNF, and granzymes were upregulated in Nr4aTKOTIL, consistent with the increased production of TNF observed upon restimulation ( Fig. 9 ). In contrast, genes that are typically upregulated in naive / memory T cells compared to effector populations, such as Sell (encoding L-selectin / CD62L) and Ccr7, were downregulated in Nr4a TKO compared to WT TILs ( Fig. 10A Compared with WT TILs, inhibitory surface receptors that are normally upregulated in hyporesponsive T cells, including Pdcd1, Havcr2, Cd244, Tigit, and Cd38, were also downregulated in Nr4a TKOs ( Fig. 10A ).

[0321] To identify transcriptional targets of individual Nr4a proteins, genes differentially expressed in Nr4aTKO TILs compared with WT TILs were considered ( Figure 10 ) and clustered according to whether their expression was altered when Nr4a was ectopically expressed ( Fig. 10B Clusters 1 and 2 contained genes that were downregulated in the absence of Nr4a and upregulated in cells ectopically expressing Nr4a, including Pdcd1, Havcr2, Cd244 in cluster 1 and Tox, Tigit, and Cd38 in cluster 2. Cluster 4 contained genes, particularly Tnf and Il21, that were upregulated in the absence of Nr4a and downregulated in cells ectopically expressing Nr4a. Notably, Runx3 was not among the genes differentially expressed in Nr4a TKO compared to WT TILs, although a previous publication has identified it as a downstream target of Nr4a during CD8+ T cell development. 33 , and its overexpression contributes to tumor regression 34 .

[0322] ATAC-seq revealed approximately 2500 differentially accessible regions between WT and Nr4a TKO TILs ( Fig. 10C Among the regions lost in Nr4a TKO TILs, a considerable portion ( 36%) contain Nr4a binding motifs, and a smaller subset contains NFAT binding sites ( Fig. 10C). Among the regions that were more accessible in Nr4a TKO than WT TILs, ~71% were enriched for consensus bZIP family motifs and ~25% were enriched for consensus Rel / NFκB binding motifs, confirming the established roles of bZIP (Fos, Jun, ATF, CREB, etc.) and Rel / NFκB family members in T cell activation and effector function. These data are also consistent with previous publications suggesting a negative crosstalk between Nr4a and NFκB 35,36 Thus, Nr4a TKO TILs exhibited robust effector functions by several independent measures: altered gene expression profiles that reduced expression of inhibitory receptors and increased cytokine production, and strong enrichment of binding motifs for transcription factors implicated in effector functions in accessible chromatin regions.

[0323] To determine whether differentially accessible regions enriched for Nr4a motifs actually bind Nr4a, individual HA-tagged Nr4a proteins ectopically expressed in CD8+ T cells were used and Nr4a binding was confirmed by chromatin immunoprecipitation using an anti-HA antibody followed by qPCR for differentially accessible regions in selected loci that were differentially expressed. For example, high expression in naive and memory T cells and reduced expression in effector cells 37 The gene Ccr7, which is less expressed in effector-like Nr4aTKO than in WT (more exhausted) TILs (Figure 20A); consistent with lower expression, the distal 5' region of Ccr7 has at least two ATAC-seq peaks, which are not as prominent in Nr4a TKO as in WT TILs, and contains adjacent NFAT and Nr4a binding motifs (Figure 20A, left, black line). ChIP-qPCR shows that ectopically expressed Nr4a binds these two Ccr7 enhancer regions (Figure 20A, left, black line; and right, bar graph). In contrast, the proximal promoter region and first intron of Ccr7 have ATAC-seq peaks, which are more prominent in Nr4a TKO than in WT TILs, and contain bZIP and NFκB motifs (Figure 20, left, blue line). Other examples (Ifng, Ccr6) are shown in Figure 19A (Ifng, Ccr6: left, genome browser view, NFAT and Nr4a motifs marked in pink, bZIP and NFkB motifs marked in blue; right, ChIP-qPCR, Nr4a binds to the accessible regions shown). Examples of genes enriched for bZIP motifs in differentially accessible regions include Il21, which encodes a cytokine involved in effector function 38 , and was more highly expressed in Nr4a TKO compared with WT TILs ( Fig. 10A); Compared with WT TIL, two regions of the Il21 promoter became accessible in Nr4a TKO, one of which contained a bZIP motif (Figure 20B). Similarly, in Nr4a TKO TIL, the cytokine TNF was expressed in mRNA ( Fig. 10A ) and protein ( Fig. 10A ) levels, and the Tnf locus showed widespread increased accessibility both throughout the promoter and throughout the gene in Nr4a TKO TILs ( FIG. 20B ).

[0324] Examination of chromatin accessibility at the Pdcd1 locus (encoding PD-1) revealed that each of the Nr4a family members is at least partially responsible for increasing the expression of chromatin at the Pdcd1 transcription start site ( Fig. 10C , gray box) Accessibility of the enhancer at 23 kb 5', which has been noted in all depletion or dysfunctional mouse models studied to date 17-19,21,29 . ATAC-seq peaks marking this enhancer were reduced in Nr4a TKO compared to WT CAR T cells, but increased in T cells ectopically expressing Nr4a1, Nr4a2, or Nr4a3 compared to cells transduced with empty vector alone. ChIP-qPCR showed that all three Nr4a family members bind to this enhancer region of the PD-1 locus ( Fig. 10C , right). It is worth noting that previous publications 21 Supporting the small decrease in PD-1 MFI observed in Nr4a TKO TILs, indicating a loss of The 23 kb enhancer region resulted in a small decrease in the mean fluorescence intensity of PD-1 staining in the EL-4 thymoma cell line.

[0325] The engineered NFAT protein CA-RIT-NFAT1 is used to mimic dephosphorylated nuclear NFAT that cannot form a cooperative transcriptional complex with AP-1 (Fos-Jun) 16 Compared with mock-treated cells, CA-RIT-NFAT1-transduced cells expressed higher levels of Nr4a transcription factors as well as inhibitory receptors and displayed a transcriptional program that mimicked in vivo depletion, especially the early stages of “dysfunctional” 16-18. Genome-wide analysis of ATAC-seq data showed that regions that were more accessible in WT compared to Nr4a TKO TILs were also more accessible in cultured cells expressing CA-RIT-NFAT1 compared to mock-transduced cells, and were also more accessible in cells expressing Nr4a compared to cells transduced with an empty vector (Figure 4E). Therefore, regulatory elements that are sensitive to reduced NR4A activity in vivo are sensitive to induction of constitutive NFAT and NR4A activity in vitro. Conversely, regions that were more accessible in Nr4a TKO TILs compared to WT TILs were also more accessible in PMA / ionomycin-stimulated cells compared to quiescent cells (Figure 4E). Fig. 10C Since PMA / ionomycin stimulation involves the activity of bZIP and NFκB family members, these data suggest that the increase in effector function and gene expression in vivo in Nr4a TKO TILs compared with WTTILs is associated with increased activity of these transcription factors.

[0326] The Nr4a transcription factor is an important redundant effector of the CD8+ T cell hyporesponsiveness program downstream of NFAT ( Fig. 10C ). Ectopic expression of each individual Nr4a protein inhibits cytokine function. In contrast, TILs lacking all three Nr4a proteins exhibited gene expression profiles characteristic of effector function, including increased expression of granzymes and cytokines. Nr4a TKO CAR TILs also showed increased chromatin accessibility in regions containing binding sites for bZIP and Rel / NFκB family transcription factors, which are associated with classical T cell activation programs. Therefore, the loss of Nr4a leads to a genomic landscape capable of T cell activation. In addition, Nr4aTKO CAR TILs exhibited a higher frequency of the TIM3-TCF1- population (Figure 18D, bottom), which may represent enhanced effector function and is consistent with TIM3 - TCF1 + Memory / precursor population 39-42 Different from the latter, which expanded after PD-1 blockade but were less numerous in Nr4a TKO CAR TILs than in WT CAR TILs 39 .

[0327] There is a clear relationship between PD-1 and Nr4a, both in our own studies and in previously published data from other laboratories. 17-19,21,29. Ectopic expression of any single Nr4a family member in vitro resulted in upregulation of PD-1 at the protein and mRNA levels, as well as increased accessibility to the -23kb upstream enhancer region of the Pdcd1 locus. Nr4a TKO CAR TILs had reduced accessibility to this enhancer compared to WTCAR TILs, and all three Nr4a family members bound to this enhancer in cells. Taken together, these data suggest that the effects of triple Nr4a deletion are functionally somewhat similar to those of PD-1 blockade ( Fig. 10C ), but Nr4a deletion has a broader effect than PD-1 blockade alone because of the wide range of regulatory elements affected. In other words, PD-1 may be just one of many genes regulated by the NFAT / Nr4a axis in mouse and human T cells, with others encoding other inhibitory receptors, including CTLA4, TIM3, LAG3, and TIGIT.

[0328] Immune cell therapy offers broad prospects for cancer treatment 43,44 In some cases, endogenous CD8+ T cells that are unable to effectively reject tumors can be made functional by antibodies that block inhibitory receptors such as PD-1 and CTLA4. 45-47 . However, complete cure is rarely achieved with treatment with blocking antibodies alone, and thus the field of cancer immunotherapy is moving toward treatment with blocking antibodies against multiple inhibitory receptors. The NFAT / Nr4a axis controls the expression of multiple inhibitory receptors, and treatment of tumor-bearing mice with CAR T cells functionally deficient in all three Nr4a transcription factors resulted in tumor regression and prolonged survival. Therefore, inhibiting the function of Nr4a family members in tumor-infiltrating T cells may be a promising strategy for cancer immunotherapy. Materials and methods Construction of a retroviral vector (MSCV-myc-CAR-2A-Thy1.1) containing a chimeric antigen receptor (CAR).

[0329] Use the public portion of the cloned FMC63 human CD19 single-chain variable fragment 22,23 and the public portions of the mouse CD28 and CD3ζ sequences 1 Chimeric antigen receptors were assembled. The sequence of the myc tag at the N-terminus was obtained from a published work 48 This chimeric antigen construct was then cloned into the MSCV-puro (Clontech) murine retroviral vector instead of PGK-puro.

[0330] The polynucleotide sequence of the CAR construct is provided in SEQ ID NO:1.

[0331] The amino acid sequence of the CAR construct is provided in SEQ ID NO:2. Construction of retroviral vector containing huCD19.

[0332] The DNA fragment encoding huCD19 was PCR amplified and cloned into the MSCV-puro (Clontech) murine retroviral vector. Construction of retroviral vectors containing Cre (MSCV-Cre-IRES-NGFR) and Nr4a1, Nr4a2, Nr4a3 (MCSV-HA-Nr4a1-IRES-NGFR, MCSV-HA-Nr4a2-IRES-NGFR, MCSV-HA-Nr4a3-IRES-NGFR).

[0333] The DNA fragment encoding Cre was PCR amplified and cloned into MSCV-IRES-NGFR (Addgene plasmid #27489). The DNA fragment encoding Nr4a1 (gift from C.-WJ Lio, La Jolla Institute for Allergy and Immunology, La Jolla, CA) was PCR amplified with a 5' HA tag and cloned into MSCV-IRES-NGFR. The DNA fragment encoding Nr4a2 (Addgene plasmid #3500) was PCR amplified with a 5' HA-tag and cloned into MSCV-IRES-NGFR. The DNA fragment encoding Nr4a3 (DNASU plasmid #MmCD00080978) was PCR amplified with a 5' HA-tag and cloned into MSCV-IRES-NGFR. Eukaryotic cell lines.

[0334] EL4 mouse thymoma cell line was purchased from the American Type Culture Collection (ATCC): EL4 ( TIB-39 TM , Mus musculus T-cell lymphoma). The ovalbumin-expressing B16-OVA mouse melanoma cell line has been described previously. 18 (gift from S. Schoenberger, La Jolla Institute for Allergy and Immunology, La Jolla, CA). 293T cell line was purchased from ATCC: 293T ( CRL-3216 TMPlatinum-E retroviral packaging cell line, thermophilic (PlatE) cell line were purchased from Cell BioLabs, Inc: RV-101. MC-38 mouse colon adenocarcinoma cell line (gift from AW Goldrath, UCSD, La Jolla, CA) was originally purchased from Kerafast, Inc (ENH204). Construction of mouse tumor cell lines expressing huCD19.

[0335] B16-OVA, EL4, and MC-38 cells were transduced with amphipathic viruses containing human CD19 (huCD19), and cells expressing high levels of huCD19 were sorted. Preparation of B16-OVA-huCD19 melanoma cells for tumor inoculation.

[0336] B16-OVA-huCD19 cells were cultured in Dulbecco's medium (DMEM) containing 10% (volume / volume) FBS, 1% L-glutamine, 1% penicillin / streptomycin and passaged 3 times before inoculation. At the time of injection, the cells were trypsinized and resuspended in Hanks' balanced salt solution without phenol red at 10 million cells per ml. 500,000 B16-OVA-huCD19 cells (50 μL per injection) were injected intradermally into C57BL / 6J male mice (8-12 weeks old). Preparation of MC38-huCD19 colon adenocarcinoma cells for tumor inoculation.

[0337] MC38-huCD19 cells were cultured in Dulbecco's medium (DMEM) containing 10% (vol / vol) FBS, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, 1% L-glutamine, 1% penicillin / streptomycin and passaged twice before inoculation. At the time of injection, the cells were trypsinized and resuspended in Hanks' balanced salt solution without phenol red at 10 million cells per ml. C57BL / 6J male mice (8-12 weeks old) were injected intradermally with 500,000 MC38-huCD19 cells (50 μL per injection). Mouse

[0338] C57BL / 6J, B6.SJL-Ptprc a Pepc b / BoyJ, Rag 1- / - mice were purchased from Jackson Laboratories. The strain with Nr4a gene disruption was obtained from Takashi Sekiya and Akihiko Yoshimura with permission from Pierre Chambon. Both male and female mice were used for the study. Mice were age-matched and used in experiments between 8-12 weeks of age. Tumor-bearing mice were first matched for tumor size and then randomly assigned to experimental groups. All mice were housed and / or maintained in the animal facility of the La Jolla Institute for Allergy and Immunology. All experiments were performed in accordance with the regulations of the LJI Institute Animal Care and Use Committee (IACUC). B16-OVA-huCD19 tumor model.

[0339] For CAR CD8 + TIL and endogenous CD8 + Analysis of TILs: On day 0, 8-12 week old C57BL / 6J mice were injected intradermally with 5x10 5 B16-OVA-huCD19 cells. After tumors became palpable, tumor measurements were recorded every other day with a manual caliper and tumor area was calculated in square centimeters (length x width). On day 13, 1.5 million CAR-transduced CD45.1 + CD8 + T cells were adoptively transferred into tumor-bearing mice with matched tumor size. On day 21, tumors and spleens of mice were harvested. For analysis of CAR CD8+ TILs lacking Nr4a family members: On day 0, 8-12 week old Rag1- / - mice were injected intradermally with 5x 10 5 B16-OVA-huCD19 cells, and tumors were measured every two days after they became palpable. On day 13, 1.5 million CAR- and empty vector pMIN-transduced Nr4a1fl / fl Nr4a2fl / fl Nr4a3+ / + or CAR- and Cre-transduced CD8+Thy1.1+NGFR+Nr4a1fl / fl Nr4a2 fl / fl Nr4a3- / - T cells were adoptively transferred into tumor-size-matched tumor-bearing mice. On day 21, tumors and spleens of mice were harvested. To monitor tumor growth for survival studies after adoptive transfer of CAR T cells lacking Nr4a family members: On day 0, 8-12 week old Rag1- / - mice were injected intradermally with 5x 10 5B16-OVA-huCD19 cells, after they become obvious, measure the tumor every two days. On the 7th day, 3 million mouse T cells of CD8+Thy1.1+NGFR+Nr4a transduced by CAR and empty vector pMIN or transduced by CAR and Cre (combined to produce Nr4a1 KO, Nr4a2 KO, Nr4a3 KO, Nr4a TKO, WT as listed in Figure 8) were adopted and transferred into tumor-bearing mice with comparable tumor size. The growth of the tumor was then monitored until the experimental endpoint or IACUC endpoint on the 90th day after tumor inoculation. Preparation of cells for adoptive transfer.

[0340] CD8+T cells were isolated and activated with 1ug / mL anti-CD3 and 1ug / mL anti-CD28 for 1d, then removed from activation and transduced with retrovirus expressing CAR, Cre, pMIN or a combination of the above for 1h at 37°C, 2000g. After transduction, the cells were immediately replaced with a medium containing 100UIL-2 / mL. On the first day after the first transduction, a second transduction was performed, and the cells were immediately replaced with a medium containing 100UIL-2 / mL after transduction. On the day of adoptive transfer (day 3 or day 5 after activation), the cells were analyzed by flow cytometry and cell counts were obtained using a hemocytometer. Using the cell counts from the hemocytometer and the population percentages obtained from the flow cytometer, the number of cells transduced by CAR was obtained. The cells were then collected, washed with PBS, and resuspended at a concentration equivalent to 1.5 million or 3 million CAR-transduced cells per 200uL PBS. The mice were then adoptively transferred by retro-orbital intravenous injection of 200uL each time. Isolation of tumor infiltrating lymphocytes (TIL) for subsequent analysis.

[0341] Sample preparation for flow cytometry of TILs from CAR and OT-I experiments: On day 21, mice were euthanized and perfused with PBS, and then tumors were removed. Tumors were collected, pooled by group, homogenized, and then dissociated using the MACS Miltenyi Mouse Tumor Dissociation Kit (Miltenyi Biotec) and the gentleMAC Dissociator (Miltenyi Biotec) with Octo Heaters according to the supplier's instructions. The tumors were then filtered through a 70uM filter and centrifuged. The supernatant was aspirated and the tumor was resuspended into 4-5 grams of tumor per 5mL 1% FBS / PBS using the Dynabeads FlowComp Mouse CD8 Isolation Kit (Invitrogen) for CD8 positive isolation. After positive isolation, the cells were divided into equal amounts, stained and phenotyped by flow cytometry, or stained for cell sorting. See the next paragraph for sample preparation for flow cytometry of TILs from Nr4a WT and Nr4a TKO experiments.

[0342] On the 21st day, mice were euthanized and perfused with PBS, and then the tumor was removed. Tumors were collected, combined by group, homogenized, and then dissociated using the MACS Miltenyi mouse tumor dissociation kit (MiltenyiBiotec) and the gentleMAC dissociator (MiltenyiBiotec) equipped with Octo Heaters according to the manufacturer's instructions. The tumor was then filtered through a 70uM filter and centrifuged. The supernatant was aspirated, and the tumor was resuspended in 40% Percoll / RPMI and the 80% Percoll / PBS below it in a 15mL conical tube to form an 80% / 40% Percoll discontinuous density gradient. In a large desktop centrifuge with a shaking bucket, the sample was centrifuged at room temperature for 30 minutes at 1363g. TILs were collected from the 80% / 40% Percoll interface and further purified using CD90.2 microbeads (Miltenyi Biotec) and magnetic separation technology. After positive isolation, cells are divided into equal parts for staining and phenotypic characterization by flow cytometry or stained for cell sorting. Transfection

[0343] According to the manufacturer's Transfections were performed in 10 cm dish format using the instructions for transfection reagent (Mirus Bio LLC) and pCL10A1 and pCL-Eco packaging vectors (the former for huCD19 virus and the latter for all other produced viruses). Retroviral transduction.

[0344] Retroviral transduction was performed in a 6-well plate format using 3 mL of 0.45 uM filtered virus and 8 ug / mL polyethylene per well. Two transductions were performed using 1.5 mL of each virus, totaling 3 mL. Cells were centrifuged at 2000 g for 1 hour at 37°C in a preheated centrifuge. Immediately after transduction, cells were replaced with medium containing 100 U IL-2 / mL. A second transduction was performed the next day. Antibody

[0345] Fluorescent dye-conjugated antibodies were purchased from Biolegend, BD Sciences, eBioscience, and Cell Signaling Technologies. Primary antibodies for chromatin immunoprecipitation were purchased from Cell Signaling Technologies. Surface marker staining

[0346] Cells were centrifuged and stained with antibodies at a final concentration of 1:200 in 50% 2.4G2 (Fc blocking) and 50% FACS buffer (PBS + 1% FBS, 2 mM EDTA) for 15 minutes. Cytokine restimulation and staining.

[0347] Prior to staining, cells were incubated at 37°C for 4 hours in a medium containing 10 nM PMA and 500 nM ionomycin and 1 ug / mL Brefeldin A. After restimulation, cell surface markers were stained with live / dead dyes as described in the surface marker staining protocol above. The cells were then fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 1X BD Perm / Wash (BD Biosciences) for 30 minutes, and then stained for cytokines at a final concentration of 1:200 in 1X BD Perm / Wash buffer. 1X BD Perm / Wash buffer was prepared according to the manufacturer's instructions. All washing steps were performed using FACS buffer (PBS + 1% FBS, 2mM EDTA). TF staining

[0348] Cell surface markers were stained with live / dead dye as described in the surface marker staining protocol above. Cells were then fixed, permeabilized, and stained using the Foxp3 / transcription factor staining kit (eBioscience) according to the manufacturer's instructions. All transcription factor antibodies were used at a final concentration of 1:200. The final concentration of the Ki67 antibody was 1:100. Flow cytometric analysis

[0349] All flow cytometric analyses were performed using LSRFortessa (BD Biosciences) or LSR-II (BD Biosciences). Flow data were analyzed using FlowJo v.10 (Tree Star, Inc). The relevant sample gates are provided in the figures. Statistical analysis.

[0350] Statistical analysis of flow cytometry data and tumor growth data for the experiments involved was performed using appropriate statistical comparisons, including paired or unpaired two-sided t-tests with Welch correction as appropriate, one-way ANOVA with multiple comparison tests (Tukey or Dunnett test), or ordinary two-way ANOVA (Prism 7, GraphPad Software). Statistical analysis of survival curves was performed using the Log-rank (Mantel-Cox) test (Prism 7, GraphPad Software). p values ​​< 0.05 were considered statistically significant. In vitro killing assay.

[0351] 10,000 B16-OVA-huCD19 cells were seeded in 100uL T cell culture medium, and target cells (or culture medium only for background) were added to each well in an E-plate 96 (ACEA Biosciences Inc., San Diego, CA). After 30 minutes, the plate was placed in an xCELLigence real-time cell analysis (RTCA) instrument (ACEA Biosciences Inc., San Diego, CA) and incubated overnight. The next day, the plate was removed from the xCELLigence RTCA machine and an additional 100uL T cell culture medium with CD8+CAR T cells added as effector cells was added for 30 minutes (for lysis of positive controls, 0.2% TritonX was used; for lysis of negative controls, only culture medium was used). The plate was then returned to the incubator and data acquisition was started. After 5 hours, the cell index (CI) was obtained from each well. The percentage of specific lysis for each well was calculated as follows: % specific lysis percentage = 100-(CI 每孔 / (CI 阳 -CI 阴 ))*100. B16-OVA-huCD19 cells were thawed 3 days before vaccination on day 4 (usually present at the time of vaccination); mouse CD8+CAR T cells were prepared before the experiment and added to the target cells on day 5 after activation of CD8+ T cells. Chromatin immunoprecipitation and quantitative real-time PCR (ChIP-qPCR).

[0352] As previously described 49ChIP was performed. Briefly, CD8+ T cells were isolated from C57BL / 6J mice as above, activated with plate-bound anti-CD3 / CD28, and transduced with an empty vector control or retrovirus expressing Nr4a1, Nr4a2, or Nr4a3 with a hemagglutinin (HA) tag at the N-terminus. Cells were cultured for a total of 5 days after transduction. For fixation, formaldehyde (16%, ThermoFisher) was added directly to the cells to a final concentration of 1% and incubated for 10 minutes at room temperature with constant stirring. Glycine (final 125 mM) was added to terminate fixation, and cells were washed twice with ice-cold PBS. Cell pellets were snap-frozen in liquid nitrogen and stored at -80°C until use. To isolate nuclei, cell pellets were thawed on ice and lysed with lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X100) supplemented with 1% Halt protease inhibitor (ThermoFisher) for 10 min at 4° C. with constant rotation. Cells were washed once with wash buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Halt protease inhibitor) and then washed twice with shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS, 1% Halt protease inhibitor). The nuclei were resuspended in 1 mL shearing buffer, transferred to a 1 mL milliTUBE (Covaris, Woburn, MA), and sonicated with a Covaris E220 for 18 minutes (duty cycle 5%, intensity 140 watts, 200 bursts per cycle). After sonication, insoluble debris was removed by centrifugation at 20,000 x g for 10 minutes at 4 ° C. The chromatin concentration was quantified using the Qubit DNABR assay (ThermoFisher). For immunoprecipitation, 25 ug of chromatin was removed and mixed with an equal volume of 2x conversion buffer (10 mM Tris-HCl pH 7.5, 280 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2% sodium deoxycholate, 0.2% Triton-X100, 1% Halt protease inhibitor) in a 2 mL low binding tube (Eppendorf). 5% or 6% of the input chromatin was saved as a control. Chromatin was pre-cleaned with 30uL washed protein A magnetic beads (ThermoFisher) at 4°C for 1h with constant rotation. The pre-cleaned chromatin was transferred to a new tube, 10ug rabbit monoclonal anti-HA (C29F4, Cell Signaling Technology) and 30uL washed protein A magnetic beads were added, and incubated at 4°C with constant rotation overnight.The chromatin bound to the magnetic beads was washed twice with RIPA buffer (50mM Tris-HCl pH 8.0, 250mM LiCl, 1mM EDTA, 1% sodium deoxycholate, 1% NP-40, 0.1% SDS), once with high salt wash buffer (50mM Tris-HCl pH 8.0, 500mM NaCl, 1mM EDTA, 1% NP-40, 0.1% SDS), once with lithium wash buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 1mM EDTA, 1% sodium deoxycholate, 1% NP-40), and once with TE (10mM Tris-HCl pH 8.0, 1mM EDTA). All washes were incubated at 4°C for 5 minutes under constant rotation. In the presence of 0.5mg / mL RNaseA (Qiagen), incubate with elution buffer (100mM NaHCO3, 1% SDS) at room temperature for 30 minutes to elute chromatin from the magnetic beads. In order to de-crosslink proteins and DNA, proteinase K (final 0.5mg / mL) and NaCl (final 200mM) were added to the recovered supernatant and incubated overnight at 65°C with continuous shaking (1000rpm) in a ThermoMixer (Eppendorf). DNA was purified using the Zymo ChIP DNA Cleaning and Concentration Kit (Zymo Research) according to the manufacturer's manual. Eluted DNA was analyzed by qPCR using Power SYBR Green PCR Master Mix (Roche) and StepOne Real Time PCR System (ThermoFisher). The signal from the ChIP sample was normalized to form the input signal and calculated as "input percentage". ChIP qPCR primers (all coordinates are mm10).

[0353] 1) chrX: 7584283-7584409 127bp: Fp3-CNS2-qF (forward) CCCAACAGACAGTGCAGGAA, (reverse) Fp3-CNS2-qRTGGTGTGACTGTGTGATGCA. 2) chr1: 94074907-94075062 156bp: Pd1.4A_qF1 (forward) ACCTTTCCTGTGCCTACGTC, Pd1.4A_qR1 (reverse) TAAGAGTGGTGGTGGTTGGGGG. 3) chr11: 99163437-99163632 196bp: CCR7_E1_F1 (forward) GGCTCTACTGCCCTGTTGTC, CCR7_E1_R1 (reverse) AACACATCATTTTGCCGTGA. 4) chr11: 99168432-99168614 183bp: CCR7_E2_F1 (forward) GGACACAGACGGGTGAGTTT, CCR7_E2_R1 (reverse) GGCCTGTGTTCAAATGAGGT. 5) chr17: 8196147-8196301 155bp: CCR6_F1 (forward) GGCAGGATGTGGCTTTGTAT, CCR6_R1 (reverse) CCTGCATGTAGTGCTGACCA 6) chr10: 118460432-118460610 179bp: IfngE_F1 (forward) GCGCCTAGAAGTTCAGTGCT, IfngE_R1 (reverse) TTTGAGATGCAGCAGTTTGG. Cell sorting.

[0354] Cell sorting was performed by LJI Flow Cytometry Core using FACSAria-I, FACSAria-II, or FACSAria-Fusion (BD Biosciences). For ATAC-seq, 50,000 cells were sorted from isolated CD8+ TILs, except for OT-I samples, of which 15,000-30,000 cells were sorted. In some cases, a second ATAC-seq repeat using 50,000 additional cells was prepared in parallel. For RNA-seq, 10,000 cells were sorted from isolated CD8+ TILs. For CAR and OT-I experiments, the sorted populations were as follows: CD8+CD45.1+Thy1.1+PD-1 高 TIM3 高 CAR (group A), CD8 + CD45.1+Thy1.1+PD-1高 TIM3 低 CAR (group B), CD8+CD45.1-Thy1.1-PD-1 高 TIM3 高 Endogenous cells (population C), CD8+CD45.1-Thy1.1-PD-1 高 TIM3 低 Endogenous cells (population D) and CD8+CD45.1-Thy1.1-PD-1 低 TIM3 低 Endogenous cells (population E) and CD8+CD45.1+PD-1 高 TIM3 高 OT-I (population F). For Nr4a experiments, the sorted populations were as follows: CD8+Thy1.1+NGFR+Nr4a WT TIL and CD8+Thy1.1+NGFR+Nr4a TKO TIL. For experiments in which Nr4a was ectopically expressed in vitro, populations were sorted by set levels of NGFR+ expression. ATAC-seq sample and library preparation.

[0355] By Literature 25 ATAC-seq samples were prepared as described in with minor modifications. Briefly, cells were sorted into 50% FBS / PBS, centrifuged, washed once with PBS, and then lysed. Transposition reactions were performed using Nextera enzyme (Illumina) and purified using the MinElute kit (Qiagen), followed by 10-12 cycles of PCR amplification using barcoded primers (KAPA Biosystems) and 2x 50 cycles of paired-end sequencing (Illumina). ATAC-seq analysis.

[0356] Sequencing reads in FASTQ format were generated from Illumina Basespace (for mouse datasets) or from published data. 19,21 Generated using bowtie (version 1.0.0 50, parameters “-p 8 -m 1 -best-strata -X 2000 -S -fr --chunkmbs 1024”) to the mouse (mm10) or human (hg19) genome. Unmapped reads were processed with trim_galore using parameters “--paired --nextera --length37 --stringency 3 -three_prime_clip_R1 1 --three_prime_clip_R2 1” before attempting to map again using the above parameters. The two bam files were merged to remove reads mapped to the mitochondrial genome and duplicate reads (using picard MarkDuplicates). For the mouse dataset, technical replicates were merged into a single biological replicate at this time. For the human dataset, samples with low coverage or that did not meet quality control metrics were excluded. For one human sample with two technical replicates, the results of these replicates matched closely, so number 1 was selected for analysis. Using the complete fragments captured by ATAIPS-seq, the biological replicates were analyzed using MEDIPS 51 The genome coverage of a single replicate was calculated on a 10 bp window and the Java Genomics Toolkit 52 Generate average coverage for each group.

[0357] To identify peaks, bam files containing unique, non-chrM reads were processed with samtools and awk using “'{if(sqrt(\$9*\$9)<100){print\$0}}'” to identify fragments of nucleosome-free DNA less than 100nt in length. These sub-nucleosomal fragments were used for peak analysis of each replicate of MACS2 using the parameters “--nomodel --keep-dup all --call-summits”. For peak calling, the q cutoff was 0.0001 for the mouse dataset and 0.01 for the human dataset. The peaks of each peak in all replicate samples were extended to a uniform size of 200bp for the mouse dataset and 300bp for the human dataset. These regions from all replicates were merged into a global set of peaks and filtered to remove peaks on chromosome Y or overlapping with ENCODE blacklisted regions. 53,54 .

[0358] Summarize Overlap is used to calculate the number of transposase insertions that overlap with each peak from all replicates 27 For differential coverage, raw ATAC-seq counts in each peak across all replicates of all samples were normalized using voom 55Pairwise contrasts were performed using limma and differentially accessible regions were filtered based on an FDR-adjusted p-value less than 0.01 and an estimated fold change of at least 4. ATAC-seq density (number of transposase insertion sites per kilobase of sequence mapped per million reads) was calculated for each peak and accessible region and accessible regions were defined as having an average of 5 normalized insertions per kilobase. HOMER 56 Motifs for identifying transcription factor binding sites enriched in different peak groups. RNA-seq sample and library preparation.

[0359] Total RNA was extracted using RNeasy Micro Kit (Qiagen). 24 The SMARTseq2 library was prepared as described. In short, the purified RNA was hybridized with polyA to enrich mRNA, and then the mRNA was reverse transcribed and template switched before 18 cycles of PCR pre-amplification steps. PCR purification was then performed using AMPure XP beads (Beckman Coulter). The cDNA library was quality checked using an Agilent high-sensitivity DNA chip, and 1 ng of input cDNA was further used for library preparation using the Nextera XT LibraryPrep kit (Illumina). The labeled DNA was amplified with 12 cycles of PCR and then purified again with AMPureXP beads. The library size distribution and yield were evaluated using an Agilent high-sensitivity DNA chip. The libraries were merged in equimolar ratios and sequenced with a fast run protocol on a HiSEq2500 (Illumina) with a 50nt single-end cycle. RNA-seq analysis.

[0360] Quality and adapter trimming were performed on raw RNA-seq reads using TrimGalore! v0.4.5 with default parameters and a minimum read length of 36 bp (http: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ). 26 The generated single-end reads were aligned to the mouse genome mm10 with an alignment parameter of outFilterMismatchNmax 4. Technical replicates were merged. RNA-seq analysis was performed at the gene level using the transcriptional annotation of the mouse genome mm10. Alignments to annotation features were performed using the summaryOverlaps function (mode = "Union") of the Bioconductor software package GenomicAlignmentsv1.10.1. 27The read sequences are counted. DESeq2 software package v1.14.1 28 Used to normalize raw counts and identify differentially expressed genes (FDR cutoff p < 0.1 unless otherwise stated). In all comparisons, genes with less than 10 total reads were pre-filtered as a starting step. Transformed values ​​(rlog) were calculated in DESeq2 for data visualization. Single-cell RNA-seq analysis.

[0361] Data were obtained from a previously published study on the tumor cell ecosystem in human melanoma 20 .in short 20 , analyzed by single-cell RNA-seq (scRNA-seq) malignant and non-malignant cells (including immune cells, stromal cells, and endothelial cells). Normalized expression values ​​(E i,j =log 2 (TPM i,j / 10+1), where TPM i,j TPM refers to the number of transcripts per million (TPM) of gene i in cell j, which is obtained from the Gene Expression Omnibus ( GSE72056 ). For analysis, only genes with non-zero expression values ​​in at least 10 cells were retained. Given the technical noise and gene deletions associated with scRNA-seq data 57 , the MAGIC algorithm was used for the distribution of normalized expression value matrices with diffusion parameter t = 2. The R implementation of the MAGIC method was obtained from ( https: / / www.krishnaswamylab.org / magic-project ) download. Based on the literature 20 The C-inferred cell type annotation was used to select tumor-infiltrating T cells. T cells were selected based on the expression of CD8A (cells with an estimated value ≥ 4) and CD4 (cells with an estimated value ≤ 1.5). The estimated values ​​were used for gene expression visualization. Gene set enrichment analysis (GSEA).

[0362] Gene Set Enrichment Analysis (GSEA) using GSEA pre-ranking function 32 , ranking genes by log2-fold change based on relevant comparisons, with a permutation number of 10,000 and allowing gene set sizes of up to 2000 genes. Gene sets were defined from differentially expressed genes from previously published studies. 17 Pairwise comparisons between effector, memory, and exhausted CD8+ T cells were obtained in . In this case, differential gene expression was identified using DESeq2 with an FDR cutoff of p < 0.01 and a log2 fold change cutoff of 1. Data reporting.

[0363] No statistical methods were used to determine sample size. The researchers were not blinded to group allocation during the experiments and outcome assessments. Data availability.

[0364] All data generated and supporting the findings are available with the article. RNA-seq and ATAC-seq data have been deposited in the Gene Expression Omnibus (GEO) and accession numbers will be available after the article is made public. Additional information and materials will be provided upon request. Materials and Methods Mouse.

[0365] C57BL / 6N mice were purchased from Charles River Laboratories. Tox 2 gene KO strain was obtained from Dr. Avinash Bhandool (NIH, Baltimore, MD). Rag 1- / - mice were obtained from Jackson Laboratories. Both male and female mice were used for the study. Mice were age matched and between 8-12 weeks at the time of experimentation, and tumor-bearing mice were first matched for tumor size and then randomly assigned to experimental groups. All mice were housed and maintained in the animal facility of the La Jolla Institute for Immunology. All experiments were performed in accordance with the regulations of the LJI Institutional Animal Care and Use Committee (IACUC). Preparation of CAR-T cells.

[0366] CD8 T cells were isolated and activated with 1ug / ml anti-CD3 and 1ug / m anti-CD28 for 1d, then removed from activation and transduced with retrovirus expressing CAR, non-targeting shRNA, shRNA targeting TOX, or a combination of the above for 1h 2000g 37℃. Immediately after transduction, cells were replaced with medium containing 100U IL-2 / ml. 1d after the first transduction, a second transduction was performed, and cells were replaced with medium containing 100U IL-2 / ml immediately after transduction. On the day of adoptive transfer, cells were analyzed by flow cytometry, and cell counts were obtained using a hemocytometer. Using the cell counts obtained from the hemocytometer and the population percentages obtained from the flow cytometer, the number of cells transduced with CAR was obtained. B16-OVA-huCD19 tumor model.

[0367] Analysis of CAR CD8+TIL: On day 0, 8-12 week old C57BL / 6N mice were intradermally injected with 500K B16-OVA-huCD19 cells. After the tumor became palpable, tumor measurements were recorded with a manual caliper every other day, and the tumor area was calculated in square centimeters (length x width). On day 12, 3 million transduced TOX2+ / +T cells or CAR- and non-targeted shRNA vectors of CAR- and TOX- / -T cells of transduced TOX2- / -T cells were adoptively transferred to tumor-bearing mice with tumor size matching. On day 24, the tumors of mice were harvested. In order to monitor tumor growth for survival studies after adoptive transfer of CAR T cells lacking TOX family members: On day 0, 8-12 week old C57BL / 6N mice were intradermally injected with 500K B16-OVA-huCD19 cells. After the tumor became palpable, the tumor measurements were recorded with a manual caliper every other day, and the tumor area was calculated in square centimeters (length x width). On day 12, 3 million transduced TOX2+ / +T cells or CAR- and non-targeted shRNA vectors of CAR- and TOX-targeted shRNA vectors of transduced TOX2- / -T cells were adoptively transferred into tumor-bearing mice with tumor size matching. The growth of the tumor was then monitored until the experimental endpoint or IACUC endpoint after tumor inoculation. Cytokine restimulation and intracellular staining.

[0368] For cytokines: Prior to staining, cells were incubated at 37 degrees for 4 hours in medium containing 10 nM PMA and 500 nM ionomycin and 1 ug / ml Brefeldin A. After restimulation, cells were then stained for surface markers with live / dead dye as described in the surface marker staining protocol above. Cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 1XBD perm / Wash for 30 minutes, and then stained for cytokines in 1XBD perm / Wash buffer at a final concentration of 1:100. 1XBD perm / Wash buffer was prepared according to the manufacturer's instructions. For transcription factors: Cell surface markers were stained with live / dead dye as described in the surface marker staining protocol above. Cells were then fixed, permeabilized, and stained using the Foxp3 / transcription factor staining kit (eBioscience) according to the manufacturer's instructions. Equivalent

[0369] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0370] The invention illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like should be understood broadly and without limitation. In addition, the terms and expressions employed herein have been used as terms of description rather than limitation, and it is not intended to use such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, but it is to be recognized that various modifications may be made within the scope of the invention claimed.

[0371] Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, those skilled in the art may modify, improve and vary the present invention disclosed herein, and these modifications, improvements and variations are considered to be within the scope of the present invention. The materials, methods and examples provided herein represent preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention.

[0372] The invention has been described broadly and generally herein. Each of the narrower classes and subclasses falling within the general disclosure also forms part of the invention. This includes a general description of the invention, whether or not the excised material is specifically described herein, and any subject matter is removed from the genus by its proviso or negative limitation. Table 1: Genes differentially expressed in Nr4a TKO relative to WT CAR TIL. Differentially expressed genes (adjusted p-value <0.1, log2 fold change ≥1 or ≤-1) are highlighted. Table 2: Differentially expressed genes in Nr4a TKO relative to WT CAR TILs. Differentially expressed genes (adjusted p-value < 0.1, log2 fold change ≥ 1 or ≤ -1) are highlighted. References 1.Kochenderfer,J.N.,Yu,Z.,Frasheri,D.,Restifo,N.P.&Rosenberg,S.A.Adoptive transfer of syngeneic T cells transduced with a chimeric antigenreceptor that recognizes murine CD19 can eradicate lymphoma and normal Bcells.Blood 116,3875-3886(2010). 2.Milone,M.C.et al.Chimeric receptors containing CD137 signaltransduction domains mediate enhanced survival of T cells and increasedantileukemic efficacy in vivo.Mol.Ther.17,1453-1464(2009). 3.Davila,M.L.et al.Efficacy and toxicity management of 19-28z CAR Tcell therapy in B cell acute lymphoblastic leukemia.Sci.Transl.Med.6,(2014). 4.Maude,S.L.et al.Chimeric Antigen Receptor T Cells for SustainedRemissions in Leukemia.N.Engl.J.Med.371,1507-1517(2014). 5.Ahmed,N.et al.Human Epidermal Growth Factor Receptor 2(HER2)-Specific Chimeric Antigen Receptor-Modified T Cells for the Immunotherapy ofHER2-Positive Sarcoma.J.Clin.Oncol.33,1688-1696(2015). 6.O’Rourke,D.M.et al.A singledose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance inpatients with recurrent glioblastoma.Sci.Transl.Med.9,(2017). 7.Pereira,R.M.,Hogan,P.G.,Rao,A.&Martinez,G.J.Transcriptional andepigenetic regulation of T cell hyporesponsiveness.J.Leukoc.Biol.jlb.2RI0317-097R(2017).doi:10.1189 / jlb.2RI0317-097R 8.Wherry,E.J.T cell exhaustion.Nat.Immunol.131,492-499(2011). 9.Wherry,E.J.et al.Molecular Signature of CD8+T Cell Exhaustionduring Chronic Viral Infection.Immunity 27,670-684(2007). 10.Blackburn,S.D.et al.Coregulation of CD8+T cell exhaustion bymultiple inhibitory receptors during chronic viral infection.Nat.Immunol.10,29-37(2009). 11.Barber,D.L.et al.Restoring function in exhausted CD8 T cellsduring chronic viral infection.Nature 439,682-687(2006). 12.Schietinger,A.et al.Tumor-Specific T Cell Dysfunction Is a DynamicAntigen-Driven Differentiation Program Initiated Early duringTumorigenesis.Immunity 45,(2016). 13.Schietinger,A.&Greenberg,P.D.Tolerance and exhaustion:Definingmechanisms of T cell dysfunction.Trends in Immunology 35,(2014). 14.Moon,E.K.et al.Multifactorial T cell Hypofunction That isReversible Can Limit the Efficacy of Chimeric Antibody Receptor-transducedHuman T cells in Solid Tumors.Clin.Cancer Res.20,4262-73(2014). 15.John,L.B.et al.Anti-PD-1 antibody therapy potently enhances theeradication of established tumors by gene-modified T cells.Clin.CancerRes.19,5636-5646(2013). 16.Martinez,G.J.et al.The Transcription Factor NFAT PromotesExhaustion of Activated CD8+T Cells.Immunity 42,(2015). 17.Scott-Browne,J.P.et al.Dynamic Changes in Chromatin AccessibilityOccur in CD8+T Cells Responding to Viral Infection.Immunity 45,1327-1340(2016). 18.Mognol,G.P.et al.Exhaustion-associated regulatory regions in CD8 + tumor-infiltrating T cells.Proc.Natl.Acad.Sci.201620498(2017).doi:10.1073 / pnas.1620498114 19.Philip,M.et al.Chromatin states define tumour-specific T celldysfunction and reprogramming.Nature 545,452(2017). 20.Tirosh,I.et al.Dissecting the multicellular ecosystem ofmetastatic melanoma by single-cell RNA-seq.Science(80-.).(2016).doi:10.1126 / science.aad0501 21.Sen,D.R.et al.The epigenetic landscape of T cellexhaustion.Science(80-.).354,(2016). 22.Nicholson,I.C.et al.Construction and characterisation of afunctional CD19 specific single chain Fv fragment for immunotherapy of Blineage leukaemia and lymphoma.Mol.Immunol.34,1157-1165(1997). 23.Brogdon,J.,June,C.H.,Loew,A.,Maus,M.&Scholler,J.Treatment ofcancer using humanized anti-cd19 chimeric antigen receptor.(2014). 24.Picelli,S.et al.Full-length RNA-seq from single cells using Smart-seq2.Nat.Protoc.9,171-181(2014). 25.Buenrostro,J.D.,Giresi,P.G.,Zaba,L.C.,Chang,H.Y.&Greenleaf,W.J.Transposition of native chromatin for fast and sensitive epigenomicprofiling of open chromatin,DNA-binding proteins and nucleosomeposition.Nat.Methods 10,(2013). 26.Dobin,A.et al.STAR:Ultrafast universal RNA-seqaligner.Bioinformatics 29,15-21(2013). 27.Lawrence,M.et al.Software for Computing and Annotating GenomicRanges.PLoS Comput.Biol.9,(2013). 28.Love,M.I.,Huber,W.&Anders,S.Moderated estimation of fold changeand dispersion for RNA-seq data with DESeq2.Genome Biol.15,(2014). 29.Pauken,K.E.et al.Epigenetic stability of exhausted T cells limitsdurability of reinvigoration by PD-1 blockade.Science 354,(2016). 30.Sekiya,T.et al.Nr4a receptors are essential for thymic regulatoryT cell development and immune homeostasis.Nat.Immunol.14,230-237(2013). 31.Kadkhodaei,B.et al.Nurr1 Is Required for Maintenance of Maturingand Adult Midbrain Dopamine Neurons.J.Neurosci.(2009).doi:10.1523 / JNEUROSCI.3910-09.2009 32.Subramanian,A.et al.Gene set enrichment analysis:A knowledge-basedapproach for interpreting genome-wide expressionprofiles.Proc.Natl.Acad.Sci.102,15545-15550(2005). 33.Nowyhed,H.N.et al.The Nuclear Receptor Nr4a1 Controls CD8 T CellDevelopment Through Transcriptional Suppression of Runx3.Sci.Rep.5,9059(2015). 34.Milner,J.J.et al.Runx3 programs CD8+T cell residency in non-lymphoid tissues and tumours.Nature(2017).doi:10.1038 / nature24993 35.Harant,H.&Lindley,I.J.D.Negative cross-talk between the humanorphan nuclear receptor Nur77 / NAK-1 / TR3 and nuclear factor-κB.Nucleic AcidsRes.(2004).doi:10.1093 / nar / gkh856 36.Saijo,K.et al.A Nurr1 / CoREST Pathway in Microglia and AstrocytesProtects Dopaminergic Neurons from Inflammation-Induced Death.Cell 137,47-59(2009). 37.Best,J.A.et al.Transcriptional insights into the CD8+T cellresponse to infection and memory T cell formation.Nat.Immunol.14,404-412(2013). 38.Spolski,R.&Leonard,W.J.Interleukin-21:Bas...

Claims

1. An immune cell engineered to reduce or eliminate the expression and / or function of NR4A transcription factor, the cell comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor (CAR) include: (a) Antigen binding domain; (b) Hinge domain; (c) Transmembrane domain; and (d) Intracellular domain.

2. The immune cell of claim 1, wherein the antigen binding domain comprises an anti-ROR1 binding domain; the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain; and the intracellular domain comprises a CD28 co-stimulatory signaling region, a 4-1BB co-stimulatory signaling region, an ICOS co-stimulatory signaling region, or an OX40 co-stimulatory region.

3. The immune cell according to claim 1 or 2, wherein the NR4A transcription factor is NR4A2 (Nurr1) or NR4A3 (NOR1).

4. The immune cell according to claim 1 or 2, wherein the immune cell is engineered to reduce or eliminate the expression and / or function of NR4A3 (NOR1).

5. The immune cell according to any one of claims 1-4, wherein the immune cell is a CD8+T cell.

6. An immune cell according to any one of claims 2-5, wherein the anti-ROR1 binding domain of the CAR comprises a single-chain variable fragment (scFv).

7. An immune cell according to any one of claims 1-6, wherein the immune cell further comprises a polynucleotide encoding the CAR.

8. The immune cell according to claim 7, wherein the polynucleotide comprises: a promoter operably linked to the polynucleotide to express the polynucleotide in the immune cell; a polynucleotide sequence encoding a 2A self-cleaving peptide (T2A) located upstream of the polynucleotide encoding the anti-ROR1 binding domain; and a polynucleotide encoding a signal peptide located upstream of the polynucleotide encoding the anti-ROR1 binding domain; or A combination of the above.

9. The immune cell according to claim 7, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO:

2.

10. The immune cell according to claim 7, wherein the polynucleotide comprises SEQ ID NO:

1.

11. The immune cell according to any one of claims 1 to 10, having a gene expression profile as shown in Table 1 and Table 2, wherein the immune cell expresses a receptor that binds to at least one tumor antigen or at least one antigen expressed by a pathogen.

12. The immune cell of any one of claims 1 to 10, wherein the immune cell expresses a receptor that binds to at least one tumor antigen or at least one antigen expressed by a pathogen.

13. The immune cell of any one of claims 1 to 10, which exhibits reduced exhaustion compared to a corresponding wild-type immune cell.

14. The immune cell of any one of claims 1 to 10, wherein the antigen binding domain comprises an anti-ROR1 binding domain.

15. The immune cell according to any one of claims 1 to 10, wherein the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain.

16. The immune cell of claim 15, wherein the intracellular domain comprises a CD28 co-stimulatory signaling region, a 4-1BB co-stimulatory signaling region, an ICOS co-stimulatory signaling region, or an OX40 co-stimulatory region.

17. The immune cell of claim 16, further comprising a CD3ζ signaling domain.

18. The immune cell of claim 17, wherein the anti-ROR1 binding domain of the CAR is a single-chain variable fragment (scFv).

19. The immune cell according to claim 10, wherein the polynucleotide further comprises: a vector having an isolated nucleic acid sequence, wherein the isolated nucleic acid sequence comprises SEQ ID NO:

1.

20. The immune cell according to claim 19, wherein the vector is a plasmid.

21. The immune cell according to claim 20, wherein the vector is a viral vector selected from a retroviral vector, a lentiviral vector, an adenoviral vector and an adeno-associated viral vector.

22. The immune cell of any one of claims 1-10, wherein the immune cell is isolated from a subject.

23. The immune cell of claim 22, wherein the subject has cancer.

24. The immune cell of claim 23, wherein cells associated with the cancer express a tumor antigen.

25. A method for producing engineered immune cells, the method comprising reducing or eliminating the expression and / or function of NR4A transcription factor in the immune cells.

26. The method of claim 25, wherein the method comprises: isolating immune cells from a subject, reducing or eliminating the expression and / or function of the NR4A transcription factor in the cells, and culturing the immune cells under conditions that are conducive to the expansion and proliferation of the cells.

27. The method of claim 26, wherein the immune cells isolated from the subject bind to a target antigen.

28. The method of claim 27, wherein the target antigen is at least one tumor antigen.

29. The method of claim 28, wherein the tumor antigen comprises mesothelin, ROR1 or EGFRvIII.

30. The method of claim 25, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (polynucleotide CAR) into the cell.

31. The method of claim 30, wherein the polynucleotide CAR comprises a polynucleotide encoding: (a) an antigen binding domain, (b) a hinge domain, (c) a transmembrane domain, and (d) an intracellular domain.

32. The method of claim 30 or 31, wherein the polynucleotide sequence comprises SEQ ID NO:

1.

33. The method of claim 30 or 31, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO:

2.

34. The method according to claim 30 or 31, wherein the polynucleotide further comprises a vector.

35. The method of claim 34, wherein the vector is a plasmid.

36. The method of claim 35, wherein the vector is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

37. An immune cell prepared by the method of any one of claims 25 to 36.

38. A substantially homogeneous population of immune cells as described in any one of claims 1-24 or 37.

39. A composition comprising a vector and one or more of the immune cells of any one of claims 1 to 24 or 37 or a cell population of claim 38.

40. The composition of claim 39, wherein the carrier is a pharmaceutically acceptable carrier.

41. The composition of claim 39 or 40, wherein the composition further comprises a cryoprotectant.

42. The immune cell of claim 37, wherein the immune cell binds to a target cell.

43. A kit comprising a vector and instructions for preparing the cell of any one of claims 1 to 24 or 37.

44. The kit of claim 43, wherein the kit further comprises instructions for its diagnostic or therapeutic use.

45. Use of the immune cell of any one of claims 1 to 24 or 37 or the immune cell colony of claim 38 in the preparation of a medicament for stimulating an immune cell-mediated immune response to a target cell colony, wherein the target cell colony is contacted with the medicament.

46. ​​The use of claim 45, wherein the contacting is in vivo and the target cell population is a cancer cell population in a subject.

47. The use of claim 45, wherein the contacting is in vivo and the target cell population is a cell population infected by a pathogen in a subject.

48. Use according to claim 47, wherein the immune cell according to any one of claims 1 to 24 or 37 specifically binds cells to a target cell population.

49. The use of claim 46, wherein the subject suffers from cancer, has suffered from cancer, or is in need of treatment for cancer.

50. Use of the immune cell of any one of claims 1 to 24 or 37 or the composition of claim 39 in the preparation of a medicament for treating cancer in a subject, a medicament for providing anti-tumor immunity in a subject, or a medicament for treating a disease, disorder or condition associated with elevated expression of a tumor antigen in a subject.

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