Inosine induces stemness features in CAR-T cells and enhances potency

Abstract

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8⁺ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.

Figure 1:. CD39 marks a subset of exhausted CAR T cells with features of regulatory T cells.

A. (Left) Flow cytometric analysis of exhaustion marker expression in gated CD19.28z-CAR⁺ (CD19) (blue), HA.28z-CAR⁺ (HA) (red) or treated starting D4 with dasatinib (HA+DB) (orange) T cells at D0 (grey) and at each indicated time-point post-activation. Representative histogram shown of n = 3–4 donors. (Right) Corresponding frequencies of exhaustion markers at D14 post-activation. Pooled data from n=3–4 donors. B. (Top) Representative flow cytometry contour plot of CD39 expression in HA-CAR⁻ vs. CAR⁺-T cells on day 10 post-activation. (Bottom) Percent of CD39⁺ cells in HA-CAR⁻ vs. CAR⁺-T cells (n=12 donors). C. (Left) Representative flow cytometry analysis of CD39 expression in CD8⁺ vs. CD4⁺ HA-CAR-T cells 10 days post-activation. (Right) Percent of CD39⁺ cells in CD4⁺ vs. CD8⁺ HA-CAR-T cells from n=9 donors. D. Luminex analysis of cytokines secreted by CD39⁺ vs. CD39⁻CD8⁺ HA-CAR-T cells sorted on day 14 and stimulated with Nalm6-GD2 for 24hrs at 1:1 E:T ratio. Data are mean ± s.e.m. of n=3 donors. E. Gene Set Enrichment Analysis (GSEA) of bulk RNA-seq collected 14 days post-activation of CD39⁺compared to CD39⁻HA-CAR-T cells using the publicly available GSE25087 gene collection. F. CyTOF analysis of CD4⁺ (bottom) and CD8⁺ (top) HA-CAR-T cells gated based on CD39 expression 10 days post-activation. Heat map represents expression of the indicated markers as median Arcsinh relative to the total CD4⁺ or CD8⁺ sample. Representative donor shown of n=3 donors. G. Schematic of immune suppression mediated by purinergic pathway. ATP- adenosine triphosphate; ADP- adenosine diphosphate; AMP- adenosine monophosphate; ADO- adenosine (red); A2aR- adenosine 2a receptor; CD39- ecto-ATP diphosphohydrolase-1; CD73- 5′-ectonucleotidase; NF-κB- Nuclear Factor kappa B. H. (Left) Representative flow cytometry contour plots showing expression of CD39 and CD73 in gated CD4⁺ or CD8⁺ HA-, CD19-CAR- or mock T cells 14 days post-activation. (Right) Percent of CD39⁺CD73⁺ T cells in CD4⁺ vs. CD8⁺ by day 14 HA-, CD19-CAR or mock T cells in 6 donors. I. Percent of ATP hydrolyzed by mock or CAR-expressing T cells with or without CD39 or CD73 after spiking with 20μM of ATP at day 15 post-activation. Representative data from n=4 donors. Average purity of the knock-out cells was > 90%. P values determined by unpaired two-tailed t-tests. J. Concentration of adenosine (ADO) produced by mock or CAR-expressing T cells with or without CD39 or CD73 spiked with 20μM of ATP at day 17 post-activation. Representative data from n=3–5 donors. Average purity of the knock-out cells was > 90%. P values determined by unpaired two-tailed t-tests. K. (Left) schematic of experimental design of immunosuppression co-culture assays. (Right) Control or A2aR KO CD19.bbz-CAR-T cells were activated with Nalm6 at 1:1 ratio in the presence of HA- or CD39KO HA-CAR-T cells. IL-2 was measured 24hrs post-stimulation. Data are mean ± s.e.m. from triplicate wells. P values determined by unpaired two-tailed t-tests. Representative result of two independent experiments. P values determined by paired two-tailed t-tests unless otherwise specified. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 See also Figure S1.

Figure 2:. Overexpression of Adenosine deaminase (ADA-OE) increases features of stemness and decreases terminal differentiation.

A. Schematic of immune suppression mediated by purinergic pathway. ADO- adenosine (red); A2aR- adenosine 2a receptor; CD39- ecto-ATP diphosphohydrolase-1; CD73- 5′-ectonucleotidase; INO- inosine (green); ADA- adenosine deaminase. B. Principal component analysis (PCA) of bulk RNA-seq from control AAVS1KO HA-CAR-T cells or overexpressing Ado deaminase (ADA-OE) or deficient for A2aR, CD39 or CD73 15 days post-activation (n=3 donors). C. Number of up-regulated and down-regulated genes in ADA-OE, CD39-KO, CD73-KO and A2aR-KO conditions compared to AAVS1 controls. D. Boxplots showing log2TPM of T cell exhaustion and effector associated genes in CAR T cells profiled by bulk RNA-seq. The top, bottom, middle and whiskers of the boxplot represents the upper quantile, lower quantile, median and the rest of the distribution, respectively *, P < 0.1; ** P < 0.05; *** P < 0.01 by DESeq2. E-G. UMAP analysis of day 15 CD8⁺ HA-CAR cells engineered as in (A). Expression of 26 markers was analyzed by CyTOF. 5,000 or maximum of CD8⁺ CAR-T cells from each donor (n=4) were combined and colored by (E) genotype, (F) marker intensity or (G) subpopulation defined by FlowSOM algorithm. Pie charts show population frequencies defined using FlowSOM for each condition. SCM; stem cell memory, EF; effector, EM; effector memory, Pexh; progenitor exhausted, Exh, exhausted. Table shows the average frequencies and corresponding p values determined by paired two-tailed t-tests. See also Figure S2.

Figure 3:. Inosine (INO) induces stemness and augments CAR T cell function in vitro.

A. Schematic of the experimental design. B. PCA of bulk RNA-seq from HA-CAR-T cells expanded in indicated media for 10 days. C. GSEA of HA-CAR-T cells expanded as in (B) using the GSE23321 gene collection. D. Schematic of the experimental design. E. Frequency of CD8⁺CD62L⁺ HA-CAR-T cells cultured in INO-media starting from the indicated days and assessed by flow cytometry at day 21 (n=5 donors). F. Co-cultures of HA, or HA-CAR-T cells grown in INO media starting from indicated days post-activation and 143B tumor (1:1 E:T) starting at day 15 post-activation. Tumor GFP fluorescence intensity was normalized to the first time point (duplicate or triplicate wells). Representative donor of n=3 donor shown. P values determined by two-way ANOVA with Dunnett’s multiple comparisons test. G. Cell expansion of HA-CAR-T cells grown in culture media containing indicated concentration of glucose and/or inosine from day 4 to 10 post-activation. Data are mean ± s.e.m. of n=3–5 donors. H. Percent of CD62L⁺CD8⁺ HA-CAR-T cells grown in culture media containing indicated concentration of glucose and/or inosine from day 4 to 21 and measured by flow cytometry. Data are mean ± s.e.m. of n=5–8 donors. I. Fold increase of IFNγ release after 24hrs of co-culture with Nalm6-GD2 by D14 HA-CAR-T cells grown in culture media containing various concentrations of glucose and/or inosine and normalized to CAR-T cells grown in control media. Data are mean ± s.e.m. of n=2–4 donors. J. Expansion of HA-CAR-T cells grown in RPMI containing 11mM of glucose and increasing concentrations of inosine for 6 days. Scatter plot showing correlation between ratio of expansion (y-axis) and inosine concentration (x-axis). Pearson correlation, r, n=2–4 donors from independent experiments. K. HA-CAR-T cells were grown in RPMI containing 11mM of glucose and increasing concentrations of inosine for 17 days. Scatter plot showing correlation between CD62L frequency (y-axis) and inosine concentration (x-axis). Pearson correlation, r, n=2–6 donors from independent experiments. L. HA-CAR-T cells were grown in RPMI containing 11mM of glucose and increasing concentrations of inosine for 10 days, and stimulated with Nalm6-GD2 for 24hrs. Scatter plot showing correlation between IFNγ secretion (y-axis) and inosine concentration (x-axis). Pearson correlation, r, n=2–4 donors from independent experiments. M. At day 10 post-activation HA-CAR-T cells were collected and cell cycle state was evaluated using BrdU and 7AAD staining. (Left) representative dot plots shown from one donor (n=3). (Right) Bar graphs represent percentage of apoptotic cells or in G0/G1, S and G2/M phase indicated for each media conditions. Data are mean+/− s.e.m. from n=3 donors. P values determined by paired two-tailed t-tests unless otherwise specified. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 See also Figures S3 and S4.

Figure 4:. Inosine enhances CAR-T cell anti-tumor function in vivo.

A. NSG mice were injected intravenously with 1×10⁶ Nalm6 leukemia cells. Three days later 2×10⁶ of mock or HA-CAR-T cells manufactured in different media from day 6 to 10, were given intravenously. B. Survival curves were compared using the log-rank Mantel–Cox test. C. Tumor progression was monitored using bioluminescent imaging. Data are mean ± s.e.m. of n=5 mice per group. P values determined at day 21 by using Mann-Whitney test. (Right) D. NSG mice were injected intravenously with 1×10⁶ Nalm6 leukemia cells. Three days later 0.2×10⁶ of mock or CD19-CAR-T cells manufactured in the presence of inosine or glucose, were given intravenously. E. Tumor progression was monitored using bioluminescent imaging. Data are mean ± s.e.m. of n=10 mice per group. F. NSG mice were inoculated with 1×10⁶ 143B osteosarcoma cells via intramuscular injection. At day 4 post-engraftment 1×10⁷ mock or Her2.bbz-CAR-T cells cultured in glucose- or inosine-containing media were given intravenously. G. 143B tumor growth monitored by caliper measurements. P values determined by unpaired two-tailed t-test with Welch’s correction at day 20. (Right) Long-term survival of CAR-treated mice. Data are mean ± s.e.m. of n=3–5 mice per group. H. Survival curves were compared using the log-rank Mantel–Cox test. I. Concentration of total CD3⁺ T cells detected in blood of mice at day 17 post-CAR-T cell injection. P values determined by unpaired two-tailed t-test with Welch’s correction. J. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Figure S5.

Figure 5:. Exhausted CAR T cells expanded in inosine undergo metabolic reprogramming.

A. Pathway Enrichment Analysis of day 14 CAR-T cells shown in (3B) using the Reactome pathway collection and DAVID algorithm. Fold enrichment, number of genes represented, and FDR are shown. B. Heat map of differentially expressed genes in HA- vs. HA-INO-CAR-T cells shown in (3B) (padj<0.01). C. Graphic illustration of metabolic changes upon inosine addition into glucose-free culture media. Upregulated genes highlighted in (B) shown in blue and downregulated genes shown in grey. D. UMAP analysis of CD8⁺ CD19-, HA- or HA-INO-CAR T cells 14 days post-activation. Expression of 28 markers (Panel 1) or 24 markers (Panel 2) was analyzed by CyTOF. 9,000 of CD8⁺ CAR-T cells from each condition and donor (n=3–4) were combined and colored by marker intensity. Graphs represent median expression of indicated markers expressed by CD8⁺CAR-T cells. P values determined by paired two-tailed t-tests. E. E-H. Seahorse analysis of mitochondrial fitness and glycolytic function was performed at day 11 post-activation. F. Oxygen consumption rate (OCR) before and after treatment with oligomycin (Oligo), FCCP, and rotenone and antimycin (R+A). Plot shows mean ±s.e.m from 7–9 technical replicates from one representative donor (n=3). G. Basal OCR, Maximal OCR and Spare Respiratory Capacity (SRC) from one representative donor. P values determined by two-way ANOVA with Dunnett’s multiple comparisons test or Mixed-effects model with Dunnett’s multiple comparisons test. G. Extracellular acidification rate (ECAR) before and after treatment with glucose (Glc), oligomycin (Oligo), and 2-DG. Plot shows mean ±s.e.m from 6–9 technical replicates from one representative donor (n=5). H. Basal Glycolysis, Glycolytic Capacity and Glycolytic Reserve from one representative donor. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Figure S6.

Figure 6.. Inosine induces epigenetic reprogramming HA-CAR-T cells epigenetic by increasing polyamine metabolism.

A. Global chromatin accessibility profile of D14 CD8⁺ HA- and HA-INO-CAR-T cells from three healthy individuals determined by ATAC-seq (p-value < 0.001 and log2 FC > 1). B. PCA of ATAC-seq chromatin accessibility from HA-CAR-T cells cultivated in glucose or inosine. C. Overlayed accessibility profiles in HA (black) and HA-INO-CAR-T cells (green) in the TCF7, IL2, IL7R, CCR7 and CXCR3 loci at day 14 post-activation. Concatenated samples from n=3 donors. D. Top transcription factor motifs enrichment in HA-INO- vs HA-CAR-T cells ranked by HOMER analysis. E. Top 25 transcription factor motif deviation scores in HA-INO- vs HA- by chromVAR analysis. Heatmap represents transcription factor deviation score. F. Levels of expression of EIF5A gene transcripts from bulk RNA samples shown in (Fig. 4B). Data are mean+/− s.e.m. from n=3 donors. P values determined by paired two-tailed t-tests. G. Graphic illustration of targeting polyamine-hypusine circuit. EIF5A- Eukaryotic initiation factor 5A; DOHH- Deoxyhypusine Hydroxylase; CPX- ciclopirox. H. Relative frequency of stem cell memory (CD45RA⁺CD62L⁺), central memory (CM; CD45RA⁻CD62L⁺), effector memory (EM; CD45RA⁻CD62L⁻) and terminally differentiated effector (TEMRA; CD45RA⁺CD62L⁻), in control AAVS1-, EIF5A-KO CD8⁺HA- or HA-INO-CAR-T cells at day 15 post-activation and measured by flow cytometry. (Right) CRISPR efficiency of indicated genes measured as Inference of CRISPR Edits (ICE). Data are mean+/− s.e.m. from n=2 donors. P values determined by paired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Figure S7.

Figure 7.. Clinical scale manufacturing of GD2-CAR-T cells using INO containing media improves CAR product quality.

A. Schematic of large-scale manufacturing of GD2.bbz-CAR⁻T cells in a semi-closed G-Rex system in glucose- or inosine-containing media. 7 days post-activation, transduction efficiency, percentage of viable cells, and CD4/CD8 ratio of GD2⁺ cells were assessed by flow cytometry. Data are mean+/− s.e.m. from n=2 donors. P values determined by paired two-tailed t-tests. B. (Left) Expansion of total viable T cells manufactured in control (black) vs. inosine (green)- containing media and (Right) corresponding number of CAR⁺ cells at day 7. Data are mean+/− s.e.m. from n=2 donors. P values determined by paired two-tailed t-tests. C. IL-2 and IFNγ secretion by CAR-T cells stimulated for 24hrs with 143B, mg63.3 or Nalm6-GD2 tumor lines. Error bars represent mean ± SD of triplicate wells from one representative donor (n=2 donors). P values determined by unpaired two-tailed t-tests. D. NSG mice were injected with 1×10⁶ Nalm6 leukemia cells. On day 3 post-tumor injection, 2×10⁶ of mock or GD2.bbz-CAR-T cells manufactured in the presence of inosine or in regular media were transferred intravenously. Tumor growth was monitored by bioluminescent imaging. Data are mean ± s.e.m. of n=5 mice per group. P values determined at day 43 by using Mann-Whitney test. Representative results of two independent experiments shown. E. Concentration of CD45⁺ T cells detected in blood of mice at D32 post-CAR-T cell injection. P values determined by unpaired two-tailed t-test with Welch’s correction. Results from one experiment shown. F. Schematic of the experimental design. G. GD2.bbz-CAR-T cells grown in media containing indicated inosine concentration between day 3 and day 10 post-activation and stimulated with 143B tumor. IL-2 and IFNγ secretion after 24hrs of co-culture was measured. Data are mean ± s.d. of duplicate or triplicate wells. Representative of n=3 donors shown. P values determined by unpaired two-tailed t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 See also Figure S8.