Extended Data Fig. 10: RASA2 ablation boosts CAR-T cell resistance to tumor rechallenge and has a similar safety profile to control CAR-T cells.
From: RASA2 ablation in T cells boosts antigen sensitivity and long-term function
a, BLI values for Nalm6-bearing mice which received 0.2x106 TRAC CAR-T cells after first Nalm6 injection without any rechallenge injections, and for Nalm6-bearing mice which received 0.2x106 CAR-T cells and then 3 further Nalm6 rechallenge injections (1x106 Nalm6/injection). Mice were monitored for tumor burden (BLI levels) and survival. n = 7 mice per arm in each cohort. Arrows depict Nalm6 leukemia rechallenges. b, mean ± SEM for BLI values shown in (a) until first mouse death in the control cohort, *p < 0.05 and **p < 0.01 for two-sided unpaired Student’s t-test. c, Survival analysis for the leukemia rechallenge model of the cohort shown in (a,b). Exact p-value by log-rank test. d, Body weights over time for mice receiving CAR-T cells with no tumor cells engrafted (mean ± SEM, n = 2 human donors and 3 mice per group). e, White blood cell (WBC; K/μL), hemoglobin (Hb; g/dL), and platelet (Plt; x104 K/μL) counts for mice receiving only CAR-T cells (n = 2 human donors, n = 3 mice per group), as well as mice receiving tumor-clearing CAR-T cell infusions (n = 1 human donor, n = 6 mice per group, box shows the upper and lower quartiles, horizontal line is median). f, Representative H&Es from bone marrow and spleens of mice from (e). Sternal bone marrows (40X magnification) showed tri-lineage hematopoiesis, which was similar in all animals. As expected, tumor naive NSG mice had few cells of lymphoid appearance in their white pulp (seen here as few mononuclear cells surrounding a blood vessel) (spleens at 20X magnification). The red pulp of NSG mice contains abundant erythroid precursors (seen here as numerous cells with dense dark nuclei) as well as megakaryocytes. In comparison to the cancer naive NSG mice, the red pulp and white pulp of recipient animals were largely similar; in some mice, independent of group, some expansion of splenic white pulp was seen, consistent with engrafted lymphoid cells. Of importance, recipients of RASA2 KO TRAC CAR-T cells did not show evidence of increased lymphoid infiltrates in comparison to matched recipients of TRAC CAR-T control cells. g, Scheme of EphA2-specific CAR retroviral vectors. 4H5: scFV recognizing EphA2. SSR: short spacer region. h. Summary data for CAR expression in gene targeted T-cells as measured by flow cytometry (n = 10 for CTRL, n = 14 for RASA2). i, Western Blot analysis of RASA2 expression RASA2 KO EphA2-CAR T cells compared to T cells treated with non-targeting guide (CTRL). Two guide RNAs targeting RASA2 were tested. j, Individual BLI traces from main Fig. 4m, using both RASA2 sgRNAs for one donor, and sgRNA1 for the second donor. k, Mice without detectable BLI from experiment in (j) were re-challenged with a second intraperitoneal (i.p.) tumor injection with 1x106 LM7-ffLuc tumor cells on Day 174. Graphs show quantitative bioluminescence imaging (total flux). Dotted vertical line indicates the second tumor injection.
