The antitumor efficacy of ACT immunotherapy with Bcma-CAR T cells was improved by Asct2 overexpression. (A) Gln concentration measured in the BM of healthy mice or mice challenged with 5080 MM cell line (25 days after tumor challenge). (B) Experimental design to evaluate antitumor activity of CAR T cells in a MM murine model based on the IV injection of 5080 MM cells and percent of survival of 5080 MM-challenged mice treated with 1 × 10⁶ CAR T cells (1:1 ratio of CD4⁺ and CD8⁺ CAR T cells; number of mice per group n = 10). (C) Total number of tumor cells (B220⁺GFP⁺) found in the BM of mice treated with Asct2 Bcma-CAR T cells and Bcma-CAR T cells. (D-F) Total numbers of CAR T cells measured in the BM (D, F), and the spleen (E) of mice treated with Asct2 Bcma-CAR T cells or Bcma-CAR T cells at day 14 (D-E), and at day 21 (F) after CAR T-cell transfer. (G-J) Asct2 Bcma-CAR T cells exert antitumor activity in a genetic model of MM. (G) Gln concentration in the BM aspirates and the sera of 170-day-old MICγt1 mice compared with the Gln levels in BM or serum of healthy mice. (H) Experimental design for testing CAR T-cell immunotherapy against the MICγt1 MM genetic model. Fc γ-globulin fraction levels and survival curves after CAR T-cell treatment (n = 7 mice per group; survival data for untreated animals corresponds to a cohort of 50 mice). (I) Number of tumor cells in both the spleen and the BM of mice treated with Bcma-CAR T cells and Asct2 Bcma-CAR T cells compared with untreated mice. (J) CAR T cells in the spleen and in the BM of MICγt1 mice treated with Bcma or Asct2 Bcma-CAR T cells analyzed in the CD4⁺ and CD8⁺ compartments. Data are representative of 2 to 3 independent experiments. Data represent mean ± SEM and were analyzed using Student t test, 2-way ANOVA, and 1-way ANOVA with Bonferroni multiple comparisons test. Statistical analysis for survival curves is a log-rank test (∗∗P < .01; ∗P < .05; ∗∗∗P < .001). ACT, adoptive cell therapy; FC, fold change; rIL, recombinant interleukin.