Figure 4. Comparison of CD19-BAFF-R(l) dual-targeting CAR with CD19 and BAFF-R single CAR in a mixed tumor model.

A. Naïve and central memory T cells (Tn/mem) cells were enriched, activated, transduced, and expanded for 13 days. FACS analysis plots CD107a positive CD4 or CD8 CAR T cells (CAR gated) comparing CD19-BAFF-R(l) dual CAR, BAFF-R single CAR, and CD19 single CAR degranulation against Nalm-6 CD19−/−BAFF-R+/+ and Nalm-6 CD19+/+BAFF-R−/− B-ALL lines. Mock T cells from the same donor were used as a control. B. Graph shows ELISA measurements of IFN-γ released by CD19-BAFF-R(l) dual CAR T cells compared to BAFF-R and CD19 single CAR T cells. CAR T cells were co-incubated with either BAFF-R−/− or CD19−/− Nalm-6 B-ALL lines. IFN-γ was measured from sampled supernatant. Experiments were conducted in triplicate and analyzed by a Student’s t-test; **P<0.01, ***P<0.001, ****P<0.0001, NS: not statistically significant. C. Specific lysis induced by CAR T cells against Nalm-6 WT, BAFF-R−/− or CD19−/− Nalm-6 B-ALL cell lines. Effector CAR T cells were CD19-BAFF-R(l) dual-targeting CARs and BAFF-R and CD19 single-targeting CARs at 10:1, 2:1, and 0.5:1 E:T ratios. Mock T cells were used as negative control. Experiments were conducted in triplicate and analyzed by a Student’s t-test. ****P<0.0001, ****P<0.0001, ***P<0.001, **P<0.01, NS: not statistically significant. D. Bioluminescent imaging of NSG Mice (n=5/group in one experiment) challenged with mixed tumor (0.1 ×10⁶ Nalm-6-CD19−/− plus 0.1 ×10⁶ Nalm-6-BAFF-R−/−). Mice were administered a single infusion of (1 × 10⁶ CAR T cells), BAFF-R, CD19 single-targeting CAR T cells or CD19-BAFF-R(l) dual-targeting CAR T cells on day 9. Mock T cells from the same donor were used as control. E. Flux (photons/sec) was determined by measuring bioluminescence every two weeks. F. Kaplan-Meier survival curve show the results after monitoring mice for up to 100 days. Log-rank test: **p<0.01, between dual CAR and CD19, BAFF-R single CAR.