Figure 6.

Antitumor efficacy by nerve growth factor receptor (NGFR)-enriched CD19 and CEA chimeric antigen receptor (CAR)-T cells. Primary T cells from healthy donors were stimulated with CD3/CD28-beads, transduced with RVs encoding for CD19 or CEA CARs spaced with NGFR wild-type long (NWL) or NGFR mutated short (NMS), enriched with immuno-magnetic beads and cultured with IL-7/IL-15. (A) FACS plots of CAR-T cells from a representative donor (of n = 3) are depicted before (pre) and after (post) enrichment. NGFR isoform-enriched CD19 (B) or CEA (C) CAR-T cells were cocultured at 1:10 E:T ratio with tumor cell lines differentially expressing their respective target antigen and/or Fc receptors (FcRs) (see caption of Figure 1). After 4 days, the elimination of tumor cells by CAR-T cells were analyzed by FACS and expressed as elimination indexes (see Materials and Methods, means ± SEM from n = 3 donors). Results from a one-way ANOVA test are indicated when statistically significant (***P ≤ 0.001; ****P ≤ 0.0001). (D) NSG mice were xeno-engrafted with ALL-CM leukemic cells and, after three days treated with CD19 CAR-T enriched through either NWL (n = 8) or NMS (n = 7), or with CTRL (CD44v6-specific) CAR-T cells (n = 6). Left: concentration of circulating ALL-CM cells over time expressed as number of T cells (identified as hCD19+/hCD3−) per microliter of blood (means ± SEM for the different cohorts). Right: ALL-CM cell infiltration in the bone marrow and in the spleen is shown for the three cohorts as mean percentages ± SEM. Results from an unpaired t-test are indicated when statistically significant (**P ≤ 0.01; ****P ≤ 0.0001).