Figure 2. C7R enhances GD2-CAR T-cell activity during serial tumor challenge.

(A) Cytokines secreted by GD2-CAR T-cells or GD2-CAR.C7R T-cells 24 hours after co-culture with LAN-1 tumor cells was determined by ELISA. (B) 4-hour luciferase-based cytotoxicity assay of T-cells killing LAN-1 tumor cells. (C) Serial co-culture schematic. The first co-culture (CC1) was initiated with 1×10⁶ GD2-CAR or GD2-CAR.C7R T-cells together with 0.5×10⁶ LAN-1 GFP-FFluc tumor cells for 7 days, in the absence of IL-15 or IL-7. For the second and third co-cultures (CC2 and CC3), T-cells were harvested from the previous co-culture and then replated in new culture medium with fresh tumor cells at the same 2:1 E:T ratio. (D) Cumulative expansion of GD2-CAR or GD2-CAR.C7R T-cells during serial co-culture. Arrows indicate timepoints of T-cell re-stimulation with tumor cells. (E) LAN-1 tumor cells remaining after CC3 with GD2-CAR T-cells and GD2-CAR.C7R T-cells, respectively. (F,G) For proliferation analysis, GD2-CAR and GD2-CAR.C7R T-cells collected at the end of CC1 were labeled with Cell Trace Violet before being rechallenged during CC2. (F) Histogram overlay represents data from a representative donor. (G) The experiment in F was repeated with multiple donors and the division indices compiled from the GD2-CAR and GD2-CAR.C7R proliferation histograms. (H) For survival analysis, GD2-CAR and GD2-CAR.C7R T-cells were stained with Annexin V and 7-AAD after 2 serial tumor challenges with LAN-1 tumor cells. Bar graphs show the frequencies of T-cells staining positive for Annexin V, 7-AAD, both, or neither. The Annexin V(+)7-AAD(−) and Annexin V(−)7-AAD(+) mean comparisons were n.s. (I) After the end of CC2, tumors were labeled with GD2-specific antibody and magnetically separated from the CAR T-cells. Total RNA was isolated from T-cells and gene expression analysis was subsequently performed using the Human Immunology Panel Version 2 and nCounter Analysis System (Nanostring). The displayed heat map shows genes with log2 fold changes (GD2-CAR.C7R/GD2-CAR) that had P values less than 0.02. Data was generated from 5 donors (10 paired samples). *P<0.05, **P<0.01, (two-tailed paired t-test, A, B, D, E, G, H). Graphs A, B, C, E, G, H represent averages from different donors ± SEM (n=6, A, D, E; n=3, G, H).