Figure 1. Combinatorial tumor targeting strategy with a CAR and a CCR results in enhanced cytotoxicity.

(A) Schematic representations of dual targeted CAR+CCR strategies are shown. First generation CARs were combined with the CD38-CCR construct bearing either CD28 (CD38-28) or both CD28 and 4-1BB costimulatory domains (CD38-28BB). Second generation CARs were combined with the CD38-CCR that contains only the 4-1BB signaling domain (CD38-BB). (B) Representative flow cytometry plots illustrating BCMA-CAR/dsRed and CD38-CCR/LNGFR expression of double transduced T cells are shown. BCMA-CAR expression was measured with an F(ab’)₂ Fragment Goat Anti-Mouse antibody. CD38-CCR expression was measured after binding of Protein L. (C) Mean fluorescence intensity (MFI) of BCMA-CAR expression on transduced T cells is shown. MFI values are reported. Mock indicates mock-transduced T cells. (D) Percentage of CAR T cells that have a naïve (CD62L⁺CD45RA⁺), central memory (CD62L⁺CD45RA⁻), or an effector memory (CD62L⁻CD45RA⁻) phenotype are shown (n=4 per group). (E) The absolute number of unstimulated CAR T cells per ml is shown at five days after transduction (n=4 per group). (F) AKT1 phosphorylation (phosho-AKT1) in unstimulated CAR T cells was assessed by flow cytometry. Representative results from one donor are shown. (G) Lysis of luciferase expressing MM1.S (BCMA⁺CD38⁺) cells is shown. Tumor cell killing was measured in a 16 hour bioluminescence (BLI) assay (n=4 per group) at the indicated effector:target (E:T) ratios. (H) Calcein-loaded 3T3 cells transduced to express CD38 were co-cultured at indicated E:T ratios with either double-targeting BCMA-CAR+CD38-CCR T cells or single targeting CD38-28ζ CAR T cells. Tumor cell killing was measured after 4 hours (n=4 per group). Statistical analysis in (G) and (H) was performed by two-way ANOVA and subsequent multiple comparison, corrected by Turkey test. *p<0.05, ****p<0.0001.