Figure 6: The CD28 hinge-transmembrane region enhances activity in a variety of tumor models and CAR architectures.

(a) Schema of first generation CD19 CARs with either a CD8 or CD28 hinge-transmembrane region (CD19-CD8H/T-ζ and CD19-CD28H/T-ζ). (b) NALM6 clones expressing either 963 or 45,851 molecules of surface CD19 were cocultured at a 1:1 ratio with either CD19-CD28ζ, CD19–4-1BBζ, CD19-CD28H/T-ζ or CD19-CD8H/T-ζ CAR T cells and tumor cell killing was measured in an Incucyte assay. Representative of three experiments with different T cell donors. Statistical analysis performed with repeated measures ANOVA between CD19-CD28H/T-ζ and CD19-CD8H/T-ζ. (c) CD19-CD28ζ, CD19–4-1BBζ, CD19-CD28H/T-ζ, and CD19-CD8H/T-ζ CAR T cells were cocultured with NALM6 clones expressing various amounts of CD19 for 24 hours and secreted IL-2 was measured in the supernatant by ELISA. Representative of three experiments with different T cell donors. Statistical comparisons performed with the student’s t-test (two sided) between CD19-CD28H/T-ζ and CD19-CD8H/T-ζ. (d) Schema of a Her2 CAR containing a CD28 hinge-transmembrane region and 4–1BB costimulatory domain (Her2-CD28H/T-4–1BBζ). (e) One million 143b osteosarcoma cells were orthotopically implanted in the hind leg of NSG mice. After seven days, mice were treated with 10 million Her2–4-1BBζ CAR T cells, Her2-CD28H/T-4–1BBζ CAR T cells, or untransduced control T cells (MOCK). Leg measurements were obtained twice weekly with digital calibers. Measurements for individual mice are shown. Statistical analysis performed with repeated measures ANOVA. (f) Survival curves for mice treated as in (e). Statistical analysis performed with the log-rank test. (e-f) are representative of two experiments with different T cell donors (n=5 mice per group). (g) Schema of a B7-H3 CAR containing a CD28 hinge-transmembrane region and 4–1BB costimulatory domain (B7-H3-CD28H/T-4–1BBζ). (h) One million CHLA255 neuroblastoma cells were engrafted into NSG mice by tail vein injection in a metastatic neuroblastoma model. Six days later, mice were injected with 10 million B7-H3–4-1BBζ CAR T cells, B7-H3-CD28H/T-4–1BBζ CAR T cells, or untransduced control T cells (MOCK). Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Representative bioluminescent images are shown. (i) Quantified tumor flux values for individual mice treated as in (h). Statistical analysis performed with repeated measures ANOVA. (j) Survival curves for mice treated as in (h). Statistical analysis performed with the log-rank test. (h-j) are representative of two experiments with different T cell donors. For in vitro experiments, error bars represent SD and for in vivo experiments, error bars represent SEM. p < 0.05 was considered statistically significant, and p values are denoted with asterisks as follows: p > 0.05, not significant, NS; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.