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. Author manuscript; available in PMC: 2021 Mar 8.
Published in final edited form as: Cancer Discov. 2020 Mar 19;10(5):702–723. doi: 10.1158/2159-8290.CD-19-0945

Figure 4: Engineering CARs to reduce downstream signaling strength sacrifices CAR efficacy against low antigen density tumors.

Figure 4:

(a) Schema of a CD19-CD28ζ CAR with only a single ITAM (CD19-CD28ζ**). (b) Schema of a CD19–4-1BBζ CAR with only a single ITAM (CD19–4-1BBζ**). (c) NALM6 clones expressing either 963 or 45,851 molecules of surface CD19 were cocultured at a 1:1 ratio with either CD19-CD28ζ or CD19-CD28ζ** 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. (d) NALM6 clones expressing either 2,053 or 45,851 molecules of surface CD19 were cocultured at a 1:1 ratio with either CD19–4-1BBζ or CD19–4-1BBζ** 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. (e) CD19-CD28ζ and CD19-CD28ζ** 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 by the student’s t-test (two sided). (f) CD19–4-1BBζ and CD19–4-1BBζ** 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 by the student’s t-test (two sided). (g) Schema of a CD19 CAR containing the CD8 hinge-transmembrane region and the CD28 and CD3ζ endodomains (CD19-CD8H/T-CD28ζ). (h) NALM6 clones expressing either 963 or 45,851 molecules of surface CD19 were cocultured at a 1:1 ratio with either CD19-CD28ζ or CD19-CD8H/T-CD28ζ 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. (i) CD19-CD28ζ and CD19-CD8H/T-CD28ζ 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 by the student’s t-test (two sided). For all experiments, error bars represent SD. 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.