Figure 5.

CER-1236 mediates cytotoxicity, cytokine secretion, and proliferation against EGFR mutation-positive NSCLC HCC827 cells, augmented by osimertinib, an EGFR inhibitor

(A) Osimertinib treatment led to dose-dependent inhibition of HCC827 target growth expansion and increase in exposure of PS (CER-T ligand) compared with medium control. PS exposure was measured by IncuCyte real-time quantitative microscopic detection of binding of recombinant human TIM-4 ECD to externalized PS. STS included as a positive control for maximal PS induction. Average object count ± SEM shown for two technical duplicates from one representative experiment (n = 3). Statistics were analyzed using a two-way ANOVA with Dunnett’s multiple comparisons test at each time point. Results for 141 h are reported. ∗∗∗∗p < 0.0001. (B) Representative IncuCyte image overlays of phase/contrast and red channel taken at 120 h post co-culture demonstrating HCC827 target abundance or lack thereof from co-cultures. (C and D) CER-1236 co-cultures demonstrates enhanced tumor elimination in combination with osimertinib treatment. Statistics were analyzed using a two-way ANOVA with Dunnett’s multiple comparisons test at each time point and results for 112 h are reported. ∗∗∗∗p < 0.0001. CER-1236 were co-cultured with HCC827 targets at an E:T ratio of 1:1, 1:2, and 1:4 for 120 h. Vehicle-treated HCC827 targets and untransduced T cells were included as controls. Percent cytotoxicity is reported as percent change in HCC827 average target cell count from T cell co-cultures at 120 h relative to HCC827 target alone average count for the respective control or osimertinib concentrations. Percent change in average object count shown (n = 3 donors). Statistics were analyzed using a two-way ANOVA with Dunnett’s multiple comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001. (E) CER-1236 T cells secrete effector cytokines in response to HCC827 target cells. Co-culture with osimertinib-treated HCC827 targets led to further enhancement of secretion of IFN-γ, TNF-α, and granzyme B compared with untreated targets. CER-1236 were co-cultured with HCC827 targets at a 1:4 E:T ratio for 120 h. Vehicle-treated HCC827 targets and untransduced T cells were included as controls. After co-culture, cytokine secretion was measured by automated ELISA. Average cytokine secretion ± SEM is shown (n = 3 donors). Statistics were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (F) Enhanced proliferation of CER-1236 T cells co-cultured with osimertinib-treated targets compared with untreated target co-cultures of CER-1236 and untransduced T cell co-cultures. Proliferation was measured by flow cytometry using precision count beads to determine the absolute counts of viable T cells at the end of the 120-h co-culture. T cell proliferation is reported as viable T cell count determined. Average viable T cell count ± SEM shown (n = 3 donors). Statistics were analyzed using a two-way ANOVA with Dunnett’s multiple comparisons test. ∗p < 0.05, ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001. Donors designated as follows: (▲) donor 1, (▪) donor 2, (◆) donor 3. PS, phosphatidylserine; ECD, extracellular domain; STS, staurosporine; CER, chimeric engulfment receptor; IFN-γ, interferon gamma; TNF-α, tumor necrosis factor alpha; SEM, standard error of the mean; E:T, effector to target ratio; NLR, nuclear localized red.