Figure 5.

Hinge truncation progressively diminishes tumor cell killing and expansion of primary sdCAR-T cells in response to EGFR expressing target cells. Concentrated lentiviral particles encoding hinge-modified EGFR-specific sdAb021 CARs as well as a GFP marker were generated. Peripheral blood T cells were isolated from 3 independent healthy human donors before polyclonal expansion and lentiviral transduction. Varying doses of sdCAR-T cells or mock transduced cells (empty CAR backbone lentivirus) were placed at an E:T ratio of 1:1 in low density co-culture (2000 sdCAR-T cells and 2000 target cells). Co-cultures were examined over 7 days via live fluorescence microscopy (Incucyte) to differentiate red-fluorescent (NLS-mKate2) target cell counts or total area of green-fluorescent (NLS-NanoGreen) CAR-T cells. (A, B) Depicts the response to EGFR-high SKOV3 targets, (C, D) depicts the response to EGFR-low MCF7 targets, (E, F) depicts the response to EGFR-high healthy donor human dermal fibroblast cells. Each graph depicts automated cell counts or fluorescent areas from a single independent experiment. (G) Day 5 mean fold change in various target cell growth at varying E:T ratio for hinge-variant EGFR-sdCAR-T cells derived from 3 donors is shown +/- SEM, P values are derived from a two-way ANOVA comparison of response curves for untransduced T cell co-cultures with CAR-T cells expressing hinge-truncated constructs. (H) Similarly the mean EGFR-sdCAR-T fold expansion at day 5 of hinge-variant CAR co-cultures from 3 donors is shown +/-SEM is shown. P values are derived from a two-way ANOVA comparison on 45CD8h-hinge containing constructs with other constructs tested in parallel.