Figure 1.

Identification of EGFR-specific sdAbs with CAR functionality. (A) The structural elements of human EGFR and anti EGFR-sdAb CAR tested in this study are shown. Three EGFR-specific sdAbs were cloned into a modular CAR backbone with 41BB and CD3z signaling domain via golden gate cloning. Jurkat cells were then electroporated with the resulting constructs. Control cells with no plasmid or with a CD19 (FMC63) scFv CAR were also tested here. Jurkat cells (30 000/well) transiently expressing various CAR plasmids as shown were co-cultured with varying doses of (B, C) EGFR-high H292 or SKOV3 cells, (D) EGFR-low MCF7 cells, (E, F) EGFR-negative Raji cells or Ramos Cells and examined for activation via staining with APC-labelled anti-human CD69 antibody. CAR-J results show the mean +/- SEM from a three independent experiments performed in duplicate. Primary human T cells were then transduced with lentivirus encoding the sdAb021-EGFR-sdCAR construct and tested for activity against mKate2-expressing target cells with varying EGFR expression. (G) Automated fluorescent counting was used to examine CAR-T mediated target cell killing and (H) expansion of EGFP-labelled CAR-T cells. Primary CAR-T results show the mean of 3 experiments performed in duplicate. NSG mice were injected with 6x10⁶ H292 human lung cancer cells subcutaneously, then treated with 5x10⁶ sdAb021-BBz CAR-T or untransduced control T cells intravenously (n=5 mice/group). (I) Growth of H292 tumors via regular caliper measurements is shown. (J) Probability of survival throughout the experiment is shown (P values are derived from a Mantel-Cox comparison of treatment groups).