From the authors

Abstract

<h3>Abstract</h3> <h3>Background</h3> Chimeric antigen receptor (CAR) technology has revolutionized the treatment of B-cell malignancies and steady progress is being made towards CAR-immunotherapies for solid tumours. Epidermal growth factor family receptors EGFR or HER2 are commonly overexpressed in cancer and represent proven targets for CAR-T therapy; given their expression in healthy tissues it is imperative that any targeting strategy consider the potential for on-target off-tumour toxicity. <h3>Methods</h3> Herein, we utilize high-throughput CAR screening to identify novel camelid single-domain antibody CARs (sdCARs) with high EGFR-specific CAR-T response. To optimize antigenic sensitivity of this EGFR-sdCAR, we performed progressive N-terminal truncation of the human CD8 hinge domain used as a spacer in many CAR constructs. Hinge truncation resulted in decreased CAR sensitivity to EGFR and improved selectivity for EGFR-overexpressing cells over EGFR-low target cells or healthy donor derived EGFR-positive fibroblasts. To investigate the molecular mechanism of hinge truncation, we test hinge-truncated scFv-based CARs targeting membrane proximal or membrane distal domains of EGFR-family proteins, HER2 and EGFRvIII. Finally, we proceed to test hinge variant EGFR-sdCAR functionality through <i>in vitro</i> and <i>in vivo</i> assessments in primary T cells derived from multiple donors. <h3>Results</h3> For CARs targeting membrane-proximal epitopes, hinge truncation by even a single amino acid provided fine control of the antigenic sensitivity, whereas CARs targeting membrane distal domains were not sensitive to even complete hinge domain removal. Hinge-modified EGFR-sdCARs showed consistent and predictable responses in Jurkat-CAR cells and primary human CAR-T cells <i>in vitro</i> and <i>in vivo</i>. <h3>Conclusions</h3> Overall, these results indicate that membrane-proximal epitope targeting CARs can be modified through hinge length tuning for programmable antigenic sensitivity and improved tumour selectivity. Single amino acid truncations of CD8-hinge domain provide precise control of CAR antigen sensitivity Truncated hinge CARs show enhanced selectivity for antigen overexpressing tumour cells and decreased activity towards healthy antigen-expressing cells Epitope location is a critical factor in determining hinge sensitivity for a CAR Hinge tuning can modulate CAR-T antigenic sensivity <i>in vitro</i> and <i>in vivo</i>