Programmable Attenuation of Antigenic Sensitivity for a Nanobody-Based EGFR Chimeric Antigen Receptor Through Hinge Domain Truncation

Epidermal growth factor family receptor (EGFR) is commonly overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR)-T therapy, but as EGFR is also expressed at lower levels in healthy tissues a therapeutic strategy must balance antigenic responsiveness against the risk of on-target off-tumor toxicity. Herein, we identify several camelid single-domain antibodies (also known as nanobodies) that are effective EGFR targeting moieties for CARs (EGFR-sdCARs) with very strong reactivity to EGFR-high and EGFR-low target cells. As a strategy to attenuate their potent antigenic sensitivity, we performed progressive truncation of the human CD8 hinge commonly used as a spacer domain in many CAR constructs. Single amino acid hinge-domain truncation progressively decreased both EGFR-sdCAR-Jurkat cell binding to EGFR-expressing targets and expression of the CD69 activation marker. Attenuated signaling in hinge-truncated EGFR-sdCAR constructs increased selectivity for antigen-dense EGFR-overexpressing cells over an EGFR-low tumor cell line or healthy donor derived EGFR-positive fibroblasts. We also provide evidence that epitope location is critical for determining hinge-domain requirement for CARs, as hinge truncation similarly decreased antigenic sensitivity of a membrane-proximal epitope targeting HER2-CAR but not a membrane-distal EGFRvIII-specific CAR. Hinge-modified EGFR-sdCAR cells showed clear functional attenuation in Jurkat-CAR-T cells and primary human CAR-T cells from multiple donors in vitro and in vivo. Overall, these results indicate that hinge length tuning provides a programmable strategy for throttling antigenic sensitivity in CARs targeting membrane-proximal epitopes, and could be employed for CAR-optimization and improved tumor selectivity.