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

• DOI: 10.3389/fimmu.2022.864868
• Publication Date: 2022-07-22T11:17:01Z
• Authors: Scott McComb; Tina Nguyen; Alex Shepherd; Kevin A. Henry; Darin Bloemberg; Anne Marcil; Susanne Maclean; Ahmed Zafer; Rénald Gilbert; Christine Gadoury; Robert A. Pon; Traian Sulea; Qin Zhu; Risini D. Weeratna
• Keywords: Jurkat cells

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