PT  - JOURNAL ARTICLE
AU  - Scott McComb
AU  - Tina Nguyen
AU  - Alex Shepherd
AU  - Kevin A. Henry
AU  - Darin Bloemberg
AU  - Anne Marcil
AU  - Susanne Maclean
AU  - Rénald Gilbert
AU  - Christine Gadoury
AU  - Rob Pon
AU  - Traian Sulea
AU  - Qin Zhu
AU  - Risini D. Weeratna
TI  - Antigenic Sensitivity of Membrane-Proximal Targeting Chimeric Antigen Receptors can be Fine-Tuned through Hinge Truncation
AID  - 10.1101/2020.10.30.360925
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2020.10.30.360925
4099  - http://biorxiv.org/content/early/2021/05/03/2020.10.30.360925.short
4100  - http://biorxiv.org/content/early/2021/05/03/2020.10.30.360925.full
AB  - Background 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.Methods 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 in vitro and in vivo assessments in primary T cells derived from multiple donors.Results 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 in vitro and in vivo.Conclusions 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 sensitivityTruncated hinge CARs show enhanced selectivity for antigen overexpressing tumour cells and decreased activity towards healthy antigen-expressing cellsEpitope location is a critical factor in determining hinge sensitivity for a CARHinge tuning can modulate CAR-T antigenic sensivity in vitro and in vivoCompeting Interest StatementThe authors have declared no competing interest.