RT Journal Article
SR Electronic
T1 Antigenic Sensitivity of Membrane-Proximal Targeting Chimeric Antigen Receptors can be Fine-Tuned through Hinge Truncation
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.10.30.360925
DO 10.1101/2020.10.30.360925
A1 Scott McComb
A1 Tina Nguyen
A1 Alex Shepherd
A1 Kevin A. Henry
A1 Darin Bloemberg
A1 Anne Marcil
A1 Susanne Maclean
A1 Rénald Gilbert
A1 Christine Gadoury
A1 Rob Pon
A1 Traian Sulea
A1 Qin Zhu
A1 Risini D. Weeratna
YR 2021
UL http://biorxiv.org/content/early/2021/05/03/2020.10.30.360925.abstract
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.