Antigen-independent activation is critical for the durable antitumor effect of GUCY2C-targeted CAR-T cells

Abstract

Background Chimeric antigen receptor (CAR)-T cells face many obstacles in solid tumor therapy, including heterogeneous antigen expression and inefficient T cell persistence. Guanylyl cyclase C (GUCY2C) has been identified as a suitable tumor antigen for targeted therapy due to its intestinal-restricted expression pattern in normal tissues and steady overexpression in gastrointestinal tumors, especially colorectal cancer. An antigen-sensitive and long-lasting CAR-T cell targeting GUCY2C was investigated in this study.Methods Using constructed tumor cell lines with various GUCY2C expression densities, we screened out an antigen-sensitive single chain variable fragment (scFv) that enabled CAR-T cells to efficiently eradicate the GUCY2C lowly expressed tumor cells. CAR-T cells with different compositions of the hinge, transmembrane and costimulatory domains were also constructed for selection of the long-lasting CAR-T format with durable antitumor efficacy in vitro and in tumor-bearing mice. The underlying mechanism was further investigated based on mutation of the hinge and transmembrane domains.Results We found that the composition of the antigen-sensitive scFv, CD8α hinge, CD8α transmembrane, and CD28 costimulatory domains boosted CAR-T cells to rapidly kill tumors, maintain high expansion capacity, and long-term efficacy in various colorectal cancer models. The durable antitumor function was attributed to the optimal CAR tonic signaling that conferred CAR-T cells with autonomous activation, proliferation, survival and cytokine release in the absence of antigen stimulation. The tonic signaling was associated with the length and the cysteine residues in the CD8α hinge and transmembrane domains.Conclusions This study demonstrated a potent GUCY2C-targeted CAR-T cell for gastrointestinal tumor therapy and highlights the importance of adequate tonic signaling for effective CAR-T cell therapy against solid tumors.