Abstract
The limited number of targetable tumor-specific antigens and the immunosuppressive nature of the microenvironment within solid malignancies represent major barriers to the success of chimeric antigen receptor (CAR)-T cell therapies. Here, using epithelial cell adhesion molecule (EpCAM) as a model antigen, we used alanine scanning of the complementarity-determining region to fine-tune CAR affinity. This allowed us to identify CARs that could spare primary epithelial cells while still effectively targeting EpCAMhigh tumors. Although affinity-tuned CARs showed suboptimal antitumor activity in vivo, we found that inducible secretion of interleukin-12 (IL-12), under the control of the NFAT promoter, can restore CAR activity to levels close to that of the parental CAR. This strategy was further validated with another affinity-tuned CAR specific for intercellular adhesion molecule-1 (ICAM-1). Only in affinity-tuned CAR-T cells was NFAT activity stringently controlled and restricted to tumors expressing the antigen of interest at high levels. Our study demonstrates the feasibility of specifically gearing CAR-T cells towards recognition of solid tumors by combining inducible IL-12 expression and affinity-tuned CAR.