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A functional screening platform for engineering chimeric antigen receptors with reduced on-target, off-tumour activation

Chimeric antigen receptor (CAR) T cell therapies have advanced substantially in the clinic for cancer immunotherapy. However, challenges related to safety persist; one major concern is when CARs respond to antigen present on healthy cells (on-target, off-tumour response). A strategy to ameliorate this consists in engineering the affinity of CARs such that they are only activated by tumor cells expressing high antigen levels. Here, we developed a CAR T cell display platform for functional screening based on cell signaling. Starting with a CAR with high affinity towards its target antigen, we used CRISPR-Cas9 genome editing to generate a library of antigen-binding domain variants. Following multiple rounds of functional screening and deep sequencing-guided selection, CAR variants were identified that were discriminatively activated by tumor cells based on antigen expression levels. Our platform demonstrates how directed evolution based on functional screening can be used to enhance the selectivity and safety of CARs.