Sequence and structural determinants of efficacious de novo chimeric antigen receptors

Sequence and structural determinants of efficacious de novo chimeric antigen receptors | Arthur Chow and Hoyin Chu https://www.biorxiv.org/content/10.64... Advances in generative protein design using artificial intelligence (AI) have enabled the rapid development of binders against heterogeneous targets, including tumor-associated antigens. Despite extensive biochemical characterization, these novel protein binders have had limited evaluation as agents in candidate therapeutics, including chimeric antigen receptor (CAR) T cells. Here, we synthesize generative protein design workflows to screen 1,589 novel protein binders targeting BCMA, CD19, and CD22 for efficacy in scalable protein-binding and T cell assays. We identify three main challenges that hinder the utility of de novo protein binders as CARs, including tonic signaling, occluded epitope engagement, and off-target activity. We develop computational and experimental heuristics to overcome these limitations, including screens of sequence variants for individual parental structures, that restore on-target CAR activation while mitigating liabilities. Together, our framework accelerates the development of AI-designed proteins for future preclinical therapeutic screening, helping enable a new generation of cellular therapies.