Chimeric antigen receptors (CARs) are synthetic protein receptors engineered for precise molecular recognition of cell surface antigens, enabling customized T cell responses. This paper presents speedingCARs, an integrated method for engineering CAR T cells using signaling domain shuffling and pooled functional screening. A library of 180 unique CAR variants is genomically integrated into primary human T cells via CRISPR-Cas9. In vitro tumor cell co-culture, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), identifies variants with potent tumor-killing properties and diverse T cell phenotypes. Mapping scRNA-seq data onto tumor-infiltrating lymphocyte data guides variant selection, expanding the CAR signaling domain combination space and establishing speedingCARs as a tool for CAR engineering.

Rocío Castellanos-Rueda, Raphaël B. Di Roberto, Florian Bieberich, Fabrice S. Schlatter, Darya Palianina, Oanh T. P. Nguyen, Edo Kapetanovic, Heinz Läubli, Andreas Hierlemann, Nina Khanna, Sai T. Reddy