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speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing

Author

Listed:
  • Rocío Castellanos-Rueda

    (ETH Zürich
    University of Zurich)

  • Raphaël B. Roberto

    (ETH Zürich)

  • Florian Bieberich

    (ETH Zürich
    University of Zurich)

  • Fabrice S. Schlatter

    (ETH Zürich)

  • Darya Palianina

    (University of Basel)

  • Oanh T. P. Nguyen

    (ETH Zürich)

  • Edo Kapetanovic

    (ETH Zürich)

  • Heinz Läubli

    (University of Basel)

  • Andreas Hierlemann

    (ETH Zürich)

  • Nina Khanna

    (University of Basel)

  • Sai T. Reddy

    (ETH Zürich)

Abstract

Chimeric antigen receptors (CARs) consist of an antigen-binding region fused to intracellular signaling domains, enabling customized T cell responses against targets. Despite their major role in T cell activation, effector function and persistence, only a small set of immune signaling domains have been explored. Here we present speedingCARs, an integrated method for engineering CAR T cells via signaling domain shuffling and pooled functional screening. Leveraging the inherent modularity of natural signaling domains, we generate a library of 180 unique CAR variants genomically integrated into primary human T cells by CRISPR-Cas9. In vitro tumor cell co-culture, followed by single-cell RNA sequencing (scRNA-seq) and single-cell CAR sequencing (scCAR-seq), enables high-throughput screening for identifying several variants with tumor killing properties and T cell phenotypes markedly different from standard CARs. Mapping of the CAR scRNA-seq data onto that of tumor infiltrating lymphocytes further helps guide the selection of variants. These results thus help expand the CAR signaling domain combination space, and supports speedingCARs as a tool for the engineering of CARs for potential therapeutic development.

Suggested Citation

  • Rocío Castellanos-Rueda & Raphaël B. Roberto & Florian Bieberich & Fabrice S. Schlatter & Darya Palianina & Oanh T. P. Nguyen & Edo Kapetanovic & Heinz Läubli & Andreas Hierlemann & Nina Khanna & Sai , 2022. "speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-34141-8
    DOI: 10.1038/s41467-022-34141-8
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