Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy

Bridge, J.; Johnson, M. J.; Kim, J.; Wenthe, S.; Krueger, J.; Wick, B.; Kluesner, M.; Crane, A. T.; Bell, J.; Skeate, J. G.; Moriarity, B. S.; Webber, B. R.

doi:10.1101/2024.09.03.611042

bioRxiv · 10.1101/2024.09.03.611042

Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy

Bridge, J.·Johnson, M. J.·Kim, J.·Wenthe, S.·Krueger, J.·Wick, B.·Kluesner, M.·Crane, A. T.·Bell, J.·Skeate, J. G.·Moriarity, B. S.·Webber, B. R.

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Abstract

Gamma delta ({gamma}{delta}) T cells are defined by their unique ability to recognize a limited repertoire of non-peptide, non-MHC-associated antigens on transformed and pathogen-infected cells. In addition to their lack of alloreactivity, {gamma}{delta} T cells exhibit properties distinct from other lymphocyte subsets, prompting significant interest in their development as an off-the-shelf cellular immunotherapeutic. However, their low abundance in circulation, heterogeneity, limited methods for ex vivo expansion, and under-developed methodologies for genetic modification have hindered basic study and clinical application of {gamma}{delta} T cells. Here, we implement a feeder-free, scalable approach for ex vivo manufacture of polyclonal, non-virally modified, gene edited chimeric antigen receptor (CAR)-{gamma}{delta} T cells in support of therapeutic application. Engineered CAR-{gamma}{delta} T cells demonstrate high function in vitro and and in vivo. Longitudinal in vivo pharmacokinetic profiling of adoptively transferred polyclonal CAR-{gamma}{delta} T cells uncover subset-specific responses to IL-15 cytokine armoring and multiplex base editing. Our results present a robust platform for genetic modification of polyclonal CAR-{gamma}{delta} T cells and present unique opportunities to further define synergy and the contribution of discrete, engineered CAR-{gamma}{delta} T cell subsets to therapeutic efficacy in vivo.

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Bridge, J., Johnson, M. J., Kim, J., Wenthe, S., Krueger, J., Wick, B., Kluesner, M., Crane, A. T., Bell, J., Skeate, J. G., Moriarity, B. S., Webber, B. R.. 2024-09-07. Efficient multiplex non-viral engineering and expansion of polyclonal γδ CAR-T cells for immunotherapy. https://doi.org/10.1101/2024.09.03.611042

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