Comprehensive genome editing confers "off-the-shelf" CAR-T cells superior efficacy against solid tumors

Murray, R.; Navarrete, N. R. R.; Desai, K.; Chowdhury, M. R.; Chilakapati, S. R.; Chong, B.; Messana, A.; Sobon, H.; Rocha, J.; Musenge, F.; Camblin, A.; Ciaramella, G.; Sitkovsky, M.; Maldini, C.; Hatfield, S.

doi:10.1101/2023.08.03.551705

bioRxiv · 10.1101/2023.08.03.551705

Comprehensive genome editing confers "off-the-shelf" CAR-T cells superior efficacy against solid tumors

Murray, R.·Navarrete, N. R. R.·Desai, K.·Chowdhury, M. R.·Chilakapati, S. R.·Chong, B.·Messana, A.·Sobon, H.·Rocha, J.·Musenge, F.·Camblin, A.·Ciaramella, G.·Sitkovsky, M.·Maldini, C.·Hatfield, S.

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Abstract

Biochemical and immunological negative regulators converge to inhibit tumor-reactive Chimeric Antigen Receptor T (CAR-T) cells, which may explain clinical failures of CAR-T cell therapies against solid tumors. Here, we developed a multifaceted approach to genetically engineer allogeneic ( off -the-shelf) CAR-T cells resistant to both biochemical (adenosine) and immunological (PD-L1 and TGF-{beta}) inhibitory signaling. We multiplexed an adenine base editor with a CRISPR-Cas12b nuclease to manufacture a CAR-T cell product comprising six gene edits to evade allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E) and resist major biochemical (ADORA2A) and immunological (PDCD1, TGFBR2) immunosuppressive barriers in solid tumors. Combinatorial genetic disruption in CAR-T cells enabled superior anti-tumor efficacy leading to improved tumor elimination and survival in humanized mouse models that recapitulated the suppressive features of a human tumor microenvironment (TME). This novel engineering strategy conferred CAR-T cells resistance to a diverse TME, which may unlock the therapeutic potential of CAR-T cells against solid tumors. One Sentence SummaryMultiplex genome engineered CAR-T cells resistant to allorejection and the convergence of biochemical and immunological negative regulators within the tumor microenvironment exhibit superior efficacy against solid tumors.

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Murray, R., Navarrete, N. R. R., Desai, K., Chowdhury, M. R., Chilakapati, S. R., Chong, B., Messana, A., Sobon, H., Rocha, J., Musenge, F., Camblin, A., Ciaramella, G., Sitkovsky, M., Maldini, C., Hatfield, S.. 2023-08-04. Comprehensive genome editing confers "off-the-shelf" CAR-T cells superior efficacy against solid tumors. https://doi.org/10.1101/2023.08.03.551705

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