Enhanced functionality of low-affinity CD19 CAR T-cells is associated with activation priming and a polyfunctional cytokine phenotype

Michelozzi, I. M.; Gomez-Castaneda, E.; Pohle, R. V. C.; Cardoso-Rodriguez, F.; Sufi, J.; Puigdevall-Costa, P.; Subraniyam, M.; Wu, S. W.; Guvenel, A.; Ghorashian, S.; Pule, M. A.; Tape, C. J.; Castellano, S.; Amrolia, P. J.; Giustacchini, A.

doi:10.1101/2020.09.22.291831

bioRxiv · 10.1101/2020.09.22.291831

Enhanced functionality of low-affinity CD19 CAR T-cells is associated with activation priming and a polyfunctional cytokine phenotype

Michelozzi, I. M.·Gomez-Castaneda, E.·Pohle, R. V. C.·Cardoso-Rodriguez, F.·Sufi, J.·Puigdevall-Costa, P.·Subraniyam, M.·Wu, S. W.·Guvenel, A.·Ghorashian, S.·Pule, M. A.·Tape, C. J.·Castellano, S.·Amrolia, P. J.·Giustacchini, A.

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Abstract

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR) CAT that showed enhanced expansion, cytotoxicity, and anti-tumour efficacy compared to the high-affinity (FMC63 based) CAR used in Tisagenlecleucel, in pre-clinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a Phase I clinical study. To understand the molecular mechanisms behind these properties of CAT CAR T-cells, we performed a systematic in vitro characterization of the transcriptomic (RNA-seq) and protein (CyTOF) changes occurring in T-cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T-cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared to FMC63 CAR T-cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T-cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B-cells present in the manufacture.

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Michelozzi, I. M., Gomez-Castaneda, E., Pohle, R. V. C., Cardoso-Rodriguez, F., Sufi, J., Puigdevall-Costa, P., Subraniyam, M., Wu, S. W., Guvenel, A., Ghorashian, S., Pule, M. A., Tape, C. J., Castellano, S., Amrolia, P. J., Giustacchini, A.. 2020-09-23. Enhanced functionality of low-affinity CD19 CAR T-cells is associated with activation priming and a polyfunctional cytokine phenotype. https://doi.org/10.1101/2020.09.22.291831

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