Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells

Ma, L.; Ramasubramanian, R.; Mehta, N.; Cossette, B.; Morgan, D.; Sukaj, I.; Bergaggio, E.; Kadauke, S.; Myers, R.; Paruzzo, L.; Ghilardi, G.; Grzywa, T.; Cozzone, A.; Schuster, S.; Frey, N.; Zhang, L.; Yousefpour, P.; Abraham, W.; Suh, H.; Ruella, M.; Grupp, S.; Chiarle, R.; Wittrup, K. D.; Irvine, D. J.

doi:10.1101/2024.04.16.589780

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bioRxiv · 10.1101/2024.04.16.589780

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells

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Abstract

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR (FMC63) as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion and memory development of CD19 CAR-T cells in both syngeneic and humanized mouse models of B-ALL/Lymphoma, and enhanced control of disease progression. This approach thus enables vaccine boosting to be applied to any clinically relevant CAR-T cell product.

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Ma, L., Ramasubramanian, R., Mehta, N., Cossette, B., Morgan, D., Sukaj, I., Bergaggio, E., Kadauke, S., Myers, R., Paruzzo, L., Ghilardi, G., Grzywa, T., Cozzone, A., Schuster, S., Frey, N., Zhang, L., Yousefpour, P., Abraham, W., Suh, H., Ruella, M., Grupp, S., Chiarle, R., Wittrup, K. D., Irvine, D. J.. 2024-04-17. Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells. https://doi.org/10.1101/2024.04.16.589780

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