Rationally designed modular STAT-activating scaffolds enforce cell-intrinsic transcriptional programs augmenting the anti-tumor potency of CAR T cells

Saxby, C. P.; Ishida, T. K.; Rosser, J. M.; Curtis, B. C.; Baldwin, M. L.; Spurrell, C. H.; Giles, J.; Valdez, A.; Johnson, A. J.; Gustafson, J.; Appelbaum, J. S.; Jensen, M. C.

doi:10.1101/2023.12.22.573170

bioRxiv · 10.1101/2023.12.22.573170

Rationally designed modular STAT-activating scaffolds enforce cell-intrinsic transcriptional programs augmenting the anti-tumor potency of CAR T cells

Saxby, C. P.·Ishida, T. K.·Rosser, J. M.·Curtis, B. C.·Baldwin, M. L.·Spurrell, C. H.·Giles, J.·Valdez, A.·Johnson, A. J.·Gustafson, J.·Appelbaum, J. S.·Jensen, M. C.

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Abstract

Chimeric antigen receptor (CAR)-expressing T cells can mediate anti-tumor responses in a variety of preclinical models and clinical settings, however, strategies to enhance anti-tumor potency is the subject of intense investigation. Signals emanating from gamma-c cytokine receptors modulate the transcriptional state of activated T cells impacting proliferation, survival, differentiation, and effector functioning through the STAT family of transcription factors. Design of ligand-independent cell-intrinsic cytokine STAT activation scaffolds is a conceptually attractive strategy to provide CAR T cells with a surrogate for exogenous cytokine support. Here, we designed a series of ligand-autonomous STAT inducer (LASI) scaffolds comprised of an extracellular identification tag, a homodimerizing transmembrane domain, and a membrane proximal IL7R Box1 domain followed by STAT5 and/or STAT3 docking sequences derived from IL7R and IL21R, respectively. We constructed LASI scaffolds having STAT5 (LASI-5), STAT3 (LASI-3), and combined STAT5 and STAT3 (LASI-5+3) docking domains and then interrogated their impact in primary human CD8+ anti-CD19 (4-1BB:zeta) CAR T cells. While LASI-5 expression had limited effects on CAR T cells, LASI-3 transcriptional programming was found to be indispensable to achieving anti-tumor functional enhancement associated with limited terminal differentiation, heightened T cell proliferation in response to antigen, and dampened expression of exhaustion-associated genes. Moreover, CAR T cells supplemented with LASI-3 or 5+3 displayed superior potency against human leukemia tumors in NSG mice. LASI-5+3 mediated the highest magnitude of CAR T cell engraftment in vivo that evolved into a fatal lymphoproliferative syndrome. However, the same efficacy enhancement was achieved with LASI-3 without the lymphoproliferative complication. Our findings provide a rationale for utilization of constitutively expressed LASI-3 to enhance the anti-tumor potency of CAR T cells, the need to regulate the activity of LASI-5+3, and a generalizable scaffold design for studying additional combinations of STAT family transcription factors.

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Saxby, C. P., Ishida, T. K., Rosser, J. M., Curtis, B. C., Baldwin, M. L., Spurrell, C. H., Giles, J., Valdez, A., Johnson, A. J., Gustafson, J., Appelbaum, J. S., Jensen, M. C.. 2023-12-26. Rationally designed modular STAT-activating scaffolds enforce cell-intrinsic transcriptional programs augmenting the anti-tumor potency of CAR T cells. https://doi.org/10.1101/2023.12.22.573170

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