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4806 A Regulated Cytokine Engineering Strategy to Augment in Vitro and In Vivo persistence of Allogeneic iPSC Derived CD8 T-Cells

Program: Oral and Poster Abstracts
Session: 702. CAR-T Cell Therapies: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Immunology, Biological Processes, Technology and Procedures, Gene editing
Monday, December 9, 2024, 6:00 PM-8:00 PM

Siddarth Chandrasekaran1*, Justin Yoo2*, Dhaarini Murugan1*, Laura Prochazka3*, Elisa Martinez3*, Michael Hunter Cadell1*, Muluken S Belew3*, Amanda AuYeung3*, Chris Saxby1*, Omar Subedar3*, Zahra Sahaf3*, Dylan Lee3*, Sommer Apelu1*, Libin Abraham1*, Valerie Wall1*, Chloe Hurling3*, Vaisakh Rajan3*, Shruti Sharma3*, Charlie Liu3*, Richard Carpenedo3*, Shri Joshi3*, Elizabeth Csaszar3*, Emily Titus3* and Chris Bond1

1Notch Therapeutics, Seattle
2Notch Therapeutics, Everett, WA
3Notch Therapeutics, Toronto, Canada

Large-scale clinical production of induced pluripotent stem cell (iPSC)-derived CAR-T cells represents a remarkable opportunity in allogeneic cell therapies. Despite the growing advantages offered by iPSCs such as multiplex gene editing and unlimited starting material, most iPSC derived CAR-T cells demonstrate limited clinical proliferation and persistence. This has been partially attributed to existing iPSC differentiation protocols that yield only cytotoxic CD8 T-cells, whereas conventional autologous and donor-derived CAR-T therapies contain a mixture of CD8 T-cells and supportive CD4 T-cells. It has been well established that cytokine signaling via the JAK/STAT pathway plays an important role in promoting CAR-T cell function. Herein we describe novel engineered receptors that can induce JAK/STAT signaling in iPSC derived CD8 T-cells. We demonstrate that iPSC derived CD8 T-cells have a unique JAK/STAT signaling requirement for long-term cytotoxicity and proliferation in our in vitro assays. We show that our engineered receptors can induce JAK/STAT signaling in the absence of exogenous cytokines in culture media. When fully differentiated CD8 T-cells are transduced with engineered cytokine receptors, they show long-term persistence in the absence of exogenous cytokines in vitro. We observed that premature activation of JAK/STAT signaling pathways can skew cells toward a myeloid or innate lymphoid cell lineage. To circumvent this, we designed iPSC clones with an engineered cytokine receptor integrated into a specific locus under the control of context-specific regulatory sequences. These designs exhibited a stringent off-state during differentiation and CAR-dependent transcriptional responsiveness when exposed to target antigen. This data reveals that a regulated strategy can control cytokine receptor expression during differentiation, to preserve phenotype and improve efficacy of differentiated cells. Further, constitutive activation of JAK/STAT signaling may drive adverse side-effects, and we demonstrate that our regulated designs show complete lack of activity in the absence of antigen stimulation. Finally, we evaluated iPSC derived CD8 T-cells harboring the regulated cytokine engineering strategy in vivo. Engineered iPSC derived CD8 T-cells showed tumor growth inhibition and persistence in the absence of exogenous cytokines at comparable levels to unmodified iPSC derived CD8 T-cells receiving exogenous cytokine support. Engineering of appropriate cytokine support and strategies to regulate expression during differentiation is an important first step to enhance the persistence of iPSC derived CD8 T-cells. One of the major advantages of using iPSC derived CD8 T-cells is their potential for drug readministration without toxic lymphodepletion and exogenous cytokine support. We believe that cell-intrinsic cytokine support is critical to develop durable and safe allogeneic T-cell therapies for patients.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH