Abstract 2902: Off-the-Shelf CAR-NK cells engineered to express calibrated release IL-15 exhibit enhanced persistence and anti-tumor activities
Authors: Lee Chen-Ting , Hung Michelle , Banicki Andrew , Song Wenqi , Almudhfar Niran , Kaveri Deepika , Wong Priscilla , Naitmazi Lawrence , Guzman Marcela , Lam Alice , Yucel Gozde , Lu Timothy , Junca Alba G. , Garrison Brian , Lee Philip 
Published: June 2023

Abstract
Chimeric antigen receptor (CAR) natural killer (NK) cell therapy is an attractive immunotherapy strategy due to potential for increased anti-tumor activity and favorable safety. Allogeneic donor derived NK cells have shown promising clinical outcomes in both hematological and solid tumor malignancies but have a short lifespan in the absence of cytokine support. Interleukin (IL)-15 is a pleiotropic cytokine that promotes the survival, proliferation, and cytotoxicity of NK cells. In this study, we engineered human peripheral blood-derived NK cells to co-express a CAR and calibrated release (cr) IL-15 and studied how crIL-15 affected CAR-NK cell functions. Senti Bio has designed the calibrated release technology to simultaneously produce membrane bound and secreted cytokines, such as IL-15 and IL-12, to provide autocrine and paracrine activity. We further validated crIL-15 activity in vitro and demonstrated enhanced NK cell persistence, tumor killing and autocrine activity in CAR NK cells as well as paracrine stimulation of neighboring T cells and NK cells via pSTAT5 activation. Additionally, we examined in vivo persistence and biodistribution of NK cells engineered with crIL-15 and a nanoluc reporter. crIL-15 NK cells were intravenously (i.v.) injected into NSG mice. These NK cells were detected in the lung, femur, spleen, liver and stomach 1 day post injection by bioluminescence imaging and polymerase chain reaction (PCR) and lasted up to 20 days in the lung. In addition, crIL-15 increased CAR-NK cells persistence in a dose-dependent manner compared to unengineered NK cells. Injection of higher CAR-NK cell numbers (15× 106 vs 7.5× 106 CAR+ cells per mouse) or multiple NK dosages (1 vs 3 doses) both contributed to prolonged persistence. Finally, we studied how crIL-15 affected CAR-NK cell anti-tumor function in acute myeloid leukemia (AML) xenograft models. MV4-11-Fluc AML tumor cells were co-injected with engineered NK cells expressing a FLT3 and/or CD33 bivalent activating CAR, a EMCN inhibitory CAR and crIL-15 into NSG-Tg (Hu-IL15) mice via i.v. injection. Our data showed that these CAR-NK cells significantly reduced MV4-11 tumor burden and prolonged mouse survival compared to unengineered NK cells. The improved anti-tumor functions correlated with IL-15 levels produced by the CAR-NK cells. In conclusion, our results demonstrated that crIL-15 can improve persistence and anti-tumor activity of CAR-NK cells.
Citation Format: Chen-Ting Lee, Michelle Hung, Andrew Banicki, Wenqi Song, Niran Almudhfar, Deepika Kaveri, Priscilla Wong, Lawrence Naitmazi, Marcela Guzman, Alice Lam, Gozde Yucel, Timothy Lu, Alba G. Junca, Brian Garrison, Philip Lee. Off-the-Shelf CAR-NK cells engineered to express calibrated release IL-15 exhibit enhanced persistence and anti-tumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2902.