Abstract 584: Driving anti-tumor activity in solid tumors with controlled arming of allogeneic CAR-NK cells
Authors: Gonzalez Alba , Hung Michelle , Guzman Marcela , Sotelo Aldo , Frankel Nicholas , Chong Yin Yin , Kaveri Deepika , Ramkumar Poornima , Leitner Elizabeth , Wong Priscilla , Ponek Ronni , Lee Kelly , Mullenix Alyssa L. , Gordley Russell M. , Lee Gary 
Published: June 2022

Abstract
Recent successes with cell therapies, including CAR-T and CAR-NK cells, for hematopoietic cancers have yet to translate into solid tumors. The major obstacle solid tumors present for cell therapy is the highly immunosuppressive tumor microenvironment (TME), which can impair the function of endogenous as well as therapeutic immune cells. To overcome this challenge, Senti Bio is developing gene circuits to arm CAR-NK cells with multiple cytokines to improve the cytotoxicity and persistence of CAR-NK product candidates. Cytokines, as well as other immune effectors, can act in autocrine or paracrine manner, benefiting the CAR-NK cells as well as potentially engaging the local immune system in the TME. Secreted cytokines can be widely distributed and stimulate other immune cells in the TME, however this is often associated with systemic toxicity as well as reduced impact on the CAR-NK cells. On the other hand, membrane-bound cytokines result in a localized and potent stimulation of the CAR-NK cells but have limited impact on the broader TME. Since IL-15 is known to stimulate NK cell survival and function, Senti Bio has created a novel calibrated release (cr) technology to specifically optimize IL-15 signaling in its CAR-NK platform. crIL-15 is tethered to the NK cells via a cleavable linker that allows for IL-15 release upon cleavage by a ubiquitously expressed protease on the cell surface of NK cells. The rate of linker cleavage can be calibrated by engineering the sensitivity of the cleavable linker sequence to the cell surface protease, thereby enabling the tuning of the ratio of membrane tethered versus fully secreted protein. We compared the function of the wildtype IL-15 protein with different variants of crIL-15 that have a faster or slower release. We demonstrated that crIL-15 has a more potent function, stimulating pSTAT5 signaling and driving expansion and survival of NK cells. CAR-NK cells armed with crIL-15 significantly outperformed CAR-NK cells that expressed the wildtype IL-15 protein, had enhanced function, and demonstrated significantly improved cytotoxicity over three rounds of serial killing (p&lt;0.0001). In parallel, we armed CAR-NK cells with gene circuits that encoded various combinations of two or more cytokines. In particular, the combination of IL-15 and IL-21 resulted in a synergistic effect, leading to longer survival in the absence of cytokines compared to NK cells armed with IL-15 alone. Furthermore, CAR-NK cells armed with IL-15 + IL-21 had significantly increased activity and cytotoxicity compared to CAR-NK cells armed with either cytokine alone (up to 4-times increased cytotoxicity, p&lt;0.0001) when co-cultured with tumor cells. Overall, our results suggest the advantage of multi-arming CAR-NK cells with cytokines, using controlled release systems and cytokine combinations, resulting in improved NK cell function in solid tumor models.
Citation Format: Alba Gonzalez, Michelle Hung, Marcela Guzman, Aldo Sotelo, Nicholas Frankel, Yin Yin Chong, Deepika Kaveri, Poornima Ramkumar, Elizabeth Leitner, Priscilla Wong, Ronni Ponek, Kelly Lee, Alyssa L. Mullenix, Russell M. Gordley, Gary Lee. Driving anti-tumor activity in solid tumors with controlled arming of allogeneic CAR-NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 584.