Background Chimeric antigen receptor (CAR) cell therapy for solid tumors is hampered by the immunosuppressive tumor microenvironment (TME), which can inhibit the function of endogenous and therapeutic immune cells, as well as a paucity of targets. The use of potent immunomodulators to transform the TME, such as interleukin (IL)-12, is limited by the need for regulation to avoid systemic toxicities. Methods To address these challenges, Senti Bio is developing CAR-NK therapies that include Multi-Arming with calibrated release (cr)IL-15 and a Regulator Dial gene circuit to control the expression of crIL-12. Senti's proprietary calibrated release technology enables cytokines to be expressed in both membrane-bound and secreted forms, providing multi-factorial activity via autocrine and paracrine stimulation to increase CAR-NK cell function and activation of endogenous immune cells in the TME. In addition, we have designed a Regulator Dial gene circuit that expresses crIL-12 under the control of grazoprevir (GRZ), an FDA-approved small molecule drug. ResultsWe have tested our Multi-Arming and Regulator Dial gene circuits in combination with a CAR targeting GPC3, a hepatocellular carcinoma (HCC)-relevant target. We observed crIL-15 to promote NK cell persistence and proliferation in an autocrine fashion, while also activating other immune cells in a paracrine manner. In addition, crIL-15 was observed to enhance GPC3 CAR-NK tumor killing in a serial killing assay compared to wild-type secreted IL-15 and showed enhanced antitumor function and increased survival over control groups in vivo.GPC3 CAR-NK cells containing the Regulator Dial gene circuit expressed low crIL-12 levels in the absence of GRZ (<100 pg/1e6 cells/48h), while induction with GRZ led to a 1000-fold increase of crIL-12 expression under the same conditions. The ON and OFF kinetics of crIL-12 induction were determined in vitro. 4 hours of GRZ-treatment was sufficient to induce >600-fold increase in crIL-12 concentrations. By day 3 post-GRZ removal, crIL-12 reverted to basal levels. Lastly, the role of crIL-12 in reversing immunosuppression was validated in a co-culture assay. Specifically, GRZ-induced crIL12 restored CAR-NK cells that were suppressed in the presence of M2 macrophages. Conclusions We have engineered off-the-shelf CAR-NK cell therapies that target GPC3 and express crIL-15. To increase the therapeutic window of the potent immune effector IL-12 in the TME, we have designed a Regulator Dial gene circuit to controllably produce crIL-12. These gene circuits have complementary mechanisms of action to enhance CAR-NK antitumor function and potentially overcome the immunosuppressive TME in solid tumors such as HCC.

Purpose: SENTI-301A is a novel off-the-shelf CAR-NK product candidate that uses gene circuits to enhance NK cell persistence and targeted cytotoxicity to address unmet needs in GPC3 expressing solid tumors such as hepatocellular carcinomas (HCC). Experimental procedures: SENTI-301A is a Multi-Armed, targeted chimeric antigen receptor (CAR) NK cell preclinical product candidate with a GPC3 CAR and calibrated-release interleukin-15 (crIL-15) engineered onto allogeneic healthy adult peripheral blood NK cells. The activating CAR is designed to target GPC3, an antigen overexpressed in several cancers, such as HCC. crIL-15 is a unique technology attaching wild type IL-15 to the membrane via a linker that can be cleaved by a ubiquitous protease. This results in membrane-bound and secreted IL-15 to provide both autocrine support to the CAR NK cells, and paracrine stimulation to other surrounding immune cells in the tumor microenvironment for enhanced antitumor response. Results: SENTI-301A exhibited increased crIL-15-driven survival and expansion (&gt;30 days) without exogenous cytokine support compared to unengineered NK cells (~6 days). SENTI-301A also showed significantly higher in vitro cytotoxic activity (~20%) against GPC3 expressing cell lines compared to isogenic cells lacking the antigen, indicating CAR-mediated activation and antigen specificity. SENTI-301A also showed significant tumor killing function, IFNg, and Granzyme-B production, when co-cultured with HCC and other GPC3 expressing tumor cell lines at different effector:target ratios. SENTI-301A maintained the antitumor function in the presence of full-length soluble GPC3, reducing the risk of competition from shedding GPC3 with surface GPC3 for CAR-NK cell binding. In HCC mouse xenograft models, SENTI-301A showed NK cells tumor infiltration, and displayed enhanced persistence, antitumor function and increased median survival (p&lt; 0.01), in comparison to unengineered NK cells. As next steps, the potential positive impact of immune checkpoint inhibitors (CPIs) in combination with SENTI-301A to enhance anticancer function is being assessed. Conclusions: SENTI-301A is a novel off-the-shelf preclinical CAR-NK cell therapy candidate that targets GPC3-expressing tumors in an antigen-specific manner and exhibits increased persistence via crIL-15. We are exploring the potential synergy between SENTI-301A and CPIs to enhance the existing antitumor functions of SENTI-301A. SENTI-301A is planned for clinical development with an investigational new drug application (IND) expected in 2023. Citation Format: Marcela Guzman Ayala, Deepika Kaveri, Enping Hong, Elizabeth Leitner, Priscilla Wong, Ronni Ponek, Lawrence Naitmazi, Pearley Chinta, Wesley Gorman, Mengxi Tian, Niran Almudhfar, Kelly Lee, Nicholas Frankel, Alba Gonzalez Junca, Russell Gordley, Philip Lee, Timothy Lu, Kanya Rajangam. SENTI-301A, an off-the-shelf multi-armed preclinical CAR-NK cell therapy, for the treatment of GPC3 expressing tumors [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 2905.