Key Points• Wnt activation, Pten loss, andMyc translocation synergize to define a novel subset of murine Notch-independent T-ALL.Wnt signaling is important for T-cell differentiation at the early CD4 2 CD8 2 stage and is subsequently downregulated with maturation. To assess the importance of this downregulation, we generated a mouse line (R26-bcat) in which high levels of active b-catenin are maintained throughout T-cell development. Young R26-bcat mice show a differentiation block at the CD4 1 CD8 1 double-positive (DP) stage. These DP cells exhibit impaired apoptosis upon irradiation or dexamethasone treatment. All R26-bcat mice develop T-cell leukemias at 5 to 6 months of age. R26-bcat leukemias remain dependent on b-catenin function but lack Notch pathway activation. They exhibit recurrent secondary genomic rearrangements that lead to Myc overexpression and loss of Pten activity. Because b-catenin activation and Myc translocations were previously found in murine T-cell acute lymphoblastic leukemias (T-ALLs) deficient for Pten, our results suggest that activation of the canonical Wnt pathway is associated with a subtype of Notch-independent T-ALLs that bear Myc gene rearrangements and Pten mutations. (Blood. 2013;122(5):694-704)

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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.

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 (>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< 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.