Human cytomegalovirus (HCMV) has evolved a number of mechanisms for long-term co-existence within its host. HCMV infects a wide range of cell types, including fibroblasts, epithelial cells, monocytes, macrophages, dendritic cells, and myeloid progenitor cells. Lytic infection, with the production of infectious progeny virions, occurs in differentiated cell types, while undifferentiated myeloid precursor cells are the primary site of latent infection. The outcome of HCMV infection depends partly on the cell type and differentiation state but is also influenced by the composition of the immune environment. In this review, we discuss the role of early interactions between HCMV and the host immune system, particularly cytokine and chemokine networks, that facilitate the establishment of lifelong latent infection. A better understanding of these cytokine signaling pathways could lead to novel therapeutic targets that might prevent latency or eradicate latently infected cells.

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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.

CD33 and FLT3 are validated targets for myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). These malignancies have poor prognosis and high clinical unmet need, and current targeted therapies present ample limitations. One challenge is that untargeted leukemic stem cells (LSCs) can contribute to eventual relapse; however, targeting the LSC markers FLT3 and CD33 can contribute to off-tumor toxicities against normal hematopoietic stem cells and progenitor cells (HSCs/HPCs), leading to bone marrow toxicities such as thrombocytopenia, neutropenia, and infectious complications. SENTI-202 is designed to address these challenges and provide a broader therapeutic window. SENTI-202 is a preclinical CAR-NK cell product candidate engineered with an OR and NOT logic gated gene circuit onto allogeneic healthy adult peripheral blood NK cells along with multi-arming via expression of calibrated release IL-15 (crIL-15) to enhance therapeutic activity and tolerability. The OR gate is implemented using a bivalent CAR (aCAR) that binds both CD33 and FLT3 to target both AML blasts and LSCs. Additionally, the NOT gate is implemented using an inhibitory CAR (iCAR) that recognizes the healthy cell protective antigen endomucin (EMCN) and targets endomucin which is designed to prevent CAR-mediated cytotoxicity against healthy cells, such as HSCs and early HPCs, potentially reducing on-target/off-tumor toxicities. Finally, crIL-15 provides NK cell activation and persistence via simultaneous autocrine, juxtracrine, and paracrine activities. Preclinical studies demonstrated the [antitumor] activity and tolerability of SENTI-202. All SENTI-202 components are stably expressed via a single g-retroviral vector. SENTI-202 preclinical pharmacokinetics and secondary pharmacodynamics demonstrated CAR target engagement, function, as well as mechanism of action for all components in different AML models. SENTI-202 CAR-NK cells demonstrated cytotoxic activity when co-cultured with AML target cells and primary patient-derived samples and had increased antitumor capacity compared to non-engineered NK cells. SENTI-202 induced the production of cytotoxic cytokines and increased expression of activation and cytotoxicity markers consistent with CAR-mediated activation of the NK cells. The presence of EMCN iCAR reduced cytotoxicity in an EMCN-dependent manner in target cells and primary HSCs. crIL-15 expression yielded functional pSTAT5 signaling and increased persistence of SENTI-202 in vitro and in vivo. These results demonstrate the antitumor activity and tolerability of SENTI-202 and warrant further clinical development as a novel therapeutic option for patients with CD33+ and/or FLT3+ tumors. Citation Format: Alba Gonzalez, Enping Hong, Gozde Yucel, Elizabeth Leitner, Pearley Chinta, Han Deng, Ian Li, Alice Lam, Abla Bakir, Brandon Lee, Papia Chakraborty, Carmina Blanco, Chen-Ting Lee, Niran Almudhfar, Mengxi Tian, Wenqi Song, Andrew Banicki, Otto Contreras, Martin Gieldin, Brian Garrison, Timothy K. Lu, Kanya Rajangam. Preclinical development of SENTI-202, an off-the-shelf logic gated CAR-NK cell therapy, for the treatment of CD33/FLT3+ hematologic malignancies including AML [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 3195.