Senti-202, a Selective, Off-the-Shelf, Preclinical CAR-NK Cell Therapy with CD33 and/or FLT3 Activating CAR, Healthy Cell Protection from Endomucin (EMCN) Inhibitory CAR and Calibrated Release IL-15 for Hematologic Malignancies Including AML

Abstract

Background: Patients with CD33 and/or FLT3 malignancies, which encompass most myeloid malignancies such as acute myeloid leukemia (AML), have a grim prognosis and a high unmet need. There are no approved cell therapies for patients with AML due to paucity of targets and an immunosuppressive bone marrow milieu. SENTI-202, a preclinical CAR-NK cell is engineered with a logic-gated gene circuit to overcome these challenges. Methods: SENTI-202 is engineered with a FLT3 OR CD33 NOT EMCN logic-gated gene circuit and a proprietary calibrated release IL-15 (crIL-15). These 3 chimeric proteins are engineered onto a natural killer (NK) cell (healthy adult peripheral blood-derived) to create an off-the-shelf chimeric antigen receptor (CAR) NK cell therapy. The bivalent CD33 OR FLT33 (OR GATE) activating CAR (aCAR) enables concurrent targeting of CD33+ and/or FLT3+ AML cells which kills both AML blasts and leukemic stem cells (LSCs) in vitro and in vivo. This concurrent targeting of two tumor antigens could potentially provide longer remission and less chance of relapse. The NOT EMCN (NOT GATE) inhibitory CAR (iCAR) protects healthy EMCN+ hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) from off-tumor toxicity to potentially aid in post-treatment reconstitution of a healthy hematopoietic system. The crIL-15 provides both autocrine and paracrine IL-15 stimulation to the CAR-NK cells (and surrounding immune cells) to promote cell expansion, persistence, and tumor killing. While previous presentations (Garrison et al., 2021) focused on the performance of individual SENTI-202 components in a cell, this presentation focuses on functional data from the complete logic-gated gene circuit and the crIL-15 together in a cell (i.e., the final CAR-NK with all 3 genetic elements). Results: We demonstrated delivery of all circuit components using a single retroviral vector. We then investigated activity of the resulting CAR-NK cells using a traditional in vitro cytotoxicity assay (E:T=1:2; 20hrs) in which SENTI-202 showed >90% killing of leukemia cells (e.g. SEM; P≤0.001), maintained significant serial killing potential (3 rounds; P≤0.0001; P≤0.001; P≤0.001), as well as robust killing against primary AML blasts and LSC-enriched target cell populations. Importantly, SENTI-202 also exhibited significant tumor cell killing and improved survival (P≤0.001) in an MV4-11 xenograft AML mouse model. We investigated the ability of SENTI-202 to reduce off-tumor toxicity toward EMCN+ healthy cells, including primary HSCs. Within in vitro assays, SENTI-202 provided >50% protection to FLT3+ CD33+ model healthy cells that also expressed the EMCN safety antigen, and similar significant protection was maintained through serial protection assays (3 rounds; P≤0.0001; P≤0.001; P≤0.0001). As further evidence of the selectivity imparted using the OR/NOT logic gating approach, in vitro assays with SENTI-202 resulted in killing of leukemia cells but not primary healthy human HSCs and HPCs (~42% protection) (P≤0.01). Finally, in the presence of SENTI-202 in vivo, the NOT GATE protected and enabled significant (P≤0.0001) expansion of EMCN+ FLT3+ CD33+ model healthy cells. Conclusions: In summary, preclinical evaluation of SENTI-202 demonstrated selective killing of CD33 and/or FLT3 expressing AML cell lines while protecting healthy HSCs and HPCs. This selectivity is imparted by the novel logic-gated gene circuit engineered into healthy adult NK cells. Clinical evaluation of SENTI-202 is planned to evaluate the safety and efficacy in patients with hematologic malignancies with high unmet need, including AML.