Calibrated Release IL15 Bivalent CD33 and/or FLT3 and NOT Emcn Logic Gated Gene Circuit CAR-NK Cell Therapy (SENTI-202) in Venetoclax Resistant Patient Derived Xenograft Acute Myeloid Leukemia Models
Authors: Muftuoglu Muharrem , Basyal Mahesh , Li Li , Lv Jiangxing , Ostermann Lauren B. , Zhao Ran , Ke Baozhen , Akiyama Hiroki , Mak Po Yee , Carter Bing Z. , Deng Han , Li Yongshuai , Garrison Brian S. , Rajangam Kanya L. , Andreeff Michael 
Published: December 2023

Venetoclax (VEN) as combination therapy has improved response rates and overall survival of patients with acute myeloid leukemia (AML). However, once AML relapse prognosis is dire with a 5.3 month median survival (Brandwein, 2020). While chimeric antigen receptor (CAR) cell therapies have revolutionized the treatment landscape for lymphoid hematological malignancies, development of such therapies for AML has been challenging, in part due to the heterogeneity of the disease. Currently, there is a paucity of individual AML targets that are consistently expressed across AML subpopulations. Furthermore, the expression of these AML targets is not restricted to tumor cell populations, often resulting in off-tumor toxicity against healthy cell populations. SENTI-202 employs OR and NOT Logic Gating to overcome these challenges.
SENTI-202 represents an innovative preclinical CAR-NK cell therapy, engineered to exploit a powerful CD33 OR FLT3 NOT EMCN Logic Gate gene circuit, in conjunction with calibrated release IL-15 (crIL15) expression. The CD33 OR FLT3 (OR GATE) activating CAR (aCAR) concurrently targets two AML antigens, thus permitting a broader therapeutic window against AML LSCs (CD33+/-, FLT3+), blasts (CD33+, FLT3+/-), and more specifically, VEN-resistant (VEN-r) AML with a CD33+ monocytic phenotype (Pei, 2020). The NOT EMCN (NOT GATE) inhibitory CAR (iCAR) is a pivotal safeguard, selectively shielding healthy EMCN+ Hematopoietic Stem Cells and Progenitor Cells (HSCs/PCs) from potential off-tumor toxicity. Previously, SENTI-202 exhibited remarkable efficacy in targeting and eliminating CD33 and/or FLT3 expressing AML cell lines, while concurrently safeguarding healthy HSCs/PCs (Garrison et al., ASH, 2022). To further validate the efficacy and broaden the utility of SENTI-202, we conducted assessments on clinically relevant preclinical AML models, along with VEN-r AML patient-derived xenograft (PDX) models. We first assessed the in vitro cytotoxicity and cytokine profiles of CAR-NK cells possessing the SENTI-202 OR Gate and crIL15 against primary R/R AML patient samples (n=11), where we observed significantly higher cytotoxicity compared to unengineered NK-cells (68% vs 19.7%) (p=0.0005, E:T ratio 1:1 for 24h) and increased TNF-a and IFN-g secretion. To further explore CAR-NK cell anti-tumor activity, we screened 10 different PDX samples and discovered that while the median frequency of CD33 and/or FLT3 expression was 99.9% (97.48-100), individual CD33 and FLT3 expression level varied, underscoring AML proteomic heterogeneity and the clinical need for OR Gate approaches. Our initial PDX studies used clinically relevant VEN/HMA-r AML PDX cells (designated as PDX1) obtained from a patient with FLT3-ITD, GATA2, and NRAS mutations, who initially responded to VEN/decitabine treatment but later relapsed. To facilitate in vivo tracking of AML and disease progression, the VEN-r PDX cells were engineered with Akaluc, an exceptionally brighter luciferase compared to conventional luciferase. Following confirmation of AML engraftment in bone marrow through bioluminescence imaging (BLI), the CAR-NK cells, along with unengineered NK-cells, were infused at a dose of 30 million cells per infusion, once a week for three consecutive weeks. The persistence of the CAR-NK cells was detectable for up to 2 weeks after the last infusion. Notably, the infusion of CAR-NK cells resulted in the clearance of bone marrow (BM) disease as evident from BLI one week after the second infusion of CAR-NK cells while unengineered NK-cells failed to clear bone marrow disease. Assessment of CAR-NK cell efficacy in two different AML PDX models is ongoing, along with additional in vitro and in vivo studies that further demonstrate the ability of the SENTI-202 NOT EMCN Logic Gate to significantly protect healthy HSCs from off-tumor toxicity.
In conclusion, CAR-NK cells expressing the SENTI-202 OR Gate and crIL15 demonstrate robust anti-tumor activity against primary AML cells within in vitro cytotoxicity assays and in vivo AML PDX models, including VEN-r AML. These promising preclinical results highlight the potential of SENTI-202 as a targeted and selective therapy for AML, offering a new avenue for overcoming the challenges posed by the heterogeneity of the AML proteomic landscape.