FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML
Authors: Garrison Brian S. , Deng Han , Yucel Gozde , Frankel Nicholas W. , Guzman-Ayala Marcela , Gordley Russell , Hung Michelle , Lee Derrick , Gainer Marcus , Loving Kathryn , Chien Jenny , Pan Tiffany , Gorman Wesley , Leemans Nelia , Lam Alice , Wood Travis , Wong Wilson , Lee Philip , Lu Tim , Lee Gary 
Published: November 2021

Abstract
Background: While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and immature leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using logic gated gene circuits, we are engineering our SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating AML patients.
Methods: To maximize clearance of AML tumor subpopulations and minimize off-tissue toxicities, we used a proprietary bioinformatics paired antigen discovery platform to identify the optimal combinations of AML tumor-associated and healthy tissue antigens to target using an OR and NOT logic gated CAR gene circuit approach. The SENTI-202 therapeutic candidate is a FLT3 OR CD33 NOT Endomucin (EMCN) gene circuit-enabled allogeneic CAR-NK cell, designed to broadly target FLT3 and/or CD33-expressing AML tumor cells (including both LSCs and blasts) but not healthy hematopoietic stem cells (HSCs).
Results: First, for the OR GATE portion of the logic circuit we demonstrated that engineered primary human NK cells expressing activating CARs (aCARs) that recognize both FLT3 and CD33 outperformed more traditional single target CAR approaches with FLT3 (p&lt;0.05) or CD33 (p&lt;0.01), and exhibited &gt;80% cytotoxicity and significant cytokine secretion (GrB, IFN-g, and TNF-a) against multiple leukemia cell lines in vitro, including MOLM13, THP1, and SEM. We successfully engineered FLT3 OR CD33 CAR-NK cells using both bicistronic and bivalent CAR configurations, where bicistronic CARs possess separate FLT3 and CD33 CARs linked via a 2A peptide, and bivalent CARs use a loop structure to connect FLT3 and CD33 scFvs within the same CAR. While both approaches demonstrated robust efficacy against AML cells, the bivalent approach enabled greater CAR expression and cytotoxicity (p&lt;0.05). Importantly, our FLT3 OR CD33 CAR-NK cells demonstrated significant cytotoxicity against primary AML patient samples (p&lt;0.01-0.001) and significantly reduced tumor burden and improved mouse survival in MOLM13 (p&lt;0.05) and MV4-11 (p&lt;0.01) xenograft AML models. We believe that our strategy of concurrently targeting FLT3 and CD33 will result in a more robust synergistic anti-tumor effect, leading to a more durable remission with decreased risk of relapse due to single antigen escape.
Second, for the NOT GATE portion of the logic circuit to protect healthy HSCs, we developed NK and T cell inhibitory CARs (iCARs) consisting of an scFv against a healthy cell antigen, hinge and transmembrane domains, and functional intracellular domains derived from inhibitory co-receptors containing immunoreceptor tyrosine-based inhibitory motifs. In the case of SENTI-202, the iCAR scFv recognizes EMCN, a surface antigen expressed on up to 76% of healthy HSCs but not on AML cells. Using two different iCAR configurations, we demonstrated that FLT3 (CD28z) aCAR-NK cells engineered with an EMCN-specific iCAR protected up to 67% (iCAR#1, p&lt;0.01) or 50% (iCAR#2, p&lt;0.01) of FLT3+ EMCN+ cells from FLT3 aCAR-mediated cytotoxicity. Next, to replicate a clinical context more closely, we mixed FLT3+ EMCN- (AML-like) and FLT3+ EMCN+ (HSC-like) target cells and demonstrated that FLT3 NOT EMCN CAR-NK cells exhibit preferential killing of FLT3+ EMCN- target cells (p&lt;0.0001), demonstrating that our NOT GATED gene circuit controls NK-mediated responses on a cell-by-cell basis.
Conclusion: SENTI-202 is a novel NK cell product candidate to be engineered with both OR and NOT logic gated CAR gene circuits, wherein the OR gate is designed to increase AML LSC/blast tumor clearance (to prevent relapse), and the NOT gate is designed to protect healthy HSCs from off-tumor toxicity, enabling regeneration of a healthy hematopoietic system and mitigating the need for a bone marrow transplant. Beyond AML, OR and NOT logic gated CAR-NK cell therapy has applicability to other cancer-associated antigen targets that are potentially limited by antigen escape and/or off-tumor toxicity, increasing the potential for enhanced efficacy and reduced risk of undesirable side effects.

Disclosures
No relevant conflicts of interest to declare.