Abstract 3207: Preclinical development of first-of-kind dual-targeted off-the-shelf CAR-NK cell product with engineered persistence for an effective treatment of B cell malignancies
Authors: Goodridge Jode , Mahmood Sajid , Zhu Huang , Gaidarova Svetlana , Blum Robert , Bjordahl Ryan , Cichocki Frank , Chu Hui-Yi , Bonello Greg , Lee Tom , Groff Brian , Malmberg Karl-Johan , Walcheck Bruce , Miller Jeffrey , Kaufman Dan , Valamehr Bahram 
Published: August 2019

Abstract
The unprecedented success of chimeric antigen receptor (CAR) and monoclonal antibody (mAb) -based immune-therapies has provided a clear indication that a system as complex as human immunity can be harnessed, even enhanced, toward a growing number of hematological cancers. Here we describe pre-clinical progress to develop a multi-functional induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cell platform that combines engineered longevity with CAR and mAb-based modalities to leverage the intrinsic polyfunctionality of NK cells. As frontrunners of immune surveillance, NK cells employ a diverse array of germline encoded receptors in distinct combinations, which engage multiple signaling pathways to deliver potent effector responses that can be directed toward tumor cells, drive rapid proliferation, and pave the way for recruitment of adaptive immunity. Specific engagement of multiple signaling pathways was achieved in iNK cells through design of an NK cell-centric CAR combining the transmembrane domain of activating receptor NKG2D with intracellular signaling domains of 2B4 and CD3ζ. Recombining an anti-CD19 scFv onto this signaling platform, CAR modified iNK cells produced specific in vitro recognition of CD19+ B cell lymphoma cells in short term and long term cytotoxicity assays (84% vs 40% clearance of tumor cells at 60H, p&lt;0.001). Further introduction of a fusion receptor consisting of Interleukin-15 (IL15) with IL15 receptor α, enabling autonomous IL15 stimulation, greatly improved iNK longevity and functional persistence in animal models. Moreover, iNK cells modified with IL15 fusion receptor showed enhanced functional maturation including KIR expression and effector molecules such as granzyme B ( ≥2 fold). While iNK cells with anti-CD19 CAR delayed tumor progression in vivo prior to relapse, iNK cells engineered with anti-CD19 CAR and IL15/IL15 receptor were curative against B cell lymphoma, (p&lt;0.002). Expression of CAR and IL15 fusion receptor was then combined with a third modality, a high affinity CD16a receptor modified to prevent proteolytic cleavage (hnCD16). These multifunctional iNK cells demonstrated enhanced directed cytotoxicity in vitro in combination with rituximab against CD19+ targets (&gt;99% vs 90% clearance of tumor cells) and CD19- targets (&gt;99% vs 50% clearance of tumor cells by iNK with anti-CD19 CAR alone, p&lt;0.0001), revealing a unique opportunity to combine CAR with a universal targeting modality to mitigate antigen escape and address heterogeneity in the tumor population by a multi-node targeting strategy. The resulting product, FT519, is designed to provide a flexible, potent and persistent engineered immune cell that utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform.
Citation Format: Jode Goodridge, Sajid Mahmood, Huang Zhu, Svetlana Gaidarova, Robert Blum, Ryan Bjordahl, Frank Cichocki, Hui-Yi Chu, Greg Bonello, Tom Lee, Brian Groff, Karl-Johan Malmberg, Bruce Walcheck, Jeffrey Miller, Dan Kaufman, Bahram Valamehr. Preclinical development of first-of-kind dual-targeted off-the-shelf CAR-NK cell product with engineered persistence for an effective treatment of B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3207.