Renewable And Genetically Engineered Natural Killer Cells For Off-The-Shelf Adoptive Cellular Immunotherapy

Abstract The unique attributes of a combinatorial tumor recognition system, diminished off-tumor cytotoxicity, and multifaceted effector function make natural killer (NK) cells a prime candidate for a universal approach to cancer immunotherapy. In addition, NK cells are the principal mediator of antibody-directed cellular cytotoxicity (ADCC). However, NK cell function is often impaired in the setting of cancer, reducing the effectiveness of the endogenous immune system and the therapeutic efficacy of monoclonal antibodies. To address the need for advanced and combinatorial cancer therapies, we developed a unique and effective strategy to create a renewable source of engineered “off-the-shelf” NK cells with augmented function, including enhanced ADCC and persistence. Key challenges associated with genetic editing, limited expansion, persistence and variability of peripheral blood (PB)-derived NK cells were overcome by utilizing our induced pluripotent stem cell (iPSC) technology as the unlimited starting material for the reproducible and consistent derivation of engineered NK cells. Through targeted transgene integration, we produced a clonal iPSC master cell line to continuously produce NK cells engineered to uniformly express a high affinity, non-cleavable version of CD16 (hnCD16-NK). In directed differentiation, the hnCD16-NK cells displayed homogeneous expression of CD16 (>95%) and a mature CD56+ NK cell phenotype, as exhibited by expression of KIR, NCRs, DNAM-1, and NKG2D. In contrast to endogenous CD16 expression, the engineered hnCD16 molecule was shown to be cleavage resistant upon NK cell activation (>95% CD16+ hnCD16-NK vs. <10% CD16+ PB-derived NK cell, upon target cell-mediated activation), and demonstrated enhanced antibody binding compared to PB-derived NK cells expressing the low-affinity variant. In addition to increased expression of the cytolytic molecules perforin and granzyme B and enhanced direct cytotoxicity against tumor targets, hnCD16-NK cells displayed superior ADCC capacity and cytokine production in response to CD16 stimulation. Importantly, manufacture of hnCD16-NK cells was proven to be highly scalable, delivering up to 107 fold expansion over a 35 day period. The maintained proliferative capacity can be in part associated with longer telomere length seen in hnCD16-NK cells. Furthermore, deletion of classical human leukocyte antigen molecules and ectopic expression of immunosuppressive proteins engineered at the iPSC level provided the ability of hnCD16-NK cells to potentially overcome the host histocompatibility barrier and to improve persistence in the allogeneic setting. In conclusion, the preclinical data presented herein highlight the therapeutic value of hnCD16-iNK cells as an ideal ADCC-mediated “off-the-shelf” NK cell-based immunotherapeutic product with augmented persistence, anti-tumor capacity, manufacturing reliability and preclinical efficacy. Citation Format: Ryan Bjordahl, Frank Cichocki, Raedun Clarke, Svetlana Gaidarova, Brian Groff, Paul Rogers, Stacey Moreno, Ramzey Abujarour, Greg Bonello, Tom Lee, Weijie Lan, Matthieu Bauer, Dave Robbins, Betsy Rezner, Sarah Cooley, Bruce Walcheck, Stewart Abbot, Bruce Blazar, Scott Wolchko, Daniel Shoemaker, Jeffrey S. Miller, Bahram Valamehr. Renewable and genetically engineered natural killer cells for off-the-shelf adoptive cellular immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3755. doi:10.1158/1538-7445.AM2017-3755