Abstract 2907: Development of a CAR-NK cellular platform for cancer immunotherapy
Authors: Gregoli Karina Di , Mode Ravindra , Mitchell Kaitlin , Cobeta-Lopez David , de Munnik Sabrina , McLaughlin Rene , Moiset Gemma , Brackenbury Louise , Schewitz-Bowers Lauren , Nunan Robert 
Published: June 2023

Abstract
Although Chimeric Antigen Receptor (CAR)-T cell therapy has proven to be successful in treating multiple hematological malignancies, their use is limited due to the potential life-threatening adverse events, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). CAR natural killer (CAR-NK) cells are rapidly emerging as a promising alternative cellular therapeutic with improved efficacy and reduced adverse effects. Moreover, due to the intrinsic characteristics of NK cells, whereby they lack antigen-specific cell surface receptors, CAR-NK cells offer the additional advantage of being an “off-the-shelf” product, thus satisfying the requirement for large-scale production for cancer immunotherapy, giving it the potential to reach more patients at a reduced cost. Here we outline the method to generate, expand and enrich human primary CD19 CAR-NK cells, and demonstrate their efficacy in-vitro against CD19-expressing target cancer cells. Human primary NK cells were isolated from healthy donors and transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV, as an empty vector transduced control. Transduction efficiency greater than 10% or 20% was achieved following transduction and expansion over a 7-day period in response to CD19 CAR LV or GFP LV respectively. Following sufficient expansion, cytotoxicity of the CD19 CAR-NK cells was tested in-vitro in a Tumor Killing Assay (TKA) against CD19+ (Raji) or CD19- (SKOV-3) target cells. As expected, CD19 CAR-NK cells displayed increased cytotoxicity towards CD19+ target cells when compared to non-transduced or GFP transduced controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting specific CD19 CAR mediated killing. Moreover, CD19 CAR-NK cells showed increased degranulation and IFNꝩ secretion when compared to both non-transduced and GFP-transduced controls, suggesting increased NK cell activation and cytotoxicity. Additionally, when degranulation of CD19 CAR positive and CD19 CAR negative NK populations was directly compared, the level of NK cell degranulation was analogous between the two populations, suggesting that CD19 CAR NK cells can enhance the cytotoxic activity of neighboring non-transduced cells, resulting in enhanced killing of CD19+ target cells by the total NK population present. Taken together these data demonstrate the generation, expansion and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This culture system provides the scientific community a platform to screen novel CAR-constructs and to test novel immunomodulators which could enhance NK cell expansion or functional potential to further develop new, more widely available cellular therapeutics to treat cancer.
Citation Format: Karina Di Gregoli, Ravindra Mode, Kaitlin Mitchell, David Cobeta-Lopez, Sabrina de Munnik, Rene McLaughlin, Gemma Moiset, Louise Brackenbury, Lauren Schewitz-Bowers, Robert Nunan. Development of a CAR-NK cellular platform for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2907.