Abstract 1335: Generating functional CAR-NK cells for cancer immunotherapy
Authors: Gregoli Karina Di , Leonard Henry , Rocca Dan , de Munnik Sabrina , Moiset Gemma , Nunan Robert , Schewitz-Bowers Lauren , Brackenbury Louise 
Published: March 2024

Abstract
Chimeric Antigen Receptor-Natural Killer (CAR-NK) cells are rapidly emerging as a promising cellular therapeutic overcoming the potential life-threatening adverse events that may result from CAR-T therapy, including cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GVHD). Due to their intrinsic characteristics, NK cells can kill tumor cells without the requirement for recognition of antigen in the context of MHC. This removes the constraint of HLA matching and supports antigen independent cytotoxicity through NK cell intrinsic mechanisms. As such CAR-NK cells kill via both CAR-dependent and -independent mechanisms. For these reasons, they offer the advantage of being an “off-the-shelf” product; satisfying the requirement for large-scale production of cancer immunotherapies, with the potential to reach more patients at a reduced cost. The frequency of NK cells in the blood is low (~ 2-5%), therefore improving their in vitro expansion is key to generating sufficient material to offer a viable CAR-NK cell therapy. Here we outline an in-vitro method to generate, expand, and test primary human CAR-NK cells using engineered feeder cells, and go on to demonstrate the efficacy of the CAR-NK against target cancer cells. Primary human NK cells were cultured and expanded in the presence of irradiated K562 cells (iK562) modified to express 4-1BBL and membrane bound IL-21. Following initial expansion, NK cells were transduced with a clinically tested CD19-targeting CAR lentiviral vector (LV) or GFP expressing LV; as an empty vector transduced control. Following transduction, CD19 CAR-NK and GFP-NK were enriched by FACS sorting to reach &gt;80% purity and further expanded in the presence of 4-1BBL/mbIL21 iK562 feeder cells. Following sufficient expansion, cytotoxicity of fresh or cryopreserved CD19 CAR-NK cells was tested in vitro in a Tumor Killing Assay (TKA) against CD19+ or CD19- target cells. As expected, CD19 CAR-NK cells showed increased on-target cytotoxicity, increased degranulation and IFNꝩ secretion compared to controls. This effect was not observed with CD19 negative cancer cells, strongly suggesting CD19 CAR engagement of the target antigen to mediate killing. Taken together these data show the generation, expansion, and enrichment of CD19 CAR-NK cells with enhanced cytotoxic activity towards CD19+ target cells. This approach provides a platform for in vitro characterization of novel CAR-constructs, allied with in-vitro safety assessment and in vivo pharmacology testing. Together, this offers the potential to further develop new and more widely available cellular therapeutics to treat cancer.
Citation Format: Karina Di Gregoli, Henry Leonard, Dan Rocca, Sabrina de Munnik, Gemma Moiset, Robert Nunan, Lauren Schewitz-Bowers, Louise Brackenbury. Generating functional CAR-NK cells for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1335.