Preclinical NK Cell Platform for CAR Directed Therapies: Functional and Phenotypic Comparison Using a Rechallenge Cytotoxicity Assay

Introduction: CAR T-cell therapy has revolutionized the treatment of patients with relapsed/refractory (R/R) acute leukemia, NHL, and multiple myeloma. However, there are still areas of improvement in their clinical activity, source of the effector cells, prevention, and management of adverse events that require particular attention. Because of those reasons, NK cells appear as a viable effector cell alternative that can help address these challenges. NK cells offer a profile of activation, expansion, persistence, and cytotoxicity that is different from T cells and, when modified to bear CAR constructs, may provide significant advantages. However, the preclinical development of NK-CARs is challenging mainly because of the difficulty of generating large quantities of cells for testing and well-established pathways for CAR optimization before in vivo evaluation. Therefore, we developed a CAR optimization platform using the NK-92 cell line. NK-92 cells conserve their cytotoxic ability and can be easily expanded in vitro and used for functional and phenotypical evaluations of novel CAR-NK constructs. Here we present a rechallenge cytotoxic assay that mimics repetitive in vivo effector interactions with the target cells and its use for optimization, comparison, and development of NK-based cellular therapies.