Cryopreservation of Natural Killer Cells Pre-Complexed with Innate Cell Engagers

Innate cell engager (ICE (R)) constructs are bispecific tetravalent antibodies targeting specific tumor antigens and simultaneously engaging natural killer (NK) cell and macrophage receptors for the destruction of tumor cells. Pre-complexing of ICE (R) constructs with adoptive NK cells is a novel approach to enhance NK cell activity. The suitability of such complexes for cryopreservation, whilst retaining the biological activity and specificity, may enable the development of off-the-shelf NK cell products. This study investigates the binding affinity of ICE (R) constructs targeting EpCAM and NK cell receptors CD16A, NKG2D, or NKp46 to the corresponding antigens, the ICE (R) antitumor activity, and feasibility of cryopreservation. Cell surface retention assays using primary NK cells confirmed a substantially slower ICE (R) construct dissociation kinetics compared with control molecules, suggesting the formation of durable complexes independently of the CD16A polymorphism. The high-affinity NK cell and EpCAM/CD16A ICE (R) complexes were superior to those engaging NKG2D or NKp46 receptors when tested for the NK-cell-mediated elimination of EpCAM-expressing tumor cells. Moreover, the potency and efficacy of these complexes were unaffected after a single freeze-thaw cycle. CD16A-selective ICE (R) drug candidates complexed with NK cells hold promise as novel cryopreserved off-the-shelf NK cell products with chimeric antigen receptor-like NK cell properties, capable of effective depletion of tumor cells.