Natural Killer (NK) cells are components of innate immune response that are involved in tumor surveillance and clearance. Due to their ability to lyse tumor targets without prior priming they have gained some notoriety in the tumor immunotherapy setting. Although NK cells sense malignant transformation of cells through a number of stochastic receptors they posses no clonotypic receptors to drive specific recognition of tumor antigens, somewhat limiting their therapeutic applications. In order to address the antigen specificity issue we generated molecules, termed Tri-Specific Killer Engagers (TriKEs), that contain three arms: one arm contains a single chain variable fragment (scFv) that binds a potent NK cell activating receptor (CD16); one arm contains an scFv that binds a tumor antigen; and one arm contains the cytokine IL-15 to drive NK cell priming, expansion and survival. Initial pre-clinical studies with these first generation molecules showed that we could use them to target hematological malignancies (via a CD33 binding arm) both in vitro and in vivo or solid malignancies (via an EpCAM binding arm) in vitro. However, to improve on this platform we altered the molecule by swapping out the anti-CD16 scFv arm with a humanized camelid single domain antibody against CD16 (termed cam16 henceforth). This second generation cam16 TriKE platform potently enhanced NK cell activation against tumor targets and induced far greater NK cell expansion when compared to the first generation TriKE. Using our bacterial expression system we generated and isolated cam16 TriKEs against a number of different tumor antigens (CD33, EpCAM, Mesothelin and ROR1) to display the ability of the second generation TriKE platform to target a variety of cancers. To test these molecules we used flow cytometry based NK cell activation assays, where NK cells co-cultured with tumor targets +/- cam16 TriKEs are evaluated for NK cell degranulation (by expression of surface CD107a) and NK cell inflammatory cytokine production (by expression of intracellular IFNγ). We also used a real-time imaging platform, the IncuCyte Zoom, to evaluate the ability of NK cells to kill tumor targets in the presence or absence of cam16 TriKEs over a 2-3 day period. Our data indicates that cam16 TriKEs drive NK cell specific degranulation, inflammation, and killing of AML (CD33), ovarian cancer (EpCAM and Mesothelin), lung cancer (Mesothelin and EpCAM) and sarcoma (ROR1) tumor targets in vitro. Furthermore cam16 TriKE in vivo efficacy could be seen in humanized mouse models using both luciferase-labeled cell lines and patient derived xenografts. Taken together, these findings clearly highlight the translational potential of cam16 TriKEs in the tumor immunotherapy setting.

Citation Format: Martin Felices, Behiye Kodal, Alexander J. Lenvik, Brianna Ettestad, Laura Bendzick, Caitlin Ryan, Dawn K. Schirm, Ron McElmurry, Craig E. Eckfeldt, Melissa A. Geller, Todd R. Lenvik, Daniel A. Vallera, Jeffrey S. Miller. Driving natural killer cell antigen-specific targeting of cancer via next generation trivalent molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4939.