PROJECT SUMMARY/ABSTRACT Natural killer (NK) cell engagers are a promising anticancer therapy that potently leverages the immune system while providing an exceptional safety profile. Trispecific killer cell engagers (TriKEs) are NK cell engagers that mediate NK cell killing of tumor cells through multispecific binding. TriKEs contain a tumor binding domain, an IL-15 cytokine, and a CD16 engager. The IL-15 and CD16 engager stimulate robust NK cell activation and tumor cell killing while the tumor binding domain targets the therapy to the tumor microenvironment. Expansion of the TriKE platform to target new tumor-associated antigens requires integration of novel TriKE tumor binding domains. This process is bottlenecked by difficulties preserving TriKE functionality and developability (i.e. sufficient stability, aggregation, and recombinant yield for therapeutic use) upon the addition of a new tumor binding domain. This project will transform the TriKE discovery process by interrogating the impact of specific tumor binding domain properties on the resultant TriKE’s functionality and developability, including use of innovative hyperstable miniproteins. Specifically, Aim 1 will investigate the impact of tumor binder scaffold architecture by comparing the performance of several different scaffolds within TriKEs, including antibody fragments and hminiproteins. Aim 2 will investigate the impact of preemptively optimizing tumor binder stability and recombinant yield before insertion into TriKEs. This will be accomplished by systematically varying tumor binder stability and recombinant yield and quantifying the impact on TriKE functionality and developability. These investigations will involve the production and characterization of experimental tumor binding domains and TriKEs targeting B7-H3, an established tumor-associated antigen. The primary goal of this work is to inform the design of TriKE tumor targeting domains, thereby streamlining the expansion of the platform to new therapeutic targets. Furthermore, this project will advance the development of TriKEs as a therapeutic option for B7-H3 positive cancers.