456142 Evolution of Antibody Agonists That Functionally Recapitulate Natural Cytokines
The monoclonal antibody platform represents a robust, engineerable scaffold that benefits from extended in vivo half-life and we thus aimed to engineer antibody-based constructs that dimerize receptors in their native conformations induced by endogenous cytokines to functionally recapitulate cytokine signaling. We devised a novel yeast display-based evolutionary strategy to isolate antibodies that use a single binding site to bridge the interface between two different receptor subunits within a dimeric signaling complex. Our approach involved screening antibody libraries against receptor-ligand complexes that served as ‘molecular casts’ of the active cytokine-receptor complex conformation and performing iterative rounds of selections to evolve ‘staplers’ that recognize a composite epitope between heterodimeric receptor subunits. Using this new engineering strategy, we identified staplers for two different cytokine systems that bound fully assembled cytokine-receptor complexes but not any of the individual components thereof. Total internal reflection fluorescence (TIRF) microscopy co-localization studies demonstrated that these molecules efficiently dimerized receptor subunits in living cell membranes in the absence of cytokine. Determination of the crystal structure of the stapler bound to the cytokine-receptor complex ECD provided biophysical evidence that staplers recognize a composite epitope formed by the conjunction of two receptor subunits and that they bind heterodimeric receptors in their active, cytokine-bound signaling conformation. Further, we demonstrated that staplers activated JAK/STAT pathway signaling in human cell lines with equivalent intensity to native cytokines and showed that staplers recapitulated functional responses such as immune cell proliferation and immunoglobulin class switching. Our evolutionary strategy can be applied to isolate stable and readily engineerable agonistic antibodies that serve as staplers for other immune receptor complexes and, more generally, for any dimeric receptor system to address a broad range of disease applications.