High-Throughput De Novo Protein Design Yields Novel Immunomodulatory Agonists

Cytokines regulate cell behavior by bringing together specific receptor subunits to trigger downstream signaling. Designed molecules that bring together non-natural receptor pairs could have novel signaling responses and cell specificities. We present a high-throughput de-novo design approach to create novel cytokines by generating and fusing pairs of computationally designed binders. By combining 33 designed receptor-binding domains, we generated over a thousand potential de novo designed "Novokines", of which 75 activated pSTAT signaling in peripheral blood mononuclear cells. We characterized 18 of these, including new pairings of established common receptors, cross-family pairings such as TrkA-γcommon, and a series of pairings with interferon receptor-1 (IFNAR1), revealing that IFNAR1 can function as a versatile common receptor similar to γcommon or βcommon. We identify novokines that drive monocyte proliferation, T cell survival and CD4⁺ T cell-specific proliferation. Our framework provides a blueprint for expanding the understanding of cytokine signaling and generating novel therapeutic proteins.