Facile Diversification of Interleukin-2 Pharmacology with Surrogate Agonists

Cytokines, such as interleukin-2, initiate signaling by dimerizing their receptors into orientations and proximities that induce intracellular signaling. We wished to explore a new pharmacological strategy for cytokine discovery based on variation of receptor dimer geometries using surrogate ligands, since natural cytokines are structurally limited as engineering scaffolds. Here, we report a structurally agonistic approach based on a modular, ‘plug and play’ class of ligands, that is amenable to high-throughout screening. We isolated nanobodies (Nb) and scFvs against human IL-2Rβ and γc, and generated a 40-member combinatorial matrix of tandem single-chain bispecific (IL-2Rβ:γc) molecules. We identified 28 surrogate IL-2 ligands exhibiting a wide range of agonist strengths and biased signaling properties, including cell-type bias for NK expansion and cytotoxity relative to T cells. Crystal structures of select Nb:receptor complexes validated that alternative receptor dimer geometries underly the functional diversification. This “cytokine med-chem” approach is generalizable to many dimeric cell surface receptor systems. RNA-seq analysis of human CD8+ T cells that were stimulated with native cytokines (IL-2, IL-15, IL-7), IL-2 surrogate agonists, or unstimulated for 24 hr.