In vitro and in vivo Evaluation of Chemically Synthesized, Receptor-Biased Interleukin-4 and Photocaged Variants

Interleukin-4 (IL-4) is a cytokine that plays a central role in type 2 immune responses and is involved in regulating pleiotropic actions in our body by engaging multiple different IL-4 receptor (IL-4R) complexes. Targeting the IL-4R system has a high potential for therapeutic intervention for allergic and autoimmune diseases. A challenge in developing this pleiotropic cytokine for clinical application is the construction of variants tailored for engagement with specific receptor IL-4R subunits, which are necessary for selective activation of specific signaling pathways to treat disease with minimum side effects. To establish a platform for preparation of tailored IL-4 variants, we developed a modular and flexible chemical synthesis of IL-4 and applied this approach to the preparation of (i) IL-4 variants that act as receptor antagonist due to presence of unnatural residues that block specific interactions, and (ii) photocaged and in vivo half-life extended IL-4 variants that can be conditionally activated using UV light, achieved by the incorporation of a photocaged Gln116 residue. We were able to show that these different cytokine variants elicit differential STAT5 or STAT6 phosphorylation in lymphocytes or neutrophils in vitro with just one amino acid substitution. Furthermore, we demonstrated that the photocaged IL-4 can be activated by UV light and effectively suppresses neutrophils in an inflammation model in vivo. Collectively, this work demonstrated the flexibility and applicability of chemical protein synthesis by allowing us to broaden the scope of protein variants that can be accessed for the preparation and evaluation of therapeutically valuable proteins.