In vitro and in vivo evaluation of chemically synthesized, receptor-biased interleukin-4 and photocaged variants.

Interleukin-4 (IL-4) plays a central role in type 2 immune responses. Despite its potential use for allergic and autoimmune diseases, its pleiotropic receptor binding complicates selective targeting of IL-4 signaling pathways. We developed a chemical synthesis of (i) IL-4 variants with atomically tailored side-chain modifications that deter specific receptor interactions and (ii) conditionally activatable IL-4 variants uncaged with 365-nanometer light. In primary cell studies, different variants elicited selective STAT5 or STAT6 phosphorylation in lymphocytes or neutrophils. In murine studies, photocaged IL-4 suppressed inflammation only upon UV irradiation, demonstrating precise on demand control. We accomplished the synthesis and folding of IL-4, a hydrophobic cytokine with three disulfide bonds, using the alpha-ketoacid-hydroxylamine (KAHA) ligation to assemble three segments. We introduced further conjugations, including PEGylation for half-life extension, through orthogonal ligations enabled by functionalized amino acid building blocks. This work highlights the flexibility of chemical protein synthesis to produce therapeutically valuable cytokines, including receptor-biased and spatiotemporally activatable IL-4 variants.