Topology-enabled long-acting interleukin-2 for advanced low-dose interleukin-2 therapy of autoimmune diseases

Abstract

The therapeutic efficacy of protein biologics is often hampered by their rapid in vivo degradation and off-target effects. Interleukin-2 (IL-2), a cytokine promising for treating autoimmune diseases via regulatory T cell (Treg) expansion, exemplifies this challenge due to its short half-life and pleiotropic activity. Herein, we report a molecular-scale topology engineering strategy to overcome these limitations. By using split intein mediated ligation and genetically encoded entangling motif, p53dim (X), we constructed cyclic (c-IL-2) and catenated (cat-IL-2) variants of IL-2 via direct expression in E. coli. These topological proteins preserve the native conformation and receptor binding capacity of IL-2 with minimal to minor exogenous motifs, while exhibiting superior proteolytic stability and a dramatically extended serum half-life (approximately 4.5–5.0-fold longer than wt-IL-2). In murine models of three distinct autoimmune diseases (lupus, rheumatoid arthritis, and Sjögren’s syndrome), weekly administration of these topological IL-2 variants effectively rebalanced immunity, as evidenced by a marked expansion of Tregs and significant amelioration of tissue injury and clinical biomarkers. Transcriptomic profiling confirmed the induction of a tolerogenic program without systemic toxicity. This work establishes protein topology engineering as a minimalist, yet robust and generalizable platform to enhance the pharmacokinetics and functional specificity of therapeutic proteins, offering a powerful new modality for biologic drug development.