Miniaturized IL-2/anti-IL-2 immunocytokines selectively activate and support the in vivo persistence of regulatory T cells

Introduction: Interleukin-2 (IL-2) is a multifunctional cytokine that potently expands regulatory T cells (Tregs) and thus has potential in mitigating autoimmune diseases and promoting transplant tolerance. However, the cytokine's concurrent activation of effector lymphocytes coupled with its short serum half-life limit therapeutic use. Previous efforts have overcome these challenges by fusing IL-2 to an engineered anti-IL-2 antibody denoted F5111, which selectively directs IL-2 towards Tregs over effector lymphocytes. The resulting molecule, denoted the F5111 immunocytokine (IC), potently and specifically expands Tregs, but its bulky size and bivalency limit diffusion and tissue penetration, while its dual-chain format complicates gene delivery and stable expression from cells. Methods: Here, we engineered a miniaturized version of F5111 IC (termed miniF5111 IC), comprising IL-2 fused to a single chain variable fragment (scFv) of the F5111 antibody. We optimized the topology of miniF5111 IC and performed biophysical, signaling, and functional studies to interrogate its immune activity. Results: , miniF5111 IC mediated selective Treg expansion with a reduced serum half-life compared to the full-length F5111 IC. Finally, we expressed miniF5111 IC from engineered Tregs and showed that stable expression of this molecule led to prolonged cell persistence and sustained FOXP3 expression following adoptive transfer. Discussion: Taken together, our findings position miniF5111 IC as a versatile platform to selectively target and activate specific immune cell subsets, demonstrating its potential as a next-generation therapeutic to treat autoimmune disorders and prevent transplant rejection.