Multivalent, asymmetric IL-2–Fc fusions show enhanced selectivity for regulatory T cells

The cytokine interleukin-2 (IL-2) has the potential to treat autoimmune disease but is limited by its modest specificity toward immunosuppressive regulatory T (T_reg) cells. IL-2 receptors consist of combinations of α, β, and γ chains of variable affinity and cell specificity. Engineering IL-2 to treat autoimmunity has primarily focused on retaining binding to the relatively T_reg-selective, high-affinity receptor while reducing binding to the less selective, low-affinity receptor. However, we found that refining the designs to focus on targeting the high-affinity receptor through avidity effects is key to optimizing T_reg selectivity. We profiled the dynamics and dose dependency of signaling responses in primary human immune cells induced by engineered fusions composed of either wild-type IL-2 or mutant forms with altered affinity, valency, and fusion to the antibody Fc region for stability. T_reg selectivity and signaling response variations were explained by a model of multivalent binding and dimer-enhanced avidity-a combined measure of the strength, number, and conformation of interaction sites-from which we designed tetravalent IL-2-Fc fusions that had greater T_reg selectivity in culture than do current designs. Biasing avidity toward IL2Rα with an asymmetrical multivalent design consisting of one α/β chain-binding and one α chain-binding mutant further enhanced T_reg selectivity. Comparative analysis revealed that IL2Rα was the optimal cell surface target for T_reg selectivity, indicating that avidity for IL2Rα may be the optimal route to producing IL-2 variants that selectively target T_regs.