Engineered Nonhormonal, Cationic Proinsulin Variants Prolong Lymphatic Drainage

Type 1 diabetes patients develop autoantibodies against pancreatic-islet-derived autoantigens, with proinsulin emerging as an early and dominant target. Existing antigen-specific immunotherapy (ASIT) strategies remain limited by poor antigen stability and rapid clearance, leading to reliance on global immunosuppression. To address these challenges, we engineered nonhormonal variants of proinsulin featuring cationic peptide "tails" at the <i>C</i>-terminus. All the expressed cationic proinsulin variants retained low-nM binding affinity to mouse anti-insulin mAb125 and exhibited low affinity for the insulin receptor. In addition, all variants exhibited physical stability profiles similar to those of proinsulin. A diffusion assay was performed in a viscous hyaluronic acid gel to simulate subcutaneous injection. The addition of a cationic binding motif to proinsulin slowed diffusion compared to that of unmodified proinsulin. Modifying autoantigens such as proinsulin with cationic "tails", therefore, slows release, providing a depot-like effect with prolonged antigen presence at the injection site. This slow-release strategy also provided a steady accumulation of proinsulin in the draining lymph nodes. This strategy may enhance the efficacy of ASITs by negating insulin receptor activity, promoting autoimmune cell engagement, and sustaining exposure to draining lymph nodes.