Site- and Size-Controlled PEGylation Modulates Biointerfaces and Immunoregulatory Functions of Bifunctional Peptides in an Autoimmune Disease Model

Autoimmune diseases arise from dysregulated autoreactive T cell activation, and current therapies rely on broad immunosuppression that predisposes patients to infection and malignancy. Antigen-specific strategies such as bifunctional peptide inhibitors (BPIs), which couple an antigenic epitope with an ICAM-1–binding sequence to block costimulation, offer promise but remain limited by rapid degradation and insufficient in vivo efficacy. Here, we report a biointerface-engineering strategy based on site- and size-controlled PEGylation to tune the structural presentation and immunomodulatory behavior of BPIs. By systematically varying PEGylation position (N-terminal vs internal spacer) and chain length (1–10 kDa), we identified the spacer-conjugated 5 kDa PEG–BPI conjugate (S-5k) as the optimal configuration balancing bioactivity, dispersion, and immune regulation. The S-5k conjugate demonstrated superior dendritic cell uptake, downregulation of costimulatory markers, and induction of regulatory immune phenotypes both in vitro and in vivo. In the Experimental Autoimmune Encephalomyelitis (EAE) mouse model, treatment with S-5k significantly ameliorated clinical symptoms and promoted immunosuppressive populations, including Foxp3 + Tregs and CD206 + macrophages. Unlike conventional PEGylation used for pharmacokinetic extension, this approach employs PEG as an active spatial modulator of peptide–cell interfaces. Overall, these results establish PEGylation-guided biointerface control as a rational design principle for polymer–peptide conjugates and demonstrate its potential for translational immunotherapy in autoimmune disease.