Control of SIINFEKL Antigen Presentation by the Immunoproteasome

The immunoproteasome (iCP) plays a central role in generating peptides for presentation in major histocompatibility complex-I (MHC-I) complexes, yet chemical tools to selectively exploit this activity for controlled antigen release and MHC-I loading have not been described. Here, we report an iCP-targeted peptide prodrug, mATMW-SIINFEKL, that undergoes selective cleavage within DC2.4 dendritic cells, releasing the model antigen SIINFEKL for efficient MHC-I loading and extracellular display. Flow cytometry and confocal microscopy confirmed dose-dependent intracellular processing and proper cross-presentation of SIINFEKL-H-2K^b complexes. These findings establish a modular platform for designing antigenic prodrugs that can selectively release defined peptides in iCP-expressing cells. Given the conservation of iCP subunits across species and prior functional validation of Ala-Thr-Met-Trp (ATMW)-based probes in human cells, this strategy may be extended to HLA class I loading and display, enabling controlled CD8⁺ T cell activation. The approach also provides opportunities for incorporating bioorthogonal handles for functionalization, imaging, or quantitation of antigen extracellular display. Overall, iCP-targeted peptide prodrugs offer a versatile chemical tool to interrogate the role of iCP activity in shaping the immunopeptidome and to facilitate the development of antigen-specific immunotherapies.