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.