Mapping the Modification Landscape of MHC-I Epitopes: A Framework for Immunogenic Peptidomimetic Antigen Design

Peptide-based cancer vaccines offer a promising strategy to target tumor-specific neoantigens, yet their clinical translation is restricted by poor metabolic stability, limited intracellular permeability, and stringent requirements for MHC-I binding and T cell receptor (TCR) recognition. Although peptidomimetic modifications have been widely explored to improve pharmacokinetics, their impact on antigen presentation and immune recognition remains poorly understood. Here, we systematically evaluate backbone N-methylation, peptoid substitution, and stereochemical inversion using the canonical MHC-I epitope from ovalalbumin (OVA), SIINFEKL. Through integrated assays measuring pMHC-I stability, T cell activation, cellular permeability, and serum stability, we demonstrate that tolerance to peptidomimetic modification is highly position-dependent. Specific N-methylated variants retained MHC binding and TCR engagement while exhibiting enhanced cytosolic accumulation, whereas peptoid and stereochemical substitutions were generally disruptive to TCR recognition and membrane permeability. Guided by these insights, we designed combinatorially modified peptides to probe the balance between immunogenicity and pharmacokinetic improvement, revealing that multiple modifications exert non-additive effects on immune recognition. Collectively, these findings establish design principles and provide a framework for balancing immune recognition with enhanced stability and permeability in peptidomimetic antigen design.