Table 2_Peptide optimization to improve antigen presentation and antitumor responses in sarcoma.docx

We developed an integrated computational and experimental workflow to identify and optimize HLA-A*02:01-restricted epitopes derived from sarcoma-associated TAAs. Candidate wild-type (WT) peptides were selected using NetMHCpan 4.1 based on predicted HLA binding affinity and subsequently engineered through systematic amino acid substitutions to generate heteroclitic variants. Optimized peptides were evaluated by structural modeling and molecular docking, followed by experimental in vitro validation using T2-cell HLA stabilization assays.

Their immunogenicity was assessed ex vivo in peripheral blood mononuclear cells (PBMCs) isolated from HLA-A*02:01-positive sarcoma patients using IFN-γ ELISpot and peptide–HLA dextramer analyses. The sequential optimization strategy generated heteroclitic variants with markedly improved predicted HLA-A*02:01 binding for most target proteins. Structural modeling indicated preservation of the peptide backbone and TCR-accessible surface for the majority of selected variants.

These predictions were supported experimentally: several heteroclitic peptides enhanced HLA-A*02:01 stabilization and prolonged peptide–MHC complex persistence in T2 cells. Ex vivo, optimized peptides induced stronger antigen-specific IFN-γ responses than their wild-type counterparts, with significant increases for SET, CAGE, PTPN1 and MAGE-A3, while multimer staining demonstrated recognition of heteroclitic peptide–HLA complexes by wild-type-primed CD8⁺ T cells.

These findings establish a transferable workflow for engineering more immunogenic, yet antigenically faithful, HLA-A*02:01-restricted epitopes. Rational heteroclitic optimization may therefore provide a useful platform for the development of next-generation therapeutic vaccines for sarcoma and other tumors with low intrinsic immunogenicity.</p>