Expanding the MAPPs Assay to Accommodate MHC-II Pan Receptors for Improved Predictability of Potential T Cell Epitopes

A critical step in the immunogenicity cascade is attributed to human leukocyte antigen (HLA) II presentation triggering T cell immune responses. The liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based major histocompatibility complex (MHC) II-associated peptide proteomics (MAPPs) assay is implemented during preclinical risk assessments to identify biotherapeutic-derived T cell epitopes. Although studies indicate that HLA-DP and HLA-DQ alleles are linked to immunogenicity, most MAPPs studies are restricted to using HLA-DR as the dominant HLA II genotype due to the lack of well-characterized immunoprecipitating antibodies. Here, we address this issue by testing various commercially available clones of MHC-II pan (CR3/43, WR18, and Tü39), HLA-DP (B7/21), and HLA-DQ (SPV-L3 and 1a3) antibodies in the MAPPs assay, and characterizing identified peptides according to binding specificity. Our results reveal that HLA II receptor-precipitating reagents with similar reported specificities differ based on clonality and that MHC-II pan antibodies do not entirely exhibit pan-specific tendencies. Since no individual antibody clone is able to recover the complete HLA II peptide repertoire, we recommend a mixed strategy of clones L243, WR18, and SPV-L3 in a single immunoprecipitation step for more robust compound-specific peptide detection. Ultimately, our optimized MAPPs strategy improves the predictability and additional identification of T cell epitopes in immunogenicity risk assessments.