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

The major histocompatibility complex class II-associated peptide proteomics assay is widely used during preclinical immunogenicity risk assessments to identify biotherapeutic-derived peptides. These potential T cell epitopes are presented by dendritic cells and may trigger CD4(+) T helper cell activation, which could lead to downstream anti-drug antibody secretion by plasma cells. Currently, the utility of this immunopeptidomics assay has often been restricted to studying human leukocyte antigen-DR receptors due to the lack of well-characterized human leukocyte antigen-DP, -DQ, and pan antibodies available. Here, we seek to accommodate major histocompatibility complex class II pan receptors by testing commonly commercially available antibody clones, and characterizing their specificity via the epitope prediction algorithm NetMHCIIpan. Although the application of these antibodies in the assay increased the identified compound-specific cluster profile, no individual antibody clone was able to recover the complete human leukocyte antigen II peptide repertoire. Our findings reveal that a mixed immunoprecipitation strategy utilizing a minimum of three antibody clones with differing specificities (human leukocyte antigen-DR-specific clone L243, pan-specific clone WR18, and -DQ-specific clone SPV-L3) leads to more robust compound-specific peptide detection in one single analysis. Ultimately, expanding the assay to leverage human leukocyte antigen pan receptors improves the predictability of additional 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 Tu39), 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.