Abstract:
Monoclonal antibodies (mAbs) have revolutionized immuno-oncology, with anti-programmed cell death protein 1 (PD1) mAbs emerging as key therapeutic agents in cancer treatment. This study presents the development and application of diethylpyrocarbonate (DEPC) covalent labeling-mass spectrometry (CL-MS) for detailed epitope mapping of anti-PD1 mAbs on PD1. By using DEPC CL-MS, we aimed to identify precise antibody binding sites on PD1 and benchmark its effectiveness against traditional X-ray crystallography. DEPC CL-MS offers high sensitivity and specificity while requiring less sample preparation and shorter analysis times, typically taking days or less, instead of months. PD1 was individually incubated with either nivolumab or a novel anti-human PD1 mAb (CU-MAB), followed by DEPC labeling, to assess DEPC modification extents under both binding and nonbinding conditions using bottom-up LC-MS/MS. Significant changes in DEPC modification at residues S27, S60, S62, S127, and K131 indicated binding sites and conformational shifts upon antibody interaction. These findings showed strong alignment with crystallography (PD1/nivolumab) and AlphaFold structural predictions (PD1/nivolumab and PD1/CU-MAB), highlighting the value of in-solution CL-MS for confirming AlphaFold predictions. This study underscores DEPC CL-MS as an efficient tool for epitope mapping, offering actionable insights into PD1–antibody interactions to drive therapeutic antibody development.
