Mass Spectrometry-Driven Epitope Mapping: Application of Diethylpyrocarbonate Covalent Labeling for the Immunotherapeutic Target Programmed Cell Death Protein 1

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.

Keywords: Covalent Labeling; Diethylpyrocarbonate; Epitope Mapping; Immuno-Oncology; Mass Spectrometry; Monoclonal Antibodies; Nivolumab; PD1.

1

Sequence coverage (yellow) and DEPC labeling structural coverage (green spheres) of PD1 (PDB ID: 3RRQ) in the presence of (a) W6/32, anti-human leukocyte antigen mAbs (nonbinding mAbs), (b) nivolumab, and (c) CU-MAB (anti-PD1 mAb). The sequence coverage of approximately 86% to 90% represents a range of pooled sequence coverages, each calculated from all replicates within each set (PD1/nivolumab complex, PD1/CU-MAB complex, or PD1 and W6/32 mAbs).

2

Dynamic light scattering (DLS) size distribution of PD1/anti-PD1 mAb complexes before and after DEPC labeling: (a) PD1/nivolumab and (b) PD1/CU-MAB.

3

DEPC CL-MS epitope mapping of PD1 bound to nivolumab (PDB ID: 5WT9). (a) Changes in DEPC modification levels (%CL) of PD1 residues upon binding to nivolumab. Red stars represent residues with increased labeling, and blue stars represent residues with decreased labeling. Statistical significance was determined using a t test with p < 0.10. (b) Structural representation of PD1 (green) bound to nivolumab (cyan and pale cyan), highlighting epitope and nonepitope residues identified by DEPC CL-MS. Red spheres indicate residues with increased %CL, while blue spheres indicate those with decreased %CL.

4

Structure of PD1 (green) upon binding with nivolumab (cyan and pale cyan) at the PD1 residues (a) S27, (b) S57, (c) S60, (d) S62, (e) K131, and (f) K135 (PDB ID: 5WT9). Red sticks indicate residues with increased %CL, while blue sticks indicate those with decreased %CL. The structure of unbound PD1 is shown in yellow (PDB ID: 3RRQ).

5

DEPC CL-MS epitope mapping of PD1 bound to CU-MAB. (a) Changes in DEPC modification levels (%CL) of PD1 residues upon binding to CU-MAB. Red stars represent residues with increased labeling, and blue stars represent residues with decreased labeling. Statistical significance was determined using a t test with p < 0.10. (b) AlphaFold structural prediction of PD1 (green) bound to CU-MAB (orange and light orange), highlighting epitope and nonepitope residues identified by DEPC CL-MS. Red spheres indicate residues with increased %CL, while blue spheres indicate those with decreased %CL.

6

Structure of PD1 (green) upon binding with CU-MAB (orange and light orange) at the PD1 residues (a) S27, (b) S57, (c) S127, and (d) K131 (from AlphaFold structural prediction). Red sticks indicate residues with increased %CL, while blue sticks indicate those with decreased %CL. The structure of unbound PD1 is shown in yellow (PDB ID: 3RRQ).