Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-based de novo sequencing
preprint · bioRxiv · 2023
| Date | 2023-07-05 |
| Type | preprint |
| Venue | bioRxiv |
| Publisher | openRxiv |
| Contribution | downstream-application |
| DOI | 10.1101/2023.07.05.547778 |
| Citations (OpenAlex) | 7 |
Peer-reviewed version: Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry–based de novo sequencing (2024-06-01, Life Science Alliance)
Abstract
Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at different mucosal surfaces including breast and intestine. In the gastrointestinal tract, MUC1 has a barrier function against bacterial invasion, but can also serve as an entry receptor for pathogenic Salmonella bacteria. Moreover, MUC1 is well known for its aberrant expression and glycosylation in adenocarcinomas The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated.. Monoclonal antibodies against the MUC1 VNTR can be powerful tools because of their multiplicity of binding and possible applications in the diagnosis and treatment of MUC1-expressing cancers. One such antibody is the hybridoma mouse monoclonal 139H2, which is also widely used as a research tool to study non-cancer MUC1. Here we report direct mass spectrometry-based sequencing of hybridoma-derived 139H2 IgG, which enabled reverse engineering of a recombinant 139H2. The performance of the reverse engineered 139H2 IgG and its Fab fragment were validated by comparison to the hybridoma-derived product in Western blot and immunofluorescence microscopy. The reverse engineering of 139H2 allowed us to characterize binding to the VNTR peptide epitope by surface plasmon resonance (SPR) and solve the crystal structure of the 139H2 Fab fragment in complex with the MUC1 VNTR peptide. These analyses reveal the molecular basis for 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostics, targeting and treatment strategies.
Methods and tools
- Anti-MUC1 139H2 reverse engineering: Reverse-engineers the anti-MUC1 antibody 139H2 by mass spectrometry-based de novo sequencing, MUC1 being a mucin whose aberrant expression and glycosylation matter in adenocarcinoma.