Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry-based de novo sequencing

Area of Science:

• Biochemistry
• Immunology
• Structural Biology

Background:

• Mucin 1 (MUC1) is a transmembrane mucin found on epithelial cells.
• Aberrant MUC1 expression and glycosylation are hallmarks of adenocarcinomas.
• MUC1's variable number of tandem repeats (VNTR) are heavily O-linked glycosylated.

Purpose of the Study:

• To enable reverse-engineering of the anti-MUC1 hybridoma-derived 139H2 IgG antibody.
• To elucidate the molecular basis of 139H2 binding specificity to MUC1.
• To understand 139H2's tolerance to MUC1 O-glycosylation.

Main Methods:

• Direct mass spectrometry-based sequencing of 139H2 IgG.
• Crystal structure determination of the 139H2 Fab fragment in complex with the MUC1 epitope.

Main Results:

• Successful reverse-engineering of the functional recombinant 139H2 monoclonal antibody.
• Elucidation of the molecular interactions between 139H2 and the MUC1 epitope.
• Demonstration of 139H2's binding specificity and tolerance to MUC1 O-glycosylation.

Conclusions:

• The sequence of 139H2 provides a foundation for developing novel MUC1-targeting strategies.
• This work facilitates advancements in diagnostics and therapeutics for MUC1-expressing cancers.
• Understanding MUC1-antibody interactions is key for cancer research and treatment development.