PT  - JOURNAL ARTICLE
AU  - Peng, Weiwei
AU  - Giesbers, Koen CAP
AU  - Šiborová, Marta
AU  - Beugelink, J Wouter
AU  - Pronker, Matti F
AU  - Schulte, Douwe
AU  - Hilkens, John
AU  - Janssen, Bert JC
AU  - Strijbis, Karin
AU  - Snijder, Joost
TI  - Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry–based de novo sequencing
AID  - 10.26508/lsa.202302366
DP  - 2024 Jun 01
TA  - Life Science Alliance
PG  - e202302366
VI  - 7
IP  - 6
4099  - http://www.life-science-alliance.org/content/7/6/e202302366.short
4100  - http://www.life-science-alliance.org/content/7/6/e202302366.full
SO  - Life Sci. Alliance2024 Jun 01; 7
AB  - Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at mucosal surfaces. MUC1 has a barrier function against bacterial invasion and 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 are powerful research tools with applications in the diagnosis and treatment of MUC1-expressing cancers. Here, we report direct mass spectrometry–based sequencing of anti-MUC1 hybridoma-derived 139H2 IgG, enabling reverse-engineering of the functional recombinant monoclonal antibody. The crystal structure of the 139H2 Fab fragment in complex with the MUC1 epitope was solved, revealing the molecular basis of 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 diagnostic, targeting, and treatment strategies.The raw LC-MS/MS files and analyses have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD043489. Coordinates and structure factors for 139H2 bound to the MUC1 epitope peptide have been deposited to the Protein Data Bank with the accession code 8P6I. The plasmids for 139H2 expression in mammalian cells are made available through Addgene, under plasmid ID 206201 and 206202 for the heavy and light chains, respectively.