Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-basedde novosequencing

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.

Direct Mass Spectrometry-Based Detection and Antibody Sequencing of Monoclonal Gammopathy of Undetermined Significance from Patient Serum: A Case Study

“…We have recently demonstrated the direct MS-based sequencing of both recombinant and serum-derived antibodies, using bottom-up proteomics methods. ,,, Owing to the high abundance of the MGUS M-protein in the serum samples of this donor, we were able to sequence the full antibody without further fractionation. First, we used an in-gel digestion protocol, using four proteases of complementary specificity in parallel, to obtain overlapping peptides for de novo sequencing by LC-MS/MS analysis (Figure ).…”

“…B) ABodyBuilder2 structural model prediction of the MGUS Fab variable domain with HexNAc(5) Hex(5) Fuc(1) NeuAc(2) grafted on CDRH1 using GLYCAM. C) Glycosylation profile of MGUS Fab compared to typical Fc glycosylation at N297, according to Bondt et al 2014 (ref ( 23 )).…”

A handle on mass coincidence errors inde novosequencing of antibodies by bottom-up proteomics

“…27 Stitch has been shown to enable the accurate reconstruction of monoclonal antibody sequences, as well as the sequencing of isolated Fab fragments from patient serum, M-proteins in monoclonal gammopathies, antibody light chains from urine, and the profiling of whole IgG from COVID-19 patient sera. [28][29][30][31] Sequence accuracies of ~99% can be obtained, which is sufficient to reverse engineer functional antibody products. 13,22,23,32 Remaining sequencing errors stem in large parts from common mass coincidences of isobaric residues like leucine/isoleucine, but also from incomplete fragmentation spectra in which the order of two or more residues remains ambiguous due to lacking fragment ions for the intermediate positions.…”

“…In contrast, mass spectrometry-based methods can probe specific antibody sequences directly from the secreted polypeptide product, thereby circumventing the need to sample the antibody-producing B-cell clone and providing a direct glimpse into the so-called serum compartment of the immunoglobulin repertoire. 13–26…”

“…In contrast, mass spectrometrybased methods can probe specific antibody sequences directly from the secreted polypeptide product, thereby circumventing the need to sample the antibody-producing B-cell clone and providing a direct glimpse into the so-called serum compartment of the immunoglobulin repertoire. [13][14][15][16][17][18][19][20][21][22][23][24][25][26] Using a bottom-up proteomics approach, accurate peptide sequences can be determined and assembled into complete heavy and light chain sequences. We have recently developed the software tool Stitch to perform template-based assembly of peptide sequences against the coding gene segments of antibodies available in immunogenetics databases.…”