Characterization of Binding Epitopes of CA125 Monoclonal Antibodies

Abstract

The most used cancer serum biomarker is the CA125 immunoassay for ovarian cancer that detects the mucin glycoprotein MUC16. Several monoclonal antibodies (mAbs) including OC125 and M11 are used in CA125 assays. However, despite considerable efforts, our knowledge of the molecular characteristics of the recognized epitopes and the role played by glycosylation has remained elusive. Here a comprehensive set of recombinant MUC16 tandem repeats (TRs) expressed in glycoengineered mammalian cells and E. coli, together with overlapping peptides, was used to probe antigen-binding epitopes. We present a complete analysis of N-and O-glycosylation sites of a MUC16 TR expressed in CHO cells and demonstrate that neither N-nor O-glycosylation appear to substantially influence binding of OC125 and M11 mAbs. A series of successive N-and C-terminal truncations of a MUC16 TR construct expressed in E. coli narrowed down the epitopes for OC125 and M11 to a segment containing parts of two consecutive SEA domains with a linker. Thus, a complete SEA domain is not required. These findings suggest that binding epitopes of mAbs OC125 and M11 are dependent on conformation but not on glycosylation. The availability of recombinant TR constructs with and without aberrant glycosylation now opens the way for vaccine studies.

Altered (neo-) lacto series glycolipid biosynthesis impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells

“…This technology was first demonstrated by Henrik Clausen and his colleagues, through the introduction of the ‘Simple Cell’ strategy, to study the role of truncated O -glycans in malignant diseases2324, specificity of anti-glycopeptide antibodies25, profile and map the human O -GlcNAc glycoproteome2627, and produce novel antibodies to defined glycopeptides28. Whilst a majority of this work is based on O -glycans, there is very limited information on the use of genome- editing for glycosphingolipids (GSLs) and in particular, the nsGSLs.…”

Epigenetic activation of MGAT3 and corresponding bisecting GlcNAc shortens the survival of cancer patients

“…In particular, in ovarian cancer CA125 is the most commonly used tumor marker and is glycosylated at threonine (T) or serine (S) and asparagine (N), resulting in O - and N -glycopeptides, respectively [4]. The synthesis of N -glycosylation at the consensus sequence, Asn-X-Ser/Thr, of a polypeptide chain involves a complex biosynthetic pathway in which the core glycan Glc 3 Man 9 GlcNAc 2 is trimmed by several glycosidases to form high mannose N -glycans, and further processed in the Golgi apparatus by the sequential action of GlcNAc T-I (MGAT1) to form hybrid N -glycans and subsequently by GlcNAc T-II (MGAT2) to form bi-antennary complex type N -glycans [5].…”

Human conjunctival goblet cells express the membrane associated mucin MUC16: Localization to mucin granules

“…To immunohistochemically localize MUC16 either of two monoclonal antibodies both specific to the SEA Module domain (Marcos-Silva et al, 2014), OC125 (Dako Corp., Carpenter, CA) and M11 (NeoMarkers, Fremont, CA), were applied (1:50 dilution in PBS for OC125 and 1:500 in PBS for M11) to frozen sections of conjunctiva (N=6 tissue samples for each antibody) as previously described (Argueso et al, 2003). Post-embedding immunoelectron microscopy using the monoclonal antibody H185, which recognizes a carbohydrate epitope on MUC16 (Argueso et al, 2003; Argueso and Sumiyoshi, 2006), or monoclonal antibody OC125 was done on LRWhite embedded conjunctival impression cytology samples (N=10) as previously described (Blalock et al, 2007; Danjo et al, 1998).…”

“…As demonstrated in Figure A and B, two different monoclonal antibodies, OC125 and M11, both of which recognize SEA modules regions of the protein backbone of the ectodomain of MUC16 (Marcos-Silva et al, 2014) bind to mucin packets of human conjunctival goblet cells. Both antibodies also bind to apical surfaces of the epithelium as have been previously described (Argueso et al, 2003), (Blalock et al, 2007).…”