Expression and Epitope Characterization of a Recombinant CA 125 Repeat: Fourth Report from the ISOBM TD-1 Workshop

Abstract

Background: CA 125 antigenic domains appear to reside within a region containing 156-amino acid sequence repeats. Surprisingly, anti-CA 125 antibodies can be classified into three families (groups A, B and C) indicating limited epitope diversity. In this study we describe the heterologous expression of a CA 125 repeat unit (R11) and an analysis of its epitope topography. Methods: R11 was expressed using a baculovirus approach and purified from culture supernatants by sequential ion exchange chromatography. Monoclonal antibody binding was assessed using antigen capture and cross-inhibition methods. Results: The recombinant repeat was purified to 2.5 × 10⁷ U/mg. Although a number of group A and B monoclonal antibodies were found to bind R11, the prototype antibody OC125 (group A) showed little reactivity. However, the prior binding of some group B monoclonal antibodies dramatically enhanced subsequent OC125 binding. Low monoclonal antibody reactivity to R11 correlated well with poor binding to SDS-denatured human ascites CA 125. Conclusion: The ability to ‘activate’ R11 epitopes indicates that some may not be displayed optimally on isolated repeats. This observation, together with the concordance between monoclonal antibody binding to R11 and denatured CA 125, suggests that a number of epitopes are preferentially displayed only when contained within multiple repeat domains.

MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress

“…Several antibodies, M11, VK8 and OV197 among them, were subsequently developed against CA125 [56,57]. Based on their binding specificities, CA125-specific antibodies are grouped as belonging to the OC125, M11 or OV197 families [58-61]. …”

“…Studies with recombinant R11 indicate that binding of OC125 to this antigen is enhanced several-fold after pre-binding of M11 [61]. Whether enhancement—or inhibition—of OC125 binding is also observed with other recombinant repeats of MUC16, or the entire molecule in patient sera, needs to be determined.…”

Overcoming Soluble Target Interference in An Anti-Therapeutic Antibody Screening Assay For An Antibody–Drug Conjugate Therapeutic

“…Monoclonal antibodies targeting CA125 have been extensively characterized and are grouped according to their binding properties to the three antigenic domains identified for this molecule: A or OC125-like, B or M11-like, and C or OV197-like [19]. In our study, three commercially available mAbs with specificities for domains A, B and C and six in-house produced mAbs (domain specificity unknown) were evaluated for their ability to block CA125 in the ATA assay.…”

Deciphering the Molecular Nature of Ovarian Cancer Biomarker CA125

“…These antibodies are classified as OC125-like (group A), M11-like (group B) or OV197 (group C) depending on the antigenic determinant recognized [31]. These antibodies are further subdivided into groups A1–A4 (OC125-like), B1/B2 (M11-like) and C1/C2 (OV197-like) [32,33]. It is important to note that although there are two antibodies classified as OV197-like, the antigenic recognition behavior of OV197 is different to that of 7C12 [32].…”