Chien-Wei Wang

Characterization of M11-like and OC125-like Monoclonal Antibody Binding to CA125 Tandem Repeats

The CA125 epitope within the MUC16 tandem repeat region is detected via the CA125 II test for ovarian cancer surveillance. This test utilizes the M11 and OC125 antibodies. A revised model of MUC16 with 19 tandem repeats has recently been identified, including splice variants that exclude entire repeats. Additionally, OC125 has exhibited gaps in coverage of the tandem repeat region. To identify antibodies that bind more repeats and are suitable for spliceoform detection, more antibodies must be characterized using the revised model. This study characterized the binding of two M11-like and two OC125-like antibodies against the updated tandem repeat numbering system. 16 individual tandem repeats were expressed and purified. Binding interactions between each of the antibodies and recombinant repeats were examined by indirect enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). The M11-like antibodies displayed different binding patterns when compared to each other, while the two OC125-like antibodies exhibited similar binding patterns. M11-like clone M77161 bound to all 16 repeats tested, indicating that it may be suitable for accurate detection of CA125. These findings demonstrate how different antibodies vary in their binding to CA125, contributing to ongoing development of improved clinical and research tools for ovarian cancer.

A Revised Molecular Model of Ovarian Cancer Biomarker CA125 (MUC16) Enabled by Long-read Sequencing

Abstract The biomarker CA125, a peptide epitope located in several tandem repeats of the mucin MUC16, is the gold standard for monitoring regression and recurrence of high-grade serous ovarian cancer in response to therapy. However, the CA125 epitope along with several structural features of the MUC16 molecule are ill defined. One central aspect still unresolved is the number of tandem repeats in MUC16 and how many of these repeats contain the CA125 epitope. Studies from the early 2000s assembled short DNA reads to estimate that MUC16 contained 63 repeats. Here, we conduct Nanopore long-read sequencing of MUC16 transcripts from three primary ovarian tumors and established cell lines (OVCAR3, OVCAR5, and Kuramochi) for a more exhaustive and accurate estimation and sequencing of the MUC16 tandem repeats. The consensus sequence derived from these six sources was confirmed by proteomics validation and agrees with recent additions to the NCBI database. We propose a model of MUC16 containing 19—not 63—tandem repeats. In addition, we predict the structure of the tandem repeat domain using the deep learning algorithm, AlphaFold. The predicted structure displays an SEA domain and unstructured linker region rich in proline, serine, and threonine residues in all 19 tandem repeats. These studies now pave the way for a detailed characterization of the CA125 epitope. Sequencing and modeling of the MUC16 tandem repeats along with their glycoproteomic characterization, currently underway in our laboratories, will help identify novel epitopes in the MUC16 molecule that improve on the sensitivity and clinical utility of the current CA125 assay. Significance: Despite its crucial role in clinical management of ovarian cancer, the exact molecular sequence and structure of the biomarker, CA125, are not defined. Here, we combine long-read sequencing, mass spectrometry, and in silico modeling to provide the foundational dataset for a more complete characterization of the CA125 epitope.