Poster Session - Abstract # 31
Characterizing the Tandem Repeat Region of Ovarian Cancer Biomarker CA125
Chien-Wei Wang2, Simon D. Weaver1, Lisa Minkoff1, Eliza Hanson2, Rebecca J. Whelan2
1Department of Chemistry and Biochemistry, University of Notre Dame, IN, USA; 2Department of Chemistry, University of Kansas, KS, USA
Current ovarian cancer monitoring relies on an FDA approved immunoassay called the CA125II test, which detects CA125, an epitope on the large mucin MUC16. However, the CA125II test can only track patient response to treatment and recurrence. The results of the CA125II test are not suitable for use in population-wide screening due to the missing knowledge of the exact location of antibody binding epitopes of anti-CA125 antibodies and the structure of mucin protein MUC16. MUC16 contains a heavily glycosylated N-terminal region, an intracellular C-terminal region and a tandem repeat region that was previously believed to contain 63 repeats and the CA125 epitopes. The highly repetitive nature of CA125 makes it a challenging subject for short-read sequencing and epitope mapping. In our study, we utilized the third-generation sequencing technique Oxford nanopore to solve the exact sequence of the CA125 tandem repeat region in three ovarian cancer cell lines and tumor tissue collected from three patients with high-grade serous ovarian cancer. The sequence was validated with bottom-up proteomics results on MUC16 that was immunoprecipitated, digested with trypsin, and analyzed with LC-MS/MS. With our new sequence results, we propose a new MUC16 model that contains only 19 tandem repeats. To better understand the binding epitopes of clinically used anti-CA125 antibodies OC125 and M11, we expressed different repeats individually as recombinant proteins. The binding affinity between different anti-CA125 antibodies were measured by a variety of affinity assays including Western blot, ELISA, and surface plasmon resonance. All three experimental methods showed variable antibody binding to all nine expressed repeats. Additionally, antibodies which have been categorized by their epitope as OC125-like group or M11-like group showed different binding patterns to their respective “-like” clones. These results help us better understand the ovarian biomarker CA125 and will be used for new affinity assay development.