Understanding the Role of Cancer Glycosylation on Antibody Binding to MUC16

Abstract

Protein glycosylation is a common post‐translation modification where carbohydrates are anchored to a protein. Glycans play critical roles in mediating cell‐cell interactions, protein‐receptor signaling, and protein folding during translation. One of the hallmarks of cancer is aberrant glycosylation, where shortened glycans specific to cancer are observed. Tumorigenesis, tumor progression, and migration correlate with abnormal glycosylation. Overexpression of the membrane‐bound glycoproteins, MUC16, has been linked to poor prognosis and metastasis in pancreatic and ovarian cancer patients. A common feature of tumor‐associated MUC16 is altered glycosylation, revealing cryptic epitopes where antibodies can bind. These tumor‐specific antibodies can form the basis of new cancer immunotherapies. This project aims to establish an expression system using genetically modified Chinese Hamster Ovary (CHO) cell lines to produce cancer‐associated MUC16 with truncated O‐glycans. A CRISPR/Cas9 modified CHO cell line (CHO‐S□cosmc/mcherry+) was obtained, which has had the gene mcherry inserted into the cosmc locus, a mutation that is hypothesized to produce truncated O‐linked glycans. Here we report the purification and Enzyme‐Linked Immunosorbent Assay (ELISA) analysis of recombinant MUC16 produced in these cell lines. MUC16 in CHO (ExpiCHOwildtype and CHO‐S□cosmc/mcherry+)cell lines were transiently transfected and purified through the Nickel affinity chromatography. The purified protein was analyzed through SDS‐PAGE. Quantitative analysis of MUC16 binding to three different therapeutic antibodies was performed using ELISA. The results revealed that MUC16 produced in CHO‐Smcherry+displayed a higher binding affinity towards the antibodies than ExpiCHOwildtype. The recombinant proteins produced will be further used for examining the role of cancer‐associated glycans binding to therapeutic antibodies.