Identification of CD24 as a therapeutic antibody target for ovarian cancer

Abstract

Ovarian cancer is the second most common gynecologic cancer. It has the highest mortality rate among gynecologic cancers, and more than 50% of patients with ovarian cancer die. CD24 is a mucin-type glycoprotein with a small protein core that is a marker of B-cell differentiation. It is highly glycosylated and is anchored on the membrane via glycosylphosphatidylinositol (GPI). Recently, the role of CD24 in tumor biology has received considerable attention. It is involved in cell adhesion, metastatic tumor spread, and p-selectin binding. It has also recently been described as a diagnostic tumor marker, a marker of neuroendocrine differentiation, and, the most intriguing of all, of patient prognosis. Because CD24 is overexpressed in a variety of carcinomas such as ovarian cancer, breast cancer, non-small cell lung cancer, prostate cancer, and pancreatic cancer, it is a promising target for cancer therapy. Here we verified the usefulness of CD24 as a target for anticancer therapy in several breast cancer and ovarian cancer cell lines. We also studied the development of CD24 as a target protein for anticancer therapy. In a previous study in our lab, we observed p-selectin binding (an important function of CD24) by ELISA. We treated mice with mAbs targeting CD24. Compared with mice in the control group, mice treated with mAbs exhibited meaningful changes in tumor growth and apoptosis. We analyzed CD24 expression in the cell lines by RT-PCR, real-time PCR, western blotting, and FACS analysis. These results suggested that CD24 is a potential molecular target for therapeutic antibody development. A second aim of this study was to generate a human scFv antibody against CD24 and evaluate its affinity for CD24 as a step toward developing a human monoclonal antibody against CD24. To screen the antibodies, we identified the optimal CD24 epitope through structural and functional analyses, and then produced recombinant CD24 in a mammal cell line to reconstruct the glycosylated form of CD24 in vivo. Isolated, single-chain variable antibody fragments (scFv) were selected after 4 rounds of biopanning and secondary screening by ELISA and sequence analysis. The affinity of the selected scFvs was measured by flow cytometry and surface plasmin resonance (SPR) with a CD24-overexpressing cancer cell line. In conclusion, in this study, we demonstrated that CD24 expression is a marker for ovarian cancer and we identified anti-CD24 scFvs that could be developed as potential therapeutic anticancer antibodies