High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening

Abstract

Isolating high-affinity antibodies against native tumor antigens on the cell surface is not straightforward using standard hybridoma procedures. Here, we describe a combination method of synthetic peptide immunization and high-throughput flow cytometry screening to efficiently isolate hybridomas for cell binding. Using this method, we identified high-affinity monoclonal antibodies specific for the native form of glypcian-3 (GPC3), a target heterogeneously expressed in hepatocellular carcinoma (HCC) and other cancers. We isolated a panel of monoclonal antibodies (YP6, YP7, YP8, YP9 and YP9.1) for cell surface binding. The antibodies were used to characterize GPC3 protein expression in human liver cancer cell lines and tissues by flow cytometry, immunoblotting and immunohistochemistry. The best antibody (YP7) bound cell surface-associated GPC3 with equilibrium dissociation constant, K_D = 0.3 nmol/L and was highly specific for HCC, not normal tissues or other forms of primary liver cancers (such as cholangiocarcinoma). Interestingly, the new antibody was highly sensitive in that it detected GPC3 in low expression ovarian clear cell carcinoma and melanoma cells. The YP7 antibody exhibited significant HCC xenograft tumor growth inhibition in nude mice. These results describe an improved method for producing high-affinity monoclonal antibodies to cell surface tumor antigens and represent a general approach to isolate therapeutic antibodies against cancer. The new high-affinity antibodies described here have significant potential for GPC3-expressing cancer diagnostics and therapy.

Keywords: :

• cell-surface glycoproteins
• flow cytometry
• heparan sulfate proteoglycans
• hepatocellular carcinoma
• high-throughput screening
• hybridoma technology
• peptide immunization

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This study was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute (NCI), Center for Cancer Research. Satoshi Nagata was supported by a NIH COBRE grant (1P20RR024219–01A2). We thank our colleagues Heungnam Kim for establishing the A431/G1 cell line, Mingqian Feng and Ruoqi Wang for producing recombinant GPC3 and GPC3ΔHS proteins, and the NIH Fellows Editorial Board for editorial assistance.