Structural And Functional Characterazation Of A Recombinant Anti-EGFR Monoclonal Antibody

The epithelial growth factor receptor(EGFR) is a transmembrane glycoprotein encoded by a proto oncogene C-erbB-1(HER-1) with approximate molecular weight of 170 kDa. The EGFR can bind specifically to the epithelial growth factor(EGF) or other related ligands and stimulate cell proliferation by activating the cellular signal transduction pathway. The EGFR is constitutively expressed in many normal epithelial tissues, including the skin and hair follicle. Overexpression of EGFR is also detected in many human cancers including those of the head and neck, colon, and rectum. It is believed that the EGFR plays an important role in tumor cell proliferation, new blood vessel formation, tumor invasion and metastasis, inhibition of apoptosis and so on.It is suggested that EGFR may be a potential therapeutic target for tumors.An anti-EGFR monoclonal antibody can bind specifically to the EGFR on both normal and tumor cells, and can competitively inhibit the binding of EGF and other ligands, such as transforming growth factor-alpha. In 2004, the FDA(the Food and Drug Administration) approved cetuximab(Erbitux?, made by Imclone Systems, Inc.), a monoclonal antibody directed against EGFR. Erbitux is approved for use, in combination with irinotecan, for the treatment of EGFR-expressing, metastatic colorectal carcinoma in patients who are refractory to irinotecan-based chemotherapy. In 2009, Erbitux was permitted into China for the same use.Erbitux is produced in mammalian cell(SP2/O murine myeloma cell line) culture.Shanghai National Engineering Research Center of Antibody Medicine(CMAB) designed and produced a recombinant anti-EGFR monoclonal antibody. It is a follow-on biologic of Erbitux. The follow-on biologic is produced in mammalian cell(the chinese hamster ovary cell line) culture. It is believed that different cell lines and cell culture conditions can affect the glycosylation of monoclonal antibodies, and the glycosylation may also influence the structure and function of proteins.In this study, we took Erbitux as control, conducted a series of research on the follow-on biologic. The detailed structures information between the follow-on biologic and Erbitux, including amino acid sequences and glycosylation profiles, were successfully characterized at levels of intact protein subunits and free oligosaccharides using liquid chromatography electrospray ionization quadrupole time-of-fight mass spectrometry(LC-ESI-QTof MS). The combined data revealed that the follow-on biologic showed the same amino acid sequences as Erbitux and distinct glycosylation patterns from Erbitux. Furthermore, it is confirmed that the major of sialylation is NANA in the follow-on biologic instead of NGNA in Erbitux. The follow-on biologic almost does not contain galactose- alpha-1,3-galactose, whereas Erbitux contains more. The high mannose glycosylation of Erbitux is 6 times more than that of the follow-on biologic.The glycosylation patterns of Erbitux indicate the risk of immunogen.We would like to reduce the risk by producing the follow-on biologic in CHO cell line which was considered close to human cells. Furthermore, the advanced structures of proteins between the follow-on biologic and Erbitux are very similar.In vitro, the follow-on biologic can mediate antibody-dependent cellular cytotoxicity(ADCC) against human epidermoid tumor A431 cells.And the follow-on biologic exhibited good performances in the binding ability, inhibition of cell proliferation and ADCC. In vitro assays and in vivo animal studies have shown that binding of the follow-on biologic to the EGFR can block the phosphorylation of EGFR, resulting in inhibition of the growth and survival of tumor cells.In conclusion, the follow-on biologic and Erbitux have different glycosylation patterns due to being producd in the different cell lines and cell culture conditions. The advanced structures of proteins between two are very similar. In vitro assays and in vivo animal studies have confirmed that the follow-on biologic was anticipated to be a candidate drug for tumors.