| Epidermal growth factor receptor (EGFR) is a trans-membrane receptor tyrosine kinase that includes three domains:an N-terminal extracellular ligand-binding domain, a single hydrophobic trans-membrane-anchoring alpha helix region and a cytosolic C-terminal domain with tyrosine enzymatic activity. EGFR belongs to HER family of receptors and also called HER-1. Binding of EGFR to its cognate ligands leads to autophosphorylation of receptor tyrosine kinase and subsequent activation of signal transduction pathways that are involved in regulating cellular proliferation, differentiation, and survival. Besides presenting in normal cells, EGFR is also overexpressed in a variety of human solid tumor and has been associated with poor prognosis and decreased survival. The expression level of EGFR has been correlated with degree of tumor differentiation, malignancy, invasion and prognosis. Therefore, EGFR as a potential target for directed tumor therapy has become a new hot pot. Some small molecule drugs and antibody drugs against EGFR has been appeared on the market, e,g. the anti-EGFR chimeric antibody Cetuximab.Cetuximab, a mouse-human chimeric antibody, was mutated to human sequences on CDR and FR of mouse variable region via simulation. Consequently seven humanized antibodies (C1-C7) against EGFR were constructed and expressed. All L and H genes of those humanized antibodies were cloned into pIRES bicistronic expression vector. Binding activity of the humanized antibody to the antigen is determined by Biacore3000. Tests illustrated that the humanized antibody C3(3# antibody) has high antigen-binding activity. Based on this, in our work four light chains and three heavy chains of the seven antibodies were cloned into the vector pcDNA3.1 (+) and pcDNA3.1 (-) respectively. Finally four kinds of antibody light chain gene expression vector (named as PL1, PL2, PL3, and PL4) and three heavy chain gene expression vector (named as PHI, PH2 and PH3) were obtained. Cotransfected light chain and heavy chain expression vectors into 293T cells and detected expression level, affinity anlysis of the twelve antibodies by ELISE. The result show that all the twelve antibodies were expressed well and had good affinity with antigen EGFR except 3# antibody. It is caused by the fact that 3# antibody has a good affinity with antigen EGFR in early research. Then 3#,7#,10# and 11# antibody light and heavy chain gene fragments were cloned into a modified high expression pcDNA3.1 vector, and cotransfected into CHO cells to select stable cell lines with G418 resistance. Meanwhile, the expression of antibodies by SDS-PAGE, Western blot and ELISA were detected. The SDS-PAGE and western blot results showed that the molecular weight of light chain and heavy chain of the four antibodies were consistent with expectations. ELISA results showed that 7#,10# and 11# antibody had higher affinity with antigen, while the lower affinity of 3#. Next we expand 7#,10# and 11# antibodies cultivation scale of stable cell lines, and collect supernatant and purified antibodies through recombinant protein A affinity chromatography. Finally, the quality of 7#,10#,11# antibodies were 110mg, 151mg,190mg. Binding activity of the humanized antibody to the antigen was detected by Biacore3000, test revealed that the humanized antibody 7#,10#,11 #and Cetuximab had antigen-binding activity 3.87×10-9M,8.16×10-8M,4.29×10-8M and 3.74×10-8M. Comparing with the positive control, our results showed that the ability of binding antigen for 7#,10# and 11# is comparable, while among which 7# is even slightly better. The three humanized antibodies suppressed growth and migration of tumor cells by tumor cells crossed experiment.To conclude,12 humanized antibodies against EGFR were successfully constructed, of which only three antibodies had better affinity with antigen. The preliminary experimental results show that the three humanized antibodies suppress the growth and migration of tumor cells. |
