Transient Expression Of Humanized AGR2 Monoclonal Antibody And The Preliminary Druggability Evaluation

Anterior gradient-2(AGR2)gent was the homologous gene of the Xenopus anterior gradient-2(XAG2), which was first identified in MCF-7. This protein plays an important role in tumor growth, metastasis, and drug resistance. And rhAGR2-mAb is a humanized monoclonal antibody which can specifically binds AGR2 to suppress proliferation and migration of tumor cells effectively. In this study, the HEK293 F was transient transfected to express the rhAGR2-mAb, followed by purified to prepare rhAGR2-mAb. This recombinant protein was compared with that expressed in CHO cells in molecular weight, isoelectric point, affinity and glycosylation. Pharmacokinetic and acute toxicitystudy of this recombinant antibody were taken for evaluation the metabolism and safety of rhAGR2-mAbIn this study, plasmids including light and heavy chains were transfected into HEK293 F cells simultaneously with PEI as the transfection reagent. The optimized conditions for rhAGR2-mAb expression in 5mL culture volume were the cell density is 6×106 cells/m L, DNA concentration is 0.5 μg/106 cells and PEI is 2 μg/106 cells, and the ratio of two plasmids of heavy and light chains is 1:1. Then transient transfection was linear amplified in 30 mL, 100 mL, 350 m L culture volume, and there were not significant loss of the protein.The purity of rhAGR2-mAb could be up to 95% after Protein A affinity chromatography, andit was found that this antibody could inhibit the proliferation of breast cancer cell MCF-7 significantly. Compared the the expression products of HEK293 F cells and CHO cells, there were no detected difference on molecular weight, isoelectric point, affinity and glycan structures. However, relative percent of glycosylation profiles were different between them.Using the validated ELISA method for detecting pharmacokinetics of the rhAGR2-mAb in rats plasma, it was found that when administration with rhAGR2-mAb of 5.6mg/kg in rat, the half-life of rhAGR2-mAb was about 11 days, and the AUC up to(30001.95 + 992.07)mu g/mL * h. With the acute toxity study in mice, histological examination of organs(heart, liver, spleen, lungs and kidneys) suggested that no visible tissue damage was induced by rhAGR2-mAb at a dose of 100mg/kg.