title: Development of a robust reporter-based assay for the bioactivity determination of anti-VEGF therapeutic antibodies.
abstract: Development of anti-VEGF based biologic agents has been a focus in cancer treatment for the past decades, and several anti-VEGF pharmaceuticals have been already approved for treatment of various medical indications especially in cancer. The first anti-angiogenic agent approved by FDA was bevacizumab (BVZ, trade name Avastin, Genentech/Roche), a humanized anti-VEGF monoclonal antibody. Accurate determination of bioactivity is crucial for the safety and efficacy of therapeutic antibodies. The current method widely used in the lot release and stability test for clinical trial batches of BVZ is anti-proliferation assay using primary human umbilical vein endothelial cells (HUVEC), which is tedious with high assay variations. We describe here the development and preliminary validation of a reporter gene assay (RGA) that is based on an HEK293 cell line stably expressing vascular endothelial growth factor receptor 2 (VEGFR-2), and a luciferase reporter under the control of nuclear factor activated T cell (NFAT) response elements. Our study shows this assay not only to be superior on precision, sensitivity and assay simplicity compared with HUVEC assay, but also applicable to other VEGF-targeted biotherapeutics. These results show for the first time that this new reporter assay, based on the VEGF-VEGFR-NFAT pathway, can be a viable supplement to the HUVEC assay and employed in potency determination of BVZ and other kinds of anti-VEGF antibody-based biotherapeutics.
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Lan
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Gang-Ling
authors.initials: GL
authors.lastname: Xu
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Gao
authors.affiliation: Promega Corporation, Madison, WI, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wilkinson
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Yu
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Chun-Yu
authors.initials: CY
authors.lastname: Liu
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Chuan-Fei
authors.initials: CF
authors.lastname: Yu
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Wen-Bo
authors.initials: WB
authors.lastname: Wang
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Li
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: Promega Corporation, Madison, WI, United States.
authors.forename: Frank
authors.initials: F
authors.lastname: Fan
authors.affiliation: Promega Corporation, Madison, WI, United States.
authors.forename: Mei
authors.initials: M
authors.lastname: Cong
authors.affiliation: Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing, China. Electronic address: jzwang_nifdc@163.com.
authors.forename: Jun-Zhi
authors.initials: JZ
authors.lastname: Wang
mesh_terms: D000068258:Bevacizumab / Q000276:immunology*; D017930:Genes, Reporter; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D015203:Reproducibility of Results; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology*
chemical_list: D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab
keywords: Anti-VEGF pharmaceuticals; Bevacizumab; Bioassay; HUVEC assay; Reporter gene assay