Anti-kdr Antibody Gene And Its Fab-like Gene Engineering Antibody Expression Vector Construction And Expression Studies

Angiogenesis is a fundamental mechanism of cancer growth and invasion, it describes the multistep process of new blood vessels formation from existing vasculature, In theory, inhibition of tumor angiogenesis may reduce tumor perfusion and increase tumor hypoxia and chemoresistance, and at last suppresses tumor growth. In Recent years, many preclinical results so far indicated that tumor-associated vasculature may be a relevant target to inhibit tumor growth. A lot of growth factors have been implicated as possible regulators in this process of angiogenesis and among them, VEGF is the key proangiogenic mediator, it acts as an endothelial cell- specific mitogen during angiogenesis. Furthermore, VEGF is the growth factor most consistently found in a wide variety of conditions associated with angiogenesis.VEGF mediates its activity mainly through two tyrosine kinase receptors, the fms-like tyrosine kinase (Flt-1, VEGFR-1) and the kinase insert domain-containing receptor (KDR, VEGFR-2). Numerous studies have showed that overexpression of VEGF and its receptors plays an important role in tumor-associated angiogenesis . Of the two receptors, KDR is generally believed to be the main receptor that mediates VEGF biological activities and thus plays a major role in tumor-associated angiogenesis. Taken together, these results suggest that KDR may represent good targets for developing anti-angiogenic therapeutics.KDR belongs to the subfamily of class III receptor tyrosine kinases (RTKs) and is characterized by 7 immunoglobulin (Ig)-like domains in the extracellular region and a split kinase intracellular domain. Deletion mutant analysis demonstrated that KDR extracellular Ig-like domain II and III are sufficient for high affinity binding of VEGF. Deletion of domain III alone caused more than 1000-fold reduction in binding affinity, indicating a critical role for this domain in VEGF/KDR interaction.Ycom1D3 (IgG1) is identified as a anti-KDR extracellular domain III specific monoclonal antibody (mAb) prepared by our lab, it could specifically bind to both the soluble protein (KDR extracellular domain III )and KDR expressed on endothelial cells and blocked VEGF binding to KDR, therefore it could indirectly inhibit the proliferation of human umbilical vein endothelial cells (HUVECs).In order to reduce the immunogenicity of murine-derived monoclonal antibody, a Fab-like engineering antibody from anti-KDR mAb Ycom1D3 was designed for its clinical application in the future. The genes encoding for the heavy- and light- chain variable regions (V_H and V_L) had been cloned by RT-PCR from murine hybridoma cells by using extensive overlap splicing PCR. The VH and VL genes were cloned into two different interstep vectors pB11 and pB12, which containing constant region ofκandγ-CH1 of human antibody, respectively. After that, the two constructed interstep vectors were transformated into E.coli and amplified for extracted plasmids. At last, light chain and heavy chain Fd were separately digested from the two interstep vectors shown as above and ligated into a expression vector, pAYZ, to construct a new expression vector pAYZKDRFab, which containing two cistrons(light chain and heavy chain Fd) controlled under one promotor. The new expression vector pAYZKDRFab was induced to express anti-KDR Fab-like engineering antibody fragment in E. Coli 16C9, The results of the expression of Fab antibody fragment were analyzed by SDS-PAGE and Western blot, it showed that the recombinant anti-KDR Fab gene could be induced to express in E. Coli 16C9, but the yield was low. ELISA tests showed that the anti-KDR Fab fragment had antigen-specific binding activity, and it shared the same binding site with its parental mAb Ycom1D3, and that it could also block VEGF/KDR interaction effectively, so it layed the foundation for anti-angiogenesis research and clinical application in the future.