Purification of VEGF-trap and its antiangiogenic effect

Abstract

Vascular endothelial growth factor (VEGF) exerts a crucial role during pathological angiogenesis and normal physiology. It has been the target of therapeutics for tumors and other diseases. Blocking VEGF signal pathway by using monoclonal antibodies targeted against VEGF or VEGFR shows the clinical promise in tumor therapeutics. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. It is reported that VEGF-Trap, fusing the second Ig domain of VEGFR1 with the third Ig domain of VEGFR2, is a promising molecule to inhibit the binding of VEGF and its receptors. VEGF-Trap binds to VEGF with substantially high affinity, and has minimal interactions with extracellular matrix. This property apparently accounts for its satisfying pharmacokinetic profile. VEGF-Trap is cloned using standard molecular cloning methods. The gene amplification procedure offered by the use of dihydrofolate reductase-deficient $(DHFR^-)$ Chinese hamster ovary (CHO) cells with DHFR-mediated gene amplification is selected to produce therapeutic VEGF-Trap proteins. This system is based on DHFR gene coding for DHFR enzyme that catalyzes the conversion of dihydrofolate to tetrahydrofolate. Methotrexate (MTX) binds to and inhibits the DHFR enzyme, leading to cell death. However, $DHFR^-$ CHO cells, which have taken up an expression vector containing the DHFR gene, can develop resistance to it by amplifying the DHFR gene. With this mammalian expression system, three parental clones selected from 14 parental clones are cultivated independently throughout the gene amplification procedure. It takes approximately 21 weeks to obtain high-producing clones such as C1 and C8. Recombinant proteins are purified by using protein A sepharose affinity chromatography. Thereafter, we inject the purified proteins to the mice to investigate the effects of VEGF-Trap on tumor-...