Study On The Anti-angiogenenis Effect Of Humanized Anti-VEGF Monoclonal Antibody BD0801 By Two Kinds Of Biological Models

Objective:To investigate the antiangiogenic effect of humanized anti-VEGF monoclonal antibody BD0801 using two types of model organisms and discuss its mechanism preliminary.Methods:1 BD0801 single-agent assayZebrafish assay:24 hpf (hours post fertilization, hpf) fluorescent transgenic zebrafish with healthy blood vessels are used as animal models. After removal of the outer membrane, the models are randomly allocated into groups namely BD0801 low concentration group, BD0801 moderate concentrated group, BD0801 high concentration group, Bevacizumab (Bev) low concentration group, Bev moderate concentration group, Bev high concentration group, and control group (negative control). After administrating the drug to the embryos which are then incubated for 48 hours, the number of intersegmental vessels (ISV) formed are observed under fluorescent microscope using quantitive RT-PCR tests of zebrafish VEGF mRNA expression.Chicken chorioallantoic membrane (CAM) assay:Nine days old chick embryos were randomly divided into seven groups:BD0801 low concentration group, BD0801 moderate concentration group, BD0801 high concentration group; bevacizumab (Bev) low concentration group, Bev moderate concentration group, Bev high concentrations; saline control group (negative control group). After drying, the embryos are implanted in the methylcellulose discs before drugs for each group are given. After 72 hours, observation for new blood vessel sprouting or inhibition of angiogenesis within the field of the implanted disks are made and subsequently compared and analyzed.2 BD0801 combined-agents assayZebrafish assay:On the basis of positive results of single drug assay,24 hpf (hours post fertilization, hpf) fluorescent transgenic zebrafish with healthy blood vessels are used as animal models. After removal of the outer membrane, the models are randomly allocated into nine groups of combined agents. After administrating the drug to the embryos which are then incubated for 48 hours, the number of intersegmental vessels (ISV) formed are observed under fluorescent microscope using quantitive RT-PCR tests of zebrafish VEGF mRNA expression.Chicken chorioallantoic membrane (CAM) assay:On the basis of positive results of single drug assay, nine days old chick embryos were randomly divided into nine groups of combined agents. After drying, the embryos are implanted in the methylcellulose discs before drugs for each group are given. After 72 hours, observation for new blood vessel sprouting or inhibition of angiogenesis within the field of the implanted disks are made and subsequently compared and analyzed.Results:BD0801 single-agent assay1 BD0801 can significantly inhibit zebrafish ISV generation (P<0.05), demonstrated stronger inhibitory effect compared to the negative control group and its inhibitory effect was positively correlated with the concentration of the drug. RT-PCR test result shows:BD0801 can downregulate the level of VEGF mRNA in zebrafish embryos (P<0.05) similarly to the postive control but with stronger inhibitory effect and being concentration-dependent.2 BD0801 shows inhibitory effect of CAM angiogenesis (P<0.05), and compared with the negative control group, the inhibition effect is stronger while being positively correlated with the drug concentration.BD0801 combined-agents assay1 BD0801 single-agent group, Bev single-agent group, OXA single-agent group,5-FU single-agent group and combined-agents groups can significantly inhibit zebrafish ISV generation (P<0.05), demonstrated stronger inhibitory effect compared to the single-agent group(P<0.05). The inhibitory effect of BD0801 combined-agents group is stronger than Bev combined-agents group. RT-PCR test result shows:BD0801 single-agent group, Bev single-agent group, OXA single-agent group,5-FU single-agent group and combined-agents groups can downregulate the level of VEGF mRNA in zebrafish embryos (P<0.05), and the downregulating effect of combined-agents group is stronger than single-agent group. Compared with Bev combined-agents group, BD0801 combined-agents group shows stronger downregulating effect.2 BD0801 single-agent group, Bev single-agent group, OXA single-agent group,5-FU single-agent group and combined-agents groups show inhibitory effect of CAM angiogenesis (P<0.05), and compared with the single-agent group, the inhibition effect of combined-agents groups is stronger. Compared with Bev combined-agents group, BD0801 combined-agents group shows stronger inhibition effect.Conclusions:BD0801 can, with stronger inhibitory effect than Bevacizumab, significantly inhibit zebrafish and CAM angiogenesis, and BD0801 combined with OXA/5-FU can significantly enhance the inhibitory effect. This study point out that its mechanism may be associated with reduced VEGF mRNA level, hence worthy of further study.