Synthesis and immune response of non-native isomers of vascular endothelial growth factor

Native proteins often lack immunogenicity and thus limit vaccine and mAb development. We described here a unique method to enhance the immunogenicity of native proteins. This is achieved by creating non-native isomers of disulfide proteins (X-isomers) using the method of disulfide scrambling. X-isomers have the potential to be developed as vaccines and effective immunogens, as they are capable of breaking the immune tolerance and eliciting antibodies that cross-react with the native protein. In this report, we describe production of X-isomers of vascular endothelial growth factor (X-VEGF). The aim is to develop X-VEGF for cancer immunotherapy targeting reduction of VEGF. The production of mouse X-VEGF is achieved by expressing the short version of VEGF (1-110) commonly shared by all VEGF isoforms, with two Cys --> Ala mutations at Cys(51) and Cys(60) to generate R-VEGF(110) (R stands for fully reduced). R-VEGF(110) was then allowed to undergo oxidative folding in the absence of denaturant to form N-VEGF(110) (N stands for native) or in the presence of denaturant to generate five fractions of X-VEGF(110) isomers. While N-VEGF(110) exhibits only marginal immunogenicity in mice, all five fractions of X-VEGF(110) isomers were shown to elicit high titers of antibodies that cross-react with N-VEGF(110). In sera of immunized mice, the amounts of anti-N-VEGF antibodies elicited by X-VEGF(110) isomers range from 54 to 186 mug/mL, which are compatible with or greater than the concentration required for effective therapy using anti-VEGF MAbs. The underlying mechanism of enhanced immunogenicity of X-VEGF(110) is investigated and elaborated. These data suggest that X-VEGF(110) isomers are potential compounds in developing active immunotherapy for treatment of VEGFR bearing tumors and the wet form of age-related macular degeneration.