An anti-VEGF designed ankyrin repeat protein (DARPin) has been successfully used in AMD models,17 and it is now being evaluated in clinical trials.18 In addition, an ODM-203 alternative class of antibodies, the camelid-derived nanobodies are in several trials (e.g., anti-IL-6R for autoimmune diseases19). lack the Fc domain name, as a safer alternative. To investigate the feasibility of this, anti-vascular endothelial growth factor (VEGF)-blocking antibodies in two formats were produced and tested and compared to larger standard antibodies, 16 thus acting on a greater area of the retina. An anti-VEGF designed ankyrin repeat protein (DARPin) has been successfully used in AMD models,17 and it is now being evaluated in clinical trials.18 In addition, an alternative class of antibodies, the camelid-derived nanobodies are in several trials (e.g., anti-IL-6R for autoimmune diseases19). However, we are unaware of any reports of anti-angiogenic antibodies in the scFv format being developed for AMD. Therefore, this is both a novel and highly relevant option to developing a safer, longer lasting, and convenient therapy for AMD. Furthermore, the combination of anti-VEGF scFv with an AAV2/8 vector may translate to an improved gene therapy for wet AMD. The purpose of this study was to provide preclinical data for an AAV2/8 vector encoding a previously described G6-31 anti-VEGF antibody20 in scFv format for possible translation as a novel therapy for AMD. Open in a separate window Physique?1 Characterization of Recombinant Anti-VEGF Antibodies (A) Schematic of the antibodies produced; to the left, a representation of the scFv format, and to the best, the full IgG format, adapted from Hansel et?al.35 (B) SDS-PAGE analysis of the anti-VEGF IgG1 format. The expected single 160-kDa band of the IgG1 antibody was observed in the absence of mercaptoethanol (?), while, in the presence of mercaptoethanol (+), dissociation into the heavy (55?kDa) and light chains (25?kDa) was seen. M refers RIEG to protein marker. (C) SDS-PAGE analysis of the anti-VEGF scFv format. The anti-VEGF scFv migrated at around 30?kDa (predicted molecular weight [MW]?= 32?kDa). (D and E) Antigen specificity of anti-VEGF antibodies. Binding of anti-VEGF antibodies to human or murine VEGF-coated ELISA plates was detected by anti-His for scFv (D) or anti-mouse HRP for IgG1 (E). Both formats of anti-VEGF and specifically bound both mouse and hVEGF. N/A refers to a control where no sample was added to the plate. N/C refers to a control where no VEGF was added to the plate. Bars represent the mean of samples that were added in duplicate, and error bars represent the SD. (F) Biological activity of anti-VEGF antibodies (bioassay). VEGF-dependent growth of HDMECs was blocked by adding increasing amounts of anti-VEGF antibody to the cells. The IgG and scFv forms of anti-VEGF as well as the positive control anti-VEGF (bevacizumab) were able to block growth in a dose-dependent fashion, indicating activity. The unfavorable control (Neg Con Ab, an anti-PDGFR- IgG1) was not active. Data points represent the mean of samples that were added in triplicate, and error bars represent the SD. Values are presented as percent relative to the first data point (0.2?ng/mL antibody). Results Production and Testing of Recombinant Anti-VEGF Antibodies The anti-VEGF antibodies used in this study were based on the G6-31 antibody described by ODM-203 Liang et?al.20 We incorporated the binding sites of G6-31 into both a standard (immunoglobulin G1 [IgG1]) and scFv format, and we evaluated these antibodies for effectiveness in treating a laser-induced choroidal neovascularization (CNV) mouse model. The IgG1 antibody protein was produced by transfecting 293F cells with a plasmid made up of both the heavy and light chains that later associate to form the mature IgG1 (see Physique?1A for illustration of antibodies used in this study). The scFv (variable heavy and variable light chains only) was encoded in a single plasmid and produced in a similar way. Both proteins gave high yields (>10?mg/L medium). After purification, SDS-PAGE analysis demonstrated the expected molecular weight (160?kDa for IgG1 and 32?kDa for scFv) and high purity of both of the antibody formats (Figures 1B and 1C). Importantly, both antibodies were able to bind both human and mouse VEGF in ODM-203 ELISA (Figures 1D and 1E). The blocking activity of both antibody formats was also confirmed in a bioassay (Physique?1F). This assay used varying amounts of antibody to block the human VEGF-dependent growth of.