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Hanieh Khalili, Steve Brocchini, Peng Khaw; Bivalent ranibizumab is more active in vitro than bevacizumab. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3279.
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Ranibizumab is a monovalent Fab therapeutic. Its complimentary binding region (CDR) has a higher apparent affinity for VEGF than does the CDR in bevacizumab which, as a full IgG, is bivalent. We have developed a practical method to make Fab-PEG-Fab conjugates that mimic IgG molecules. Conjugation of the Fab to PEG occurs essentially in the same place where the Fab is linked within a native IgG. We hypothesised that two ranibizumab (Fabrani) molecules could be conjugated to give Fabrani-PEG-Fabrani which would achieve equivalent or better binding than bevacizumab.
Ranibizumab was conjugated with a thiol-specific bis-alkylation PEG to give Fab-PEG-Fab. Fabbeva was obtained from papain digestion of bevacizumab. Purification gave pure Fab-PEG-Fab (silver stain). SPR binding analyses and in vitro angiogenesis assay were performed on Fabrani-PEG20-Fabrani (N=5), Fabrani (N=4) and bevacizumab (N=5).
Fabrani-PEG20-Fabrani (Figure 1, A, lane 2) was obtained from Fabrani (lane 1). Fabbeva-PEG20-Fabbeva was prepared for comparison. SPR analysis indicates that Fabrani-PEG20-Fabrani conjugates bind to VEGF. The dissociation rate constant (kd) of Fabbeva-PEG20-Fabbeva was slower than bevacizumab and Fabbeva (Figure 1, B). Fabrani-PEG-Fabrani inhibited angiogenesis in vitro to a greater extent than bevacizumab as determined by the reduction in tubule formation in a concentration-dependent manner. The average number of junctions made in the wells treated with Fabrani-PEG20-Fabrani was 6, where wells treated with VEGF only (positive control), bevacizumab and Fabrani resulted in an average 830, 382 and 233 number of junctions respectively. The average number of junctions calculated for the wells treated with medium (negative control) was only 11.
Fabrani-PEG20-Fabrani inhibited angiogenesis in vitro better than bevacizumab and ranibizumab. This work has the potential to optimise new protein-based therapeutics, particularly antibodies.
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