June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Bivalent ranibizumab is more active in vitro than bevacizumab
Author Affiliations & Notes
  • Hanieh Khalili
    UCL School of Pharmacy, London, United Kingdom
    National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfield Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, United Kingdom
  • Steve Brocchini
    UCL School of Pharmacy, London, United Kingdom
    National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfield Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, United Kingdom
  • Peng Khaw
    National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfield Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, United Kingdom
  • Footnotes
    Commercial Relationships Hanieh Khalili, None; Steve Brocchini, None; Peng Khaw, University College Moorfields (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3279. doi:
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    • Get Citation

      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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

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.

 
Methods
 

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).

 
Results
 

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.

 
Conclusions
 

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.

 
 
Figure 1. A; SDS-PAGE gels of the disulfide bridging PEGylation of the Fab; Novex Bis-Tris 4-12% gel stained with colloidal blue for protein (lanes M-2) and silver staining (lane 3) to detect impurity. M: Standard protein markers. Lane 1: purified Fab. Lanes 2: 3: purified Fabrani-PEG20-Fabrani. B; The dissociation rate profiles for Fabbeva, Fabbeva-PEG20-Fabbeva and bevacizumab using CM3 chip with 53 RU VEGF at 25 0C.
 
Figure 1. A; SDS-PAGE gels of the disulfide bridging PEGylation of the Fab; Novex Bis-Tris 4-12% gel stained with colloidal blue for protein (lanes M-2) and silver staining (lane 3) to detect impurity. M: Standard protein markers. Lane 1: purified Fab. Lanes 2: 3: purified Fabrani-PEG20-Fabrani. B; The dissociation rate profiles for Fabbeva, Fabbeva-PEG20-Fabbeva and bevacizumab using CM3 chip with 53 RU VEGF at 25 0C.
 
Keywords: 658 protein purification and characterization • 657 protein modifications-post translational  
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