March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
VEGF Trap Exhibits VEGF Binding Stoichiometry Distinct From Antibodies, and Does Not Support Platelet Aggregation
Author Affiliations & Notes
  • Douglas A. MacDonald
    Regeneron Pharmaceuticals, Tarrytown, New York
  • Jiann-Kae Luo
    Regeneron Pharmaceuticals, Tarrytown, New York
  • Nicholas Papadopoulos
    Regeneron Pharmaceuticals, Tarrytown, New York
  • Erica Pyles
    Regeneron Pharmaceuticals, Tarrytown, New York
  • Stanley Wiegand
    Regeneron Pharmaceuticals, Tarrytown, New York
  • Françoise Bono
    Sanofi, France, Toulouse, France
  • Nathalie Delesque
    Sanofi, France, Toulouse, France
  • Pierre Savi
    Sanofi, France, Toulouse, France
  • John Francis
    Center for Thrombosis Research, Orlando, Florida
  • Todd Meyer
    Center for Thrombosis Research, Orlando, Florida
  • Footnotes
    Commercial Relationships  Douglas A. MacDonald, Regeneron Pharmaceuticals (E); Jiann-Kae Luo, Regeneron Pharmaceuticals (E); Nicholas Papadopoulos, Regeneron Pharmaceuticals (E); Erica Pyles, Regeneron Pharmaceuticals (E); Stanley Wiegand, Regeneron Pharmaceuticals (E); Françoise Bono, Sanofi France (E); Nathalie Delesque, Sanofi France (E); Pierre Savi, Sanofi France (E); John Francis, None; Todd Meyer, Funding grant from Regeneron (F)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 440. doi:
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      Douglas A. MacDonald, Jiann-Kae Luo, Nicholas Papadopoulos, Erica Pyles, Stanley Wiegand, Françoise Bono, Nathalie Delesque, Pierre Savi, John Francis, Todd Meyer; VEGF Trap Exhibits VEGF Binding Stoichiometry Distinct From Antibodies, and Does Not Support Platelet Aggregation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):440.

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

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Abstract

Purpose: : VEGF Trap (aflibercept) is a novel, soluble decoy receptor that binds VEGF-A, VEGF-B and placental growth factor. Like anti-VEGF antibodies, VEGF Trap contains an Fc domain. However, given its otherwise distinct structure, VEGF Trap may bind VEGF with different stoichiometry than antibodies. The present studies were conducted to compare binding stoichiometries of bevacizumab (Bev, an anti-VEGF antibody) and VEGF Trap to VEGF, as well as FcγRIIA -mediated platelet activating capability of Bev:VEGF and VEGF Trap:VEGF complexes.

Methods: : Stoichiometry of binding for Bev and VEGF Trap to VEGF165 was measured using multi-angle laser light scattering. Platelet activation was measured using platelet aggregometry & 14C-serotonin release assays (SRA). Animal studies used human FcγRIIA transgenic mice. Pre-formed 1:1 molar mixtures of Bev or VEGF Trap with VEGF165 plus heparin were injected into the tail vein (n=10/group). After 10 min, blood drawn by cardiac puncture was used to measure platelet number. Lungs were dissected en bloc, PBS washed and formalin-fixed. H&E sections were analyzed for evidence of thrombosis.

Results: : Bev:VEGF165 complexes were heterogeneous and predominantly multimeric (~400kDa to >2,000 kDa). In contrast, VEGF Trap formed a homogeneous 1:1 binding complex with VEGF165, with a M.W. of 155 kDa. Platelet aggregometry and SRA showed that preformed equal molar Bev:VEGF165 complexes (100-500 nM) plus heparin (0.5-50 ug/ml) activated platelets, while VEGF Trap:VEGF165 complexes did not. Increasing the molar excess of Bev (≥ 4 fold relative to VEGF) resulted in smaller complexes that did not trigger platelet activation. Injection of pre-formed VEGF Trap:VEGF165 complexes with heparin into FcγRIIA transgenic mice did not significantly affect platelet count (966±168) vs saline-injected controls (1173±179). Animals receiving Bev+VEGF165 complexes with heparin exhibited behavior consistent with thrombotic shock and experienced profound thrombocytopenia (platelet count of 331±217; P < 0.001). Lung sections from these animals showed patterns of occlusive thrombi.

Conclusions: : Bev when mixed at near equal molar ratios with VEGF165, in the presence of heparin, can support platelet aggregation. This is not the case for VEGF Trap. The clinical relevance of these observations remains to be determined.

Keywords: vascular endothelial growth factor • age-related macular degeneration • protein structure/function 
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