title: Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes.
abstract: Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: MacDonald
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Martin
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Kathir K
authors.initials: KK
authors.lastname: Muthusamy
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Jiann-Kae
authors.initials: JK
authors.lastname: Luo
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Erica
authors.initials: E
authors.lastname: Pyles
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Ashique
authors.initials: A
authors.lastname: Rafique
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Tammy
authors.initials: T
authors.lastname: Huang
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Terra
authors.initials: T
authors.lastname: Potocky
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Jingtai
authors.initials: J
authors.lastname: Cao
authors.affiliation: Sanofi, Toulouse, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Bono
authors.affiliation: Sanofi, Toulouse, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Delesque
authors.affiliation: Sanofi, Toulouse, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Savi
authors.affiliation: Center for Thrombosis Research, Florida Hospital, Orlando, FL, USA.
authors.forename: John
authors.initials: J
authors.lastname: Francis
authors.affiliation: Center for Thrombosis Research, Florida Hospital, Orlando, FL, USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Amirkhosravi
authors.affiliation: Center for Thrombosis Research, Florida Hospital, Orlando, FL, USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Meyer
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Carmelo
authors.initials: C
authors.lastname: Romano
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Meredith
authors.initials: M
authors.lastname: Glinka
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: George D
authors.initials: GD
authors.lastname: Yancopoulos
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Neil
authors.initials: N
authors.lastname: Stahl
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
authors.forename: Stanley J
authors.initials: SJ
authors.lastname: Wiegand
authors.affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA. nicholas.papadopoulos@regeneron.com.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Papadopoulos
mesh_terms: D020533:Angiogenesis Inhibitors / Q000009:adverse effects / Q000378:metabolism / Q000627:therapeutic use; D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000378:metabolism; D000068258:Bevacizumab / Q000009:adverse effects / Q000378:metabolism* / Q000627:therapeutic use; D002460:Cell Line; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D066298:In Vitro Techniques; D008268:Macular Degeneration / Q000276:immunology / Q000378:metabolism / Q000628:therapy; D051379:Mice; D008822:Mice, Transgenic; D015539:Platelet Activation; D011485:Protein Binding; D055503:Protein Multimerization; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor / Q000378:metabolism* / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000009:adverse effects / Q000378:metabolism* / Q000627:therapeutic use; D013921:Thrombocytopenia / Q000209:etiology; D013927:Thrombosis / Q000209:etiology; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism*
chemical_list: D020533:Angiogenesis Inhibitors; D000936:Antigen-Antibody Complex; C080568:Fc gamma receptor IIA; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C533178:aflibercept; D000068258:Bevacizumab; D040262:Receptors, Vascular Endothelial Growth Factor
keywords: Aflibercept; Bevacizumab; Immune complexes; VEGF