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Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes.

MacDonald DA, Martin J, Muthusamy KK, Luo JK, Pyles E, Rafique A, Huang T, Potocky T, Liu Y, Cao J, Bono F, Delesque N, Savi P, Francis J, Amirkhosravi A, Meyer T, Romano C, Glinka M, Yancopoulos GD, Stahl N, Wiegand SJ, Papadopoulos N - Angiogenesis (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Cell surface binding of bevacizumab is directly proportional to endogenous VEGF165 concentration. ARPE-19 cells, cultured on human fibronectin-coated coverslips in six-well plates, were treated with equimolar concentration (1.68 μM) of bevacizumab, aflibercept, or hFc for 3 days starting from different time points (Day 2, 4, 9, and 14) followed by immunofluorescence staining of cell surface-bound inhibitor (red fluorescence, detected with mouse antihuman IgG Fc-specific, and secondary Ab, goat anti-mouse IgG-Alexa flour 594. Nuclei were counterstained with DAPI in blue). Cells and culture media at various culture times (Days 2, 6, 9, and 28) were collected for next-generation sequencing and ELISA of VEGF expression levels. Cells treated with bevacizumab (a–d) showed an increased cell surface binding in confluent ARPE-19 cell culture, coincident with the upregulation of VEGF expression (m, n). Cells treated with aflibercept (e–h) or control protein hFc (i–l) showed minimal binding at all time points. Scale Bar = 50 μm
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Fig7: Cell surface binding of bevacizumab is directly proportional to endogenous VEGF165 concentration. ARPE-19 cells, cultured on human fibronectin-coated coverslips in six-well plates, were treated with equimolar concentration (1.68 μM) of bevacizumab, aflibercept, or hFc for 3 days starting from different time points (Day 2, 4, 9, and 14) followed by immunofluorescence staining of cell surface-bound inhibitor (red fluorescence, detected with mouse antihuman IgG Fc-specific, and secondary Ab, goat anti-mouse IgG-Alexa flour 594. Nuclei were counterstained with DAPI in blue). Cells and culture media at various culture times (Days 2, 6, 9, and 28) were collected for next-generation sequencing and ELISA of VEGF expression levels. Cells treated with bevacizumab (a–d) showed an increased cell surface binding in confluent ARPE-19 cell culture, coincident with the upregulation of VEGF expression (m, n). Cells treated with aflibercept (e–h) or control protein hFc (i–l) showed minimal binding at all time points. Scale Bar = 50 μm

Mentions: ARPE-19 cells were used to test cell surface binding of bevacizumab and aflibercept in the presence of VEGF endogenously produced by these cells. ARPE-19 cells have been shown to express VEGF after several days in culture [31]. Cells in culture were incubated with aflibercept, bevacizumab, or hFc, for 3 days starting at day 2, 4, 9, and 14, and surface binding was then examined 3 days later. Aflibercept and the hFc control showed minimal surface binding at all time points. In contrast, bevacizumab showed significant cell surface binding at the Day 9–12 and Day 14–17 time points (Fig. 7c–l and 7d–1).Fig. 7


Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes.

MacDonald DA, Martin J, Muthusamy KK, Luo JK, Pyles E, Rafique A, Huang T, Potocky T, Liu Y, Cao J, Bono F, Delesque N, Savi P, Francis J, Amirkhosravi A, Meyer T, Romano C, Glinka M, Yancopoulos GD, Stahl N, Wiegand SJ, Papadopoulos N - Angiogenesis (2016)

Cell surface binding of bevacizumab is directly proportional to endogenous VEGF165 concentration. ARPE-19 cells, cultured on human fibronectin-coated coverslips in six-well plates, were treated with equimolar concentration (1.68 μM) of bevacizumab, aflibercept, or hFc for 3 days starting from different time points (Day 2, 4, 9, and 14) followed by immunofluorescence staining of cell surface-bound inhibitor (red fluorescence, detected with mouse antihuman IgG Fc-specific, and secondary Ab, goat anti-mouse IgG-Alexa flour 594. Nuclei were counterstained with DAPI in blue). Cells and culture media at various culture times (Days 2, 6, 9, and 28) were collected for next-generation sequencing and ELISA of VEGF expression levels. Cells treated with bevacizumab (a–d) showed an increased cell surface binding in confluent ARPE-19 cell culture, coincident with the upregulation of VEGF expression (m, n). Cells treated with aflibercept (e–h) or control protein hFc (i–l) showed minimal binding at all time points. Scale Bar = 50 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930479&req=5

Fig7: Cell surface binding of bevacizumab is directly proportional to endogenous VEGF165 concentration. ARPE-19 cells, cultured on human fibronectin-coated coverslips in six-well plates, were treated with equimolar concentration (1.68 μM) of bevacizumab, aflibercept, or hFc for 3 days starting from different time points (Day 2, 4, 9, and 14) followed by immunofluorescence staining of cell surface-bound inhibitor (red fluorescence, detected with mouse antihuman IgG Fc-specific, and secondary Ab, goat anti-mouse IgG-Alexa flour 594. Nuclei were counterstained with DAPI in blue). Cells and culture media at various culture times (Days 2, 6, 9, and 28) were collected for next-generation sequencing and ELISA of VEGF expression levels. Cells treated with bevacizumab (a–d) showed an increased cell surface binding in confluent ARPE-19 cell culture, coincident with the upregulation of VEGF expression (m, n). Cells treated with aflibercept (e–h) or control protein hFc (i–l) showed minimal binding at all time points. Scale Bar = 50 μm
Mentions: ARPE-19 cells were used to test cell surface binding of bevacizumab and aflibercept in the presence of VEGF endogenously produced by these cells. ARPE-19 cells have been shown to express VEGF after several days in culture [31]. Cells in culture were incubated with aflibercept, bevacizumab, or hFc, for 3 days starting at day 2, 4, 9, and 14, and surface binding was then examined 3 days later. Aflibercept and the hFc control showed minimal surface binding at all time points. In contrast, bevacizumab showed significant cell surface binding at the Day 9–12 and Day 14–17 time points (Fig. 7c–l and 7d–1).Fig. 7

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.

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.

No MeSH data available.


Related in: MedlinePlus