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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

Aflibercept forms 1:1 complexes with VEGF165. The molar masses of aflibercept:VEGF165 and bevacizumab:VEGF165 complexes were analyzed by multi-angle laser light scattering detection coupled to SEC. The differential refractive index (righty axis) and the measured molar mass (lefty axis) of peaks are indicated as a function of elution volume for each sample. The experimentally determined molar masses are indicated by horizontal lines. Cartoons of free VEGF165 and complexes of aflibercept or bevacizumab bound to VEGF165 are shown. Complexes of VEGF165 with bevacizumab (a) or aflibercept (b) at various molar ratios were incubated for 12 h at ambient temperature. Following incubation, the samples were kept at 4 °C in the autosampler prior to injection (~100–200 µg per sample) onto a Superose 12 column pre-equilibrated in 10 mM phosphate containing 500 mM NaCl buffer (pH 7.0) with a flow rate of 0.3 mL/min. Chromatograms of VEGF165 and bevacizumab (a) or aflibercept (b) are superimposed to indicate the elution profiles of the unbound proteins. The 1:1 molar ratio complexes yielded similar elution profiles and are not shown for the purposes of clarity
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Fig1: Aflibercept forms 1:1 complexes with VEGF165. The molar masses of aflibercept:VEGF165 and bevacizumab:VEGF165 complexes were analyzed by multi-angle laser light scattering detection coupled to SEC. The differential refractive index (righty axis) and the measured molar mass (lefty axis) of peaks are indicated as a function of elution volume for each sample. The experimentally determined molar masses are indicated by horizontal lines. Cartoons of free VEGF165 and complexes of aflibercept or bevacizumab bound to VEGF165 are shown. Complexes of VEGF165 with bevacizumab (a) or aflibercept (b) at various molar ratios were incubated for 12 h at ambient temperature. Following incubation, the samples were kept at 4 °C in the autosampler prior to injection (~100–200 µg per sample) onto a Superose 12 column pre-equilibrated in 10 mM phosphate containing 500 mM NaCl buffer (pH 7.0) with a flow rate of 0.3 mL/min. Chromatograms of VEGF165 and bevacizumab (a) or aflibercept (b) are superimposed to indicate the elution profiles of the unbound proteins. The 1:1 molar ratio complexes yielded similar elution profiles and are not shown for the purposes of clarity

Mentions: Multiple-angled laser light scattering (MALLS) coupled to size exclusion chromatography (SEC) was used to calculate the stoichiometry of bevacizumab:VEGF165 and aflibercept:VEGF165 complexes. VEGF165 was mixed with either bevacizumab or aflibercept in solution at molar ratios of inhibitor to ligand of between 5:1 and 1:5. Bevacizumab was found to form a heterogeneous mixture of multimeric complexes in the presence of VEGF165, with molar masses ranging from ~330 to 700 kDa (Fig. 1a). In contrast, aflibercept at a 1:5 molar ratio showed two discrete peaks (Fig. 1b), as expected based on previous results [21]. The earlier eluting peak, with an apparent molar mass of 157 kDa, corresponded to a 1:1 complex between aflibercept (~115 kDa) and VEGF165 (~40 kDa). The latter eluting peak corresponded to the expected excess of free VEGF165 dimer (based on the average molar mass of the peak and comparison with the elution times from VEGF165 injections). Analysis of a preformed aflibercept:VEGF165 complex at a 5:1 molar ratio showed a single peak of 119 kDa (Fig. 1b), which is attributed to incomplete separation between the 1:1 aflibercept:VEGF165 complex (~157 kDa) and the excess free aflibercept (~115 kDa).Fig. 1


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)

Aflibercept forms 1:1 complexes with VEGF165. The molar masses of aflibercept:VEGF165 and bevacizumab:VEGF165 complexes were analyzed by multi-angle laser light scattering detection coupled to SEC. The differential refractive index (righty axis) and the measured molar mass (lefty axis) of peaks are indicated as a function of elution volume for each sample. The experimentally determined molar masses are indicated by horizontal lines. Cartoons of free VEGF165 and complexes of aflibercept or bevacizumab bound to VEGF165 are shown. Complexes of VEGF165 with bevacizumab (a) or aflibercept (b) at various molar ratios were incubated for 12 h at ambient temperature. Following incubation, the samples were kept at 4 °C in the autosampler prior to injection (~100–200 µg per sample) onto a Superose 12 column pre-equilibrated in 10 mM phosphate containing 500 mM NaCl buffer (pH 7.0) with a flow rate of 0.3 mL/min. Chromatograms of VEGF165 and bevacizumab (a) or aflibercept (b) are superimposed to indicate the elution profiles of the unbound proteins. The 1:1 molar ratio complexes yielded similar elution profiles and are not shown for the purposes of clarity
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Aflibercept forms 1:1 complexes with VEGF165. The molar masses of aflibercept:VEGF165 and bevacizumab:VEGF165 complexes were analyzed by multi-angle laser light scattering detection coupled to SEC. The differential refractive index (righty axis) and the measured molar mass (lefty axis) of peaks are indicated as a function of elution volume for each sample. The experimentally determined molar masses are indicated by horizontal lines. Cartoons of free VEGF165 and complexes of aflibercept or bevacizumab bound to VEGF165 are shown. Complexes of VEGF165 with bevacizumab (a) or aflibercept (b) at various molar ratios were incubated for 12 h at ambient temperature. Following incubation, the samples were kept at 4 °C in the autosampler prior to injection (~100–200 µg per sample) onto a Superose 12 column pre-equilibrated in 10 mM phosphate containing 500 mM NaCl buffer (pH 7.0) with a flow rate of 0.3 mL/min. Chromatograms of VEGF165 and bevacizumab (a) or aflibercept (b) are superimposed to indicate the elution profiles of the unbound proteins. The 1:1 molar ratio complexes yielded similar elution profiles and are not shown for the purposes of clarity
Mentions: Multiple-angled laser light scattering (MALLS) coupled to size exclusion chromatography (SEC) was used to calculate the stoichiometry of bevacizumab:VEGF165 and aflibercept:VEGF165 complexes. VEGF165 was mixed with either bevacizumab or aflibercept in solution at molar ratios of inhibitor to ligand of between 5:1 and 1:5. Bevacizumab was found to form a heterogeneous mixture of multimeric complexes in the presence of VEGF165, with molar masses ranging from ~330 to 700 kDa (Fig. 1a). In contrast, aflibercept at a 1:5 molar ratio showed two discrete peaks (Fig. 1b), as expected based on previous results [21]. The earlier eluting peak, with an apparent molar mass of 157 kDa, corresponded to a 1:1 complex between aflibercept (~115 kDa) and VEGF165 (~40 kDa). The latter eluting peak corresponded to the expected excess of free VEGF165 dimer (based on the average molar mass of the peak and comparison with the elution times from VEGF165 injections). Analysis of a preformed aflibercept:VEGF165 complex at a 5:1 molar ratio showed a single peak of 119 kDa (Fig. 1b), which is attributed to incomplete separation between the 1:1 aflibercept:VEGF165 complex (~157 kDa) and the excess free aflibercept (~115 kDa).Fig. 1

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