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Aflibercept
Aflibercept
Aflibercept is a recombinant fusion protein that binds to and inhibits vascular endothelial growth factor (VEGF), a key regulator of angiogenesis.
It is used to treat various ocular conditions, including wet age-related macular degeneration, diabetic macular edema, and retinal vein occlusion.
Aflibercept works by blocking the activity of VEGF, which helps prevent the growth of abnormal blood vessels and reduces fluid leakage in the eye.
This MeSH term provides a concise, informative overview of Aflibercept and its clinical applications.
Pucompare.ai can optimize your Aflibercept reserach by improving reproducibility and accuracy, helping you locate the best protocols and products from literature, pre-prints, and patents.
It is used to treat various ocular conditions, including wet age-related macular degeneration, diabetic macular edema, and retinal vein occlusion.
Aflibercept works by blocking the activity of VEGF, which helps prevent the growth of abnormal blood vessels and reduces fluid leakage in the eye.
This MeSH term provides a concise, informative overview of Aflibercept and its clinical applications.
Pucompare.ai can optimize your Aflibercept reserach by improving reproducibility and accuracy, helping you locate the best protocols and products from literature, pre-prints, and patents.
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OIR was induced as previously described (Connor et al., 2009 (link); LeBlanc et al., 2016 (link)). In brief, mice (C57BL/6; male and female) at postnatal day 7 (P7) were exposed to 75% oxygen in a regulated chamber. At P12, anti-Scg3 pAb, control IgG, anti-Scg3 mAb, aflibercept, or PBS was intravitreally injected into anesthetized mice, which were returned to room air after the injection. At P17, mice were euthanized by CO2 inhalation. Isolated retinas were stained with Alexa Fluor 488–isolectin B4, flat-mounted, and analyzed by confocal microscopy. Neovascularization and related tufts were quantified as previously described (Connor et al., 2009 (link)). Additionally, the number of branching points within a defined area was quantified in the same region of each retina. All data were normalized against noninjection OIR eyes.
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We have developed a whole‐body PK model to predict the effect of intravenous administration of an anti‐VEGF agent in cancer. The model, illustrated in Figure 1 , predicts interstitial and plasma concentrations of VEGF and aflibercept in three compartments: normal tissue (“normal compartment,” represented by skeletal muscle), the vasculature (“blood compartment”), and diseased tissue (“tumor compartment”). The tumor compartment is parameterized as a breast tumor; however, the model is broadly applicable to any solid tumor. The geometric parameters used to characterize the compartments are taken from the literature, and compiled in the Supplementary Materials .
The model includes molecular interactions between two major VEGF isoforms (VEGF121 and VEGF165), VEGF receptors (VEGFR1 and VEGFR2), and co‐receptors neuropilins (NRP1 and NRP2). The VEGF ligands have specific interactions with the receptors because of differential exon splicing, and the kinetic rates of these interactions are based on experimental data. VEGF is secreted by parenchymal cells (muscle fibers and tumor cells, in the normal and diseased compartments, respectively), as well as by endothelial cells (both luminal secretion into the blood compartment and abluminal secretion into the tissue compartments). The isoform secretion ratios for the different cell types are taken from the literature.
VEGFRs are present on endothelial cells (both the luminal and abluminal surfaces) and tumor cells. Additionally, the co‐receptor NRP1 is present on muscle fibers, endothelial cells, and tumor cells, whereas NRP2 is only expressed in tumor cells in the model. The density of VEGF receptors and co‐receptors is based on quantitative flow cytometric measurements, which determine the number of receptors on a cell‐by‐cell basis.19 , 20 The model also includes soluble factors: soluble VEGFR1 and α‐2‐macroglobulin. Soluble α‐2‐macroglobulin is present in two forms: native (α2M) and active (α2Mfast). These species, which are present at nanomolar to micromolar concentrations in plasma, are ∼720 kDa in size. Because of their large size, these species are assumed to be confined to the blood compartment. Both soluble VEGFR1 and α‐2‐macroglobulin are secreted by endothelial cells, where the secretion rate is set to match the plasma and tissue concentrations reported in the literature.
Many of the model parameters are based on in vitro measurements (for example, kinetic binding constants, clearance, and degradation rates). Geometric parameters are required to characterize the body compartments and enable conversion of the concentrations in units used in the model (moles/cm3 tissue) to more standard units (pM). The number of VEGFRs and co‐receptors are based on quantitative flow cytometry measurements from in vitro and in vivo studies in our laboratory.19 , 20 In our previous publications,12 , 13 , 15 , 21 , 22 we have performed extensive sensitivity analyses to quantify how the model outputs (namely, the concentrations of VEGF in the three compartments) are affected when model parameters are varied. We found that most parameters do not significantly change the predicted VEGF concentrations over a wide range of values (i.e., up to an order of magnitude above or below the baseline value). In the current article, we build on the baseline model to understand the effects of specific molecular interactions involving aflibercept.
The model includes molecular interactions between two major VEGF isoforms (VEGF121 and VEGF165), VEGF receptors (VEGFR1 and VEGFR2), and co‐receptors neuropilins (NRP1 and NRP2). The VEGF ligands have specific interactions with the receptors because of differential exon splicing, and the kinetic rates of these interactions are based on experimental data. VEGF is secreted by parenchymal cells (muscle fibers and tumor cells, in the normal and diseased compartments, respectively), as well as by endothelial cells (both luminal secretion into the blood compartment and abluminal secretion into the tissue compartments). The isoform secretion ratios for the different cell types are taken from the literature.
VEGFRs are present on endothelial cells (both the luminal and abluminal surfaces) and tumor cells. Additionally, the co‐receptor NRP1 is present on muscle fibers, endothelial cells, and tumor cells, whereas NRP2 is only expressed in tumor cells in the model. The density of VEGF receptors and co‐receptors is based on quantitative flow cytometric measurements, which determine the number of receptors on a cell‐by‐cell basis.
Many of the model parameters are based on in vitro measurements (for example, kinetic binding constants, clearance, and degradation rates). Geometric parameters are required to characterize the body compartments and enable conversion of the concentrations in units used in the model (moles/cm3 tissue) to more standard units (pM). The number of VEGFRs and co‐receptors are based on quantitative flow cytometry measurements from in vitro and in vivo studies in our laboratory.
Most recents protocols related to «Aflibercept»
Intravitreal administration of aflibercept was performed in a sterile laminar flow operating room following sterile surgical procedures. A 30-gauge needle was inserted from the superotemporal quadrant, 3.5 to 4.0 mm from the corneoscleral limbus, to inject 3-mg/0.075-mL aflibercept into the vitreous cavity. The injection was administered once every 4 weeks until the retina became dry (no evidence of IRF or SRF on OCT images). Since all eyes were not treatment-naive and had received an average of 4.9 (range, 3–10) ranibizumab or conbercept injections and a minimum of 3 injections within 3 months prior to switching to aflibercept, 3 to 5 loading doses of aflibercept were not considered.
When there was at least one of the following conditions, patients continued receiving aflibercept injections on an individualized basis as a modified or extended treatment regimen: recurrent or persistent IRF or SRF on OCT images, the appearance of new hemorrhage on color fundus photographic or ophthalmoscopic images, or decreased visual acuity as compared to the previous examination. Retinal pigment epithelial detachment (PED) was not an indication for further treatment.
When there was at least one of the following conditions, patients continued receiving aflibercept injections on an individualized basis as a modified or extended treatment regimen: recurrent or persistent IRF or SRF on OCT images, the appearance of new hemorrhage on color fundus photographic or ophthalmoscopic images, or decreased visual acuity as compared to the previous examination. Retinal pigment epithelial detachment (PED) was not an indication for further treatment.
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Aflibercept-coding DNA (1296 bp) was inserted into a magnICON® vector pICH26211α—a derivative of pICH26211 (Icon Genetics GmbH (Halle/Saale, Germany)) [23 (link)] carrying a barley α-amylase signal sequence—using BsaI restriction sites, leading to the expression vector pICH26211α-aflibercept (Figure S2A ). Sequence information is available in the Supplementary Materials Section (Figure S2B) . The construct was transformed into Agrobacterium fabrum GV3101(pMP90) and the resulting strain used for the subsequent agroinfiltration experiments.
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Intravitreal injections of aflibercept were administered in the operating room according to the standard procedure for intravitreal injections.
Patients were treated according to the modified PRN regimen, after a loading phase of three-monthly intravitreal injections followed by monthly observations during control visits. The criteria for treatment resumption included detecting disease activity such as the appearance of any amount of SRF or IRF on the macular OCT scan, subretinal hemorrhages observed in the macular region, or visual loss. When the absence offluid was observed at one visit, the injection was given, while the absence of fluid at two consecutive visits indicated a non-injection monitoring visit.
Patients were treated according to the modified PRN regimen, after a loading phase of three-monthly intravitreal injections followed by monthly observations during control visits. The criteria for treatment resumption included detecting disease activity such as the appearance of any amount of SRF or IRF on the macular OCT scan, subretinal hemorrhages observed in the macular region, or visual loss. When the absence offluid was observed at one visit, the injection was given, while the absence of fluid at two consecutive visits indicated a non-injection monitoring visit.
Glycoengineered N. benthamiana plants [18 (link),24 (link)] were grown in a growth chamber under controlled conditions at 24 °C, 60% humidity, with a 16 h light/8 h dark photoperiod.
For agroinfiltration, respective recombinant bacterial strains were grown at 29 °C for 24 h, centrifuged at 4000× g for 10 min at room temperature, and resuspended in infiltration buffer (10 mM MES, pH 5.6; 10 mM MgSO4). The optical density of each strain was measured by light absorption at 600 nm (OD600) of an adequate dilution. The final OD600 was set to 0.1 by dilution with infiltration buffer. The agrobacterial suspensions were delivered to leaves of 4–5-week-old plants using a syringe. The infiltrated leaves were harvested 4 days post-infiltration, flash-frozen in liquid nitrogen, and ground to fine powder.
Total soluble protein (TSP) was extracted with extraction buffer (0.5 M NaCl, 0.1 M Tris, 1 mM EDTA, 40 mM ascorbic acid; pH 7.4) in a ratio of 1:2 w/v (fresh leaf weight: buffer) for 90 min at 4 °C on an orbital shaker. Subsequently, the solution was centrifuged twice at 14,000× g for 20 min at 4 °C and the supernatant was vacuum filtered using 8–12 µm and 2–3 µm filters (ROTILABO® Typ 12A and 15A) (Karl Roth GmBH, Karlsruhe, Germany).
To collect intercellular fluid, the infiltrated intact leaves were harvested 4 days post-infiltration (dpi) and submerged in a beaker containing IF extraction buffer (100 mM Tris·HCl, pH 7.5, 10 mM MgCl2, 2 mM EDTA). The leaves were infiltrated with the IF buffer by applying vacuum; excess liquid was removed with a paper towel and the leaves were placed inside separate 50 mL conical tubes equipped with a flat plastic mesh installed in the circular cross-section plane between the cylindrical and the conical parts of the tube to keep the leaves above the collected IF. The tubes were centrifuged at 800× g for 5 min at 4 °C to release the IF from the leaves.
To determine aflibercept expression, 10 µg of TSP extracted from the infiltrated leaf material was separated on a 12% SDS-PAA gel followed by immunoblotting using anti-human IgG (Goat anti-hIgG-HRPO, Thermofischer Scientific, Waltham, MA, USA Invitrogen 62-8420) at the dilution 1:5000.
Recombinant proteins were purified by affinity chromatography using protein A (rProA Amicogen, Cat no: 1080025, Amicogen, Inc., Gyeongsangnam-do, Republic of Korea). The TSP extracts were loaded at a flow rate of 1.5 mL/min onto a manually packed column which was pre-equilibrated with 10 column volumes (CV) of PBS (137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4). Washing was carried out with 20 CV of PBS. Aflibercept was eluted in 1 mL fractions with 0.1 M Glycine·HCl (pH 3.0); eluates were immediately neutralized with 25 µL of 1 M Tris (pH 9.0) and dialyzed overnight against PBS.
The dialyzed and concentrated protein was subjected to size-exclusion chromatography using BioLogic DuoFlo FPLC system (Bio-Rad, South Granville, NSW, Australia) equipped with a QuadTec UV–vis detector (Bio-Rad) and Superdex 200 Increase 10/300 GL column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Elution was performed at room temperature in an isocratic flow of PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4) at 0.75 mL/min and proteins were detected by UV absorbance at 280 nm.
For agroinfiltration, respective recombinant bacterial strains were grown at 29 °C for 24 h, centrifuged at 4000× g for 10 min at room temperature, and resuspended in infiltration buffer (10 mM MES, pH 5.6; 10 mM MgSO4). The optical density of each strain was measured by light absorption at 600 nm (OD600) of an adequate dilution. The final OD600 was set to 0.1 by dilution with infiltration buffer. The agrobacterial suspensions were delivered to leaves of 4–5-week-old plants using a syringe. The infiltrated leaves were harvested 4 days post-infiltration, flash-frozen in liquid nitrogen, and ground to fine powder.
Total soluble protein (TSP) was extracted with extraction buffer (0.5 M NaCl, 0.1 M Tris, 1 mM EDTA, 40 mM ascorbic acid; pH 7.4) in a ratio of 1:2 w/v (fresh leaf weight: buffer) for 90 min at 4 °C on an orbital shaker. Subsequently, the solution was centrifuged twice at 14,000× g for 20 min at 4 °C and the supernatant was vacuum filtered using 8–12 µm and 2–3 µm filters (ROTILABO® Typ 12A and 15A) (Karl Roth GmBH, Karlsruhe, Germany).
To collect intercellular fluid, the infiltrated intact leaves were harvested 4 days post-infiltration (dpi) and submerged in a beaker containing IF extraction buffer (100 mM Tris·HCl, pH 7.5, 10 mM MgCl2, 2 mM EDTA). The leaves were infiltrated with the IF buffer by applying vacuum; excess liquid was removed with a paper towel and the leaves were placed inside separate 50 mL conical tubes equipped with a flat plastic mesh installed in the circular cross-section plane between the cylindrical and the conical parts of the tube to keep the leaves above the collected IF. The tubes were centrifuged at 800× g for 5 min at 4 °C to release the IF from the leaves.
To determine aflibercept expression, 10 µg of TSP extracted from the infiltrated leaf material was separated on a 12% SDS-PAA gel followed by immunoblotting using anti-human IgG (Goat anti-hIgG-HRPO, Thermofischer Scientific, Waltham, MA, USA Invitrogen 62-8420) at the dilution 1:5000.
Recombinant proteins were purified by affinity chromatography using protein A (rProA Amicogen, Cat no: 1080025, Amicogen, Inc., Gyeongsangnam-do, Republic of Korea). The TSP extracts were loaded at a flow rate of 1.5 mL/min onto a manually packed column which was pre-equilibrated with 10 column volumes (CV) of PBS (137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4). Washing was carried out with 20 CV of PBS. Aflibercept was eluted in 1 mL fractions with 0.1 M Glycine·HCl (pH 3.0); eluates were immediately neutralized with 25 µL of 1 M Tris (pH 9.0) and dialyzed overnight against PBS.
The dialyzed and concentrated protein was subjected to size-exclusion chromatography using BioLogic DuoFlo FPLC system (Bio-Rad, South Granville, NSW, Australia) equipped with a QuadTec UV–vis detector (Bio-Rad) and Superdex 200 Increase 10/300 GL column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Elution was performed at room temperature in an isocratic flow of PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4) at 0.75 mL/min and proteins were detected by UV absorbance at 280 nm.
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Example 7
To generate a heavy chain with aflibercept-derived VEGF-binding domains added at the C-terminus, an amino acid sequence was generated comprising the following appended amino acid sequences, from N-terminus to C-terminus:
1) SEQ ID NO: 14 (heavy chain of antibody HC2:LC1);
2) SEQ ID NO: 31 (linker peptide, underlined); and
3) SEQ ID NO: 22 (aflibercept-derived sequence).
To generate a light chain with aflibercept-derived VEGF-binding domains added at the C-terminus, an amino acid sequence was generated comprising the following appended amino acid sequences, from N-terminus to C-terminus:
1) SEQ ID NO: 17 (light chain of antibody HC2:LC1);
2) SEQ ID NO: 31 (linker peptide, underlined); and
3) SEQ ID NO: 22 (aflibercept-derived sequence).
The resulting polypeptides, SEQ ID NO: 149 and SEQ ID NO: 219 were co-expressed to provide a hexavalent, bispecific antibody HC2-AFL:LC1-AFL shown in TABLE 39. Amino acids 1-19 of SEQ ID NO: 149 are the heavy chain signal peptide (SEQ ID NO: 11). Amino acids 1-20 of SEQ ID NO: 219 are the light chain signal peptide (SEQ ID NO: 12). In some embodiments, a mature HC2-AFL:LC1-AFL does not comprise the signal peptides. For example, a mature HC2-AFL:LC1-AFL of the disclosure can comprise SEQ ID NO: 254 and SEQ ID NO: 259.
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Top products related to «Aflibercept»
Sourced in Germany, Switzerland, United States
Aflibercept is a laboratory-produced protein that binds to and blocks the activity of vascular endothelial growth factor (VEGF), a substance that promotes the growth of new blood vessels. This product is used for research purposes to study the role of VEGF in various biological processes.
Sourced in Germany, Switzerland
Eylea is a laboratory equipment product manufactured by Bayer. It is designed for use in scientific and medical research applications. The core function of Eylea is to facilitate the analysis and examination of samples, but its specific intended use should not be interpreted or extrapolated beyond the provided information.
Sourced in United States
Aflibercept is a recombinant fusion protein that acts as a soluble decoy receptor for vascular endothelial growth factor (VEGF). It is designed to bind to and inhibit the biological activity of VEGF-A, VEGF-B, and placental growth factor (PlGF).
Sourced in Switzerland, Germany, China
Ranibizumab is a biopharmaceutical product that functions as an anti-vascular endothelial growth factor (anti-VEGF) agent. It is a recombinant humanized monoclonal antibody fragment designed to bind to and inhibit the activity of human VEGF protein.
Sourced in Germany, United States, United Kingdom, Japan, Switzerland, Ireland
The Spectralis is an optical coherence tomography (OCT) imaging device developed by Heidelberg Engineering. It captures high-resolution, cross-sectional images of the retina and optic nerve using near-infrared light. The Spectralis provides detailed structural information about the eye, which can aid in the diagnosis and management of various eye conditions.
Sourced in United States, Switzerland
Ranibizumab is a recombinant humanized monoclonal antibody fragment that binds to and inhibits the vascular endothelial growth factor (VEGF). It is designed for use in the treatment of various eye conditions.
Sourced in United States, Cameroon
Bevacizumab is a recombinant humanized monoclonal antibody that binds to and inhibits the biological activity of human vascular endothelial growth factor (VEGF).
Sourced in United States, Cameroon, United Kingdom
Avastin is a laboratory-produced monoclonal antibody used in various scientific research applications. It functions by targeting and binding to a specific protein involved in the process of angiogenesis, which is the formation of new blood vessels. Avastin's core function is to inhibit this process, but its precise application and intended use are not included in this response.
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Lucentis is an ophthalmic solution used for the treatment of various eye conditions. It contains the active ingredient ranibizumab, which is a recombinant humanized monoclonal antibody fragment. Lucentis is designed to inhibit vascular endothelial growth factor (VEGF) and reduce the growth of abnormal blood vessels in the eye.
More about "Aflibercept"
Aflibercept, also known by the brand name Eylea, is a recombinant fusion protein medication used to treat various ocular (eye) conditions.
It works by inhibiting vascular endothelial growth factor (VEGF), a key regulator of angiogenesis (the growth of new blood vessels).
This anti-VEGF therapy is primarily used to treat wet age-related macular degeneration (AMD), diabetic macular edema (DME), and retinal vein occlusion (RVO).
Wet AMD is characterized by the growth of abnormal blood vessels in the eye, leading to fluid leakage and vision loss.
Aflibercept helps prevent this by blocking VEGF, which reduces the formation of these problematic blood vessels.
Similarly, in DME, Aflibercept helps reduce fluid buildup in the macula, the part of the eye responsible for central vision.
And in RVO, it can help improve vision by limiting the growth of new, leaky blood vessels.
Aflibercept is often compared to other anti-VEGF medications like Ranibizumab (Lucentis) and Bevacizumab (Avastin).
While these drugs share a similar mechanism of action, Aflibercept may have some advantages, such as a longer duration of action, potentially requiring fewer injections.
Additionally, the Spectralis imaging system can be used to monitor the effects of Aflibercept treatment.
Ultimately, Aflibercept is an important tool in the management of various sight-threatening eye conditions, helping to preserve and even improve vision for many patients.
By understanding its unique properties and applications, healthcare providers can optimize its use and provide the best possible care for their patients.
It works by inhibiting vascular endothelial growth factor (VEGF), a key regulator of angiogenesis (the growth of new blood vessels).
This anti-VEGF therapy is primarily used to treat wet age-related macular degeneration (AMD), diabetic macular edema (DME), and retinal vein occlusion (RVO).
Wet AMD is characterized by the growth of abnormal blood vessels in the eye, leading to fluid leakage and vision loss.
Aflibercept helps prevent this by blocking VEGF, which reduces the formation of these problematic blood vessels.
Similarly, in DME, Aflibercept helps reduce fluid buildup in the macula, the part of the eye responsible for central vision.
And in RVO, it can help improve vision by limiting the growth of new, leaky blood vessels.
Aflibercept is often compared to other anti-VEGF medications like Ranibizumab (Lucentis) and Bevacizumab (Avastin).
While these drugs share a similar mechanism of action, Aflibercept may have some advantages, such as a longer duration of action, potentially requiring fewer injections.
Additionally, the Spectralis imaging system can be used to monitor the effects of Aflibercept treatment.
Ultimately, Aflibercept is an important tool in the management of various sight-threatening eye conditions, helping to preserve and even improve vision for many patients.
By understanding its unique properties and applications, healthcare providers can optimize its use and provide the best possible care for their patients.