Limits...
Highly potent VEGF-A-antagonistic DARPins as anti-angiogenic agents for topical and intravitreal applications.

Stahl A, Stumpp MT, Schlegel A, Ekawardhani S, Lehrling C, Martin G, Gulotti-Georgieva M, Villemagne D, Forrer P, Agostini HT, Binz HK - Angiogenesis (2012)

Bottom Line: The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations.In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model.Even at elevated doses, DARPins were safe to use.

View Article: PubMed Central - PubMed

Affiliation: Universitäts-Augenklinik Freiburg, Freiburg, Germany.

ABSTRACT
The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.

Show MeSH

Related in: MedlinePlus

Cellular screening for DARPins inhibiting VEGF-induced phosphorylation of VEGFR-2. VEGF-A (1.4 nM) was pre-incubated with 1.4 nM of different purified DARPins (#1–#10), isotype (negative) control DARPins (I1 and I2), as well as the controls ranibizumab (Lucentis, L), bevacizumab (Avastin, A). The signal of PBS (NI non-inhibited) and the signal obtained without addition of VEGF-A (NV) were used as controls
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3526737&req=5

Fig2: Cellular screening for DARPins inhibiting VEGF-induced phosphorylation of VEGFR-2. VEGF-A (1.4 nM) was pre-incubated with 1.4 nM of different purified DARPins (#1–#10), isotype (negative) control DARPins (I1 and I2), as well as the controls ranibizumab (Lucentis, L), bevacizumab (Avastin, A). The signal of PBS (NI non-inhibited) and the signal obtained without addition of VEGF-A (NV) were used as controls

Mentions: To confirm the potency of selected DARPins, we performed a VEGF-receptor-2 (VEGF-R2) phosphorylation-inhibition cell culture assay (Fig. 2) as well as a human umbilical vein endothelial cell (HUVEC) spheroid sprouting assay (Fig. 3). In the VEGF-R2 phosphorylation assay, the ten VEGF-binding DARPins as well as controls (Isotype DARPin I1 and I2 as negative controls [18]; bevacizumab and ranibizumab as positive controls) were used at a fixed concentration of 1.4 nM in combination with 1.4 nM VEGF-A. Maximal stimulation (PBS added to VEGF-A) and no stimulation (PBS, no VEGF-A used) were used to illustrate the assay window. The results demonstrated that all DARPins and the positive controls are able to suppress VEGF-A induced phosphorylation of VEGFR-2, whereas isotype controls do not affect its phosphorylation (Fig. 2). Overall, some of the selected DARPins showed inhibition potency in the range of ranibizumab and bevacizumab in this assay. These data were confirmed in a spheroidal model of in vitro sprouting angiogenesis [27–29]. In this assay, anti-VEGF-A DARPins potently suppressed endothelial cell sprout formation. Similar to what was observed in the phosphorylation assay, some of the DARPin candidates blocked sprouting very potently, similar to or better than ranibizumab, while the isotype control DARPins showed no effect. These cellular assays confirm that a large number of VEGF-A antagonists was generated, some of which exhibit very high potency in vitro. Note that for both cellular assays, VEGF-A has to be used at 8–70 times higher concentrations than the one used in the Quantikine assay to reliably induce VEGF receptor phosphorylation or spheroid sprouting. Consequently, potency values are limited by the concentration of human VEGF-A used and are thus underestimated.Fig. 2


Highly potent VEGF-A-antagonistic DARPins as anti-angiogenic agents for topical and intravitreal applications.

Stahl A, Stumpp MT, Schlegel A, Ekawardhani S, Lehrling C, Martin G, Gulotti-Georgieva M, Villemagne D, Forrer P, Agostini HT, Binz HK - Angiogenesis (2012)

Cellular screening for DARPins inhibiting VEGF-induced phosphorylation of VEGFR-2. VEGF-A (1.4 nM) was pre-incubated with 1.4 nM of different purified DARPins (#1–#10), isotype (negative) control DARPins (I1 and I2), as well as the controls ranibizumab (Lucentis, L), bevacizumab (Avastin, A). The signal of PBS (NI non-inhibited) and the signal obtained without addition of VEGF-A (NV) were used as controls
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526737&req=5

Fig2: Cellular screening for DARPins inhibiting VEGF-induced phosphorylation of VEGFR-2. VEGF-A (1.4 nM) was pre-incubated with 1.4 nM of different purified DARPins (#1–#10), isotype (negative) control DARPins (I1 and I2), as well as the controls ranibizumab (Lucentis, L), bevacizumab (Avastin, A). The signal of PBS (NI non-inhibited) and the signal obtained without addition of VEGF-A (NV) were used as controls
Mentions: To confirm the potency of selected DARPins, we performed a VEGF-receptor-2 (VEGF-R2) phosphorylation-inhibition cell culture assay (Fig. 2) as well as a human umbilical vein endothelial cell (HUVEC) spheroid sprouting assay (Fig. 3). In the VEGF-R2 phosphorylation assay, the ten VEGF-binding DARPins as well as controls (Isotype DARPin I1 and I2 as negative controls [18]; bevacizumab and ranibizumab as positive controls) were used at a fixed concentration of 1.4 nM in combination with 1.4 nM VEGF-A. Maximal stimulation (PBS added to VEGF-A) and no stimulation (PBS, no VEGF-A used) were used to illustrate the assay window. The results demonstrated that all DARPins and the positive controls are able to suppress VEGF-A induced phosphorylation of VEGFR-2, whereas isotype controls do not affect its phosphorylation (Fig. 2). Overall, some of the selected DARPins showed inhibition potency in the range of ranibizumab and bevacizumab in this assay. These data were confirmed in a spheroidal model of in vitro sprouting angiogenesis [27–29]. In this assay, anti-VEGF-A DARPins potently suppressed endothelial cell sprout formation. Similar to what was observed in the phosphorylation assay, some of the DARPin candidates blocked sprouting very potently, similar to or better than ranibizumab, while the isotype control DARPins showed no effect. These cellular assays confirm that a large number of VEGF-A antagonists was generated, some of which exhibit very high potency in vitro. Note that for both cellular assays, VEGF-A has to be used at 8–70 times higher concentrations than the one used in the Quantikine assay to reliably induce VEGF receptor phosphorylation or spheroid sprouting. Consequently, potency values are limited by the concentration of human VEGF-A used and are thus underestimated.Fig. 2

Bottom Line: The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations.In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model.Even at elevated doses, DARPins were safe to use.

View Article: PubMed Central - PubMed

Affiliation: Universitäts-Augenklinik Freiburg, Freiburg, Germany.

ABSTRACT
The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.

Show MeSH
Related in: MedlinePlus