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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.

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Screening for potent VEGF-A-binding DARPins and characterization of their affinities by sandwich inhibition ELISA (Quantikine; see “Materials and methods” section). a Screening for quantitative VEGF-A-inhibiting DARPins. Numbers 1–10 represent 10 different VEGF-A binding DARPins used in this screening assay. L represents Lucentis (ranibizumab), A represents Avastin (bevacizumab), I1 and I2 represent two non-binding DARPin isotype controls. V1, V2, V3, and V0 represent the signals obtained for 25, 12.5, 6.25, and 0 pM VEGF-A, respectively, applied without any inhibitor. The dashed line represents the signal obtained with free 12.5 pM VEGF-A, corresponding approximately to the equivalent of 50 % inhibition. b IC50 analysis of DARPin #6 in the identical assay as a. DARPin #6 is displayed as one VEGF-binding DARPin representative; similar curves were obtained for other anti-VEGF DARPins identified in a
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Fig1: Screening for potent VEGF-A-binding DARPins and characterization of their affinities by sandwich inhibition ELISA (Quantikine; see “Materials and methods” section). a Screening for quantitative VEGF-A-inhibiting DARPins. Numbers 1–10 represent 10 different VEGF-A binding DARPins used in this screening assay. L represents Lucentis (ranibizumab), A represents Avastin (bevacizumab), I1 and I2 represent two non-binding DARPin isotype controls. V1, V2, V3, and V0 represent the signals obtained for 25, 12.5, 6.25, and 0 pM VEGF-A, respectively, applied without any inhibitor. The dashed line represents the signal obtained with free 12.5 pM VEGF-A, corresponding approximately to the equivalent of 50 % inhibition. b IC50 analysis of DARPin #6 in the identical assay as a. DARPin #6 is displayed as one VEGF-binding DARPin representative; similar curves were obtained for other anti-VEGF DARPins identified in a

Mentions: Consistent with previous publications [17, 25, 26], target-binding DARPins from naïve DARPin libraries could rapidly be enriched using ribosome display. To guarantee high-affinity binding, three standard selection rounds were performed followed by three consecutive off-rate selection rounds followed by a collection round. Importantly, no additional randomization was applied during the selection process and a proof-reading DNA-polymerase was used for DNA amplifications. The resulting DNA pools were screened for VEGF-A binders by crude extract ELISA. Binders with strong ELISA signal were further characterized. The identified candidates were expressed in E. coli and purified from the soluble fraction using described methods (see “Materials and methods”). Expression levels were comparable to previously published DARPins and in the range of 200 mg expressed protein per liter shake-flask culture using LB-Lennox medium supplemented with 1 % glucose and using E. coli XL-1 Blue as expression strain. Strong interaction of the DARPins with VEGF-A165 was confirmed by ELISA. A competition assay showed that the DARPins interact well with both VEGF-A121, and VEGF-A165 (of human, dog, mouse, and rabbit), but not with VEGF-C. After purification, anti-VEGF-A DARPins were analyzed in more detail using a Quantikine sandwich ELISA (Fig. 1). In this assay, human VEGF-A is incubated with either a DARPin or controls and then applied to a plate which is pre-coated with a monoclonal anti-VEGF-A antibody. VEGF-A binding to the plate is then detected using a polyclonal anti-VEGF-A-HRP conjugate. Strong VEGF-A binders thus quantitatively reduce the ELISA signal compared to controls. Our results showed that all DARPins tested induced strong signal suppression of more than 50 % (Fig. 1), even when using only 25 pM (monomer) VEGF-A, whereas the isotype controls (i.e. non-binding DARPins) were not affecting the signals. This indicates that the affinity of these DARPins tested is at least KD = 25 pM and that the chosen selection strategy led to a panel of highly potent anti-VEGF-A DARPins. As most DARPins showed inhibited signal down to background (Fig. 1), the potency of DARPin #4 was analyzed in more detail by performing the Quantikine experiment with varying DARPin concentrations (Fig. 1). An apparent IC50 value of 10 pM was derived by fitting the observed values. Note that in this experiment, the VEGF-A concentration (20 pM) is limiting exact affinity determination, as the DARPin titrates the amount of VEGF-A, indicating that the effective IC50 may be below 10 pM for DARPin #4. A more accurate determination of the DARPin affinity is limited by the detection limit of the Quantikine assay. The low picomolar anti-VEGF-A affinity of the selected DARPins was further confirmed by surface Plasmon resonance (SPR), where the detection system similarly works at its detection limits due to very slow off-rates. Also, we could show that by binding to VEGF-A, the DARPins block binding of VEGF-A to its receptor VEGFR-2, similar to Lucentis (see Supplementary Figure 1).Fig. 1


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)

Screening for potent VEGF-A-binding DARPins and characterization of their affinities by sandwich inhibition ELISA (Quantikine; see “Materials and methods” section). a Screening for quantitative VEGF-A-inhibiting DARPins. Numbers 1–10 represent 10 different VEGF-A binding DARPins used in this screening assay. L represents Lucentis (ranibizumab), A represents Avastin (bevacizumab), I1 and I2 represent two non-binding DARPin isotype controls. V1, V2, V3, and V0 represent the signals obtained for 25, 12.5, 6.25, and 0 pM VEGF-A, respectively, applied without any inhibitor. The dashed line represents the signal obtained with free 12.5 pM VEGF-A, corresponding approximately to the equivalent of 50 % inhibition. b IC50 analysis of DARPin #6 in the identical assay as a. DARPin #6 is displayed as one VEGF-binding DARPin representative; similar curves were obtained for other anti-VEGF DARPins identified in a
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526737&req=5

Fig1: Screening for potent VEGF-A-binding DARPins and characterization of their affinities by sandwich inhibition ELISA (Quantikine; see “Materials and methods” section). a Screening for quantitative VEGF-A-inhibiting DARPins. Numbers 1–10 represent 10 different VEGF-A binding DARPins used in this screening assay. L represents Lucentis (ranibizumab), A represents Avastin (bevacizumab), I1 and I2 represent two non-binding DARPin isotype controls. V1, V2, V3, and V0 represent the signals obtained for 25, 12.5, 6.25, and 0 pM VEGF-A, respectively, applied without any inhibitor. The dashed line represents the signal obtained with free 12.5 pM VEGF-A, corresponding approximately to the equivalent of 50 % inhibition. b IC50 analysis of DARPin #6 in the identical assay as a. DARPin #6 is displayed as one VEGF-binding DARPin representative; similar curves were obtained for other anti-VEGF DARPins identified in a
Mentions: Consistent with previous publications [17, 25, 26], target-binding DARPins from naïve DARPin libraries could rapidly be enriched using ribosome display. To guarantee high-affinity binding, three standard selection rounds were performed followed by three consecutive off-rate selection rounds followed by a collection round. Importantly, no additional randomization was applied during the selection process and a proof-reading DNA-polymerase was used for DNA amplifications. The resulting DNA pools were screened for VEGF-A binders by crude extract ELISA. Binders with strong ELISA signal were further characterized. The identified candidates were expressed in E. coli and purified from the soluble fraction using described methods (see “Materials and methods”). Expression levels were comparable to previously published DARPins and in the range of 200 mg expressed protein per liter shake-flask culture using LB-Lennox medium supplemented with 1 % glucose and using E. coli XL-1 Blue as expression strain. Strong interaction of the DARPins with VEGF-A165 was confirmed by ELISA. A competition assay showed that the DARPins interact well with both VEGF-A121, and VEGF-A165 (of human, dog, mouse, and rabbit), but not with VEGF-C. After purification, anti-VEGF-A DARPins were analyzed in more detail using a Quantikine sandwich ELISA (Fig. 1). In this assay, human VEGF-A is incubated with either a DARPin or controls and then applied to a plate which is pre-coated with a monoclonal anti-VEGF-A antibody. VEGF-A binding to the plate is then detected using a polyclonal anti-VEGF-A-HRP conjugate. Strong VEGF-A binders thus quantitatively reduce the ELISA signal compared to controls. Our results showed that all DARPins tested induced strong signal suppression of more than 50 % (Fig. 1), even when using only 25 pM (monomer) VEGF-A, whereas the isotype controls (i.e. non-binding DARPins) were not affecting the signals. This indicates that the affinity of these DARPins tested is at least KD = 25 pM and that the chosen selection strategy led to a panel of highly potent anti-VEGF-A DARPins. As most DARPins showed inhibited signal down to background (Fig. 1), the potency of DARPin #4 was analyzed in more detail by performing the Quantikine experiment with varying DARPin concentrations (Fig. 1). An apparent IC50 value of 10 pM was derived by fitting the observed values. Note that in this experiment, the VEGF-A concentration (20 pM) is limiting exact affinity determination, as the DARPin titrates the amount of VEGF-A, indicating that the effective IC50 may be below 10 pM for DARPin #4. A more accurate determination of the DARPin affinity is limited by the detection limit of the Quantikine assay. The low picomolar anti-VEGF-A affinity of the selected DARPins was further confirmed by surface Plasmon resonance (SPR), where the detection system similarly works at its detection limits due to very slow off-rates. Also, we could show that by binding to VEGF-A, the DARPins block binding of VEGF-A to its receptor VEGFR-2, similar to Lucentis (see Supplementary Figure 1).Fig. 1

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