Abstract
Background
Ocular neovascularization is a hallmark of retinal diseases including neovascular age-related macular degeneration and diabetic retinopathy, two leading causes of blindness in adults. Neovascularization is driven by the interaction of soluble vascular endothelial growth factor (VEGF) ligands with transmembrane VEGF receptors (VEGFR), and inhibition of the VEGF pathway has shown tremendous clinical promise. However, anti-VEGF therapies require invasive intravitreal injections at frequent intervals and high doses, and many patients show incomplete responses to current drugs due to the lack of sustained VEGF signaling suppression.
Methods
We synthesized insights from structural biology with molecular engineering technologies to engineer an anti-VEGF antagonist protein. Starting from the clinically approved decoy receptor protein aflibercept, we strategically designed a yeast-displayed mutagenic library of variants and isolated clones with superior VEGF affinity compared to the clinical drug. Our lead engineered protein was expressed in the choroidal space of rat eyes via nonviral gene delivery.
Results
Using a structure-informed directed evolution approach, we identified multiple promising anti-VEGF antagonist proteins with improved target affinity. Improvements were primarily mediated through reduction in dissociation rate, and structurally significant convergent sequence mutations were identified. Nonviral gene transfer of our engineered antagonist protein demonstrated robust and durable expression in the choroid of treated rats one month post-injection.
Conclusions
We engineered a novel anti-VEGF protein as a new weapon against retinal diseases and demonstrated safe and noninvasive ocular delivery in rats. Furthermore, our structure-guided design approach presents a general strategy for discovery of targeted protein drugs for a vast array of applications.
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Abbreviations
- NVAMD:
-
Neovascular age-related macular degeneration
- DR:
-
Diabetic retinopathy
- VEGF:
-
Vascular endothelial growth factor
- PDGF:
-
Platelet derived growth factor
- VEGFR:
-
Vascular endothelial growth factor receptor
- RTK:
-
Receptor tyrosine kinase
- IgG1:
-
Immunoglobulin G1
- AAV:
-
Adeno-associated virus
- LV:
-
Lentivirus
- HEK:
-
Human embryonic kidney
- FPLC:
-
Fast protein liquid chromatography
- HBS:
-
HEPES-buffered saline
- BAP:
-
Biotin-acceptor protein
- PDBePISA:
-
Protein data bank in Europe proteins, interfaces, structures, and assemblies
- MACS:
-
Magnetic-activated cell sorting
- SA:
-
Streptavidin
- BSA:
-
Bovine serum albumin
- EDTA:
-
Ethylenediaminetetraacetic acid
- FACS:
-
Fluorescence-activated cell sorting
- PBAE:
-
Poly(beta-amino ester)
- THF:
-
Tetrahydrofuran
- DMSO:
-
Dimethyl sulfoxide
- PDI:
-
Polydispersity index
- PTFE:
-
Polytetrafluoroethylene
- GPC:
-
Gel permeation chromatography
- DLS:
-
Dynamic light scattering
- NTA:
-
Nanoparticle tracking analysis
- PBS:
-
Phosphate-buffered saline
- TEM:
-
Transmission electron microscopy
- BLI:
-
Bio-layer interferometry
- scFv:
-
Single-chain variable fragment
References
Avery, R. L., et al. Systemic pharmacokinetics and pharmacodynamics of intravitreal aflibercept, bevacizumab, and ranibizumab. Retina 37:1847–1858, 2017.
Boder, E. T., and K. D. Wittrup. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15:553–557, 1997.
Boyer, D. S., J. S. Heier, D. M. Brown, S. F. Francom, T. Ianchulev, and R. G. Rubio. A phase IIIb study to evaluate the safety of ranibizumab in subjects with neovascular age-related macular degeneration. Ophthalmology 116:1731–1739, 2009.
Brown, D. M., et al. Ranibizumab vs. verteporfin for neovascular age-related macular degeneration. N. Engl. J. Med. 355:1432–1444, 2006.
Brozzo, M. S., et al. Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization. Blood 119:1781–1788, 2012.
Campochiaro, P. A. Ocular neovascularization. J. Mol. Med. 91:311–321, 2013.
Campochiaro, P. A., et al. Lentiviral vector gene transfer of endostatin/angiostatin for macular degeneration (GEM) study. Hum. Gene Ther. 28:99–111, 2017.
CATT Research Group. Ranibizumab and bevacizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 364:1897–1908, 2011.
Chao, G., W. L. Lau, B. J. Hackel, S. L. Sazinsky, S. M. Lippow, and K. D. Wittrup. Isolating and engineering human antibodies using yeast surface display. Nat. Protoc. 1:755–768, 2006.
Daien, V., et al. Incidence and outcomes of infectious and noninfectious endophthalmitis after intravitreal injections for age-related macular degeneration. Ophthalmology 125:66–74, 2018.
Diabetic Retinopathy Clinical Research Network. Aflibercept, bevacizumab, or ranibizumab for diabetic macular edema. N. Engl. J. Med. 372:1193–1203, 2015.
Ding, K., et al. AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression. J. Clin. Invest. 129:4901–4911, 2019.
Dixon, J. A., S. C. N. Oliver, J. L. Olson, and N. Mandava. VEGF trap-eye for the treatment of neovascular age-related macular degeneration. Expert Opin. Investig. Drugs. 18:1573–1580, 2009.
Falavarjani, K. G., and Q. D. Nguyen. Adverse events and complications associated with intravitreal injection of anti-VEGF agents: a review of literature. Eye 27:787–794, 2013.
Heier, J. S., et al. Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial. Lancet 390:50–61, 2017.
Ho, C. C. M., et al. “Velcro” engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis. J. Biol. Chem. 290:12650–12663, 2015.
Holz, F. G., et al. Multi-country real-life experience of anti-vascular endothelial growth factor therapy for wet age-related macular degeneration. Br. J. Ophthalmol. 99:220–226, 2015.
Holz, F. G., S. Schmitz-Valckenberg, and M. Fleckenstein. Recent developments in the treatment of age-related macular degeneration. J. Clin. Invest. 124:1430–1438, 2014.
Jager, R. D., W. F. Mieler, and J. W. Miller. Age-related macular degeneration. N. Engl. J. Med 358:2606–2617, 2008.
Kieran, M. W., R. Kalluri, and Y.-J. Cho. The VEGF pathway in cancer and disease: responses, resistance, and the path forward. Cold Spring Harb. Perspect. Med. 2:1–17, 2012.
Klein, R., et al. Prevalence of age-related macular degeneration in 4 racial/ethnic groups in the multi-ethnic study of atherosclerosis. Ophthalmology 113:373–380, 2006.
Klein, R., B. E. Klein, S. E. Moss, M. D. Davis, and D. L. DeMets. The Wisconsin epidemiologic study of diabetic retinopathy. II. Prevalence and risk of diabetic retinopathy when age at diagnosis is less than 30 years. Arch. Ophthalmol. 102:520–526, 1984.
Klein, R., B. E. K. Klein, S. C. Tomany, S. M. Meuer, and G.-H. Huang. Ten-year incidence and progression of age-related maculopathy: the Beaver Dam eye study. Ophthalmology 109:1767–1779, 2002.
Kotterman, M. A., L. Yin, J. M. Strazzeri, J. G. Flannery, W. H. Merigan, and D. V. Schaffer. Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates. Gene Ther. 22:116–126, 2015.
Krissinel, E., and K. Henrick. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372:774–797, 2007.
Krohl, P. J., S. D. Ludwig, and J. B. Spangler. Emerging technologies in protein interface engineering for biomedical applications. Curr. Opin. Biotech. 60:82–88, 2019.
Kwak, N., N. Okamoto, J. M. Wood, and P. A. Campochiaro. VEGF is major stimulator in model of choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 41:3158–3164, 2000.
MacDonald, D. A., et al. Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes. Angiogenesis 19:389–406, 2016.
Markovic-Mueller, S., et al. Structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A. Structure 25:341–352, 2017.
Pandey Arvind, K., et al. Mechanisms of VEGF (Vascular Endothelial Growth Factor) inhibitor-associated hypertension and vascular disease. Hypertension 71:e1–e8, 2018.
Papadopoulos, N., et al. Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis 15:171–185, 2012.
Patel, S. R., D. E. Berezovsky, B. E. McCarey, V. Zarnitsyn, H. F. Edelhauser, and M. R. Prausnitz. Targeted administration into the suprachoroidal space using a microneedle for drug delivery to the posterior segment of the eye. Invest. Ophthalmol. Vis. Sci. 53:4433–4441, 2012.
Patel, S. R., A. S. P. Lin, H. F. Edelhauser, and M. R. Prausnitz. Suprachoroidal drug delivery to the back of the eye using hollow microneedles. Pharm. Res. 28:166–176, 2011.
Pennington, K. L., and M. M. DeAngelis. Epidemiology of age-related macular degeneration (AMD): associations with cardiovascular disease phenotypes and lipid factors. Eye Vis. (Lond) 3:1–20, 2016.
Rakoczy, E. P., et al. Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial. Lancet 386:2395–2403, 2015.
Raman, S., et al. Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies. Proc. Natl. Acad. Sci. USA 116:6812–6817, 2019.
Reichel, F. F., et al. AAV8 can induce innate and adaptive immune response in the primate eye. Mol. Ther. 25:2648–2660, 2017.
Rosenfeld, P. J., et al. Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med. 355:1419–1431, 2006.
Saaddine, J. B., K. M. V. Narayan, and F. Vinicor. Vision loss: a public health problem? Ophthalmology 110:253–254, 2003.
Schlothauer, T., et al. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Eng. Des. Sel. 29:457–466, 2016.
Shen, J., et al. Suprachoroidal gene transfer with nonviral nanoparticles. Sci. Adv. 6:1–10, 2020.
Shmueli, R. B., J. C. Sunshine, Z. Xu, E. J. Duh, and J. J. Green. Gene delivery nanoparticles specific for human microvasculature and macrovasculature. Nanomedicine 8:1200–1207, 2012.
Silva, D.-A., et al. De novo design of potent and selective mimics of IL-2 and IL-15. Nature 565:186–191, 2019.
Singer, M. A., et al. HORIZON: an open-label extension trial of ranibizumab for choroidal neovascularization secondary to age-related macular degeneration. Ophthalmology 119:1175–1183, 2012.
Sunshine, J. C., S. B. Sunshine, I. Bhutto, J. T. Handa, and J. J. Green. Poly(β-Amino Ester)-nanoparticle mediated transfection of retinal pigment epithelial cells in vitro and in vivo. PLoS ONE 7:e37543, 2012.
Tzeng, S. Y., L. J. Higgins, M. G. Pomper, and J. J. Green. Biomaterial-mediated cancer-specific DNA delivery to liver cell cultures using synthetic poly(beta-amino ester)s. J. Biomed. Mater. Res. 101A:1837–1845, 2013.
Vitt, U. A., S. Y. Hsu, and A. J. W. Hsueh. Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules. Mol. Endocrinol. 15:681–694, 2001.
Weiskopf, K., et al. Engineered SIRPα variants as immunotherapeutic adjuvants to anticancer antibodies. Science 341:88–91, 2013.
Wrenbeck, E. E., M. S. Faber, and T. A. Whitehead. Deep sequencing methods for protein engineering and design. Curr. Opin. Struct. Biol. 45:36–44, 2017.
Xiong, W., et al. AAV cis-regulatory sequences are correlated with ocular toxicity. Proc. Natl. Acad. Sci. USA 116:5785–5794, 2019.
Yang, J., et al. Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A. Mol. Pharm. 11:3421–3430, 2014.
Yorston, D. Anti-VEGF drugs in the prevention of blindness. Community Eye Health 27:44–46, 2014.
Acknowledgments
The authors thank Patrick James Krohl for technical assistance with the project. The authors also acknowledge the NIH (R01EY031097, R01CA228133), the E. Matilda Ziegler Foundation for the Blind, the Louis B. Thalheimer Translational Fund, and Research to Prevent Blindness (Dr. H. James and Carole Free Catalyst Award and unrestricted grant) for support.
Conflict of interest
The authors declare that they have no conflicts of interest.
Ethical Approval
All animals were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research, and protocols were reviewed and approved by the Johns Hopkins University Animal Care and Use Committee. No human studies were carried out by the authors for this article.
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RK, AZ, JS, SYT, LRA, and PRS designed, conducted, and analyzed experiments. PAC, JJG, and JBS conceptualized the study, supervised all research, interpreted data, and provided funding. RK, PRS, and JBS wrote the manuscript with input from co-authors.
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12195_2020_641_MOESM1_ESM.pptx
Supplementary Figure 1: VEGF-A affinity characterization of receptor variants. Bio-layer interferometry-based kinetic titrations of soluble Fc-fused aflibercept (a), Fc-fused VEGFR-1 domains 2 and 3 (VEGFR-1-Fc) (b), and Fc-fused VEGFR-2 domains 2 and 3 (VEGFR-2-Fc) (c) against immobilized VEGF-A. Fivefold titrations of soluble protein were used starting at 100 nM for aflibercept and VEGFR-1-Fc and starting at 250 nM for VEGFR-2-Fc. Binding constants were determined using Octet Data Analysis HT Software and are presented in Table 1.
Supplementary Figure 2: VEGF-A affinity characterization of enhanced-affinity aflibercept variants. Bio-layer interferometry-based kinetic titrations of soluble Fc-fused aflibercept variants C5 (a), D4 (b), and F3 (c) against immobilized VEGF-A. Fivefold dilutions of soluble protein were used starting at 100 nM for all aflibercept variants. Binding constants were measured using Octet Data Analysis HT Software and are presented in Table 2.
Supplementary Figure 3: DNA encapsulation in NPs. PBAE/DNA NPs were made by diluting DNA in 25 mM sodium acetate buffer at pH 5 (NaAc), and mixing with diluted PBAE at increasing polymer-to-DNA mass ratios (w/w). After 10 min of incubation for NP formation, sucrose was added, and the NPs were then diluted 1:11 (v/v) in additional NaAc (a) or PBS (b). Samples were mixed with 30% glycerol as a loading buffer at a 1:5 ratio (v/v) of loading buffer to NPs, then loaded into a 1% agarose gel with 1 µg/mL ethidium bromide. Each well contained 110 ng DNA. The gel was run for 30 min under 80 V, then visualized by UV exposure. DNA was completely bound in the NPs at 2 w/w or higher at pH 5, and even after dilution in PBS, DNA was completely bound at 5 w/w or higher.
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Kureshi, R., Zhu, A., Shen, J. et al. Structure-Guided Molecular Engineering of a Vascular Endothelial Growth Factor Antagonist to Treat Retinal Diseases. Cel. Mol. Bioeng. 13, 405–418 (2020). https://doi.org/10.1007/s12195-020-00641-0
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DOI: https://doi.org/10.1007/s12195-020-00641-0