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Structure-Guided Molecular Engineering of a Vascular Endothelial Growth Factor Antagonist to Treat Retinal Diseases

  • 2020 CMBE Young Innovators issue
  • Published:
Cellular and Molecular Bioengineering Aims and scope Submit manuscript

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

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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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Authors and Affiliations

  1. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Rakeeb Kureshi, Stephany Y. Tzeng, Leilani R. Astrab, Jordan J. Green & Jamie B. Spangler

  2. Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Angela Zhu, Leilani R. Astrab, Paul R. Sargunas, Jordan J. Green & Jamie B. Spangler

  3. Department of Ophthalmology, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Jikui Shen, Jordan J. Green & Peter A. Campochiaro

  4. Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Jikui Shen & Peter A. Campochiaro

  5. Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Stephany Y. Tzeng, Jordan J. Green & Jamie B. Spangler

  6. Insititute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD, USA

    Stephany Y. Tzeng & Jordan J. Green

  7. Department of Materials Science & Engineering, Johns Hopkins University, Baltimore, MD, USA

    Jordan J. Green

  8. Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Jordan J. Green

Authors
  1. Rakeeb Kureshi
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  2. Angela Zhu
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Contributions

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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Correspondence to Jamie B. Spangler.

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Associate Editor Scott Simon oversaw the review of this article.

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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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