| Title:
|
Design, engineering and construction of a therapeutic anti-VEGF chimeric protein, similar to sFLT01, with a prolonged half-life
|
| Author(s):
|
Edris Rezaei, Mehdi Sadeghi, Zahra-soheila soheili, Shahram Samiei,Ali Kashanian
|
| Presentation Type:
|
Oral
|
| Subject:
|
Molecular Biology and Genetics
|
| Others:
|
|
|
Presenting Author:
|
|
| Name:
|
Edris Rezaei
|
| Affiliation :(optional)
|
Department of Genetic Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran. Iran
|
| E mail:
|
edrisrezaei.88@ut.ac.ir
|
| Phone:
|
|
|
Mobile: |
09181707379
|
|
Abstract (Max 200 words)
|
|
| Purpose:
|
Age-related macular degeneration (AMD), the leading cause of blindness, irreversible vision loss in individuals over the age of 50 years in the world, is a degenerative disorder of the retinal pigment epithelium (RPE) and neurosensory retina. Choroidal neovascularization (CNV), the hallmark of ‘wet’, ‘exudative’ or ‘neovascular’ AMD, is responsible for approximately 90% of the cases of severe vision loss due to AMD. Vascular endothelial growth factor (VEGF) has been shown to play a key role in the regulation of CNV and vascular permeability. We constructed an adeno-associated virus harboring human soluble VEGF receptor, similar to sFLT01, fused to Fc portion of human IgG1. The neonatal FcR (FcRn) binds to the Fc domain of sFLT01 at acidic pH in the endosome and protects sFLT01 from degradation, thereby contributes to the long serum half-life of sFLT01. Modulation of the interaction between Fc and FcRn through protein engineering is one of the desired methods for improving the pharmacokinetics of therapeutic proteins.
|
| Methods:
|
To improve the neutralization ability of anti-VEGF chimeric protein, we used a bioinformatics method for fast in silico mutagenesis of protein–protein complexes and examined the effects of mutations on sFLT01 half-life. The free energy differences between the wild type and mutants were evaluated from a thermodynamic stability prediction upon mutation model. Gene splicing by overlap extension (gene SOEing) was used for two variants N163A/T341Q and T330Q/M347L in the sFLT01.
|
| Results:
|
The sFLT01 mutants were expressed in human embryonic kidney cells 293(HEK 293). These two variants, displayed increased binding affinity to FcRn at pH 6 and negligible binding to FcRn at pH 7.4. They also exhibited increased exposure, and increased half-life when compared to the wild type anti-VEGFs.Two variants N163A/T341Q and T330Q/M347L, seemed to exert different extents of affinity improvement over binding to a wild type anti-VEGF.
|
| Conclusion:
|
These results suggest that, if proven to be clinically safe and effective, a modified version of sFLT01 could potentially provide clinical benefits to patients for its long lasting effects, anti-VEGF therapy through less-frequent dosing and improved compliance with the drug therapy.
|
| Attachment:
|
|