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3 results on '"Mason Wolak"'

1. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Author

Ying Fu, Juliana da Fonseca Rezende E Mello, Bryan D Fleming, Alex Renn, Catherine Z Chen, Xin Hu, Miao Xu, Kirill Gorshkov, Quinlin Hanson, Wei Zheng, Emily M Lee, Lalith Perera, Robert Petrovich, Manisha Pradhan, Richard T Eastman, Zina Itkin, Thomas B Stanley, Allen Hsu, Venkata Dandey, Kedar Sharma, William Gillette, Troy Taylor, Nitya Ramakrishnan, Shelley Perkins, Dominic Esposito, Eunkeu Oh, Kimihiro Susumu, Mason Wolak, Marc Ferrer, Matthew D Hall, Mario J Borgnia, and Anton Simeonov

Subjects
Medicine, Science
Abstract

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

Published
2022
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2. High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

Author

Kirill Gorshkov, Mason Wolak, Kimihiro Susumu, Eunkeu Oh, and Bruce Nguyen Tran

Subjects
HEK293 Cells, General Immunology and Microbiology, SARS-CoV-2, General Chemical Engineering, General Neuroscience, Quantum Dots, Spike Glycoprotein, Coronavirus, Humans, Endocytosis, General Biochemistry, Genetics and Molecular Biology
Abstract

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

Published
2022

3. A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike

Author

Ying Fu, Juliana da Fonseca Rezende e Mello, Bryan D. Fleming, Alex Renn, Catherine Z. Chen, Xin Hu, Miao Xu, Kirill Gorshkov, Quinlin Hanson, Wei Zheng, Emily M. Lee, Lalith Perera, Robert Petrovich, Manisha Pradhan, Richard T. Eastman, Zina Itkin, Thomas B. Stanley, Allen Hsu, Venkata Dandey, Kedar Sharma, William Gillette, Troy Taylor, Nitya Ramakrishnan, Shelley Perkins, Dominic Esposito, Eunkeu Oh, Kimihiro Susumu, Mason Wolak, Marc Ferrer, Matthew D. Hall, Mario J. Borgnia, and Anton Simeonov

Subjects
Mutation, Multidisciplinary, biology, SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19, Spike Protein, Human airway, Single-Domain Antibodies, Antibodies, Viral, medicine.disease_cause, Antibodies, Neutralizing, Virology, Article, Neutralization, Spike Glycoprotein, Coronavirus, medicine, biology.protein, Humans, Bacteriophages, Spike (software development), Antibody, Ex vivo, Protein Binding
Abstract

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: A cost-effective, high-throughput, adaptable pipeline capable of identifying effective humanized nanobodies against SARS-CoV-2.

Published
2021

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