COVID-19: Kyoto Prefectural University of Medicine, Neutralizing protein preparation: Binding inhibition / protein development
“Spike protein” of the new corona:
The new corona uses the “spike protein on the surface of the virus” to enter the cell.
This protein binds to the protein “ACE2” on the surface of human cells.
Produce proteins that interfere with binding:
The research team modified the ACE2 gene to create a protein that interferes with binding.
Binding power of modified protein:
The ability to bind to spike proteins is 100 times higher.
If you administer this as a medicine,
The new corona binds to the produced protein first,
It is said that it can prevent invasion into living cells.
At the time of practical use, we are considering a method of injecting protein intravenously.
Life Science Institute:
In collaboration with the Life Science Institute under the umbrella of Mitsubishi Chemical Holdings, we will confirm the safety through animal experiments.
If all goes well, we will proceed to clinical trials and aim for practical application.
Nihon Keizai Shimbun
https://www.nikkei.com/article/DGXMZO66383590Y0A111C2000000/
Development of new coronavirus neutralizing protein preparation
Kyoto Prefectural University of Medicine: Assistant Professor Atsushi Hoshino,
Osaka University: Junichi Takagi Teaching,
Institute for Microbial Diseases: Professor Toru Okamoto,
Research group:
We have succeeded in increasing the “virus binding capacity of the ACE2 protein, which is a receptor for the new coronavirus,” more than 100 times.
ACE2 protein:
The new coronavirus binds to and infects the ACE2 protein in human cells.
By using “high affinity modified ACE2 protein with enhanced binding force”, it is expected to have the effect of inhibiting infection to human cells.
Drug discovery of virus-neutralizing protein preparations:
“Drug discovery of virus-neutralizing protein preparation with this high-affinity modified ACE2 protein” is planned.
In the future, we will collaborate with Life Science Institute Co., Ltd.
Research methods and results:
The joint research group
We used the directional evolution method (2), a method of evolving the ACE2 protein so that it easily binds to the virus, so to speak, in vitro.
First,
The ACE2 gene is amplified under conditions that allow errors to easily enter.
Then, we will create a library of 100,000 ACE2 mutants.
next,
Each mutant is expressed on the cell surface.
Then, the one that binds well to the spicy component of the virus is recovered.
In addition
Further mutations are introduced into the recovered ACE2 mutant.
In the same flow, collect the one that binds to the spin component.
By repeating this, we finally succeeded in increasing the binding force of ACE2 to the virus spike more than 100 times (Fig. 1).
Dissociation constant (KD value):
The dissociation constant (KD value), which is an index of binding force, is
Wild type is 41.4nM,
Modified ACE2 is 0.37nM,
Achieved a binding force equal to or higher than that of antibody preparations.
next,
A “protein preparation in which the Fc region (3) of the antibody is bound to this high-affinity modified ACE2” is synthesized.
Then, we evaluated the function.
By combining Fc,
Stabilization of pharmacokinetics in the body,
It becomes a dimer and can be expected to improve the neutralization activity.
Neutralization experiment against Pseudovirus (4):
50% infection inhibition concentration (IC50)
In the wild type, it is 12.6 μg / mL, whereas it is 12.6 μg / mL.
In the modified version, 0.055 μg / mL
It was found to be about 200 times more effective.
Also,
In the neutralization experiment against the new coronavirus,
In the blood concentration area of general antibody preparations
Good virus neutralizing activity was confirmed in the modified ACE2-Fc (Fig. 2).
【the next deployment】
Using the high affinity modified ACE2 protein developed in this study,
Has high virus neutralizing activity,
Due to a genetic mutation in the virus
Drug resistance is not an issue,
With a new modality,
We will work with Life Science Institute Co., Ltd. on the development of therapeutic agents.
It is hoped that this drug will be safely administered to humans, disseminated, and put an end to COVID-19.
https://www.kpu-m.ac.jp/doc/news/2020/files/25474.pdf
High affinity modified ACE2 receptors prevent SARS-CoV-2 infection
Yusuke Higuchi, Tatsuya Suzuki, Takao Arimori, Nariko Ikemura, Yuhei Kirita, Eriko Ohgitani, Osam Mazda, Daisuke Motooka, Shota Nakamura, View ORCID ProfileYoshiharu Matsuura, Satoaki Matoba, Toru Okamoto, Junichi Takagi, Atsushi Hoshino doi: https://doi.org/10.1101/2020.09.16.299891
ABSTRACT
The SARS-CoV-2 spike protein
binds to the human angiotensin-converting enzyme 2 (ACE2) receptor via receptor binding domain (RBD) to enter into the cell.
Inhibiting this interaction
is a main approach to block SARS-CoV-2 infection and it is required to have high affinity to RBD independently of viral mutation for effective protection.
To this end,
we engineered ACE2 to enhance the affinity with directed evolution in human cells.
Three cycles of random mutation and cell sorting
achieved more than 100-fold higher affinity to RBD than wild-type ACE2.
The extracellular domain of modified ACE2 fused to the Fc region of the human immunoglobulin IgG1
had stable structure and neutralized SARS-CoV-2 pseudotyped lentivirus and authentic virus with more than 100-fold lower concentration than wild-type.
Engineering ACE2 decoy receptors with directed evolution
is a promising approach to develop a SARS-CoV-2 neutralizing drug that has affinity comparable to monoclonal antibodies yet displaying resistance to escape mutations of virus
bioRxiv