COVID-19 Research: Japanese Researchers Engineer High-Affinity ACE2 Receptors To Serve As Decoys To Neutralize SARS-CoV-2
: Japanese researchers from the Kyoto Prefectural University of Medicine and Osaka University have engineered to create soluble ACE2 (sACE2) in human cells that have enhanced affinity to receptor-binding domain (RBD) of the SARS-CoV-2 thus serving as decoy while also neutralizing the novel coronavirus.
The research findings were published on a preprint serve and are currently being peer-reviewed. https://www.biorxiv.org/content/10.1101/2020.09.16.299891v1
The SARS-Cov-2 is a single-stranded RNA virus that binds to host cells with its trimeric spike glycoprotein S, which has two subunits. The S1 subunit facilitates receptor binding, and S2 is responsible for membrane fusion.
It is known that during infection, the receptor-binding domain (RBD) of the S1 subunit directly binds with the human angiotensin-converting enzyme 2(ACE2) receptor. RBD is shown to be the common binding site of neutralizing antibodies from convalescent patients. Thus, the RBD can serve as a key target for drugs that inhibit viral infection.
It must be noted that RNA viruses have high mutation rates and evolvability that help them acquire anti-viral drug resistance. Evidence shows that neutralizing antibodies have efficacy in fighting COVID-19 and monoclonal antibodies taken from convalescent COVID-19 patients have been shown to have potency in neutralizing viruses. However, spike gene mutation can cause SARS-CoV-2 adaptation to such antibodies. Hence this is what motivated the researchers to look at alternatives besides antibodies.
The extracellular domain of ACE2 can also serve as a decoy receptor to neutralize SARS-CoV-2 similar to the anti-RBD antibodies.
The therapeutic potency of ACE2 against COVID-19 has been confirmed by a few studies. Also, fusing sACE2 to the human IgG1 Fc region has been shown to increase neutralization capacity and boost pharmacokinetics to human IgG levels in mice. Increasing the affinity of ACE2 to RBD is crucial for adequate protection against viral mutation.
The Japanese researchers in this study engineered ACE2 to enhance its affinity to RBD in human cells.
The stud team introduced random mutations in the protease domain, having the interface to the RBD. Full length ACE2 mutant library expressed in 293T cells was incubated with fluorescence-labeled RBD.
Subsequently cells that showed high binding activity were sorted, and DNA extracted from these cells was further induced with random mutations for the next selection cycle.
The team carried out three such cycles of random mutation and the cell sorting produced ACE2 that had over 100-fold higher affinity to RBD compared to wild-type ACE2. This protein engineering system generated a virus-neutralizing drug with high affinity comparable with that of antibodies and can provide a solution to drug resistance due to escape mutation.
Co-corresponding author Dr Junichi Takagi from the Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University told Thailand Medical News, “Significantly, when the neutralization potential against the authentic SARS-CoV-2 in TM
PRSS2- expressing VeroE6 cells was evaluated, wild-type sACE2-Fc showed no efficiency even at 100μg/ml, whereas 3N39 sACE2-Fc demonstrated significant neutralizing effect in 6.3μg/ml.”
It was found that when the extracellular domain of the engineered ACE2 fused to the human immunoglobulin IgG1 Fc region, a more stable structure resulted that was able to neutralize SARS-CoV-2 pseudotyped lentivirus and authentic virus at over 100-fold lower concentration than wild-type ACE2.
It must be noted that engineering decoy receptors with improved affinity has been previously reported in cancer-related molecules. These decoy receptor drugs are used to neutralize various cytokines such as vascular tumor necrosis factor-alpha, endothelial growth factor, and CTLA-4 and are approved for the treatment of rheumatoid arthritis and orbital vascular diseases.
It was found that although recombinant sACE2 or sACE2-Fc fusion protein has the ability to neutralize the SARS-CoV-2 virus, due to its modest binding affinity, a higher dose is required than a monoclonal antibody.
The new mutant ACE2s developed by the team not only had affinity comparable to anti-spike antibodies, but they also had a more extensive Interface to the RBD compared to that of antibodies, which increases their efficacy.
Dr Takagi added, “We developed the screening system based on the cycle of random mutation and sorting of high-affinity population in 293T cells followed by validation of neutralizing activity in a soluble form.”
The study team concluded that engineering decoy ACE2 receptors with directed evolution could be an effective approach in the development of a SARS-CoV-2 neutralizing drug that has an RBD affinity comparable to monoclonal antibodies, yet resist escape mutation of the virus.
The study team says that high-affinity, engineered ACE2 fused with Fc protein is a promising approach to neutralizing the SARS-CoV-2 virus. Also, the system they have developed can rapidly generate therapeutic candidates effective against many different viral diseases and may help fight future pandemics caused by viruses.
They added, “The time frame for running one cycle of mutagenesis and sorting was just one week in our system, and we succeeded in developing optimized mutants in a couple of months without depending on patients derived cells or tissues.”
The study team is planning vivo studies involving animal models to test out their engineered ACE2 decoys’ effectiveness before proceeding to human clinical trials.
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