A promising therapeutic solution to COVID-19 - using ACE2 decoy

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Andrew Mason
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Summary:

The SARS-CoV-2 virus enters cells through the ACE2 surface protein, which is expressed mainly in cells of the lung and of the small intestine. Using recombinant technology, researchers have created a protein that mimics ACE2. The SARS-CoV-2 virus cannot distinguish the human recombinant soluble ACE2 (hrsACE2) from the ACE2 protein on cell surfaces so the hrsACE2 acts as a decoy diverting the virus from infecting cells.

Main Question or Discussion Point

I came across this article about a promising COVID 19 therapy developed by an international team led by researchers from the University of British Columbia.

Since the SARS-CoV-2 virus that causes COVID-19 gets into cells by attaching to the ACE2 surface protein (which is expressed mainly in epithelial cells of the lung and cells in the lining of the small intestine but also in blood vessels and kidneys and other organs), the idea is to create a molecule that mimics ACE2 but is not attached to a cell to draw the virus away from cells. This very clever idea appears to work:

"The team’s study reveals that clinical grade human recombinant soluble ACE2 (hrsACE2) reduces SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. ... Based on this and other findings, the team declared that hrsACE2 can materially block early stages of SARS-CoV-2 infections. "

The drug that has been developed to deliver hrsACE2 is APN01 or APN001. It will be interesting to see what comes of this approach.

AM
 
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  • #2
Ygggdrasil
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Here's a link to the paper, published in the journal Cell: https://www.cell.com/pb-assets/products/coronavirus/CELL_CELL-D-20-00739.pdf

Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2
Abstract:
We have previously provided the first genetic evidence that Angiotensin converting enzyme 2 (ACE2) is the critical receptor for SARS-CoV and that ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growthof SARS-CoV-2. Here we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5 ,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.
The soluble ACE2 has gone through phase I and II clinical trials for other indications (e.g. see https://link.springer.com/article/10.1007/s40262-013-0072-7 and https://ccforum.biomedcentral.com/articles/10.1186/s13054-017-1823-x), and although it did not seem effective as a cardiovascular drug or treatment for acute respiratory distress syndrome, it was well tolerated by people in the trials, suggesting that it can be administered safely to people. It definitely seems like a promising candidate for further testing as a COVID-19 treatment.

However, because the drug is a biologic, it could be difficult to manufacture large quantities and cost could be an issue.
 
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Andrew Mason
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However, because the drug is a biologic, it could be difficult to manufacture large quantities and cost could be an issue.
Yes, it would likely take a bit of time to ramp up production. I expect that it would be produced by growing it in some fast-replicating bacteria such as E.coli or a yeast into which the hrsACE2 gene has been inserted. That kind of manufacturing system supplies insulin users which are somewhere around .02-.05% of the population of western countries. The demand for a COVID-19 anti-viral treatment would be much less, I would expect, because it would be for short-term use by a patient and there would be many fewer patients than insulin users (we hope...). If it worked, I don't think cost would be a concern.

AM
 
  • #4
Ygggdrasil
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Yes, it would likely take a bit of time to ramp up production. I expect that it would be produced by growing it in some fast-replicating bacteria such as E.coli or a yeast into which the hrsACE2 gene has been inserted. That kind of manufacturing system supplies insulin users which are somewhere around .02-.05% of the population of western countries. The demand for a COVID-19 anti-viral treatment would be much less, I would expect, because it would be for short-term use by a patient and there would be many fewer patients than insulin users (we hope...). If it worked, I don't think cost would be a concern.

AM
According to the Cell paper, they produced the drug in CHO cells (Chinese hamster ovary), which is a common cell line used for the manufacture of biologic drugs.
 
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Andrew Mason
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According to the Cell paper, they produced the drug in CHO cells (Chinese hamster ovary), which is a common cell line used for the manufacture of biologic drugs.
Yes. Thanks for pointing that out. From what I can gather CHO cells reproduce very quickly. Would there be anything to prevent scaling up production to a sufficient level, other than cost?

AM
 
  • #6
BillTre
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I looked CHO cells up last night.

I was concerned that they might only grow as adherent cells (stuck on the substrate).
Apparently they can be grown in suspension which makes production much easier in many ways (I used to grow non-adherent hybridomas to make monoclonal antibodies).
What I don't know is if the CHO cells will produce their product as non-adherent cells.

In graduate school, it was quite easy for me to make decent quantities of hybridoma produced antibody in large non-aggitated TC flasks (in a primitive TC room I set-up, when I was using my DIY plywood hood).
A modern lab should be able to do much better.
 
  • #7
Ygggdrasil
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Now commercial biopharmaceutical manufacturing utilizes bioreactors 10,000 – 25,000 liters and larger in size. Media and nutrients for the cells are monitored continuously as well as oxygen levels, CO2 levels, and waste product. This constant information about cell health has led to more efficient and productive culture with average production yields from 1-6 grams/liter.
https://cellculturedish.com/cho-cells-the-top-expression-system-of-best-selling-biologic-drugs/ (though note that this article is from 2012)

I don't know much about the time needed or cost to scale to these levels of production. Potentially, existing capacity could be diverted toward production of a COVID-19 treatment, but I am not familiar with the logistics here.
 
  • #8
Andrew Mason
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This may help to explain why significantly more men than women experience severe outcomes from COVID19: they seem to have more ACE2 receptors:

“When we found that one of the strongest biomarkers, ACE2, was much higher in men than in women, I realised that this had the potential to explain why men were more likely to die from COVID-19 than women,” said Iziah Sama, a doctor at UMC Groningen who co-led the study.

This follows from earlier studies such as this one from researchers in Wuhan Province, China that:

"the distribution of ACE2 is also more widespread in male donors than females: at least 5 different types of cells in male lung express this receptor, while only 2~4 types of cells in female lung express the receptor. This result is highly consistent with the epidemic investigation showing that most of the confirmed 2019-nCov infected patients were men (30 vs. 11, by Jan 2, 2020)."

That suggests that limiting virus access to cells expressing ACE2 receptors may be a key to limiting the damage.

AM
 
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  • #9
Ygggdrasil
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This may help to explain why significantly more men than women experience severe outcomes from COVID19: they seem to have more ACE2 receptors:

“When we found that one of the strongest biomarkers, ACE2, was much higher in men than in women, I realised that this had the potential to explain why men were more likely to die from COVID-19 than women,” said Iziah Sama, a doctor at UMC Groningen who co-led the study.

This follows from earlier studies such as this one from researchers in Wuhan Province, China that:

"the distribution of ACE2 is also more widespread in male donors than females: at least 5 different types of cells in male lung express this receptor, while only 2~4 types of cells in female lung express the receptor. This result is highly consistent with the epidemic investigation showing that most of the confirmed 2019-nCov infected patients were men (30 vs. 11, by Jan 2, 2020)."

That suggests that limiting virus access to cells expressing ACE2 receptors may be a key to limiting the damage.

AM
I think there are other plausible reasons that could explain the differences in the responses of men and women. Some are behavioral (e.g. smoking is more common in men than women) and some are biological (men tend to have weaker immune responses than women). See https://www.nytimes.com/2020/02/20/health/coronavirus-men-women.html for a good discussion.

I am skeptical of the ACE2 explanation because of other data that suggests relative expression of ACE2 is not a major risk factor for the disease. For example, ACE inhibitors are commonly prescribed to treat high blood pressure. While the drug affects only the ACE enzyme and not ACE2, treatment with the drug was observed increase the expression of ACE2 in animal studies. A similar effect was seen in animal studies of angiotensin II receptor blockers, another class of drugs to treat hypertension. However, despite these drugs promoting higher expression of ACE2, observational studies have found slightly decreased mortality among those taking these drugs. If increased ACE2 expression were a major risk factor, one would expect to have seen worse outcomes among those taking these drugs.
 
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Andrew Mason
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I am skeptical of the ACE2 explanation because of other data that suggests relative expression of ACE2 is not a major risk factor for the disease. For example, ACE inhibitors are commonly prescribed to treat high blood pressure. While the drug affects only the ACE enzyme and not ACE2, treatment with the drug was observed increase the expression of ACE2 in animal studies. A similar effect was seen in animal studies of angiotensin II receptor blockers, another class of drugs to treat hypertension. However, despite these drugs promoting higher expression of ACE2, observational studies have found slightly decreased mortality among those taking these drugs. If increased ACE2 expression were a major risk factor, one would expect to have seen worse outcomes among those taking these drugs.
There may be explanations for the apparent slightly decreased mortality among COVID19 patients using ACE inhibitors such as the one offered in this report.

As that report explains, ACE2 breaks down the Angiontensin II enzyme (ANG II) and ACE stimulates production of ANG II. Although they act in different ways, ACE inhibitors and ACE2 both serve a similar function in regulating the amount of ANG II (ACE inhibitors slow production of ANG II while ACE2 breaks it down). If not regulated, ANG II can produce excessive inflammatory response to injury and lead to severe tissue and organ damage, which is what appears to be occurring in COVID patients.

The Conversation: Canadian edition said:
"Of greatest relevance to COVID-19, ANG II can increase inflammation and the death of cells in the alveoli which are critical for bringing oxygen into the body; these harmful effects of ANG II are reduced by ACE2.

When the SARS-CoV-2 virus binds to ACE2, it prevents ACE2 from performing its normal function to regulate ANG II signaling. Thus, ACE2 action is “inhibited,” removing the brakes from ANG II signaling and making more ANG II available to injure tissues. This “decreased braking” likely contributes to injury, especially to the lungs and heart, in COVID-19 patients.
...
When the amount of ACE2 is reduced because the virus is occupying the receptor, individuals may be more susceptible to severe illness from COVID-19. That is because enough ACE2 is available to facilitate viral entry but the decrease in available ACE2 contributes to more ANG II-mediated injury. In particular, reducing ACE2 will increase susceptibility to inflammation, cell death and organ failure, especially in the heart and the lung.
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hello :


a small question about the ACE2 decoy , would that decoy bind also with blood plasma chemicals?

best regards
hagop
 
  • #12
Ygggdrasil
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As that report explains, ACE2 breaks down the Angiontensin II enzyme (ANG II) and ACE stimulates production of ANG II. Although they act in different ways, ACE inhibitors and ACE2 both serve a similar function in regulating the amount of ANG II (ACE inhibitors slow production of ANG II while ACE2 breaks it down). If not regulated, ANG II can produce excessive inflammatory response to injury and lead to severe tissue and organ damage, which is what appears to be occurring in COVID patients.
In the The Conversation article you cite, the authors argue that lower amounts of ACE2 would lead to more severe disease:
When the amount of ACE2 is reduced because the virus is occupying the receptor, individuals may be more susceptible to severe illness from COVID-19. That is because enough ACE2 is available to facilitate viral entry but the decrease in available ACE2 contributes to more ANG II-mediated injury. In particular, reducing ACE2 will increase susceptibility to inflammation, cell death and organ failure, especially in the heart and the lung.
However, the Reuters piece you cited earlier reports on a study that shows that men have higher levels of ACE2 (also relevant to our discussion, it notes that people taking ACE inhibitors or angiotensin receptor blockers don't have elevated levels of ACE2). If the ACE2 hypothesis is correct, higher levels of ACE2 should be protective against COVID-19, yet this is opposite of the observed poorer outcomes for men vs women. Similarly, the results showing no changes to ACE2 expression when taking ACEi or ARB drugs suggests that changes to ACE2 expression do not underlie the mild protective effect of these drugs on COVID-19. One caveat of the study is that the researchers measured the levels of circulating ACE2 in blood plasma, which may not reflect the changes in lung, which would be most relevant to COVID-19.
 
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Andrew Mason
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In the The Conversation article you cite, the authors argue that lower amounts of ACE2 would lead to more severe disease:

However, the Reuters piece you cited earlier reports on a study that shows that men have higher levels of ACE2 (also relevant to our discussion, it notes that people taking ACE inhibitors or angiotensin receptor blockers don't have elevated levels of ACE2). If the ACE2 hypothesis is correct, higher levels of ACE2 should be protective against COVID-19, yet this is opposite of the observed poorer outcomes for men vs women. Similarly, the results showing no changes to ACE2 expression when taking ACEi or ARB drugs suggests that changes to ACE2 expression do not underlie the mild protective effect of these drugs on COVID-19. One caveat of the study is that the researchers measured the levels of circulating ACE2 in blood plasma, which may not reflect the changes in lung, which would be most relevant to COVID-19.
According to this paper from 2009 dealing with the SARS-CoV virus, which operates in a similar way to SARS-CoV-2, it seems that the virus knocks out the ACE2 function when it gains entry via the ACE2 receptor.

Jia et al (Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia-July 2009) said:
"Additional data indicate that SARS-CoV S protein or SARS virus infection directly downregulates pulmonary ACE2 expression. Together these studies indicate that loss of ACE2 catalytic function perturbs the pulmonary renin-angiotensin system, enhancing inflammation and vascular permeability. In addition to loss of ACE2 cleavage of renin-angiotensin system components, the failure to inactivate other ACE2 targets such as bradykinin metabolites and other vasoactive peptides might also contribute to SARS lung disease."
If I understand it correctly, for ACE2 to function it must be expressed on the cell surface. Since the virus gains entry to the cell by receptor-mediated endocytosis, the virus entry removes both the ACE2 extra-cellular and cytoplasmic domains. So, the more cells expressing ACE2 receptors leads to more cells being infected by the virus, which results in more initial tissue damage, which ramps up production of ANG II, which is unregulated because of the loss of ACE2 function, which leads to catastrophic tissue damage.

AM
 
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Andrew Mason
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a small question about the ACE2 decoy , would that decoy bind also with blood plasma chemicals?
The ability of a protein to bind with other molecules is highly specific to the shape of the protein. It so happens that the spikes on the SARS-CoV-2 virus are complementary to the ACE2 receptor protein so those spikes fit into it and bind to ACE2.

Presumably, the ANG II enzyme also has a similar ability to bind with the ACE2 receptor. I am not sure if that means that hrsACE2 (i.e. present in the blood, unattached to a cell) would bind to ANG II enzymes. I don't know enough to comment further, but it is a good question.

AM
 
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Andrew Mason
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This report in Nature May 15:
https://www.nature.com/articles/d41587-020-00013-z
updates the progress of trials for several antiviral drug candidates that show promise in thwarting SARS-Cov-2, including hrsACE2 (APN01).

The various approaches taken are quite ingenious. It would not surprise me at all if one or more of these drug candidates should end up being effective in neutralizing the virus long before an effective vaccine is found.

AM
 
  • #16
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This paper:
https://www.sciencedirect.com/science/article/pii/S2329050120301005
looks at differences in COVID19 susceptibility in the population. The amount of ACE2 expression seems to be key. It offers this as an explanation for the fact that children are less susceptible to SARS-Cov-2 infection. The authors comment:
Airways Expression of SARS-CoV-2 Receptor ACE2 said:
However, interestingly, the expression levels of both ACE2 and TMPRSS2 were significantly lower in nasal as well as bronchial epithelial tissue of children compared with those of adults (p < 0.0001; Figure 1B). Notably, these receptors were found to be differentially expressed between upper and lower airways. In children, the expression levels of ACE2 (p = 0.032) and TMPRSS2 (p = 0.002) were both significantly higher in upper respiratory nasal tissue compared with the bronchial epithelial brushing (Figure 1C). Similarly, in adults, we found significantly higher expression of these genes in upper nasal epithelial tissue compared with bronchial and small airway epithelial brushings (p < 0.0001; Figure 1D). Moreover, the expression of these two genes was found to be significantly higher in nasal compared with blood or saliva of adult subjects (p < 0.0001; Figure S2D). This significant differential expression of both ACE2 and TMPRSS2 between children and adults could contribute to the lower infectivity and disease severity observed in younger populations.
....
Therefore, the reduced airway tissue expression of ACE2 and TMPRSS2 reported here may contribute to the lower risk for infection14,15 and the reduced disease severity observed in the younger population."
AM
 
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