NIH-CoVid-b112 nano antibody stuck directly to the ACE2 receptor binding portion of the SARS CoV-2 spike protein

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NIH researchers have isolated a set of promising, tiny antibodies, or “nanobodies,” against SARS-CoV-2 that were produced by a llama named Cormac.

Preliminary results published in Scientific Reports suggest that at least one of these nanobodies, called NIH-CoVnb-112, could prevent infections and detect virus particles by grabbing hold of SARS-CoV-2 spike proteins.

In addition, the nanobody appeared to work equally well in either liquid or aerosol form, suggesting it could remain effective after inhalation. SARS-CoV-2 is the virus that causes COVID-19.

The study was led by a pair of neuroscientists, Thomas J. “T.J.” Esparza, B.S., and David L. Brody, M.D., Ph.D., who work in a brain imaging lab at the NIH’s National Institute of Neurological Disorders and Stroke (NINDS).

“For years TJ and I had been testing out how to use nanobodies to improve brain imaging. When the pandemic broke, we thought this was a once in a lifetime, all-hands-on-deck situation and joined the fight,” said Dr. Brody, who is also a professor at Uniformed Services University for the Health Sciences and the senior author of the study.

“We hope that these anti-COVID-19 nanobodies may be highly effective and versatile in combating the coronavirus pandemic.”

A nanobody is a special type of antibody naturally produced by the immune systems of camelids, a group of animals that includes camels, llamas, and alpacas. On average, these proteins are about a tenth the weight of most human antibodies.

This is because nanobodies isolated in the lab are essentially free-floating versions of the tips of the arms of heavy chain proteins, which form the backbone of a typical Y-shaped human IgG antibody. These tips play a critical role in the immune system’s defenses by recognizing proteins on viruses, bacteria, and other invaders, also known as antigens.

Because nanobodies are more stable, less expensive to produce, and easier to engineer than typical antibodies, a growing body of researchers, including Mr. Esparza and Dr. Brody, have been using them for medical research.

For instance, a few years ago scientists showed that humanized nanobodies may be more effective at treating an autoimmune form of thrombotic thrombocytopenic purpura, a rare blood disorder, than current therapies.

Since the pandemic broke, several researchers have produced llama nanobodies against the SARS-CoV-2 spike protein that may be effective at preventing infections.

In the current study, the researchers used a slightly different strategy than others to find nanobodies that may work especially well.

“The SARS-CoV-2 spike protein acts like a key. It does this by opening the door to infections when it binds to a protein called the angiotensin converting enzyme 2 (ACE2) receptor, found on the surface of some cells,” said Mr. Esparza, who is also an employee of the Henry M. Jackson Foundation for the Advancement of Military Medicine and the lead author of the study.

“We developed a method that would isolate nanobodies that block infections by covering the teeth of the spike protein that bind to and unlock the ACE2 receptor.”

To do this, the researchers immunized Cormac five times over 28 days with a purified version of the SARS-CoV-2 spike protein. After testing hundreds of nanobodies they found that Cormac produced 13 nanobodies that might be strong candidates.

This shows how the antibody works to combat covid-19
Infections happen when SARS-CoV-2 virus spike proteins (yellow) latch onto ACE2 receptors (blue) that line the outside of a cell. The NIH nanobodies (grey) may prevent infections by covering spike proteins, which blocks binding to the ACE2 receptor. Credit: Brody lab, NIH/NINDS

Initial experiments suggested that one candidate, called NIH-CoVnb-112, could work very well. Test tube studies showed that this nanobody bound to the ACE2 receptor 2 to 10 times stronger than nanobodies produced by other labs. Other experiments suggested that the NIH nanobody stuck directly to the ACE2 receptor binding portion of the spike protein.

Then the team showed that the NIH-CoVnB-112 nanobody could be effective at preventing coronavirus infections.

To mimic the SARS-CoV-2 virus, the researchers genetically mutated a harmless “pseudovirus” so that it could use the spike protein to infect cells that have human ACE2 receptors.

The researchers saw that relatively low levels of the NIH-CoVnb-112 nanobodies prevented the pseudovirus from infecting these cells in petri dishes.

Importantly, the researchers showed that the nanobody was equally effective in preventing the infections in petri dishes when it was sprayed through the kind of nebulizer, or inhaler, often used to help treat patients with asthma.

“One of the exciting things about nanobodies is that, unlike most regular antibodies, they can be aerosolized and inhaled to coat the lungs and airways,” said Dr. Brody.

The team has applied for a patent on the NIH-CoVnB-112 nanobody.

“Although we have a lot more work ahead of us, these results represent a promising first step,” said Mr. Esparza. “With support from the NIH we are quickly moving forward to test whether these nanobodies could be safe and effective preventative treatments for COVID-19. Collaborators are also working to find out whether they could be used for inexpensive and accurate testing.”

Funding: This study was supported by NIH Intramural Research Programs at the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Environmental Health Sciences (NIEHS); Dr. Brody is an employee of the Uniformed Services University of the Health Sciences. The views expressed here do not represent those of the Department of Defense.


There are currently few approved effective treatments for SARS‑CoV‑2, the virus responsible for the COVID‑19 pandemic. Nanobodies are 12–15 kDa single‑domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals.

We have isolated nanobodies that bind to the SARS‑CoV‑2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1–5 nM affinity.

The lead nanobody candidate, NIH‑CoVnb‑112, blocks SARS‑CoV‑2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL.

NIH‑CoVnb‑112 retains structural integrity and potency after nebulization. Furthermore, NIH‑ CoVnb‑112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein.

These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

Coronaviruses are positive sense, single-stranded RNA viruses. There are seven types of coronaviruses known to infect humans, including the recent 2019 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1,2.

Patients infected with these viruses develop respiratory symptoms of various severity and outcomes. Since the beginning of the century, there have been three major world-wide health crises caused by coronaviruses: the 2003 SARS-CoV-1 outbreak, the 2012 MERS-CoV outbreak, and the 2019 SARS-CoV-2 outbreak3. To date, hundreds of thousands of people have succumbed to the virus during these outbreaks.

The SARS-CoV-2 virus gains entry to human cells via the angiotensin converting enzyme 2 (ACE2) receptor by the interaction of the receptor binding domain (RBD) of the spike protein on the viral surface4–8. This RBD- ACE2 interaction provides a clear therapeutic target for binding and prevention of infection (Fig. 1a).

Many of the antibodies considered for diagnostic or therapeutic applications have been conventional immu- noglobulins (IgG). The use of IgGs as therapeutics, while successful in many diseases, is known to have potential pitfalls due to the risk of receptor-mediated immunological reactions9 .Treatment or prophylaxis of a pulmonary virus can be delivered via aerosolization and inhalation; thus, the size and biophysical characteristics are para- mount considerations.

The camelid family, which includes llamas, produce additional subclasses of IgGs which possess an unpaired heavy-chain variable domain10–12. This heavy-chain variable domain has demonstrated the ability to function as an independent antigen-binding domain with similar affinity as a conventional IgG.

These heavy chain variable domains can be expressed as a single domain, known as a VHH or nanobody, with a molecular weight 10% of the full IgG. Nanobodies generally display superior solubility, solution stability, temperature stability, strong penetration into tissues, are easily manipulated with recombinant molecular biology methods, and possess robust environmental resilience to conditions detrimental to conventional IgGs13–15.

In addition, nanobodies are weakly immunogenic which reduces the likelihood of adverse effects compared to other single domain antibodies such as those derived from sharks or synthetic platforms. Successful framework modifications have been implemented to humanize VHH sequences without significant alteration of biophysical properties16.

Importantly, the recent approval of the first-in-class nanobody treatment for thrombotic thrombocytopenic pupura, Caplacizumab, bolsters the therapeutic potential of VHH derivatives17. Therefore, nanobodies that bind to the SARS-CoV-2 RBD and block the ACE2 interaction are an attractive therapeutic for prevention and treatment of viral infection18.

Figure 1. Overview of the therapeutic potential and isolation of nanobodies targeting the SARS-CoV-2 RBD:ACE2 interaction. (a) Illustration of the structure of SARS-CoV-2 spike protein, with receptor binding domain in contact with the human ACE2 receptor on the surface of a lung epithelial cell. A major therapeutic goal is to develop inhibitory agents that disrupt the interaction between the spike protein and the human ACE2 receptor. (b) Isolation of nanobodies binding SARS-CoV-2 spike protein. An adult llama was immunized 5 times over 28 days with purified, recombinant SARS-CoV-2 spike protein. On day 35 after first immunization (7 days after last immunization), llama blood was obtained through a central line, B-cells were isolated, the single heavy-chain variable domains (nanobodies) of the llama antibodies were amplified and cloned to construct a recombinant DNA library containing more than 108 clones. The library of clones was expressed in a phage display format, in which each phage expresses between 1–5 nanobody copies on its surface and contains the DNA sequence encoding that nanobody. Immuno-panning was performed to isolate candidate nanobodies for expression and validation studies. (c) Selection strategy for isolation of nanobody candidates which bind
to the RBD:ACE2 interaction surface. Using a phage display library from an adult llama immunized with full length S1 spike protein, nanobodies were isolated which block the interaction between RBD and ACE2. (ci) In a standard radioimmunoassay tube, ACE2 was immobilized and the surface blocked with non-specific protein. Biotinylated-RBD, was incubated with the nanobody phage library and then added to the immuno-tube and allowed to interact with the immobilized ACE2. Biotinylated-RBD with no associated nanobodies, or with nanobody associations which do not block the ACE2 binding domain, bound the immobilized ACE2. (cii)
Biotinylated-RBD with associated nanobodies that blocked the ACE2 binding domain remained in solution and were recovered using streptavidin-coated magnetic particles that bind to the biotin. (ciii) Nanobodies which did not bind to RBD were removed during washing of the magnetic beads. This method allowed for specific enrichment of nanobodies which both bind to the RBD and compete for the RBD-ACE2 binding surface. (Figure generated using BioRender.com).

Discussion
Here, we report several nanobodies that bind to the SARS-CoV-2 spike protein RBD and block spike protein interaction with the ACE2 receptor. The affinity of NIH-CoVnb-112 in monomeric form is substantially better than the monomeric form of previously reported candidate nanobody therapeutics for SARS-CoV-2: 39 nM for monomeric VHH7224, ~ 50 nM for monomeric Ty123, 30.7 nM for monomeric Sb#1421, 10 nM for monomeric Sb2322, and incompletely reported but likely lower than 1B and 3F27. The affinity and blocking potency of NIH- CoVnb-112 will likely be even higher when formulated into dimeric, trimeric, or other protein engineered constructs27,30,32,33.

Importantly, these nanobody therapeutics may be delivered via inhalation34. Inhalation has major advantages over other routes of administration and could be one of the most important potential uses for a nanobody thera- peutic for SARS-CoV-2. The most directly analogous study was performed by Larios Mora et al., who showed that nebulized treatment of newborn lambs with ALX-0171 reduced clinical, virological, and pathological mani- festations of respiratory syncytial virus35. ALX-0171 is a trimeric nanobody therapy produced in Pischia pastoris that binds to the respiratory syncytial virus fusion protein. Nebulization was performed using a commercially

available human-use Aeroneb Solo system (https://www.aerogen.com/aerogen-solo-3/), interfaced with a nose mask. The Aeroneb Solo nebulizer produced ~ 3.3 micron particles, appropriate for deep lung delivery. The authors demonstrated that once daily nebulization resulted in concentrations of ALX-0171 in lung epithelial lining fluid that were > 10 times higher than the in vitro EC50 for respiratory syncytial virus. All treated lambs had undetectable infectious virus in lung epithelial lining fluid, which supports that the proposed route of administra- tion could reduce infectivity.

After nebulization, blood concentrations were ~ 1000 fold lower than lung epithelial lining fluid concentrations, which is important because low blood concentrations likely reduce systemic toxicity and risk of host antibody formation. Of note, the parent monomeric nanobody to the respiratory syncytial virus fusion protein had an affinity of 17 nM, whereas the nanobody trimer ALX-0171 had an affinity of 0.113 nM. The trimeric ALX-0171 was produced by fermentation in Pischia pastoris with a yield of 7.5 g per liter32.

Thus, there is great potential for an inhaled therapeutic for SARS-CoV-2 derived from the NIH-CoVnb-112 nanobody, and other similar nanobodies. There are many other potential therapeutic uses for the NIH-CoVnb-112 nanobody. These include intravenous, intramuscular, or subcutaneous treatment formulations for early stage disease, as well as prevention in high risk individuals.

It appears that late-stage disease is mediated more by immunological responses and less by virological patho- genesis, making it important to initiate virologically-targeted therapy early. Importantly, it is likely that nanobody treatments would be substantially less expensive to produce than conventional monoclonal antibodies such as those currently in clinical development pipelines (e.g. REGN-CoV-2 “antibody cocktail” https://www.regeneron. com/covid19 and LY-CoV555: https://investor.lilly.com/news-releases/news-release-details/lilly-begins-world s-first-study-potential-covid-19-antibody).

Thus, it would be reasonable to initiate treatment with an inexpensive and widely available therapeutic early, even before it is clear whether or not an infection will become severe. Furthermore, even as vaccines are successfully developed, preventatives and treatments for acute illness would be beneficial, as vaccines may not be 100% effective, especially in those who do not mount adequate immune responses. By analogy, oseltamivir (Tamiflu) is an essential medicine even in the context of successful influenza vaccines. Thus, there is a substantial need for inexpensive and safe preventative and acute candidate treatments such as the NIH-CoVnb-112 nanobody.

In addition to therapeutic applications, an inexpensive binding reagent such as NIH-CoVnb-112 nanobody could be used diagnostically. Antibody-based tests could be used to assess for SARS-CoV-2 spike protein in body fluids and the environment. The low cost and temperature stability of nanobodies would be advantageous in some settings, though it is unlikely that antibody-based tests will replace current PCR-based testing for routine clinical use.

Substantial effort will be required to fully characterize and develop NIH-CoVnb-112. We have demonstrated successful neutralization assays involving pseudotyped lentivirus and authentic SARS-CoV-2 isolate neutrali- zation assays are currently underway.

Future activities include performing detailed structural and biophysical characterization involving Cryo-EM, X-ray crystallography, in vivo pharmacokinetics and stability assessment, immunogenicity determination, framework humanization, assessing multimerized formats, and further storage and delivery stability assessments. It is encouraging that NIH-CoVnb-112 binds to and blocks three recently reported RBD variants, but it will be of great interest to determine whether the candidate nanobodies provide broad spectrum blocking against other variant forms of the SARS-CoV-2 spike proteins31.

Additional assays will be required to determine whether NIH-CoVnb-112 protects against mutation-based viral escape alone or as part of a therapeutic cocktail.
Animal testing will be critically important. The optimized nanobody construct will be tested for in vivo safety and efficacy in an established small animal model of SARS-CoV-2 infection such as human ACE2 transgenic mouse or Syrian hamster36,37. Subsequently, testing for in vivo safety and efficacy in an established large animal model of SARS-CoV-2 infection such as rhesus macaque or baboon will be conducted to further support an investigational new drug application38.

Although in vitro stability and efficacy appears to be excellent, there is no guarantee that NIH-CoVnb-112 will have appropriate in vivo stability and pharmacokinetics. If NIH-CoVnb-112 proves sub-optimal in vivo, we plan to further characterize other candidate nanobodies from the initial screen, as well as performing additional screening and protein engineering. While other nanobodies have been produced in large scale current Good Manufacturing Practice facilities by fermentation, there is no guarantee that NIH- CoVnb-112 constructs can be produced in sufficient quantities at reasonable cost. Exploration of the manufac- turing parameters will be critical for product development.

There are several limitations of the current results. First, the characterization of NIH-CoVnb-112 and the other nanobodies is not yet complete. Under the current circumstances, we have opted to move forward with dissemination of our findings earlier than might otherwise be typical. For example, we have not fully character- ized all of the candidate nanobodies, nor nanobodies that do not block the interaction of the RBD with ACE2. Likewise, we have not exhaustively tested epitope binding overlap between NIH-CoVnb-112 and all of the other SARS-CoV-2 spike nanobodies developed to date.

Second, a potential new therapeutic such as this nanobody would, even under the best circumstances, come to market relatively slowly compared to a repurposed approved drug. Third, it is possible that NIH-CoVnb-112 and other nanobodies may provoke host immune responses. NIH-CoVnb-112 is a llama protein fragment that is similar to human proteins yet cannot be fully humanized and maintain desirable biophysical characteristics. Fourth, NIH-CoVnb-112 and other nanobodies may have unex- pected toxicity.

There is only one United States Food and Drug Administration-approved nanobody therapeutic, Caplacizumab, and there has not been exposure to a sufficient number of human patients to fully characterize the safety of this class of therapeutics.

In conclusion, while a substantial amount of additional characterization will be required, the novel nanobod- ies that bind to the spike protein RBD have potential widespread utility in the battle against the SARS-CoV-2 pandemic.

We are especially optimistic about the possibility that low-cost, stable, and safe nanobody-based therapeutics will be developed for inhaled use in the home and outside of formal healthcare environments.


Original Research: Open access.
High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme” by Thomas J. Esparza, Negin P. Martin, George P. Anderson, Ellen R. Goldman & David L. Brody. Scientific Reports

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