Winter is a recluse now, hidden from the eyes of the relentless media who sensationalized her contributions to the war against COVID-19. She’s retired on a farm in Belgium where she lives with a resident artist and her two newborns. “She’s happy on her own,” said Xavier Saelens, a virologist from Ghent University. He smiled as he described her soft, brown coat and gentle nature.

Winter produced the first nanobodies against COVID-19.

© KOEN VANMECHELEN

As a llama, Winter has a potential COVID-19 therapeutic coursing through her veins: nanobodies. Nanobodies are like antibodies’ smaller, scrappier cousins. They are half the size of the fragmented antibodies often used in therapy, allowing them to squeeze into hard-to-reach spaces on a virus that antibodies struggle to access. Scientists can readily make and tinker with them in a lab, and they have the potential to be delivered via inhalers and nasal sprays, an insurmountable feat for run-of-the-mill antibodies. 

Last year, Saelens developed the first nanobodies against the SARS-CoV-2 spike protein, which the virus uses to enter cells, opening the proverbial flood gates for nanobody researchers everywhere (1). Now, nanobodies against SARS-CoV-2 are in clinical trials for treating early onset infection and protecting vulnerable populations that can’t get the vaccine, such as those who recently received an organ transplant. 

However, not everyone is convinced that nanobodies can compete with vaccines and the human antibody therapeutics currently or soon-to-be available to the public. In the race to develop revolutionary new anti-viral treatments, are nanobodies a horse worth backing?

“We’ll find out in a short period of time either with our [nanobodies] or others’,” said Raymond Owens, a molecular and structural biology researcher at the University of Oxford and Rosalind Franklin Institute who is developing an inhalable nanobody therapeutic. “It’s kind of surprising in a way that there are so few nanobody treatments out there. They have some impressive properties, and there’s a lot of activity and promising research about them in the COVID space.”

Off to the races

The recent pandemic isn’t the first time researchers turned to nanobodies as a treatment option. Nanobodies were first identified at the Free University of Brussels in the late 1980s. A group of undergraduate biology students were tasked with isolating antibodies from blood in a teaching lab. The students were underwhelmed to complete the menial task, and concerned since the samples may have contained HIV, until they found a frozen bottle of camel serum deep in a freezer to use instead.

This rebellious group of undergraduate students made a surprising discovery while they sorted through the antibodies they found in the camel serum; some of the antibodies were half the expected size. Rather than chalk the finding up to the poor skills of naïve researchers, Raymond Hamers and Cecile Casterman, researchers at the Free University of Brussels at the time, took a closer look at the strange antibodies, eventually publishing the first description of these heavy chain only antibodies (HcAbs) in Science in 1993 (2). 

While conventional antibodies produced by humans contain a light and heavy chain, HCAbs produced by camelids are composed only of heavy chains. Both conventional antibodies and HCAbs have two primary regions composed of several domains: constant and variable. Constant domains, found in heavy (CH) and light (CL) chains, have amino acid sequences that remain relatively unchanged amongst different antibodies. The variable domains in the heavy (VH) and light (VL) chains of a conventional antibody vary between antibodies and mediate the antibodies interaction with its specific target. HCAbs also have a variable domain, the single variable domain on a heavy chain (VHH), which researchers isolate to make therapeutically viable nanobodies.

Designed by Natalya Ortolano. Illustrated by Ashleigh Campsell

Classical antibodies are shaped like a capital “Y” composed of two types of protein chains — heavy and light chains. The chains run parallel to each other in the top “V” segment of the “Y.” The tips of both the light and heavy chains are known as variable domains because their sequence varies significantly between different antibodies to determine what the antibody targets. 

HcAbs lack the light chain component, making them half the size of antibodies. Lacking light chain variable domains may seem like a disadvantage, but the variable domains on each heavy chain are longer, acting like an extended hand, able to reach buried sites the short-handed classical antibodies can’t. Researchers produce nanobodies by isolating the long variable domain alone.

In 2019, the first nanobody therapeutic, caplacizumab, was approved by the FDA for treating thrombotic thrombocytopenic purpura, a rare and deadly condition that causes excessive blood clot formation (3). Sanofi acquired Ablynx, the company that originally produced the antibody. Sanofi now has a NANOBODY Technology Platform, where their researchers focus on leveraging nanobodies to develop new therapeutics. 

Saelens considers nanobodies to be the perfect therapeutic for respiratory viruses since their high stability offers potential for intranasal or nebulized delivery.

“When a respiratory virus must be tackled by therapy, it might as well be a therapy that is inhalable. That type of delivery is harsh for a protein or antibody, and it can lead to loss of function or stickiness. Nanobodies are probably capable of withstanding these nebulizing conditions much better than antibodies,” said Saelens.

An obstacle course

Targeting SARS-CoV-2 with a nanobody or an antibody can be tricky. Viruses mutate, and although SARS-CoV-2 mutates more slowly than other virus, targeting a part of the virus that isn’t likely to mutate is key to developing lasting therapies.

Almost all of the antibodies and nanobodies in development target the spike protein of SARS-CoV-2, which is how the virus attaches to and enters cells. The spike protein has two main parts; one contains the receptor binding domain (RBD) that binds the ACE2 receptor on cells, and the other component helps SARS-CoV-2 enter the cell. Most antibodies and nanobodies are designed against the RBD.

This image depicts the spike protein in its “up” conformation, with the RBD (white) exposed. The image on the left shows the protein without glycosylation, and the image on the right shows the glycan sites (blue).

Credit: Elisa Fadda

“Mutations in the RBD are rather limited, and they also have a limited impact on the structure of the spike protein,” said Bing Chen, a virologist and structural biologist at Harvard Medical School. “Its function is critical, so it can’t afford to change much. In all the existing variants, the number of mutations in the RBD seem to be limited.”

The RBD is often buried inside the virus, making it virtually inaccessible to antibodies. Before SARS-CoV-2 binds cells, the spike protein is closed like a door to the inner most structure of the virus, keeping the RBD hidden inside. But once the virus is ready to enter a cell, it opens the door, allowing the RBD to extend from its tip and bind ACE2. This is when SARS-CoV-2 is most vulnerable.

“There are nanobodies published that also bind to the closed confirmation,” said Saelens. “But if one looks hard enough, you find the same epitopes or similar epitopes targetable by conventional antibodies. It just seems a little easier for a nanobody to squeeze into the hidden RBD epitopes.”

Elisa Fadda, a structural biologist from Maynooth University, studies the glycosylation of proteins. She recently characterized the spike protein of SARS-Cov-2.

credit: Elisa Fadda

Another obstacle for any antibody or nanobody developed to target the conserved RBD region of the spike protein is sugar. Elisa Fadda, a structural biologist from Maynooth University, said that students shouldn’t be taught that cells are balloons, but rather fuzzy balloons decorated with sugars on the surface. There more than 600 enzymes involved in making and breaking sugars in the cell, and these sugars, also known as glycans, have a lot to do. They help cells talk to each other, move, and stay off of the immune system’s radar.

“Our immune system, which constantly monitors our bodies for foreign material — bacteria, toxins, viruses — is used to sugar, so it recognizes the sugars as self,” said Fadda. “Viruses have evolved a particular strategy to evade the immune system by hijacking the glycosylation machinery in our cells. They dress up in a sugary coat that allows them to camouflage themselves from the immune system. It’s a very effective strategy.”

Antibodies often have a hard time penetrating these sugary coats. But nanobodies can sometimes wiggle through the small, fabric-like weavings to access the viral components underneath. Some nanobody researchers claimed that this was an advantage for nanobodies in the COVID-19 therapeutic space. However, researchers developing classical antibodies retorted that SARS-CoV-2’s coat isn’t as fuzzy as some other viruses, so it’s not a large concern when designing antibody therapeutics.

“Some viruses are better at this than others. For example, HIV is incredibly good at it. It’s very densely coated, so you cannot even see the proteins underneath. That’s one of the reasons we don’t have a vaccine for HIV,” said Fadda. “[SARS-CoV-2’s] receptor binding domain is also glycosylated, but it doesn’t have many glycans because it needs to bind ACE2.”

Fadda added that the mutations SARS-CoV-2 has developed over time resulted in less glycosylation at the RBD, making it more accessible to antibodies.

But nanobodies are more than just smaller antibodies that can navigate tight spaces. Researchers developing nanobody therapeutics contend that the biologics’ malleability, stability, and easy, low-cost production make them a strong contender in the COVID-19 therapeutic race.

Bring out the horses

When the pandemic started, Saelens and his collaborators sprinted to the farm to develop nanobodies against SARS-CoV-2 (1). They injected Winter the llama with spike proteins from SARS-CoV-1 and MERS-CoV since the spike proteins all have common structures and sequences.

Winter is a retired COVID-19 researcher who now lives on a farm with her two children.

© Koen Vanmechelen

They collected serum from the animals and analyzed how the nanobodies interacted with the spike proteins using electron microscopy. They determined that the antibodies against each respective virus targeted the RBD site that critically interacts with a cell receptor to promote viral entry. 

One nanobody that targeted SARS also effectively bound the RBD of the SARS-CoV-2 spike protein where it binds ACE2. The nanobody essentially put a door stop under SARS-CoV-2’s front door, freezing it in an open conformation, making it unable to bind its receptor.

“Nanobodies are small proteins, so when you inject them into a patient or an animal model, they wash from circulation very quickly, which can be a disadvantage if one has to treat, for example, an infection that is lingering for days in the body,” said Saelens.

Xavier Saelens, a virologist from Ghent University, helped produce the first nanobodies against COVID-19 from Winter the llama.

Credit: Xavier Saelens

To increase the stability of the nanobody the team identified, they gave the small v-shaped protein a stalk to turn it back into a “y.” They used the stalk of a human antibody known as a fragment crystallizable region (Fc) to increase the protein’s size, allowing it to stay in circulation for days, if not weeks.

Adding a human component to this llama-derived nanobody also reduced the chance that the body would have a negative reaction to it. Even with the added Fc domain, the nanobody could still be encoded in a single cassette, making it easy to produce in cell lines or yeast, something that is more challenging when working with larger antibodies.

Since last year, Saelens' team has demonstrated that their nanobody can protect animals, specifically the commonly used COVID-19 animal model Syrian hamsters, from severe COVID-19 infection (4). They recently completed a Phase Ia study showing that the nanobody isn’t toxic, and they are now testing the nanobody in a small group of patients with COVID-19 symptoms to determine the proper dose. Although they are testing the nanobody by intravenous delivery now, they hope to develop intranasal or nebulizer delivery in the future.

Antibodies capable of targeting several coronaviruses at a similar site to Saelens’ nanobody are out there. David Martinez and Alexandra Schaefer, virologists from the University of North Carolina at Chapel Hill and co-first authors of a recent paper in Science Translational Medicine described a human antibody that protects against COVID-19 and other SARS-related viral infections. This is only the most recently reported broadly neutralizing antibody (5).

Martinez and Schafer didn’t search for an antibody that could target multiple viruses; they searched through nearly 2000 monoclonal antibodies from patients previously infected with SARS or COVID-19. The researchers compared the target of their antibody to that of other broadly neutralizing antibodies and discovered that the site was nearly identical to that of a nanobody.

Just because a site may be hard to reach doesn’t mean that an antibody can’t reach it. According to Martinez, rather than looking for nanobodies that target conserved, hidden sites, scientists should look for antibodies that are less likely to be made in humans. 

Finding more horses

Finding antibodies that humans are less likely to make is a good strategy for finding a treatment that withstands viral mutations. Natural antibody production or continued antibody treatment towards a particular site in a virus can encourage it to mutate to evade the immune system, a phenomenon akin to antibiotic resistance in bacteria (6).

Kai Xu, a vaccine and antibody researcher at The Ohio State University, utilized a “camelized” mouse to produce nanobodies, making the therapeutic development easier in the lab.

Credit: Kai Xu

Kai Xu, a vaccine and antibody researcher at The Ohio State University, thinks that the nanobodies that target those uncommon sites are key to fighting COVID-19 if vaccines fail to protect against a new variant. He teamed up with researchers across the United States to identify nanobodies targeting highly conserved regions of SARS-CoV-2 and to develop a way to easily produce new nanobodies against any virus (7).

Researchers sometimes use humanized mice to make human antibodies against proteins. These mice have genes that produce antibodies with human components rather than mouse components. 

Xu’s collaborators Jianliang Xu and Rafael Casellas, nanobody researchers from the National Institute of Health, used CRISPR to replace mouse antibody-producing genes with those of camels, alpacas, and llamas to make a “camelized” mouse. In addition to adding genes, the researchers removed genes expressing the part of antibodies that normally connect the heavy and light chains of an antibody, so that only single chain antibodies are made by antibody-producing B cells.

“In theory, it may have a higher diversity in the antibody pool. And the mouse model is convenient to use in the lab, so you can have multiple animals in an experimental group. Each injection into each mouse will have some variation, which in the end, will generate more diversity,” said Xu.

To test their model, Xu and his collaborators injected both the SARS-CoV-2 spike protein and an isolated RBD into camelized mice and llamas. They identified nanobodies with high binding affinity to the RBD, but the nanobodies were unable to bind to certain mutated forms of the spike protein. To overcome this limitation, they merged multiple nanobodies together into trimers, which were better able to bind the spike protein and block ACE2 binding. In fact, the researchers claim that these merged nanobodies are more potent than other reported antibodies to date.

Xu thinks that linking nanobodies together can take them to the next level. “We can engineer a nanobody molecule that combines nanobodies that target different regions so that the combined specificity may enhance the breadth of this nanobody therapy,” said Xu.

Placing in the race

Even with these advances, researchers still have concerns about nanobodies, the largest of which is that no matter how human-like scientists make them, these nanobodies aren’t human. 

There are human antibody therapies available for COVID-19 treatment now. Regeneron’s antibody cocktail, REGEN-COV, is approved for emergency use by the FDA, and is highly effective for preventing severe COVID-19 infection after exposure, as well as providing protection to those who can’t get a vaccine. However, this cocktail, and any other cocktail in development using classical antibodies, is administered intravenously.

Jeremy Kamil, a virologist at Louisiana State University Health Sciences Center, Shreveport, thinks that the potential for inhaled therapies not only gives nanobodies an edge over antibodies due to their ease of use, but lessens concerns about an immune response to the nanobodies.

“It’s one thing to put a foreign compound outside of yourself; it’s another to put it into your blood stream,” said Kamil. “If it really works as an inhaler, and you could really deliver it to the respiratory tract that way, I think it would have a unique niche. I mean, it’s nice to not have to use a needle, right?”

Several groups have reported nanobodies that fended off severe COVID-19 symptoms in infected hamsters via intranasal delivery at low doses (8-9). The nanobodies prevented infection and even lowered the amount of SARS-CoV-2 virus in the infected hamsters. How effective these treatments will fare in humans remains to be seen, but the researchers are excited about the future.

“The number one solution is going to still be a vaccine. For normal people who are healthy, if you give them a COVID vaccine, they're going to come up with a pretty enormous repertoire of antibodies,” said Kamil.

Nanobody researchers like Yi Shi from the University of Pittsburgh, who helped develop an inhalable nanobody therapeutic dubbed PiN-21 (9), agrees that vaccines are useful, but stresses the importance of pursuing other options against COVID-19. 

“When I talk to people, they think that the vaccine will solve the problem. Because of the scale of the pandemic, the diversity of the virus, and the issues with vaccine rollout, we realize that we need different strategies, combined strategies, to really minimize the impact of this pandemic,” said Shi.

12/21/2021: This article previously stated that Kai Xu, a vaccine and nanobody researcher at The Ohio State University, developed the camelized mouse model, however, his collaborators Jiangliang Xu and Rafael Casella, nanobody researchers from the NIH, developed the model.

References

1. Wrapp, D. et al. Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell  181, P1004-1015 (2020).
2. Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Science  3, 446-448 (1993).
3. Scully, M. et al. Caplacizumab Treatment for Acquired Thrombotic Thrombocytopenic Purpura. N Engl J Med   380, 335-346 (2019).
4. Schepens, B. et al. An affinity-enhanced, broadly neutralizing heavy chain-only antibody protects against SARS-CoV-2 infection in animal models. Sci Transl Med  13, eabi7826 (2021).
5. Martinez D. et al. A broadly cross-reactive antibody neutralizes and protects against sarbecovirus challenge in mice. Sci Transl Med (2021).
6. Moore, J.P. and Offit, P.A. SARS-CoV-2 Vaccines and the Growing Threat of Viral Variants. JAMA   325, 821-822 (2021).
7. Xu, J. et al. Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants. Nature   595, 278-282 (2021).
8. Huo, J. et al. A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19. Nat Commun   12, 5469 (2021).
9. Nambulli, S. et al. Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses. Sci Advances   7, eabh0319 (2021).