New Nanobodies Stop SARS-CoV-2 and Its Dangerous Variants

The image shows how two of the newly developed nanobodies (blue and magenta) bind to the receptor binding domain (green) of the coronavirus spike protein (grey), preventing infection with Sars-CoV-2 and its variants. The nanobodies come from alpacas and are smaller and simpler than conventional antibodies. Credit: Max Planck Institute for Biophysical Chemistry

Göttingen researchers have developed mini-antibodies that efficiently block the coronavirus SARS-CoV-2 and its dangerous new variants. These so-called nanobodies bind and neutralize the virus up to 1000 times better than previously developed mini-antibodies. In addition, the scientists optimized their mini-antibodies for stability and resistance to extreme heat. This unique combination makes them promising agents to treat COVID-19. Since nanobodies can be produced in large quantities at low cost, they could meet the global demand for COVID-19 therapies. The new nanobodies are currently in preparation for clinical trials.

Antibodies help our immune system to fight off pathogens. For example, the molecules attach themselves to viruses and neutralize them so that they can no longer infect cells. Antibodies can also be industrially produced and administered to acutely ill patients. They then act as drugs, relieving symptoms and shortening recovery from the disease. This is an established practice for the treatment of hepatitis B and rabies. Antibodies are also used to treat COVID-19 patients. However, producing these molecules on an industrial scale is too complex and expensive to meet global demand. Nanobodies could solve this problem.

Scientists from the Max Planck Institute (MPI) for Biophysical Chemistry in Göttingen (Germany) and the University Medical Center Göttingen (UMG) have now developed mini-antibodies (also known as VHH antibodies or nanobodies) that unite all the properties necessary for a powerful drug against COVID-19. “For the first time, they combine extreme stability and excellent efficacy against the virus and its Alpha, Beta, Gamma and Delta mutants,” emphasizes Dirk Görlich, MPI’s director for Biophysical Chemistry.

At first glance, the new nanobodies hardly differ from anti-SARS-CoV-2 nanobodies developed by other labs. All of them target a critical component of the coronavirus spikes, the receptor-binding domain that the virus uses to invade host cells. The nanobodies block this binding domain and thus prevent the virus from infecting cells.

“Our nanobodies can withstand temperatures up to 95°C without losing their function or forming aggregates,” explains Matthias Dobbelstein, professor and director of the Institute of Molecular Oncology at UMG. “For starters, this tells us that they can remain active in the body long enough to be effective. On the other hand, heat-resistant nanobodies are easier to manufacture, process and store.”

Single, double and triple nanobodies

The simplest mini-antibodies developed by the Göttingen team already bind to the spike protein up to 1000 times stronger than previously reported nanobodies. They also bind very well to the mutated receptor binding domains of the Alpha, Beta, Gamma and Delta strains. “Our single nanobodies are potentially suitable for inhalation and thus for direct virus neutralization in the respiratory tract,” says Dobbelstein. “In addition, because they are very small, they can easily penetrate tissues and prevent the virus from spreading further at the site of infection.”

A ‘nanobody triad’ further improves binding: the researchers bundled three identical nanobodies according to the symmetry of the spike protein, which consists of three identical building blocks with three binding domains. “With the nanobody triad, we are literally joining forces: In an ideal scenario, each of the three nanobodies attaches to one of the three binding domains,” reports Thomas Güttler, a scientist on Görlich’s team. “This creates an almost irreversible bond. The triple does not release the spike protein and even neutralizes the virus up to 30,000 times better than the single nanobodies.” Another benefit: The larger size of the nanobody triad is expected to slow down renal excretion. This keeps them in the body longer and promises a longer lasting therapeutic effect.

As a third design, the scientists made tandems. These combine two nanobodies that target different parts of the receptor binding domain and together can bind the spike protein. “Such tandems are extremely resistant to virus mutations and the resulting ‘immune flight’ because they bind the viral peak so strongly,” explains Metin Aksu, a researcher in Görlich’s team.

For all nanobody variants – monomeric, double and triple – the researchers found that very small amounts are enough to stop the pathogen. When used as a medicine, this would allow a low dosage and thus fewer side effects and lower production costs.

Alpacas provide blueprints for mini-antibodies

“Our nanobodies come from alpacas and are smaller and simpler than conventional antibodies,” says Görlich. To generate the nanobodies against SARS-CoV-2, the researchers immunized three alpacas — Britta, Nora and Xenia from the herd at the MPI for Biophysical Chemistry — with parts of the coronavirus spike protein. The mares then made antibodies and the scientists took a small blood sample from the animals. For the alpacas, the mission was then complete, as all further steps were performed using enzymes, bacteria, so-called bacteriophages, and yeast. “The total burden on our animals is very low, comparable to vaccination and blood tests in humans,” explains Görlich.

Görlich’s team extracted about a billion blueprints for nanobodies from the blood of the alpacas. What followed was a lab routine that was perfected over many years: The biochemists used bacteriophages to select the very best nanobodies from the initially huge pool of candidates. These were then tested for their efficacy against SARS-CoV-2 and further improved in successive rounds of optimization.

Not every antibody is ‘neutralizing’. Researchers from Dobbelstein’s group therefore determined whether and how well the nanobodies prevent the viruses from multiplying in cells cultured in the laboratory. “By testing a wide range of nanobody dilutions, we find out which amount is sufficient to achieve this effect,” explains Antje Dickmanns of Dobbelstein’s team. Her colleague Kim Stegmann adds: “Some nanobodies were really impressive. Less than one millionth of a gram per liter of medium was sufficient to completely prevent infection. In the case of the nanobody triads, even a 20-fold dilution was sufficient.”

Also effective against current coronavirus variants

Over the course of the coronavirus pandemic, new virus variants have emerged that quickly became dominant. These variants are often more contagious than the strain that first appeared in Wuhan (China). Their mutated spike protein can also ‘escape’ neutralization by some originally effective antibodies from infected, recovered or vaccinated individuals. This makes it harder for even an already trained immune system to eliminate the virus. This problem also affects previously developed therapeutic antibodies and nanobodies.

This is where the new nanobodies show their full potential, as they are also effective against the main coronavirus variants of concern. The researchers had inoculated their alpacas with part of the spike protein of the first known SARS-CoV-2 virus, but remarkably, the animals’ immune systems also made antibodies that are active against the different virus variants. “If our nanobodies are not effective against a future variant, we can re-immunize the alpacas. Because they have already been vaccinated against the virus, they would very quickly make antibodies against the new variant,” says Güttler confidently.

Therapeutic application in sight

Göttingen’s team is currently preparing the nanobodies for therapeutic use. Dobbelstein emphasizes: “We want to test the nanobodies for safe use as medicine as soon as possible, so that they can be of use to seriously ill people with COVID-19 and those who have not been vaccinated or cannot build up effective immunity. The team is supported by technology transfer experts: Dieter Link (Max Planck Innovation), Johannes Bange (Lead Discovery Center, Dortmund, Germany) and Holm Keller (kENUP Foundation).

The receptor binding domain of SARS-CoV-2 is known to be a good candidate for a protein vaccine, but has so far been difficult to produce economically on a large scale and in a form that activates the immune system against the virus. Bacteria programmed accordingly produce misfolded material. The Göttingen researchers discovered a solution to this problem: they identified special nanobodies that force correct folding in bacterial cells, without interfering with the crucial neutralizing part of the receptor-binding domain. This could enable vaccines that can be produced cheaply, quickly adapted to new virus variants and distributed with simple logistics, even in countries with little infrastructure. “The fact that nanobodies can aid in protein folding was previously unknown and is extremely interesting for research and pharmaceutical applications,” says Görlich.

Reference: “Neutralization of SARS-CoV-2 by highly potent, hyperthermostable and mutation-tolerant nanobodies” by Thomas Güttler, Metin Aksu, Antje Dickmanns, Kim M. Stegmann, Kathrin Gregor, Renate Rees, Waltraud Taxer, Oleh Rymarenko, Jürgen Schünemann, Christian Dienemann, Philip Gunkel, Bianka Mussil, Jens Krull, Ulrike Teichmann, Uwe Groß, Volker C. Cordes, Matthias Dobbelstein and Dirk Görlich, July 24, 2021, The EMBO Journal.
DOI: 10.15252 / embj.2021107985