Researchers at Gttingen have created mini-antibodies to effectively block the coronavirus SARSCoV-2 and dangerous new variants. These nanobodies, also known as mini-antibodies, bind to the virus and neutralize it up to 1000x better than any previously-developed mini-antibodies. The scientists also optimized the mini-antibodies to resist extreme heat and stability. These unique combinations make them potential agents for COVID-19 treatment. Nanobodies are affordable and can be made in large quantities. This could allow them to meet the worldwide demand for COVID-19 therapeutics. Clinical trials are underway for the new nanobodies.Antibodies are a way for our immune system fight off pathogens. The molecules attach to viruses and are neutralized so they cannot infect cells. Also, antibodies can be manufactured industrially and given to patients who are critically ill. These antibodies act as drugs and reduce the time it takes to recover from the disease. This is a well-established practice to treat hepatitis B or rabies. For COVID-19 patients, antibodies can also be used. Producing these molecules on an industrial scale would be too difficult and costly to meet global demand. This problem could be solved by nanobodies.Researchers at the Max Planck Institute for Biophysical Chemistry (MPI) in Gttingen (Germany), and the University Medical Center Gttingen have developed mini-antibodies, also known as VHH antibodies (or nanobodies), that combine all of the properties necessary to make a powerful drug against COVID-19. Dirk Grlich (director at the MPI for Biophysical Chemistry) says that they are the first to combine extreme stability with outstanding efficacy against the virus as well as its Alpha, Beta and Gamma mutants.The new nanobodies are very similar to anti-SARS-CoV-2 drugs developed in other labs. All of them are directed at a key part of coronavirus spikes. This is the receptor-binding area that the virus uses to invade host cells. Nanobodies prevent the virus infecting cells by blocking this binding domain.Matthias Dobbelstein is professor and director of UMG's Institute of Molecular Oncology. "Our nanobodies can withstand temperature up to 95 C without losing function or forming aggregates," he says. This tells us, for one, that they may remain active in the body long sufficient to be effective. It is easier to make, store, and process heat-resistant nanobodies.Nanobodies in single, double and triple sizesThe Gttingen team has already discovered that the simplest mini-antibodies can bind to spike protein up to 1000x stronger than any previously reported nanobodies. They also bind well to the modified receptor-binding domains for the Delta, Alpha, Beta, Gamma and Gamma strains. Dobbelstein states that single nanobodies could be inhaled and used to direct virus neutralization in a person's respiratory tract. "They are also very small and could easily penetrate tissues, preventing the virus spreading further at the point of infection."AdvertisementA 'nanobody triad' improves binding. The researchers combined three identical nanobodies based on the symmetry of spike protein, which is made up of three identical building blocks and three binding domains. "The nanobody triad is a way to literally unite: Each of the three nanobodies should attach to one of three binding domains in an ideal situation," Thomas Gttler reports, a Grlich scientist. This creates an almost irreversible bond. Triple will not allow release of spike protein and neutralizes virus up to 30,000 times better than single nanobodies. Another benefit: The nanobody triad's larger size expectedly slows down renal excretion. They are kept in the body longer, which promises a longer-lasting therapeutic effect.The scientists also created tandems as a third design. These nanobodies combine two nanobodies which target different parts the receptor-binding domain, and can bind to the spike protein together. Metin Aksu, a Grlich researcher, says that such tandems are very resistant to virus mutations.Researchers found that nanobody variants of monomeric, double and triple were all effective in stopping the pathogen. This would make it possible to use the drug in a lower dosage, with fewer side effects, and lower production costs.Alpacas are blueprints for miniantibodiesGrlich states that nanobodies are derived from alpacas, and are simpler and smaller than traditional antibodies. The researchers immunized Britta, Nora and Xenia, three alpacas from the MPI for Biophysical Chemistry, with parts of the coronavirus spike proteins to create the nanobodies. After the mares produced antibodies, scientists took a small amount of blood from them. The mission for the alpacas was completed with enzymes, bacteria and so-called "bacteriophages" yeast. Grlich says that the overall burden of vaccinations and blood testing on animals is comparable to human vaccines.AdvertisementGrlich's team retrieved around one billion blueprints from the blood of alpacas. It was then a long-term laboratory procedure that took many years to perfect: Biochemists used bacteria to pick the best nanobodies out of the initial large pool of candidates. They were then tested against SARS-CoV-2, and further optimized in subsequent rounds.Some antibodies are not 'neutralizing'. Dobbelstein's team determined whether and how the nanobodies prevented the virus from reproducing in lab cultured cells. Antje Dickmanns from Dobbelstein’s team says, "We test a wide variety of nanobody concentrations to determine which amount is sufficient to achieve this effect." Kim Stegmann, her colleague adds that "some of the nanobodies are really impressive. To prevent infection, the medium contained less than one millionth of an gram. Even a twentyfold increase in dilution was enough for the nanobody triangulars.It is also effective against the current coronavirus variantsNew virus variants emerged rapidly during the coronavirus pandemic. These new variants can be more dangerous than the original strain from Wuhan (China). The mutated spike protein may also be able to 'escape' from neutralization by previously effective antibodies that have been vaccinated or recovered. Even for a trained immune system, it is more difficult to eradicate the virus. This also applies to previously created therapeutic antibodies and nanobodies.These are the areas where nanobodies really shine, since they also have the ability to fight major coronavirus strains. Researchers had given their alpacas a portion of the spike protein from the first SARS-CoV-2 virus. However, it was remarkable that the animals also developed antibodies that were active against different variants of the virus. We can reimmunize alpacas if our nanobodies are ineffective against future variants. Gttler confidently asserts that the alpacas have been vaccinated against the virus and would quickly develop antibodies against the new variant.Therapeutic applications in viewThe Gttingen team is currently working on the preparation of nanobodies for therapeutic purposes. Dobbelstein emphasizes that the team wants to quickly test the nanobodies for safety as a drug in order to benefit those with COVID-19, as well as those who are not vaccinated or have no immunity. Experts in technology transfer support the team: Dieter Link, Johannes Bange, Holm Keller (kENUP Foundation), and Johannes Bange (Max Planck Innovation).SARS-CoV-2's receptor-binding domain is a promising candidate for a vaccine, but it has been difficult to produce economically on a large scale. Accordingly, bacteria produces incorrectly folded material. Researchers at Gttingen discovered a solution to this problem. They created special nanobodies that force correct folding in bacteria cells without blocking the critical neutralizing domain. This could allow vaccines to be made cheaply, easily adapted to new viruses, and distributed in countries that have little infrastructure. Grlich states that the fact that nanobodies are capable of helping with protein folding is a new discovery and an exciting prospect for both research and pharmaceutical applications.