Inspired by a unique kind of infection-fighting antibody found in llamas, alpacas, and other camelids, a research team at the University of California, San Francisco, has synthesized a molecule that they say is among the most potent anti-coronavirus compounds tested in a lab to date.
Called nanobodies because they are about a quarter of the size of antibodies found in people and most other animals, these molecules can nestle into the nooks and crannies of proteins to block viruses from attaching to and infecting cells.
The lab-made one created by the UCSF team is so stable it can be converted into a dry powder and aerosolized, meaning it would be much easier to administer than Covid-19 treatments being developed using human monoclonal antibodies. While the work is still very preliminary, the goal is to deliver the synthetic nanobody via simple inhaled sprays to the nose or lungs, allowing it to potentially be self-administered and used prophylactically against Covid-19 — if it’s shown safe and effective in both animal tests and clinical trials.
After four months of working nearly around the clock, the team posted the results Monday to the bioRxiv preprint server. The paper has not yet been peer-reviewed, but the researchers said they are already in talks to find a partner that can quickly test, manufacture, and distribute the new compound in hopes it can prevent new infections and mitigate disease in those who are already infected. “Every day, 5,000 people die of this disease. We’d like as soon and as fast as possible to find a partner to make this,” said Peter Walter, a veteran biochemist who permanently resides on many short lists of those expected to win a Nobel Prize and who co-led the project with structural biologist Aashish Manglik.
Well-aware of the furor over early announcements for coronavirus therapies, the duo do not want to oversell their findings and acknowledge the nanobody, called Aeronab 6, needs to be tested in clinical trials. But they are enthusiastic because of both how stable the compound is and how well it has responded in lab tests where it inhibits the infection of cells by binding ferociously to the infamous spike proteins that allow coronavirus particles to enter and infect cells. “It’s almost like a mousetrap that never lets go,” said Walter.
While the lab results look promising, experts in the field advise caution because important work has not been done to test the compound in animals. “The critical thing is animal data. We’ve found things that are very potent in vitro that do nothing in vivo,” said Dimiter Stanchev Dimitrov, a professor of medicine who directs the Center for Antibody Therapeutics at the University of Pittsburgh and has created antibody-based therapeutics for numerous viruses including SARS and MERS, two other coronaviruses. He said it can take months to collect the needed data in animals. “Once these are tested in animal models, then I can get excited.”
Dimitrov said that while administering an antibody therapy through an inhaler was an exciting idea that other groups are also investigating, there could be difficulties getting a drug delivered uniformly within the lung. “It’s tricky to deliver antibodies to the lung,” he said.
The nanobody’s potency against coronavirus was tested at the Institut Pasteur in Paris by Veronica Rezelj, a postdoctoral researcher in the institute’s viral populations and pathogens unit. When Rezelj mixed coronavirus and small amounts of the nanobody on cell plates, the Vero-E6 cells (derived from the kidney cells of African green monkeys and commonly used in lab work) were protected from infection and stayed alive.
“Within four days from the day the package from the U.S. arrived, we knew we had a very potent nanobody,” she said. “Very little nanobody was needed to completely abolish virus infectivity.” She said the nanobody’s effectiveness was much higher than that in published data about neutralizing antibodies taken from the blood of Covid-19 patients, or other synthetically made nanobodies.
Rezelj has tested many antivirals against the coronavirus in recent months; many won’t even work in cell culture or animal models, let alone humans. The nanobodies, she said, are more promising: “We are very confident that they could work from a therapeutic standpoint after human clinical trials are completed.”
Dozens of related therapies using human monoclonal antibodies are being developed for use against the coronavirus, with some already in clinical trials, but Walter and Manglik are skeptical about how widespread and practical those approaches will be: They’re expensive to manufacture in mammalian cells, must be administered intravenously by medical personnel, and require high doses because they travel through the bloodstream before reaching the lungs. The UCSF researchers think an effective approach may be to target nasal passages, where the virus may first become established before it is seeded into the lungs.
Their compound, they say, could be cheaply made in enormous quantities using bacteria or yeast, and would require low doses because it is so potent against the virus and can be administered directly to the lungs and or nasal passages. “We’d like this to be made available to developing countries,” Walter said. “This is possible because it can be shipped as a dry powder and it should be very inexpensive to produce.”
Other labs around the world, including at the University of Oxford and the Rosalind Franklin Institute, have recently published work on synthetic nanobodies. In May, a University of Texas group showed true llama antibodies that were engineered in the lab helped prevent coronavirus infection in lab studies. And this week, a team from China reported in bioRxiv that it produced camel nanobodies active against the Covid-19 virus.
Markus Seeger, an assistant professor at the Institute for Medical Microbiology at the University of Zurich, was among the first to publish work on synthetic nanobodies, or sybodies as he calls them, for use against coronavirus, and he recently signed a partnership with Absolute Antibody to make the compounds available for research. Seeger said many labs are at work on the compounds because they are cheap to manufacture, simpler to optimize than human antibodies, and potentially powerful for therapeutics, prophylactics, and even use on masks and surfaces to curb viral spread.
“We’re just at the tip of the iceberg,” he said. “I think with many companies there will be a lot of activity.”
While he noted that there was a big difference between having a potent nanobody and an actual drug, Seeger said he thought the new compound looked promising because it not only blocked the virus from binding to cells but actually changed the conformation of the spike protein, locking down the receptor binding domains that allow it to attach to cells.
The UCSF team members, who normally work on research like deciphering the shape of receptors on cell membranes and understanding how proteins unfold, jumped at the chance to work on coronavirus therapies in March. “When UCSF shut down all research operations with the exception of coronavirus work, we stopped our regular work and really pivoted,” Manglik said.
The project took advantage of a massive library of synthetic nanobodies manufactured in yeast that Manglik had helped create a few years ago, long before the pandemic, to better understand the structure of proteins to aid in drug design and further basic research. “The library would not have been there without the drive for basic research,” said Walter.
It took Walter and his graduate student Michael Schoof to press Manglik to use the library to hunt for a weapon against the coronavirus. Manglik, who is also an M.D., had recently started his lab at UCSF and said he saw the tools he was developing to engineer and optimize proteins for basic research as uniquely positioned “to directly tackle the pandemic death toll.”
They rapidly screened the library’s collection of 2 billion nanobodies for ones that might work against the Covid-19 coronavirus by using its spike protein to fish out nanobodies that bound to it. They then culled that group to about 20 nanobodies that worked especially well in preventing the virus from entering and infecting cells. Aeronab 6 rose to the top because it bound to the spike protein in a really strong and unique manner and because it was so stable. Walter, Manglik, and Schoof hold a patent on the compound.
Even though the nanobody worked well from the start, Manglik led an effort to optimize the molecule so it would be even more effective. The team created a version that both prevented the coronavirus spike proteins from binding to the cell and locked the spikes down so they remain in a position that makes them unavailable for binding. They also “humanized” the nanobody so it closely resembled a human protein and thus would be less likely to create an allergic or immune response.
Manglik is not a fan of working with actual llamas; there is the hassle and veterinary bills, not to mention the fact it takes months of waiting for an animal to produce nanobodies after they are infected with a pathogen. But he does give the animals credit: “We’ve been dealing with the challenge of building molecules that are as beautiful as those produced in nature and we take deep inspiration from the kind of antibodies that exist in camels, alpacas, and llamas.”
He said the work was only possible because of the drive of the many trainees on the project, some of them routinely working until 3 or 4 in the morning. The trainees, Manglik said, lacked experience but made up for it in enthusiasm and by “working at the limits of their physical capacity.”
Those trainees say they were inspired by Manglik’s willingness to work endless hours in the lab on routine bench work, even pipetting during Zoom meetings. “Here’s a full-fledged professor, coming in, gloving up, and purifying proteins,” Schoof said.
The UCSF scientists envision Aeronab 6 as something that could be offered to people who have recently tested positive for coronavirus to prevent disease progression. It could also be offered to people who have been exposed to the disease to prevent infection, or used as a daily prophylactic for those at high risk of infection, such as health care workers, first responders, and prison guards.