Highly potent, stable nanobodies stop SARS-CoV-2

Date:
July 29, 2021
Source:
Max-Planck-Gesellschaft
Summary:
Researchers have developed nanobodies that efficiently block the
coronavirus SARS-CoV-2 and its new variants.



FULL STORY ========================================================================== Go"ttingen 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 at low costs in
large quantities, they could meet the global demand for COVID-19
therapeutics. The new nanobodies are currently in preparation for
clinical trials.


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

Scientists at the Max Planck Institute (MPI) for Biophysical Chemistry
in Go"ttingen (Germany) and the University Medical Center Go"ttingen
(UMG) have now developed mini-antibodies (also known as VHH antibodies
or nanobodies) that unite all the properties required for a potent drug
against COVID-19. "For the first time, they combine extreme stability
and outstanding efficacy against the virus and its Alpha, Beta, Gamma,
and Delta mutants," emphasizes Dirk Go"rlich, director at the MPI for Biophysical Chemistry.

At first glance, the new nanobodies hardly differ from anti-SARS-CoV-
2 nanobodies developed by other labs. They are all directed against
a crucial part of the coronavirus spikes, the receptor-binding domain
that the virus deploys for invading host cells. The nanobodies block
this binding domain and thereby prevent the virus from infecting cells.

"Our nanobodies can withstand temperatures of up to 95 DEGC without losing their function or forming aggregates," explains Matthias Dobbelstein,
professor and director of the UMG's Institute of Molecular Oncology. "For
one thing, this tells us that they might remain active in the body long
enough to be effective.

For another, heat-resistant nanobodies are easier to produce, process,
and store." Single, double, and triple nanobodies The simplest
mini-antibodies developed by the Go"ttingen team already bind up to
1000 times more strongly to the spike protein 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," Dobbelstein says. "In
addition, because they are very small, they could readily 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 is composed of three identical building blocks with three binding domains. "With the nanobody triad, we literally join forces: In an ideal scenario, each of the three nanobodies attaches to one of the three
binding domains," reports Thomas Gu"ttler, a scientist in Go"rlich's
team. "This creates a virtually irreversible bond. The triple will not let release the spike protein and neutralizes the virus even up to 30,000-fold better than the single nanobodies." Another advantage: The larger size
of the nanobody triad expectedly delays renal excretion. This keeps them
in the body for longer and promises a longer-lasting therapeutic effect.

As a third design, the scientists produced 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 escape' because
they bind the viral spike so strongly," explains Metin Aksu, a researcher
in Go"rlich's team.

For all nanobody variants -- monomeric, double as well as triple --
the researchers found that very small amounts are sufficient to stop
the pathogen.

If used as a drug, this would allow for a low dosage and thus for fewer
side effects and lower production costs.

Alpacas provide blueprints for mini-antibodies "Our nanobodies originate
from alpacas and are smaller and simpler than conventional antibodies," Go"rlich says. 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 produced antibodies, and the scientists drew a small blood sample from the animals. For the alpacas,
the mission was then complete, as all further steps were carried out with
the help of enzymes, bacteria, so-called bacteriophages, and yeast. "The overall burden on our animals is very low, comparable to vaccination
and blood testing in humans," Go"rlich explains.



========================================================================== Go"rlich's team extracted around one billion blueprints for nanobodies
from the alpacas' blood. What then followed was a laboratory routine
perfected over many years: The biochemists used bacteriophages to select
the very best nanobodies from the initially vast 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 of Dobbelstein's group therefore determined if and how well the nanobodies prevent the viruses
from replicating in cultured cells in the lab. "By testing a wide range of nanobody dilutions, we find out which quantity suffices to achieve this effect," explains Antje Dickmanns from Dobbelstein's team. Her colleague
Kim Stegmann adds: "Some of the nanobodies were really impressive. Less
than a millionth of a gram per liter of medium was enough to completely
prevent infection. In the case of the nanobody triads, even another
twenty-fold dilution was sufficient." Also effective against current coronavirus variants Over the course of the coronavirus pandemic, new
virus variants have emerged and rapidly became dominant. These variants
are often more infectious than the strain that first appeared in Wuhan
(China). Their mutated spike protein can also 'escape' neutralization
by some originally effective antibodies of infected, recovered, or
vaccinated persons. This makes it more difficult even for 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 major 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
system also produced antibodies that are active against the different
virus variants. "Should our nanobodies prove ineffective against a future variant, we can reimmunize the alpacas. Since they have already been
vaccinated against the virus, they would very quickly produce antibodies against the new variant," Gu"ttler asserts confidently.

Therapeutic application in view The Go"ttingen team is currently preparing
the nanobodies for therapeutic use.

Dobbelstein emphasizes: "We want to test the nanobodies as soon as
possible for safe use as a drug so that they can be of benefit to those seriously ill with COVID-19 and those who have not been vaccinated
or cannot build up an effective immunity." The team is supported by
experts in technology transfer: 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 so far difficult to manufacture economically
on a large scale and in a form, which activates the immune system against
the virus.

Bacteria programmed accordingly produce incorrectly folded material. The Go"ttingen researchers discovered a solution for this problem:
They identified special nanobodies that enforce correct folding in
bacterial cells, without obstructing the crucial neutralizing part of
the receptor-binding domain. This might allow for vaccines that can be
produced inexpensively, can be quickly adapted to new virus variants, and
can be distributed with simple logistics even in countries with little infrastructure. "The fact that nanobodies can help with protein folding
was previously not known and is extremely interesting for research and pharmaceutical applications," Go"rlich says.

========================================================================== Story Source: Materials provided by Max-Planck-Gesellschaft. Note:
Content may be edited for style and length.


========================================================================== Journal Reference:
1. Thomas Gu"ttler, Metin Aksu, Antje Dickmanns, Kim M. Stegmann,
Kathrin
Gregor, Renate Rees, Waltraud Taxer, Oleh Rymarenko, Ju"rgen
Schu"nemann, Christian Dienemann, Philip Gunkel, Bianka Mussil,
Jens Krull, Ulrike Teichmann, Uwe Gross, Volker C. Cordes, Matthias
Dobbelstein, Dirk Go"rlich. Neutralization of SARS‐CoV‐2
by highly potent, hyperthermostable, and mutation‐tolerant
nanobodies. The EMBO Journal, 2021; DOI: 10.15252/embj.2021107985 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/07/210729122158.htm

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