Science: Reveals the mechanisms of super-powerful synthetic nanoantibodies and new coronavirus

Nov 19, 2020 // --- In the past 20 years, three zoonotic β coronavirus have entered the human population, causing severe respiratory symptoms and high mortality rates.
the COVID-19 pandemic is caused by SARS-CoV-2, the most easily transmitted of the three coronavirus.
so far, no preventive treatments for coronavirus have been approved, and the timetable for the launch of an effective and widely available vaccine for SARS-CoV-2 remains uncertain.
, the development of new treatments and prevention methods remains crucial.
of the coronavirus are surrounded by membranes containing the congenerous tripolymer trans-membrane glycoprotein ---superin) --- responsible for the virus entering the host cell.
exposed part of the S protein consists of two domains--- S1 and --- S2.
S1 binds to the host cell-like hemangiotrophic conversion enzyme 2 (ACE2), while S2 catalytics the fusion of the virus and the host cell membrane.
included in S1 are the subject binding domain (RBD) and the N-end domain (NTD), where RBD binds directly to ACE2.
RBD is connected to the body of the S protein through a soft region and can exist as an inalienable down-state or an accessible up-state.
with ACE2 requires RBD to be in an upward configuration and allows it to be cleavaged by the host protease, triggering the change in the S2 configuration required for the virus to enter the host cell.
in SARS-CoV-2 virus particles, the S protein exchanges between an active open image (at least one RBD is in the upward configuration) and an inactive closed configuration (all three RBDs are in the downward configuration).
In a new study, researchers from research institutions such as the University of California, San Francisco, isolated single-domain antibodies (nanoantibodies) that are mediated and SARS-CoV-2 by screening the yeast surface display library, which contains more than 2×109 synthetic nanoantibodies sequences×
the authors used SpikeS2P, a mutant form of the SARS-CoV-2 S protein, as an antigen.
SpikeS2P lacks one of the two protein cutting bits between the S1 and S2 domains, and introduces two mutations and a trimerization domain to stabilize the pre-fusion composition of the S protein.
they labeled Spikes2P with biotin or fluorescent dyes and selected yeasts that show nanosomes through multiple rounds of screening ---in first by bead binding and then by fluorescently active cell sorting--- (Figure 1A).
study was recently published in the Journal of Science under the title "An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike".
1. Two different classes of anti-S protein nanoantibodies were found.
images from Science, 2020, doi:10.1126/science.abe3255.
three rounds of screening produced 21 unique nanoantibodies that bind to Spices2P, and the aCE2 extracellary domain (ACE2-Fc) of the djumer structure can reduce this binding.
these nanoantibodies fall into two categories.
class I nanoantibodies bind to RBD and compete directly with ACE2-Fc (Figure 1B).
example of this class is nanoantibody Nb6, which binds to SpickS2P and RBD separately, combining constants KD 210nM and 41nM, respectively (Figure 1C).
class II nanoantibodies, using nanoantiant Nb3 as an example, which binds to Spixes2P (KD-61nM), but does not appear to bind separately with RBD.
in the presence of excessive ACE2-Fc, the binding of Nb6 and other Class I nanoantibodies is completely blocked, while the binding of Nb3 and other Class II nanoantibodies is moderately reduced (Figure 1B).
results show that Class I nanoantibodies target RBD to block ACE2 binding, while Class II nanoantibodies target other temsters.
fact, surface plasma resonance (SPR) experiments have shown that Class I and Class II nanoantibodies can bind to Spices2P at the same time (Figure 1D).
the combination of Class I nanosomes with separated RBDs presents a consistently faster binding rate constant (ka) compared to SpeedS2P, indicating that RBD accessability affects KD.
, the authors tested the effectiveness of Class I and Class II nanoantibodies inhibiting the binding of fluorescently labeled SpikeS2P to HEK293 cells expressing ACE2 (Figure 1E).
Class I nanoantibodies Nb6 and Nb11 appear as two of the most effective clones, with IC50 values of 370 and 540nM, respectively.
Class II nanoantibodies showed little activity in the trial.
they preferred two Class I nanoantibodies: Nb6 and Nb11, which strongly bind to SpiceS2P, with a relatively small ka difference between SpikeS2P or RBD binding.
for Class II nanoantibodies, they prioritize Nb3, which is based on its high relative yield during purification.
strategy to prevent SARS-CoV-2 from entering the host cell is designed to block the interaction of ACE2-RBD.
high-affinity monoclonal antibodies are a leader in potential treatments, they are costly to express through mammalian cells and require intravenous injection by healthcare professionals.
preventive use requires a large dose because only a small number of antibodies can pass through the cortical cell layer in the air.
, nanoantibodies can be produced cheaply in bacteria or yeast.
stability of nanoantibodies allows them to be delivered directly to the nasal cavity and the upper part of the lungs.
fact, aerosol delivery of trijust nanoantibodies (ALX-0171) targeting respiratory syncytial viruses has recently been shown to be effective in reducing measurable viral load in hospitalized infants.
, the potential immunogenicity of camel-derived nanoantibodies can be reduced by established humanization strategies.
nomethic polyormerization has been shown to improve target affinity by affinity.
for Nb6 and mNb6, structurally guided polymer constructs that bind to all three RBDs of the S protein at the same time can result in significantly improved pots.
addition, since RBD must be in an upward configuration to bind to ACE2, configuration control of RBD accessability can function as an additional neutral mechanism.
fact, when mNb6 trimer (mNb6-tri) binds to S proteins, it prevents ACE2 binding by directly closing binding points and locking RBD in inactive compositions.
class II and nanoantibodies show a potentially new mechanism for disrupting the function of the S protein.
the use of Class I and Class II nanoantibodies in preventive or therapeutic cocktails may be effective in neutralization and blocking the escape variant of SARS-CoV-2.
therefore, the combination of combination stability, effectiveness and diversity of anti-S protein nanoantibodies screened by these authors provides a unique potential prevention and treatment strategy for limiting the persistent deaths caused by the COVID-19 pandemic.
(Bioon.com) Reference: Michael Schoof et al. An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike. Science, 2020, doi:10.1126/science.abe3255.<!--/ewebeditor:page->