As the world struggles to find effective therapies to combat severe and critical COVID-19, one more piece is added to the armamentarium of potential severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapies.
Study: A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to known RBD mutations. Image Credit: Kateryna Kon/Shutterstock.com
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
In a promising new study published in the pre-print server bioRxiv*, researchers describe the identification of a neutralizing antibody that binds to the RBD at the same site that is used to bind to the human host cell receptor, and which is not inhibited by any of the known mutations at the RBD.
Study Details
The study began with the construction of libraries of antibodies from 6 patients with COVID-19 infection, confirmed by reverse transcriptase-polymerase chain reaction (RT PCR), who had high levels of immune antibodies. The peripheral blood mononuclear cells (PBMCs) and active plasma cells were used to construct the libraries of immune molecules.
The antibodies were sorted according to their binding site, and those reacting against epitopes in the RBD were selected, while still ensuring these sites were present in the right conformation of the intact trimeric spike protein. This revealed ~200 unique antibodies.
Among these, 135 were selected as being easy to develop and converted to the bivalent scFv-Fc format. The bivalent format of the IgG accelerates antibody experiments as a single cloning step is required with high yields from small-scale experiments. They were then expressed in Expi293F cells.
Inhibition of Viral Binding
Their ability to inhibit viral binding was tested in a cell-based format, using the full-length spike protein ectodomain. They found that among these, 30 antibodies showed high (>80%) inhibition, the human germline V-gene VH3-66 was most highly used among the heavy chain V genes.
This shows this gene is important in the immune response against this virus. It also agrees with earlier studies, that identify this gene, along with the closely related VH3-53 gene, as enriched in SARS-CoV-2 infection. The VH CDRs on all antibodies from this gene were closely related, but with different light chains.
On a cytopathic effect (CPE) neutralization assay, 19 of these were further selected and produced as human IgG. With four of these antibodies, the half-maximal effective concentrations (EC50) values were in the subnanomolar range for all tested antigens.
One antibody, STE90-C11, was further validated in ACE2-expressing cells and showed half-maximal inhibition of binding (IC50) at 2.6-15 nM and 1-3 nM for anti-S1S2 and RBD IgG, respectively.
These had the best molar ratios between 0.02-0.3:1, for the proportion of antibody binding sites to the antigen. This is probably because the three RBDs on the same spike trimer are in different conformations, ‘up’ and ‘down’, with only the former being able to bind ACE2. In turn, this means the ‘down’ RBDs cannot bind to the antibody either.
When tested in the presence of the actual virus, this antibody showed IC50 of 0.56 nM for IgG.
Selectivity for SARS-CoV-2 RBD
This antibody binds specifically to SARS-CoV-2 RBD, but not to that of other coronaviruses. This may be because it interacts with a sequence uninvolved in ACE2 binding, a sequence also used by other leading neutralizing antibodies like CB6 and B38.
This sequence is in a different location within the RBD of SARS-CoV due to the different conformational folding of this protein, presenting steric hindrance to the binding of these antibodies.
High Mutational Tolerance
When tested against S1 subunits containing 7 of the single point mutations in the RBD that are being frequently encountered, binding was unaffected. The antibody also prevented all the mutants from binding to ACE2 in a cell-based assay. This was observed even in a “worst-case” mutant where all seven of these mutations were present in the RBD. This is probably possible because of the wide area of interaction, which is more than double that of REGN antibodies REGN10933 and REGN10987, and larger even than that of CB6 or CR3022. Secondly, the number of contacts occurring between the RBD and the light chains, which makes up for the substitution of a number of amino acids. The
The implications of such susceptibility to RBD mutations are obvious, as a mutational escape will be inevitable once these mutations become more widespread. This will occur because of positive selection pressures acting on the virus as these therapeutic antibodies are used in a greater number of people.
In this scenario, it presents distinct advantages over the better-known REGN10933, which is inhibited by several of these mutations.
an antibody combination like REGN10933 + REGN10987 where both are sensitive to some but different mutations or an antibody-like STE90-C11 which is tolerant to mutations is needed.”
Can This Antibody Cause ADE?
The current study focuses on antibodies binding to the RBD in order to prevent the occurrence of antibody-dependent enhancement (ADE) of disease. It is defined as "enhancement of disease severity in an infected person or animal when an antibody against a pathogen...worsens its virulence by a mechanism that is shown to be antibody-dependent.”
In fact, anti-spike antibodies have been reported to induce immune dysregulation and lung inflammation during acute SARS-CoV infection.
Biochemical studies show that it binds to a conformational epitope at the RBD-ACE2 binding interface. The interface at which its interactions with the host cell ACE2 receptor take place is almost completely identical to that of the RBD on the virus. About 60% of the area of binding is due to the VH segment, and hydrophobic interactions predominate. Up to 10 hydrogen bonds are formed simultaneously. The V light chains contribute another 8 hydrogen bonds, forming the remaining 40%.
No significant changes occurred in the RBD after the binding of STE90-C11, and it neutralizes the virus by direct competition with it for the ACE2 binding site.
This was not observed with animals immunized against the virus using RBD as the antigen. To protect against this, they used the Fc part of the antibody, which does not bind to the Fc-gamma receptors of immune cells, and is hence called “silent Fc”. They also used antibodies directed against the RBD in particular. They note that it is difficult to predict ADE accurately in humans from in vitro or animal studies, making it of paramount importance to take steps to avoid it as far as possible while designing or selecting therapeutic antibodies.
What are the Benefits?
The antibody STE90-C11 is therefore highly inhibitory against live SARS-CoV-2 in vitro and shows high mutational tolerance. It directly competes with ACE2 for viral RBD sites. It also has a high germinability index, a parameter which indicates it is not likely to be immunogenic. This monoclonal antibody STE90-C11 is therefore ideal for further development as a therapeutic.
This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources
Article Revisions
- Apr 3 2023 - The preprint preliminary research paper that this article was based upon was accepted for publication in a peer-reviewed Scientific Journal. This article was edited accordingly to include a link to the final peer-reviewed paper, now shown in the sources section.
Researchers develop new antibody with potential to treat several types of cancer