A team of scientists from the United States recently developed a pre-existing neutralizing antibody to Severe Acute Respiratory Syndrome (SARS-CoV) to increase its binding affinity for the spike receptor binding domain (RBD) of severe coronavirus with acute respiratory syndrome 2 ( SARS-CoV-2). The engineered antibody shows high effectiveness in neutralizing SARS-CoV-2 and preventing infection in animals.
A preprint version of the study is available on the website bioRxiv* Server while the article is being peer reviewed
Since the emergence of SARS-CoV-2, the causative agent of coronavirus disease (COVID-19), considerable efforts have been made to reuse existing antiviral drugs and to discover novel therapeutic monoclonal antibodies against SARS-CoV-2.
Regarding antibody reuse, studies have shown that monoclonal antibodies isolated from individuals infected with SARS-CoV or the Coronavirus of Respiratory Syndrome in the Middle East (MERS-CoV) are not effective for SARS-CoV-2 can neutralize. Such a lower degree of cross-neutralization could be due to the reduced binding affinity of existing antibodies for novel epitopes in SARS-CoV-2.
In the current study, the scientists investigated a hybrid “refocusing” approach that combines conventional discovery and conversion strategies to develop an already existing neutralizing antibody against a related but resistant virus.
The scientists selected a monoclonal neutralizing anti-SARS-CoV antibody for reuse. Namely CR3022, which was isolated from a convalescent donor in 2006.
Previous studies analyzing the crystal structure of CR3022 have shown that the antibody binds to an epitope in the periphery of the binding site of angiotensin converting enzyme 2 (ACE2), which is highly conserved between SARS-CoV and SARS-CoV-2 . There are only 4 amino acid differences in or around the epitope. Of 4 mutations, the reversal of one (P384A) was found to decrease the binding affinity of CR3022 for SARS-CoV-2 by 100-fold.
In light of these observations, the scientists developed CR3022 to increase its binding affinity for the corresponding epitope on SARS-CoV-2. In addition, they tested how efficiently the manipulated antibody SARS-CoV-2 can neutralize and prevent infections.
Using a strategy to rapidly mature antibody affinity, scientists developed CR3022 variants with high affinity for SARS-CoV-2 spike protein. By introducing individual mutations into the complementarity-determining regions of the antibody, a number of CR3320 variants were created, which were then displayed as a molecular Fab on the yeast surface and screened to identify the clones with increased epitope-binding affinity.
Epitope binding affinity
The screening resulted in the selection of 25 engineered CR3022 antibodies. With regard to the parental CR3022, all manipulated antibodies showed comparable affinities for the RBD of SARS-CoV. Interestingly, all manipulated antibodies showed a 100 to 1000-fold induction of binding affinities against the monomeric RBD of SARS-CoV-2.
Virus neutralization efficiency
Using a mouse leukemia virus-based pseudovirus system, the scientists confirmed that all manipulated antibodies achieved improved neutralization efficiency against SARS-CoV compared to the parents’ CR3022. In addition, they observed that parental CR3022 could not neutralize SARS-CoV-2 even at a very high concentration, but that all constructed antibodies effectively neutralized both authentic and pseudotyped virus at low micromolar concentrations. By specifically selecting three of 25 manipulated antibodies, they confirmed that these antibodies can neutralize RBD mutation-carrying variants of SARS-CoV-2 (B.1.1.7 and B.1.351) with a similar effectiveness as the wild-type virus.
Structural analysis of the constructed antibody – SARS-CoV-2 RBD complex
The scientists performed a crystal structure analysis of the complexes of the parents’ CR3022 or an engineered CR3022 with SARS-CoV-2 RBD. The analysis showed that both parental and genetically engineered antibodies on the SARS-CoV-2-RBD bind to the same epitope.
Compared to CR3022, the binding of genetically modified antibodies to SARS-CoV-2-RBD resulted in the shift of two RBD regions. The amino acid residues in these regions (Y369, F377 and P384) formed a hydrophobic pocket that interacted with the antibodies. Upon further analysis, the scientists showed that a replacement paratope residue that targets the P384 residue in the epitope can significantly improve the antibody’s epitope-binding affinity.
Infection prevention ability of genetically modified antibodies
Based on the virus-neutralizing degrees of effectiveness, the scientists selected CR3022.7 as a candidate for a constructed antibody for further evaluation. To the in vivo In experiments they injected six hamsters intraperitoneally with different concentrations of the engineered antibody. In addition, they injected a separate parent subset of hamsters with a fixed dose of CR3022. All test animals were then infected with SARS-CoV-2 via the intranasal route.
The hamsters that received the highest dose of the engineered antibody showed either no weight loss or 3% weight loss seven days after infection. In contrast, administration of parental CR3022 resulted in an 11% weight loss in hamsters.
With regard to SARS-CoV-2 infection, a significantly lower lung virus load was observed in hamsters that received engineered antibodies than in hamsters that were treated with CR3022 from their parents. By measuring serum antibody levels at the time of injection and at the end of the experiments, the scientists confirmed that the observed effects were not due to the different pharmacokinetic properties of the two antibodies.
The study describes the development of an effective anti-SARS-CoV-2 neutralizing antibody through the development of an existing monoclonal anti-SARS-CoV antibody. The engineered antibody can prevent SARS-CoV-2 infection in hamsters without causing adverse events.
* Important NOTE
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and should therefore not be considered conclusive, guide clinical practice / health-related behavior, or are treated as established information.