Most studies testing monoclonal antibodies specific for severe acute coronavirus 2 respiratory syndrome (SARS-CoV-2) use plasmablast antigen probes and memory B cells from donated plasma from recovered SARS-CoV-2 individuals. However, given the rise of new worrying variants around the world, the motivation to produce more tools based on anti-SARS-CoV-2 antibodies is high.
New bioRxiv* A preprint study led by Joshua Tan of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, involves the creation of bispecific antibodies by a new method of cross-linking antibodies to neighboring spike proteins, using the dual specificity of the N-terminal domain / domain binding receptor.
How they did it
The researchers collected SARS-CoV-2-specific monoclonal antibodies from the plasmablasts and memory B cells of 126 patients with COVID-19 infection. Samples were collected in April 2020 in New York City, indicating that the immune response reflects the first wave of the pandemic. They were collecting. A total of 119 antibodies targeted the SARS-CoV-2 spike protein, 106 targeted the receptor-binding domain, and 122 targeted the N-terminal domain.
Plasma was tested for neutralization potential if a wide range of neutralizing titers from <40 to 765 were detected. Potency was weakly correlated with antibody levels for spike protein, receptor binding domain, and N-terminal domain. Also, some plasma samples did not have the ability to neutralize despite high antibody levels.
To develop monoclonal antibodies with high specificity for SARS-CoV-2 epitopes, the researchers used a single-cell assay to examine antibodies in plasmablasts and memory B cells. A total of 169 monoclonal antibodies targeting SARS-CoV-2 and 47 from the memory B cells of 12 individuals were extracted from the plasmablast assays. Approximately 59 monoclonal antibodies target the receptor-binding domain, 64 target the N domain, and 46 neither, suggesting that the antibodies can detect the entire spike protein.
Plasmablasts and memory B cells generate highly effective neutralizing antibodies
The researchers found that most monoclonal antibodies did not have neutralizing potency, but 21 antibodies showed strong potency against SARS-CoV-2.
Immunoglobulin A (IgA) isotype antibodies showed superior neutralization. Twenty-one antibodies listed under the immunoglobulin G (IgG) isotype targeted several regions of the protein classes — 16 targeted the receptor-binding domain and 5 targeted the N-terminal domain.
The average strength of antibodies generated from plasmablasts or memory B cells was similar, indicating that both produced strong antibodies.
Using high-throughput surface plasmon resonance, antibodies specific for the receptor-binding domain showed higher affinity than monoclonal antibodies specific for the N-terminal domain. Monoclonal antibodies specific for the N-terminal domain from plasmablasts had lower affinities than antibodies from memory B cells.
The researchers found that the most potent monoclonal antibody had low affinity for the N-terminal domain.
Ability to bind antibodies to COVID-19 variants
About 28 of the 37 monoclonal antibodies specific for the receptor binding domain and 3 of the 20 antibodies specific for the N-terminal domain had over 90% binding to variant B.1.1.7.
Only 10 antibodies specific for the receptor binding domain and 2 antibodies specific for the N-terminal domain retained complete binding to variant B.1.351. The researchers suggest that the results provide more evidence that variant B.1.351 is more successful in immune escape.
Bispecific antibodies to SARS-CoV-2
Since most monoclonal antibodies bind to sites that do not overlap, they then tested for possible synergy between antibodies. They combined antibody specificities to produce ten bispecific antibodies by combining variable regions of potent neutralizers with two types of linkers.
Bispecific antibodies that can target the receptor-binding domain and the N-terminal domain have retained binding to both domains. When testing bispecific antibodies in tests for SARS-CoV-2, five antibodies – CV503_521_GS, CV521_1182_GS, CV1206_521_GS, CV521_503_GS and CV503_664_EL neutralized SARS-CoV-2.
“Significantly, CV1206_521_GS neutralized SARS-CoV-2 with> 100 times the potency of the CV1206 and CV521 component antibody cocktails. We found that CV1206_521_GS uses its inner and outer Fab domains to cross-link NTD and RBD in adjacent spike proteins, a mode of action that is inaccessible to conventional mAbs, even when used in combination.”
The researchers suggest that the results appear to promise that combining bispecific antibodies could further enhance the neutralization potential.
When spike proteins of variants B.1.1.7 and B.1.351 were tested, only bispecific antibodies whose two components lost binding to the variants could not bind. This suggests that this form of antibody is more resistant to spike mutations than typical monoclonal antibodies.
All bispecific antibodies neutralized strain D614G, most bispecific antibodies with CV521 neutralized variant B.1.1.7, and six of the nine bispecific antibodies neutralized B.1.351.
* bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered definitive, guide clinical practice / health-related behavior, or treat it as established information.