Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced pandemic of coronavirus disease 2019 (COVID-19), effective and safe antivirals are being sought with increasing urgency. One promising pathway has been the use of monoclonal antibodies (mAbs) like palivizumab, or REGN-EB3, for respiratory syncytial virus or Ebola virus prevention and treatment, respectively.
Against the current virus, too, bamlanivimab was the first mAb to receive emergency use approval by the United States Food and Drug Administration (FDA).
It is now known that mAbs directed against the receptor-binding domain (RBD) are most effective at neutralizing the virus as they disrupt virus-receptor binding and thus prevent infection. However, it is time-consuming and expensive to identify the most effective antibody clone, while emerging new strains often hinder neutralization.
A new study in the journal Nature Communications describes the process of converting an antibody into a multivalent form, thus enabling it to bind at various different points with its binding site on the antigen – also known as its epitope.
This increases antibody avidity, or overall binding affinity, for the epitope, and enhances its neutralization potency.
Avidity determines neutralizing capacity
The researchers used a human apoferritin protomer as an antibody scaffold, on which multiple antibody fragments could be attached as multimers. Called a Multabody, this molecule has potency higher by four orders of magnitude compared to the immunoglobulin G (IgG) antibodies in terms of SARS-CoV-2 neutralization.
The choice of human apoferritin is because its light chain has the property of self-assembly. This allows folded apoferritin protomers to form a symmetrical octahedral nanocage-like multimeric structure. The nitrogenous end of each protomer points outwards so that it can be fused easily to 24 identical proteins of interest.
The single chain variable domain VHH-72 of the antibody, which binds to the antigenic site on the virus, can be fused to the crystallizable fragment (Fc) of the antibody to acquire neutralizing capacity. However, it lacks the ability to neutralize the virus as a monovalent entity. When displayed in 24 copies on the light chain of human apoferritin, VHH-72 showed 10,000-fold increased neutralizing potency compared to the bivalent Fc-fused format. This shows the role of avidity in determining neutralizing capacity.
Increasing similarity to IgGs
The IgG molecule’s half-life and effector functions are mediated via the binding of the Fc with the neonatal Fc receptor (FcRn) and Fc gamma receptors (FcγR), respectively. By creating the single-chain Fab and Fc constructs, the researchers fused them to the apoferritin light chain N terminal ends.
A similar experiment conducted in mice showed the Multabody molecule to be able to bind mouse FcRn at acidic pH, as the natural mouse IgG, with higher binding to the mouse FcγR1 because of its higher affinity relative to the natural IgG. By reducing the FcγR binding of the multimeric antibody, they were able to increase the half-life, confirming that the Fc portion is implicated in the bioavailability of the molecule in vivo.
Increasing Fab number for enhanced neutralization
Protein engineering was carried out to increase the number of single-chain Fab fragments in the multimer, thus improving its neutralization potency still further. The scientists sought to construct a heterodimer of the fused apoferritin molecule.
To do this, the single-chain Fab was attached to the two C-terminal α helices of the apoferritin protomer (called C-ferritin, in short). The single-chain Fc was attached, meanwhile, to the N-terminal α helices (N-Ferritin).
The end result was a self-assembled apoferritin multimer that had even more single-chain Fab but fewer single-chain Fc on its outside. This allows the Multabody to be purified in a single step similar to IgG, but with 1,600- and 2,000-fold increased neutralization potency compared to the natural anti-SARS-CoV-2 IgG mAbs BD23 and 4A8.
Converting non-neutralizing to neutralizing mAbs
The researchers found that binding mAbs increased in potency by up to four orders of magnitude in 18 of 20 cases when converted to Multabody format. Of these, 11 were initially non-neutralizing but became neutralizing in this format, and seven became potently neutralizing antibodies in pseudovirus neutralization assays.
In fact, they were comparably potent to the approved Regeneron mAbs REGN10933 and REGN10987, indicating their clinical applicability. In the case of the most potent neutralizing mAbs, the Multabody format was also confirmed to confer similarly potent neutralizing ability against the authentic virus.
Overall, this platform allows the rapid formulation of ultrapotent IgG-like neutralizing molecules beginning from mAbs with limited neutralization potency. This increase in potency is via their enhanced avidity, linked to the fact that the Multabodies target two main epitopes on the receptor-binding domain (RBD) of the SARS-CoV-2 spike.
Prevention of mutational escape
Secondly, the researchers found that four specific antibodies, when converted to Multabody format, continued to show high neutralizing potency even in the presence of any of four naturally occurring RBD mutations. A combination of three Multabodies, each with a different specificity, neutralized all spike variants at similar potency to the wildtype spike. The overall potency was 100-1000-fold higher than for the corresponding IgGs in combination.
Even more, they were able to combine antibody-binding fragments (Fab) with three different specificities in a single Multabody format, which resulted in the same high level of neutralization. The combination of the three antibodies 298-324-46 combination was the most potent, even higher than that of the most potent IgGs that have been identified so far by one to two orders of magnitude against pseudovirus as well as authentic SARS-CoV-2.
This tri-specific Multabody was even able to neutralize the B.1.351 variant of concern of SARS-CoV-2, which has shown resistance to several mAbs so far. Moreover, the ability to use mAbs of varying specificity in a single Multabody may allow fine-tuning of the combination to achieve the highest avidity and synergistic neutralization. In fact, it may be possible one day to produce a single multimeric particle with pan-betacoronavirus neutralizing capacity.
What are the implications?
The Multabodies are formed of stable self-assembling particles that form at temperatures similar to the parent IgGs. These bodies also make use of an Fc fragment, enhancing their bioavailability by allowing them to bind to FcRn. This aspect will need further consideration during further drug development to ensure optimal avidity.
Alternatively, other functional groups may be used to extend the half-life if other antibody effector functions are not required, such as human serum albumin.
The advantages of this platform include the ability to include any antibody, irrespective of sequence, format or epitope, even using multiple Fabs targeting different epitope bins on the spike protein. The ability to use VHH domains is especially valuable as these are small and hence very efficient at neutralization.
This platform, therefore, shows how avidity-enhancing protein engineering can help to shorten the time to development of powerful neutralizing biologics against viral threats.
The researchers write:
The Multabody offers a versatile IgG-like “plug-and-play” platform to enhance antiviral characteristics of mAbs against SARS-CoV-2, and demonstrates the power of avidity as a mechanism to be leveraged against viral pathogens.”
Phase 2 study evaluates safety and efficacy of asunercept in COVID-19 patients