Multivalent Antibodies Show High Potency Against SARS-CoV-2 in New Study

Since the onset of the 2019 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) (COVID-19) coronavirus disease pandemic, effective and safe antivirals have been sought with increasing urgency. One promising avenue has been the use of monoclonal antibodies (mAbs) such as palivizumab, or REGN-EB3, for the prevention and treatment of respiratory syncytial virus or Ebola virus, respectively.

Against the current virus as well, bamlanivimab was the first mAb to receive emergency use approval from the US Food and Drug Administration (FDA).

It is now known that mAbs directed against the receptor binding domain (RBD) are the most effective in neutralizing the virus because they disrupt virus-receptor binding and thus prevent infection. However, it is time consuming and expensive to identify the most effective antibody clone, while new emerging strains often hamper neutralization.

A new study in the journal Nature Communication describes the process of converting an antibody into a multivalent form, thereby allowing it to bind at different points with its binding site on the antigen – also known as an epitope.

This increases antibody avidity, or overall binding affinity, for the epitope, and improves its neutralizing power.

Greed determines the ability to neutralize

The researchers used a human apoferritin protomer as an antibody scaffold, on which several antibody fragments could be attached as multimers. Called Multabody, this molecule is four orders of magnitude more potent than immunoglobulin G (IgG) antibodies in terms of neutralizing SARS-CoV-2.

The choice of human apoferritin is due to the fact that its light chain has the property of self-assembly. This allows the folded apoferritin protomers to form a symmetric octahedral nanocage multimeric structure. The nitrogenous end of each protomer points outward so that it can be easily fused to 24 identical proteins of interest.

The VHH-72 single chain variable domain of the antibody, which binds to the antigenic site of the virus, can be fused to the crystallizable fragment (Fc) of the antibody to acquire neutralizing ability. However, it does not have the ability to neutralize the virus as a monovalent entity. When displayed in 24 copies on the human apoferritin light chain, VHH-72 showed 10,000 times the neutralizing potency compared to the bivalent format fused with Fc. This shows the role of greed in determining the ability to neutralize.

Growing similarity to IgG

The half-life and effector functions of the IgG molecule are mediated by binding of Fc to neonatal Fc receptor (FcRn) and gamma Fc receptors (FcγR), respectively. By creating the single-chain Fab and Fc constructs, the researchers fused them to the N-terminus of the apoferritin light chain.

A similar experiment in mice showed that the Multabody molecule was able to bind to mouse FcRn at acidic pH, like natural mouse IgG, with higher binding to mouse FcγR1 due to its higher affinity. compared to natural IgG. By reducing the FcγR binding of multimeric antibody, they were able to increase the half-life, confirming that the Fc part is involved in the bioavailability of the molecule in vivo.

Increased Fab count for improved neutralization

Protein engineering was performed to increase the number of single chain Fab fragments in the multimer, thus further improving its neutralizing power. Scientists sought to construct a heterodimer of the fused apoferritin molecule.

To do this, the single-stranded Fab was attached to the two C-terminal helices of the apoferritin protomer (called C-ferritin, for short). The single-stranded Fc was attached to the N-terminal helices (N-Ferritin).

The end result was a self-assembled apoferritin multimer which had even more single-stranded Fab but less single-stranded Fc on the outside. This allows the Multabody to be purified in a single step similar to IgG, but with a neutralizing power 1,600 and 2,000 times greater than that of the natural anti-SARS-CoV-2 IgG mAbs BD23 and 4A8.

Conversion of non-neutralizing mAbs to neutralizing mAbs

The researchers found that the power of the binding mAbs increased by up to four orders of magnitude in 18 out of 20 cases when converted to Multabody format. Of these, 11 were initially non-neutralizing but became neutralizing in this format, and seven became potent. neutralizing antibodies in pseudovirus neutralization tests.

In fact, they were comparable in potency to the approved Regeneron mAbs REGN10933 and REGN10987, indicating their clinical applicability. In the case of the more powerful neutralizing mAbs, it has also been confirmed that the Multabody format confers an equally powerful neutralizing ability against the genuine virus.

Overall, this platform allows for the rapid formulation of ultra potent IgG-like neutralizing molecules from mAbs with limited neutralization power. This increase in potency is due to their increased avidity, linked to the fact that Multabodies target two main epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 peak.

Prevention of mutational escape

Second, the researchers found that four specific antibodies, when converted to the Multabody format, continued to show high neutralizing potency even in the presence of one of the four naturally occurring RBD mutations. A combination of three Multbodies, each with a different specificity, neutralized all of the spike variants to similar power to the wild-type spike. The overall potency was 100-1000 times higher than for the corresponding IgGs in combination.

More so, they were able to combine antibody binding fragments (Fabs) with three different specificities into a single Multabody format, resulting in the same high level of neutralization. The combination of the three antibodies 298-324-46 was the most potent, if not superior to that of the most potent IgGs which have so far been identified by one to two orders of magnitude against the pseudovirus as well as the genuine SARS- CoV-2.

This tri-specific Multabody was even able to neutralize the disturbing B.1.351 variant of SARS-CoV-2, which has so far shown resistance to several mAbs. Additionally, the ability to use mAbs of varying specificity in a single Multabody may allow the combination to be refined to achieve the highest avidity and synergistic neutralization. In fact, it might one day be possible to produce a single multimeric particle with pan-betacoronavirus neutralizing capacity.

What are the implications?

Multibodies are made up of stable self-assembling particles that form at temperatures similar to those of the parent IgGs. These bodies also use an Fc fragment, improving their bioavailability by allowing them to bind to FcRn. This aspect will need to be further considered during subsequent drug development to ensure optimal avidity.

Alternatively, other functional groups can be used to prolong 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, regardless of sequence, format or epitope, even using multiple Fabs targeting different epitope pools on the spike protein. The possibility of using VHH domains is particularly interesting because these are small and therefore very effective for neutralization.

This platform therefore shows how the engineering of avidity-enhancing proteins can help shorten the development time of potent biologics neutralizing viral threats.

The researchers write:

The Multabody provides a versatile plug-and-play IgG-like platform to enhance the antiviral characteristics of mAbs against SARS-CoV-2, and demonstrates the power of greed as a mechanism to be exploited against agents. viral pathogens. “