Since the pandemic of coronavirus disease 2019 (COVID-19) induced by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) began, effective and safe antiviral drugs have been urgently sought. Has been done. One of the promising routes is to use monoclonal antibodies (mAbs) such as palivizumab (REGN-EB3) for the prevention and treatment of respiratory syncytial virus or Ebola virus, respectively.
Against the current virus, gamlanivimab was the first mAb to receive an emergency use authorization from the US Food and Drug Administration (FDA).
It is currently known that mAbs to the receptor binding domain (RBD) are most effective in neutralizing the virus because it interferes with the binding of the virus to the receptor and prevents infection. However, while identifying the most effective antibody clones can be time consuming and costly, the emergence of new strains often interferes with neutralization.
Journal new research Nature Communications Describes the process of converting an antibody into a polyvalent form. This allows an antibody to bind to a binding site (also called an epitope) on an antigen at many different points.
This increases the binding strength of the antibody to the epitope, or the overall binding affinity, and enhances the neutralizing ability.
Avidity determines neutralization ability
Researchers were able to use the human apoferritin protomer as an antibody scaffold and attach multiple antibody fragments as multimers on it. This molecule, called Multabody, is four orders of magnitude more potent in SARS-CoV-2 neutralization than immunoglobulin G (IgG) antibodies.
The choice of human apoferritin is due to its light chain’s self-assembling properties. This allows the folded apoferritin protomer to form a multimeric structure, such as a symmetric octahedral nanocage. The nitrogen ends of each protomer point outwards for easy fusion to 24 identical proteins of interest.
The antibody’s single-stranded variable domain VHH-72, which binds to the antigenic site of the virus, can fuse with the crystallizable fragment (Fc) of the antibody to obtain its neutralizing ability. 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 a 10,000-fold increase in neutralizing potency compared to the divalent Fc fusion format. This shows the role of avidity in determining the ability to neutralize.
Improved similarity with IgG
The half-life and effector function of the IgG molecule is mediated by the binding of Fc to the neonatal Fc receptor (FcRn) and Fc gamma receptor (FcγR), respectively. By creating single-stranded Fab and Fc constructs, researchers fused them to the N-terminus of the apoferritin light chain.
Similar experiments performed on mice showed that the Multabody molecule can bind to mouse FcRn as a natural mouse IgG at acidic pH and has a higher affinity to mouse FcγR1 due to its higher affinity than natural IgG. I did. By reducing the FcγR binding of multimeric antibodies, they were able to prolong the half-life, confirming that the Fc moiety is involved in the bioavailability of the molecule in vivo.
Increase the number of Fabs to enhance neutralization
Protein engineering was performed to increase the number of single-stranded Fab fragments in the multimer and further improve its neutralizing potency. Scientists sought to build a heterodimer of fused apoferritin molecules.
To do this, a single-stranded Fab was attached to two C-terminal α-helices of an apoferritin protomer (abbreviated as C-ferritin). On the other hand, the single-chain Fc was bound to the N-terminal α-helix (N-ferritin).
The end result was a self-assembled apoferritin multimer, with more single-chain Fabs and less outer single-chain Fc. This allows the Multabody to be purified in a single step similar to IgG, but with a 1,600-fold and 2,000-fold increase in neutralizing potency compared to the natural anti-SARS-CoV-2 IgG mAb BD23 and 4A8.
Conversion from non-neutralization to neutralized mAb
Researchers have found that conversion to the Muturabody format increases the potency of bound mAbs by up to 4 orders of magnitude in 18 of 20 cases.Of these, 11 were initially unneutralized, but were neutralized in this form, and 7 became more powerful. Neutralizing antibody To Pseudovirus Neutralization assay.
In fact, they are as powerful as the approved Regeneron mAbs REGN10933 and REGN10987, demonstrating their clinical applicability. For the strongest neutralizing mAbs, the Muturabody format has been found to provide equally strong neutralizing abilities against real viruses.
Overall, this platform enables the rapid formulation of ultra-strong IgG-like neutralizing molecules starting with mAbs with limited neutralizing potency. This increased potency is due to their enhanced binding strength, which is related to the fact that Multabodies targets two major epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike.
Prevention of mutation escape
Second, researchers found that when converted to the Muturabody format, the four specific antibodies continued to exhibit high neutralizing potency in the presence of any of the four naturally occurring RBD mutations. .. The combination of three Multabodies, each with different specificity, neutralized all spike variants with similar potency to wild-type spikes. The overall potency was 100-1000-fold higher than the corresponding IgG combination.
In addition, antibody binding fragments (Fabs) could be combined with three different specificities in a single Mutabody format, resulting in the same high level of neutralization. The combination of the three antibodies 298-324-46 was the most potent, even higher than the most potent IgG combination ever identified by 1-2 orders of magnitude against pseudovirus and real SARS-CoV-2. .. ..
This trispecific Maltese body was even able to neutralize the feared B.1.351 mutant of SARS-CoV-2, which has previously shown resistance to several mAbs. In addition, the ability to use mAbs of varying specificities in a single Multabody may allow fine-tuning of combinations for best binding and synergistic neutralization. In fact, one day it may be possible to produce a single multimeric particle with the ability to neutralize pan-beta coronavirus.
What is the impact?
Multabodies are formed by stable self-assembled particles that form at temperatures similar to those of the parent IgG. These bodies also utilize Fc fragments to increase their bioavailability by allowing them to bind to FcRn. This aspect needs to be further considered during further drug development to ensure optimal avidity.
Alternatively, other functional groups can be used to extend the half-life if other antibody effector functions such as human serum albumin are not required.
Advantages of this platform include the ability to include any antibody, regardless of sequence, format, or epitope, even when using multiple Fabs targeting different epitope bins above. Spike protein.. The ability to use VHH domains is especially valuable because they are very efficient in neutralizing due to their small size.
Therefore, this platform shows how avidity-enhancing protein engineering can help reduce the time to development of powerful neutralizing biologics against viral threats.
The researcher wrote:
Multabody provides a versatile IgG-like “plug and play” platform for enhancing the antiviral properties of mAbs against SARS-CoV-2, providing avidity as a mechanism utilized against viral pathogens. Show power.. “
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