New peptide-antibody fusion molecule improves neutralization of SARS-CoV-2 variant
The current 2019 coronavirus disease (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has not responded as expected to vaccines developed at “warp speed” in the year after its emergence, mainly due to worrying viral variants which have shown a remarkable ability to evade the host’s immune response and spread rapidly between people.
This highlights the need for other effective treatments that can be used to treat infected people or prevent infection as post-exposure prophylaxis. A recent study described in bioRxiv* Preprint server reports dramatically improved neutralizing efficacy for fused peptide antibody molecule against multiple variants of SARS-CoV-2, raising hopes that therapeutic neutralizing antibodies (nAbs) may soon be available to protect against this scourge modern.
The surface of the virus is dotted with the characteristic “crown” of spike proteins that gives the family its name coronavirus. The peak or S protein mediates viral receptor-host binding and viral entry into host cells. It exists as homotrimers, with about 25 to 100 of these trimers on the surface of each virus particle.
The S protein has two subunits, S1 and S2, which are respectively responsible for receptor binding and membrane fusion. The S1 subunit has an N-terminal domain (NTD) with a successive receptor binding domain (RBD), which mediates recognition and binding to the angiotensin converting enzyme protein 2 (ACE2) to initiate l ‘infection.
Most neutralizing antibodies prevent this step, targeting the S1 subunit by binding to RBD to stop its binding to the receptor. Some nAbs, however, bind to neutralizing epitopes on the NTD itself. Recombinant technology has helped produce and transform several natural nAbs into effective therapeutic antibodies. However, these must be used in high doses to have adequate neutralization capacity.
In addition, they do not work well against many VOCs, which contain mutations in RBD nAb epitopes that spare ACE2 binding sites. Thus, it has been shown that beta VOC is resistant to many natural and vaccine-induced nAbs.
To overcome these limitations of bivalent nAbs that bind to the RBD peak, multivalent inhibitors are being investigated, either using small modular antibody domains or antibody-free scaffolds. The multivalent nature of these molecules allows all RBD monomers to be engaged simultaneously, thus improving neutralizing power.
The fusion of additional antigen-binding (Fab) fragments of the antibody molecule to the immunoglobulin (Ig) G antibody SARS-CoV-2 produces a tetravalent antibody-like molecule that is more effective and potent against VOCs than bivalent antibodies.
Yet another development involves small synthetic peptides that bind to the spike protein at sub-micromolar affinities. If these could bind to the spike protein, they could be used in place of the bulky Fab arms to increase the valence of IgGs and thus improve their neutralizing power –
tetravalent peptide-IgG fusions that combine strong nAb binding sites with small modular peptide binding sites. “
What did the study show?
Scientists explored the ability of synthetic peptides to bind to the spike protein in order to extend the range of therapeutic nAbs against the virus. They examined libraries of phage-displayed peptides for 16-residue peptides that bind to either RBD or NTD of the S1 subunit. They found four peptides, N1 and N2 which bind to NTD, and R1 and R2 which bind to RBD. All four probably bind to sites that overlap nAb epitopes.
The researchers further identified several variants of N1 and R1 that could be chemically synthesized with 22 residues. These variants showed moderate to high apparent binding affinities, possibly due to the ability to bind to several peak trimers. This reflected their potential performance in the versatile neutralizing hybrid molecules that this experiment aimed to produce.
Scientists designed these fusion molecules by attaching peptides to the N-terminus of an nAb with moderate tip binding affinity. This resulted in tetravalent peptide-IgG fusion molecules, which exhibited very low kill rates compared to native 15033 IgG. As a result, affinities were much improved, as expected, because the presence of a ligand peptide generally increases the binding energy and thus reduces the off-rate.
The highest affinity, with dissociation constants below the picomolar level, was found with IgGs fused with R1 and N1. The affinity for the peak trimer appeared to be 100 times higher than IgG 15033.
Next came the cell-based infection assays, where investigators tested the ability of an RBD binding peptide (33-7HR1) to inhibit cell culture invasion by six variants of SARS-CoV-2 ( the B.1.1, the Alpha, Beta, B 1.525 and the P.1, as well as the ancestral variant of Wuhan). The same fusion molecule also inhibited viral entry of SARS-CoV-2 variants that were resistant to the same nAb in its natural bivalent format.
They found that even with a high affinity nAb, the peptide increased neutralizing power over 100 times. At low nanomolar range, it neutralized all six variants, while IgG 15033-7 failed to neutralize three VOCs and showed much lower efficacy against the other three.
Finally, they discovered that the already approved therapeutic nAb REGN10933 could be successfully fused to the R1 peptide. The resulting molecule, 10933HR1, outperformed the original antibody in neutralizing the D614G variant. More interestingly, he was able to neutralize the beta variant with high potency, although REGN10933 was totally ineffective against this variant – the half-maximal inhibitory concentration, IC50, being 1000 ng / mL, for the fusion molecule against the original antibody.
Thus, he saved the neutralization against a resistant variant.
What are the implications?
Second, the trimeric structure of the tip helps design highly neutralizing antibodies that bind to three neutralizing epitopes per trimer. Previously, neutralizing IgGs have been shown to engage two RBDs on the peak trimer, which is improved to three by adding one Fab to the IgG, thereby improving the neutralizing potency.
The current study replaces large Fabs with small peptides to produce tetravalent fusion molecules from neutralizing IgGs, with much better affinities and potencies than the bivalent molecule. Peptide-IgG 33-7HR1 was a potent inhibitor of resistant variants of SARS-CoV-2, and peptide R1 rescued the already approved neutralization of VOC beta which had so far completely resisted neutralization by RGN10933.
It appears that one of the R1 peptides could bind to the third RBD on the tip trimer, with two bound by the bivalent IgG Fabs. With the approved therapeutic nAb REGN10933, compared to the native IgG 15033-7, the neutralizing epitopes overlap, but the Fabs of the first would allow one of the peptides bound in the fusion molecule 10933HR1 to bind to the third RBD epitope. .
NTD-binding peptides, which can allow binding to other neutralizing epitopes, also greatly improved the binding affinity of the fusion peptide-IgG. Interestingly, NTD and RBD binding peptides exhibit the highest binding affinity when fused to IgG light and heavy chains, respectively. Since these peptides bind to quite different epitopes, the possibility of fusing one of each type to the IgG molecule should be explored for potential improvements in neutralizing potency at even higher levels.
In addition to the COVID-19 pandemic, for which these fusion molecules could ultimately be developed as biologics, such molecules are being tested in clinical trials in cancer patients. So far, they have shown good tissue penetration and good pharmacokinetics, with high tolerance.
Taken together, our results establish peptide-IgG fusions as a potent means to dramatically improve the potency and coverage of next-generation biologics for the treatment of disease caused by SARS-CoV-2 and its variants.. “
bioRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.