Peptide-antibody Fusions Engineered by Phage Display Exhibit Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants (preprint)

The COVID-19 pandemic has been exacerbated by the emergence of variants of concern (VoCs). Many VoC mutations are found in the viral spike protein (S-protein), and are thus implicated in host infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but are limited due to low potency and vulnerability to RBD mutations found in VoCs. To address these issues, we used naive phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate affinity nAb to generate tetravalent peptide-IgG fusions, and showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD <1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrate that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug, and showed that it rescued neutralization against a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies (preprint)

Neutralizing antibodies (nAbs) that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S-protein) are promising therapeutics for COVID-19. However, natural bivalent nAbs suffer from limited potency and are vulnerable to SARS-CoV-2 variants with mutated RBDs. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The diabody-Fc-Fab format consists of a central Fc with a bivalent diabody fused to its N-terminus and two Fabs fused to its C-terminus. The diabody and Fab modules do not interfere with each other, and thus, any diabody can be combined with any Fab in a facile manner. We engineered a diabody-Fc-Fab that contained the paratopes of two distinct nAbs derived from a phage-displayed library of synthetic Abs. The tetravalent nAb was purified in high yields with methods used to produce conventional IgGs, and it exhibited favorable biophysical characteristics comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the S-protein trimer at least 100-fold more tightly than the bivalent IgGs (apparent KD <1 pM). Most importantly, the tetravalent nAb exhibited extremely high potencies in neutralization assays across a panel of pseudoviruses representing seven natural SARS-CoV-2 variants (IC50 <5 ng/mL), including several that resisted IgGs and are known to evade approved IgG drugs. Taken together, our results showed that the tetravalent diabody-Fc-Fab is a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that far exceed those of conventional bivalent IgGs.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations (preprint)

Recombinant neutralizing antibodies (nAbs) derived from recovered patients have proven to be effective therapeutics for COVID-19. Here, we describe the use of advanced protein engineering and modular design principles to develop tetravalent synthetic nAbs that mimic the multi-valency exhibited by IgA molecules, which are especially effective natural inhibitors of viral disease. At the same time, these nAbs display high affinity and modularity typical of IgG molecules, which are the preferred format for drugs. We show that highly specific tetravalent nAbs can be produced at large scale and possess stability and specificity comparable to approved antibody drugs. Moreover, structural studies reveal that the best nAb targets the host receptor binding site of the virus spike protein, and thus, its tetravalent version can block virus infection with a potency that exceeds that of the bivalent IgG by an order of magnitude. Design principles defined here can be readily applied to any antibody drug, including IgGs that are showing efficacy in clinical trials. Thus, our results present a general framework to develop potent antiviral therapies against COVID-19, and the strategy can be readily deployed in response to future pathogenic threats.

Synthetic Antibodies neutralize SARS-CoV-2 infection of mammalian cells (preprint)

Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.

Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2. (preprint)

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly disrupt epidemic transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, yet there is no consensus as to which assay should be used for such measurements. Using an infectious molecular clone of vesicular stomatitis virus (VSV) that expresses eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput imaging-based neutralization assay at biosafety level 2. We also developed a focus reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. We compared the neutralizing activities of monoclonal and polyclonal antibody preparations, as well as ACE2-Fc soluble decoy protein in both assays and find an exceptionally high degree of concordance. The two assays will help define correlates of protection for antibody-based countermeasures including therapeutic antibodies, immune {gamma}-globulin or plasma preparations, and vaccines against SARS-CoV-2. Replication-competent VSV-eGFP-SARS-CoV-2 provides a rapid assay for testing inhibitors of SARS-CoV-2 mediated entry that can be performed in 7.5 hours under reduced biosafety containment.