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

Abstract

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 naïve 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.

Synergistic drug combinations designed to fully suppress SARS-CoV-2 in the lung of COVID-19 patients

“…1 hour later cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 and cultured for 72 hours. Four strains were used: 1) the human 2019-nCoV strain 2019-nCoV/Italy-INMI1, isolated in Italy (ex-China) from a sample collected on January 29, 2020, kindly provided by the Istituto Lazzaro Spallanzani, Rome, Italy [19], 2) GZ69 (genomic data are available at EBI under study accession n. PRJEB38101), 3) Delta variant B.1.167.2 and 4) Omicron BA.1 variant, the last three viral strains kindly provided by the Department of Molecular and Translational Medicine, Section of Microbiology and Virology, University of Brescia Medical School, Brescia, Italy [20][21][22][23]. The 2019-nCoV/Italy-INMI1 strain was used for initial experiments and for drug combination studies, GZ69 was used as confirmatory strain as it was the main circulating variant in Italy at the time experiments were performed.…”