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

Abstract

The spread of COVID-19 has been exacerbated by the emergence of variants of concern (VoC). Many VoC contain mutations in the spike protein (S-protein) and are implicated in infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but they are limited by low potency and vulnerability to RBD mutations in VoC. 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 we showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent K D < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrated 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, including the dominant B.1.1.529 (Omicron) variant that is resistant to most clinically approved therapeutic nAbs. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug and showed that it enabled the neutralization of 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.

Receptor-binding domain-anchored peptides block binding of severe acute respiratory syndrome coronavirus 2 spike proteins with cell surface angiotensin-converting enzyme 2

“…Frontiers in Microbiology 06 frontiersin.org pathogen invasions in this lab (Zhao et al, 2012) or others (Hall et al, 2009;Wei et al, 2020). This study and several other efforts (Anand et al, 2021;Sokullu et al, 2021;Ballmann et al, 2022;Labriola et al, 2022;Petrenko et al, 2022;Sevenich et al, 2022;Yang et al, 2022) demonstrated that PhD was a plausible method for generating possible therapeutics to treat COVID-19 as well. For example, Petrenko used phage-displayed spike S1 protein mimotopes to search for "all" cellular receptors, including authentic and alternative ones.…”

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 – 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.…”

Cell-based passive immunization for protection against SARS-CoV-2 infection

“…While combining separate RBD-directed antibodies into a single molecule using similar mAb design principles [ 73 ] represents a promising approach to withstand viral resistance, some of the broadest RBD-directed antibodies described to date that even recognize divergent sarbecoviruses [ 24 , 25 ] are evaded by recent Omicron sublineages [ 11 , 13 , 14 , 74 ]. Thus, additional strategies to further expand valency [ 75 ], target subdominant RBD epitopes [ 76 ] or other ‘cold spot’ regions of spike entirely—such as the highly conserved S2 stem helix [ 77 , 78 ]—and more rigorously analyze the potential for escape may be needed to generate more resilient coverage across current and future viral variants. We envision that a detailed analysis of the epitopic landscape, coupled with rational antibody design strategies, will make it possible to engineer ‘escape-proof’ mAbs in such a way that their neutralization potency need not be sacrificed for their neutralization breadth.…”