Monoclonal Human Antibodies That Recognise the Exposed N and C Terminal Regions of the Often-Overlooked SARS-CoV-2 ORF3a Transmembrane Protein

Abstract

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the “pairing potential” of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the “inside” or “outside” of the infected cell. Their therapeutic potential will be discussed.

SARS-CoV-2 ORF3a-Mediated NF-κB Activation Is Not Dependent on TRAF-Binding Sequence

“…This topology is supported by the positive-inside rule, which describes the tendency of loops between transmembrane helices to face the cytosol when they contain positively charged residues (Arg and Lys) because of the negative potential of the cytosol relative to the extracellular space [43]. However, ORF3a localizes not only to the plasma membrane but also to intracellular membranes (lysosomes, endosomes, Golgi, endoplasmic reticulum) [9,11,13,[44][45][46][47], and while there is experimental evidence to support the proposed ORF3a topology at the plasma membrane [48], it is unclear whether its orientation in intracellular membranes is the same. Additionally, ORF3a has been reported to disrupt and reorganize membrane structures in infected cells or when overexpressed [11,15,18,19].…”

Methods for Engineering Binders to Multi-Pass Membrane Proteins

“…The best binder, G7 scFv, specifically bound to CD20 expressed on Raji cells with an estimated affinity of ~64 nM. Similarly, Glumac and co-workers panned a naïve human scFv phage library against the extracellular N terminal (amino acids 1-34) and C-terminal (amino acid 126-275) domains of SARS-CoV-2 ORF3a transmembrane protein, obtaining N3aB02 and 3aCA03 single-chain antibodies that were able to bind the full-length ORF3a in transfected cells [34]. Despite these successes, it is worth noting that many multi-pass transmembrane proteins have small extracellular loops that are dependent on the transmembrane region for proper folding, and as such are not suitable targets for affinity selection using this method.…”

“…transmembrane protein, obtaining N3aB02 and 3aCA03 single-chain antibodies that were able to bind the full-length ORF3a in transfected cells [34]. Despite these successes, it is worth noting that many multi-pass transmembrane proteins have small extracellular loops that are dependent on the transmembrane region for proper folding, and as such are not suitable targets for affinity selection using this method.…”