Abstract
Among the therapies against the pandemic SARS-CoV-2 virus, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry. Since anti-spike mAbs, used individually, might be unable to block the virus entry in the case of resistant mutations, we designed an innovative strategy for the isolation of multiple novel human scFvs specific for the binding domain (RBD) of Spike. By panning a large phage display antibody library on immobilized RBD, we obtained specific binders by eluting with ACE2 in order to identify those scFvs recognizing the epitope of Spike interacting with its receptor. We converted the novel scFvs into full size IgG4, differently from the previously isolated IgG1 mAbs, to avoid unwanted potential side effects of IgG1 potent effector functions on immune system. The novel antibodies specifically bind to RBD in a nanomolar range and interfere in the interaction of Spike with ACE2 receptor, either used as purified protein or when expressed on cells in its native conformation. Furthermore, some of them have neutralizing activity for virus infection in cell cultures by using two different SARS-CoV-2 isolates including the highly contagious VOC 202012/01 variant and could become useful therapeutic tools to fight against the SARS-CoV-2 virus.
Highlights
The novel coronavirus SARS-CoV-2, responsible for the Severe Acute Respiratory Syndrome known as Covid[19], infects epithelial cells of the respiratory tract, causing typical signs such as fever, dry cough, fatigue and dyspnea[1,2]
The Spike protein is a large transmembrane glycoprotein comprising two subunits: S1 containing a receptor binding domain (RBD), which is responsible for the infectivity of the virus through the interaction with the cell surface Angiotensin-Converting Enzyme 2 (ACE2) receptor on human respiratory epithelial cells, and S2 responsible for the membrane fusion in the later step of infection[11,13]
We performed 4 panning rounds (3 for Next Generation Sequencing (NGS)) on human Spike RBD-Fc recombinant target protein followed by two parallel elution methods: classical acidic elution obtained by lowering the pH and a selective elution, by using the receptor ACE2-Fc chimeric protein which binds to Spike RBD with high affinity to elute those phages that recognize the same epitope
Summary
The novel coronavirus SARS-CoV-2, responsible for the Severe Acute Respiratory Syndrome known as Covid[19], infects epithelial cells of the respiratory tract, causing typical signs such as fever, dry cough, fatigue and dyspnea[1,2]. Three vaccines have been recently approved by FDA for preventative use: BNT162b2 (from Pfizer), AZD1222 (from Astra Zeneca), mRNA-1273 (from Moderna); and several other candidates are being tested by ongoing phase III clinical t rials[16,17,18,19]. Among these approaches, monoclonal Antibodies (mAbs) targeting the Spike glycoprotein represent good candidates to interfere in the Spike/ACE2 interaction, preventing virus cell entry[20]. The U.S Food and Drug Administration (FDA) issued an emergency use authorization (EUA) for the mAb Bamlanivimab (by Eli Lilly and Company’s) for the treatment of mild-to-moderate SARS-CoV-2 adult and pediatric p atients[21]
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