Neutralizing multivalent antibodies against coronaviruses

The COVID-19 pandemic, induced by the emergent SARS coronavirus (CoV)-2 (SARS-CoV-2), creates a global health emergency. The SARS-CoV-2 cell-entry process is mediated by the trimeric Spike (S) glycoprotein, which also represents a major viral antigen naturally targeted by the immune system. In this proposal, we propose to develop neutralizing multivalent single-chain antibodies (nmAb) to efficiently block viral cell-entry. The initial asymptomatic phase induced by the virus may offer a window of opportunity to establish post-exposure prophylaxis and prevention of human-to-human transmissions. Engineering of nmAbs will be performed in two main steps: (i) selection of neutralizing synthetic single-domain antibodies (sybodies) targeting various functional domains of S-trimers, and (ii) linking of sybodies together with or without grafting to a human immunoglobulin Fc-region. As sybodies are generated entirely in vitro, we can precisely steer the binder selection process towards targeting three, non-overlapping epitopes: i) Sybody 1 blocks ACE2-interaction via the RBD. We have already generated such sybodies [34]; ii) using the prefusion-stabilized and postfusion SARS-CoV-2 soluble S-protein, we will identify sybody 2 that stabilizes the prefusion-state and thereby prevent conformational changes required to complete the viral entry process; iii) using prefusion-stabilized soluble S-protein of SARS-CoV-2, SARS-CoV-1 and MERS, we will generate sybody 3 addressing highly conserved epitope among sarbecoviruses and even among ß-coronaviruses (Seeger lab). Membrane fusion- and cell entry-inhibition by the sybodies and derived antibody-like formats will be initially investigated in cell-based fusion assays as well as neutralization assays using pseudo-typed VSVs (Plattet lab), and later on against recombinant-live viruses. Finally, epitope mapping of best neutralizing sybody candidates will be tackled by cryo-electron microscopy (cryo-EM) and single particle 3D-reconstruction (Fotiadis lab). This will enable for the rationale selection of attractive sybody combinations for our nmAb engineering approach (Plattet lab). We envisage developing nmAbs towards two different formulations: (i) three fused sybodies (triSy), devoid of an added Fc-region, for nebulization (inhaled mainly as prophylaxis); and (ii) nmAbs grafted onto engineered human Fc-region for injection as either a preventive measure or treatment of further-progressed disease. In our judgement, nmAbs exhibit the following clinical advantages. Firstly, they may outcompete conventional antibody potency, because they bind to multiple epitopes of the Spike simultaneously. Secondly, multivalent binding of nmAbs may mitigate the rapid emergence of drug-resistant viral variants. Thirdly, treatment with nmAbs may reduce antibody-dependent enhanced (ADE) illness, since Fc-mediated effector functions can be rationally attenuated. In conclusion, we are confident that the design of next-generation neutralizing multivalent single-chain antibodies may represent an essential first line of defense against this and future emerging coronavirus epidemics.