Broadening a SARS-CoV-1 neutralizing antibody for potent SARS-CoV-2 neutralization through directed evolution

Abstract

The emergence of SARS-CoV-2 underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing neutralizing antibody to a related but resistant virus. Using a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1 neutralizing antibody, to bind SARS-CoV-2 receptor binding domain with >1000-fold improved affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of an enhanced antibody elucidated the molecular mechanisms by which engineered CR3022 can accommodate sequence differences in the epitope between SARS-CoV-1 and SARS-CoV-2. The workflow described provides a blueprint for rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses

“…However, their relatively low neutralization potency may limit their clinical utility. Using an in vitro affinity maturation strategy, CR3022 has been re-engineered to neutralize SARS-CoV-2 more potently [ 36 ]. Similarly, Rappazzo et al engineered a SARS-CoV-2 and SARS-CoV mAb into a better version, ADG-2, with enhanced neutralization breadth and potency [ 37 ].…”

Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape

“…To better understand the molecular contacts of antibodies, we used cryoelectron microscopy (cryoEM) to solve the structures of two of the antibodies in complex with stabilized SARS-CoV-2 S trimers: 1) nAb CC6.30, which targets the RBS-B or Class 2 epitope site, binds RBD with an affinity of 1.7 nM (IgG format) and directly competes with ACE2, and 2) nAb CC6. 33, which targets the Class 3 epitope site that is distinct from the ACE2 binding site and binds RBD with an affinity of 257 nM (Fig. 1 and Fig.…”

“…Antibody library generation CC12.1, CC6.30 and CC6.33 heavy chain and light chain affinity maturation libraries were synthesized as Oligopools (Integrated DNA Technologies). Mutations were included in the CDR loops and the CDR1/2/3 mini-libraries were assembled into combinatorial heavy chain and light chain libraries as previously described 33 . The libraries were displayed on the surface of yeast as molecular Fab using the yeast display vector pYDSI containing the bidirectional Gal1-10 promoter.…”

“…via Gibson assembly and amplified. The resulting LC-Gal1-10-HC product was cloned into an empty pYDSI vector by homologous recombination 33 . Yeast library labeling and sorting After yeast transformation, yeast cells were passaged 1:20 the following day.…”

Cross-neutralization of SARS-CoV-2 by HIV-1 specific broadly neutralizing antibodies and polyclonal plasma

“…Understanding cross-reactive neutralization epitopes of antibodies generated in divergent viral infections can provide key evidence for engineering so called super-antibodies (antibodies that can potently neutralize diverse pathogens with similar antigenic features). Recently, using a rapid affinity maturation strategy, Zhao et.al., engineered the CR3022, a neutralizing antibody against SARS-CoV-1, to potently neutralize SARS-CoV-2 and highlighted the potential of engineering approaches that can be leveraged to refocus an existing neutralizing antibody to target a related but resistant virus [54]. Furthermore, generating libraries using the starting sequence of such cross-reactive antibodies using approaches such as rapid affinity maturation, can provide candidate antibodies in a short frame of time upon which synthetic variants can be generated in the face of future pandemics.…”