Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617

Abstract

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identify two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generate a bispecific antibody. Lead antibodies show strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solve several cryo-EM structures at ~3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and reveal distinct epitopes, binding patterns, and conformations. The lead clones also show potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generate and characterize a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

Function and Cryo-EM structures of broadly potent bispecific antibodies against multiple SARS-CoV-2 Omicron sublineages

“…in the down conformation (Fig.6B, left panel). The density for the Fab-bound up-RBD fitted well with the overlaid model of spike RBD with the MB.02 and Clone 13A Fab fragments29 , while the density of the Fab-bound down RBDs matched with the model of the MB.02 Fab-bound RBD (Fig.6B, right panel). This means that we detected three MB.02 and one Clone 13A Fabs bound to the same S trimer, potentially from three 0213 bsAbs with one of them having both Fab arms bound.…”

“…Bispecific antibodies were cloned and expressed as previously described 29 . In brief, indicated mAb variable regions for each bispecific antibody were amplified and subcloned into separate mammalian expression vectors using Gibson assembly 29 . To express recombinant bispecific antibodies, four expression vectors were transiently transfected into Expi293 cells using ExpiFectamine 293 transfection kit according to the manufacturer’s protocol (ThermoFisher).…”

“…The ACE2 (Cat. No.10108-H08H) used in ACE2 competition assay was purchased from Sino Biological 29 .…”

“…To develop potential broad spectrum SARS-COV-2-specific functional bispecific antibodies, we engineered five bispecific antibodies in this study by using an IgG1 knob-into-hole bispecific CrossMab antibody technique ( Fig. 2A ), utilizing the Fab regions from four of our previously developed fully human or largely humanized monoclonal antibodies, Clones MB.02, MB.08, PC.03 18 and Clone 13A 29 . The resultant bsAb clones were named as CoV2-0208, CoV2-0203, CoV2-0803, CoV2-0213 and CoV2-0813, respectively, based on the compositions of their respective parental monospecific mAb clones.…”

“…To better understand the mechanism of action of the bsAb CoV-0213, we performed cryo-electron microscopy (cryo-EM) studies to solve the three-dimensional (3D) structures of the bsAb and its major Fab arm (MB.02) in complexes with the Omicron spike variants (we previously reported the cryo-EM structure of the other Fab arm, 13A in complex with WT spike 29 ). We first sought to map the molecular basis for the broad specificity of MB.02 and its interaction with the RBD epitope, and determined the cryo-EM structures of MB.02 Fab in complex with the ectodomain of Omicron BA.1 spike trimer (S trimer) at ~3.2 Å resolution ( Table 1 ).…”