The COVID-19 pandemic caused by SARS-CoV-2 has resulted in millions of deaths worldwide. While vaccines are available, breakthrough infections, particularly with emerging variants, remain a concern. Therefore, effective therapeutic options are crucial. Monoclonal antibodies (mAbs) have shown promise in treating COVID-19, but the emergence of new variants necessitates the development of mAbs with broad neutralizing activity. Variants of concern (VoCs), such as Alpha, Beta, and Delta, exhibit increased transmissibility and reduced susceptibility to existing therapies and vaccines. The B.1.617 lineage, including Delta, is particularly challenging due to its increased transmissibility and resistance to authorized mAb therapies. Traditional hybridoma screening is a slow process, prompting the use of high-throughput single-cell technologies for rapid identification of neutralizing antibodies. These technologies directly sequence B cell receptor (BCR) repertoires, enabling the isolation of potent mAbs against various pathogens. The SARS-CoV-2 spike protein, which mediates cell entry, is the primary target for neutralizing antibodies. This protein is composed of S1 and S2 subunits, with the S1 subunit containing the receptor-binding domain (RBD) that interacts with the human ACE2 receptor. The RBD is flexible, transitioning between "up" and "down" conformations, only binding ACE2 in the "up" conformation. This study aims to rapidly identify potent SARS-CoV-2 neutralizing mAbs using high-throughput single-cell BCR sequencing from mice immunized with the SARS-CoV-2 RBD, characterize their binding and neutralizing activity, generate bispecific and humanized versions, and determine their efficacy in vivo.

The majority of preclinical and clinical SARS-CoV-2 mAbs were initially discovered using the blood of COVID-19 patients. However, the immunization of animals followed by hybridoma screening remains a standard method for discovering therapeutic mAbs against viruses. The advent of high-throughput single-cell technologies has revolutionized this process, enabling direct sequencing of fully recombined VDJ sequences of BCR repertoires from single cells. This approach has successfully yielded human neutralizing mAbs against various pathogens, including HIV, Ebola, and SARS-CoV-2. Previous research established that the SARS-CoV-2 spike glycoprotein is a primary target for neutralizing antibodies. The spike protein, in its pre-fusion form, is a trimer consisting of three copies of S1 and S2 subunits. The S1 subunit comprises the N-terminal domain (NTD) and the RBD, which recognizes the host ACE2 receptor. The S2 subunit is involved in membrane fusion. The RBD's flexibility allows it to switch between "up" and "down" conformations, and only the "up" conformation can bind ACE2. Studies have indicated that potent neutralizing antibodies predominantly target the spike RBD, often through interfaces overlapping with the ACE2-binding site.

This study employed a multi-faceted approach. First, C57BL/6J and BALB/c mice were immunized with the SARS-CoV-2 RBD protein. Progenitor B cells and plasma B cells were isolated from the spleen, lymph nodes, and bone marrow of immunized mice using anti-mouse CD138 beads. High-throughput single-cell VDJ sequencing was performed on these isolated B cells to identify enriched BCRs encoding strong mAbs. The VDJ sequences from top-ranked clones were cloned into human IgG1 heavy and light chain backbone vectors for antibody reconstruction using the Expi293F mammalian expression system. After expression and purification, the antibodies' reactivity against the SARS-CoV-2 spike RBD was assessed using ELISA. Neutralizing ability was evaluated using HIV-1-based and VSV-based SARS-CoV-2 pseudovirus systems. Two highly potent mAbs (Clones 2 and 6) were selected, and a bispecific antibody (Clone 16) was generated using the "knobs into holes" (KiH) methodology. Binding affinity and avidity were characterized using biolayer interferometry (BLI) and surface plasmon resonance (SPR). Cryo-EM structures of the lead clones' Fab fragments in complex with the SARS-CoV-2 spike ectodomain trimer were determined to define epitopes and binding conformations. The neutralization ability of the monospecific and bispecific antibodies was assessed using a cell fusion assay and pseudovirus neutralization assays with various SARS-CoV-2 variants (Wuhan-1, B.1.351, and B.1.617). To enhance clinical translation, a humanized version of Clone 2 (Clone 13A) was generated using standard antibody humanization techniques. The in vivo efficacy of the lead antibodies was evaluated using a prophylactic and therapeutic model with K18-hACE2 transgenic mice challenged with authentic SARS-CoV-2. Homology models of the Beta and Delta variants were generated to assess the impact of RBD mutations on antibody binding. Additional in vitro and in vivo studies were performed with Clone 13A, and compared to other EUA antibodies.

Single-cell BCR sequencing identified two highly potent SARS-CoV-2 neutralizing mAb clones (Clones 2 and 6) with picomolar binding affinity to the RBD. A bispecific antibody (Clone 16) combining both clones showed potent neutralization. Cryo-EM structures revealed distinct epitopes and binding modes, with Clones 2 and 6 recognizing the RBD in various conformations. All three antibodies potently neutralized the Wuhan-1 strain and the B.1.617 lineage variants, although neutralization of the B.1.351 variant was somewhat reduced. In vivo studies in K18-hACE2 transgenic mice demonstrated potent prophylactic and therapeutic efficacy against authentic SARS-CoV-2 (Wuhan-1). A humanized version of Clone 2 (Clone 13A) maintained high binding affinity and potent neutralization activity against both Wuhan-1 and the Delta variant, both in vitro and in vivo. Structural analysis indicated that mutations in the Beta variant (K417N and E484K) could reduce binding affinity compared to the wild type, while the Delta variant mutations had a more moderate impact. Comparisons were made to existing EUA antibodies.

This study successfully leveraged high-throughput single-cell BCR sequencing to rapidly identify potent SARS-CoV-2 neutralizing mAbs. The identified antibodies exhibit high affinity, broad neutralizing activity against multiple variants, including the challenging B.1.617 lineage, and demonstrate potent in vivo efficacy. The cryo-EM structures provided insights into the distinct binding modes and epitopes, suggesting mechanisms of neutralization that may contribute to their resistance to variant escape. The development of a humanized version of Clone 2 enhances the potential for clinical translation. The differences in neutralization potency against different variants highlight the importance of understanding variant-specific mutations and their impact on antibody binding. The comparison of the activity of these antibodies with existing EUA antibodies underscores the need for ongoing development of new therapeutic options to address the challenge of emerging SARS-CoV-2 variants.

This study successfully developed and characterized potent, broadly neutralizing monoclonal antibodies against SARS-CoV-2, including the concerning Delta variant. The use of high-throughput single-cell sequencing technology proved efficient in identifying these antibodies. The generation of a bispecific and humanized antibody further enhances their clinical potential. Future research could focus on further optimizing these antibodies for clinical use, exploring combinations with other therapeutics, and investigating their efficacy against newly emerging SARS-CoV-2 variants.

The study primarily used mouse models, which may not fully recapitulate the human immune response. While the humanized antibody showed promise, further preclinical and clinical studies are needed to confirm its safety and efficacy in humans. The in-vivo studies were conducted using a relatively high antibody dose, and further research could investigate the efficacy of lower doses. The study focused on specific variants and may not fully represent the full spectrum of SARS-CoV-2 variability.