Abstract
Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are useful for patients' treatment of the coronavirus disease 2019 (COVID-19). We report here affinity maturation of monobodies against the SARS-CoV-2 spike protein and their neutralizing activity against SARS-CoV-2 B.1.1 (Pango v.3.1.14) as well as four variants of concern. We selected matured monobodies from libraries with multi-site saturation mutagenesis on the recognition loops through in vitro selection. One clone, the C4-AM2 monobody, showed extremely high affinity (K D < 0.01 nM) against the receptor-binding domain of the SARS-CoV-2 B.1.1, even in monomer form. Furthermore, the C4-AM2 monobody efficiently neutralized the SARS-CoV-2 B.1.1 (IC 50 = 46 pM, 0.62 ng/ml), and the Alpha (IC 50 = 77 pM, 1.0 ng/ml), Beta (IC 50 = 0.54 nM, 7.2 ng/ml), Gamma (IC 50 = 0.55 nM, 7.4 ng/ml), and Delta (IC 50 = 0.59 nM, 8.0 ng/ml) variants. The obtained monobodies would be useful as neutralizing proteins against current and potentially hazardous future SARS-CoV-2 variants.
Highlights
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a trimer that binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface to initiate infection of the virus (Ke et al, 2020; Wrapp et al, 2020; Yan et al, 2020)
Other nanobodies obtained by immunization of llamas showed neutralizing activity against the pseudovirus (IC50 = 0.3 nM in monomer form, IC50 = 0.9 pM in trimer form), they were produced as an Fc fusion protein by Expi293 cells (Xu et al, 2021)
By using the transcription-translation coupled with puromycin-linker (TRAP) display (Ishizawa et al, 2013; Kondo et al, 2020, 2021a), we developed three monobodies (C4, C6b, and C12b), which simultaneously bind to a distinct epitope in the receptor-binding domain (RBD) with nM or sub-nM affinity (Kondo et al, 2020)
Summary
The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a trimer that binds the angiotensin-converting enzyme 2 (ACE2) on the host cell surface to initiate infection of the virus (Ke et al, 2020; Wrapp et al, 2020; Yan et al, 2020). Numerous research studies and clinical tests prove that neutralizing antibodies effectively treat COVID-19 patients, the use of antibodies for common diseases’ treatment is challenging because it requires low-cost and large-scale antibody production (Buyel et al, 2017; Corti et al, 2021). To solve this problem, single-domain antibodies (Hamers-Casterman et al, 1993), called nanobodies that bound to the RBD have been developed (Esparza et al, 2020; Huo et al, 2020; Schoof et al, 2020; Xiang et al, 2020; Güttler et al, 2021; Koenig et al, 2021; Li et al, 2021; Sun et al, 2021; Xu et al, 2021; Ye et al, 2021; Zebardast et al, 2021; Chen et al, 2021b).
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