Antibody-like proteins that capture and neutralize SARS-CoV-2

Abstract

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

Fig. 1. Selection of monobodies against the SARS-CoV-2 spike protein using the TRAP display.

(A) Schematic representation of the TRAP display. Monobody/mRNA complexes were synthesized simply by adding the template DNA to the TRAP system. After reverse transcription (RT), selection, and PCR, the amplified DNAs were added to the TRAP system to reproduce a monobody library for the subsequent round of selection. (B) Structure of the monobody backbone (10th type III domain of fibronectin, 1TTG). The BC and FG loops (labeled in blue) were randomized with the indicated number of residues. (C) Progress of the TRAP display selection. After each round of selection, the recovered cDNA was quantified by real-time PCR. The recovery of cDNA was calculated by dividing the amount of recovered cDNA by the theoretical amount of mRNA/PuL (1 μM). At the fourth round, the selection pressure was increased by decreasing the target concentration from 20 to 2 nM. After the sixth-round selection, the recovered DNA (*) was sequenced. The sequences of the monobodies are shown in Table 1. Stv, streptavidin; Bio, biotin; NTD, N-terminal domain; Pu, puromycin.

Fig. 2. Binding activity of each clone against the SARS-CoV-2 spike protein S1 subunit and the RBD.

(A) Determination of the binding domain. A schematic representation of the ELISA is provided on the left. S1-HRP or RBD-HRP (10 nM) was added to the monobody-immobilized microplate. Error bars indicate SD of each experiment (in triplicate). (B) Determination of kinetic parameters by BLI. A schematic representation of the BLI is provided at the top. S1-biotin was immobilized on a streptavidin-sensor chip, and monobodies (2.5 to 40 nM) were used in the kinetic analysis. Some mutations were identified in clones 6, 11, and 12. These mutations were reversed in the clones 6b, 11b, and 12b. The data are depicted in blue, and the fit data to a 1:1 binding model are shown in black. The determined kinetic parameters of the monobodies are provided in Table 1. WT, 10th type III domain of fibronectin; RLU, relative luminescence units; Nus, Nus-Tag fused at the C terminus of the monobody.

Fig. 3. Characterization of monobodies and the application for the sandwich ELISA.

(A) A schematic representation of the pull-down assay is provided at the top. The SARS-CoV S1 subunit (100 nM), SARS-CoV-2 S1 subunit (100 nM), or SARS-CoV-2 S protein trimer (100 nM) were pulled down by monobody-immobilized beads. The supernatant and the heat elution from the beads were loaded onto SDS–polyacrylamide gel electrophoresis (PAGE), followed by staining with SYPRO Ruby. (B) A schematic representation of the ELISA is provided on the left. ACE2-HRP (1 nM) was mixed with each monobody (100 nM) and was added to an S1-immobilized microplate. (C) ACE2-HRP (1 nM) was mixed with each monobody (10 nM clones 4, 6, 9, or 10) and was added to an S1-immobilized microplate (before). Alternatively, a monobody was added to an ACE2-HRP–incubated S1-immobilized microplate (after). (D) A schematic representation of the sandwich ELISA is provided on the left. The S1 subunit (10 nM) was added to the monobody-immobilized microplate. The S1 subunit bound with the capturing monobody was identified by detecting monobody-HRP (10 nM). All possible combinations were tested. (E) Titration curves of the SARS-CoV-2 S1 subunit, SARS-CoV-2 spike protein trimer, and SARS-CoV S1 subunit. After incubation of the analyte with the detecting monobody-HRP (clone 12, 1 nM), the solution was added to the capturing monobody (clone 10)–immobilized microplate. Error bars indicate SD of each experiment (in triplicate). S1, SARS-CoV or SARS-CoV-2 spike protein S1 subunit or SARS-CoV-2 spike protein trimer; N.D., not determined; MK, protein maker (kDa).

Fig. 4. Monobody binding to SARS-CoV-2 from culture and patient samples.

(A) Monobody binding activities toward SARS-CoV-2 particles. SARS-CoV-2 particles bound with monobody-biotin were pulled down by M280-streptavidin beads. The RNA genomes in the supernatant and on the beads were quantified by RT-qPCR after RNA extraction. (B) The detection limits of the traditional RT-qPCR method and the pull-down RT-qPCR method. Various concentrations of SARS-CoV-2 (0.1 to 10,000 particles/μl) were assayed. SARS-CoV-2 particles were collected from 700 μl of solution by pull down using monobody clone 4, and viral RNA was quantified by RT-qPCR after RNA extraction. Alternatively, the original solution (14 μl) was subjected to RT-qPCR. (C) Pull down of SARS-CoV-2 particles from nasal swab samples (35 μl) of patients. The number of RNA genomes in the supernatant and on the beads was quantified by RT-qPCR after RNA extraction. (D) Pull-down direct RT-qPCR using nasal swab samples of patients. SARS-CoV-2 particles were collected from 400 μl of nasal swab samples by pull down and were directly added to an RT-qPCR reaction mixture. Alternatively, an original nasal swab solution (2.5 μl) was added to an RT–droplet digital PCR (ddPCR) reaction mixture. Error bars indicate SD of RT-qPCR results (in triplicate). N.D., not detected.

Fig. 5. The affinity of monobody tandem dimers and the neutralization of SARS-CoV-2 infection.

(A) Determination of kinetic parameters of tandem dimers by BLI. S1-biotin was immobilized on a streptavidin-sensor chip, and the tandem dimers (2.5 to 40 nM) were used in the kinetic analysis. (B) The biotinylated tandem dimers of monobody were immobilized on a streptavidin-sensor chip (*), and the S1 subunit (2.5 to 40 nM) was used in the kinetic analysis. TD4, TD6b, and TD12b are tandem dimers of the corresponding clones 4, 6b, and 12b connected with a (GGGGS)₃ linker. The data are depicted in blue, and the fit data to a 1:1 binding model are shown in black. The determined kinetic parameters of the monobodies are provided in Table 1. (C) Monobody-mediated neutralization of SARS-CoV-2 infection in VeroE6/TMPRSS2 cells. The x-axis value indicates the final concentration of the indicated monobody (6b, TD6b, or WT) for each assay well. The experiment was performed with triplicate samples. The copy number of each RNA in the supernatant is plotted.