Structure-guided affinity maturation of a novel human antibody targeting the SARS-CoV-2 nucleocapsid protein

Abstract

The continuous mutation of SARS-CoV-2 has presented enormous challenges to global pandemic prevention and control. Recent studies have shown evidence that the genome sequence of SARS-CoV-2 nucleocapsid proteins is relatively conserved, and their biological functions are being confirmed. There is increasing evidence that the N protein will not only provide a specific diagnostic marker but also become an effective treatment target. In this study, 2G4, which specifically recognizes the N protein, was identified by screening a human phage display library. Based on the computer-guided homology modelling and molecular docking method used, the 3-D structures for the 2G4 scFv fragment (VH-linker-VL structure, with (G₄S)₃ as the linker peptide in the model), SARS-CoV-2 N protein and its complex were modelled and optimized with a suitable force field. The binding mode and key residues of the 2G4 and N protein interaction were predicted, and three mutant antibodies (named 2G4-M1, 2G4-M2 and 2G4-M3) with higher affinity were designed theoretically. Using directed point mutant technology, the three mutant antibodies were prepared, and their affinity was tested. Their affinity constants of approximately 0.19 nM (2G4-M1), 0.019 nM (2G4-M2) and 0.075 nM (2G4-M3) were at least one order of magnitude lower than that of the parent antibody (3 nM; 2G4, parent antibody), as determined using a biolayer interferometry (BLI) assay. It is expected that high-affinity candidates will be used for diagnosis and even as potential therapeutic drugs for the SARS-CoV-2 pandemic.

Figure 1

Screening of anti-SARS-CoV-2 nucleocapsid protein clones. (a) Comparison of the recovery ratios in different rounds of phage biopanning. (b) Phage ELISA analysis of 48 clones picked from the 2nd round of panning. (c) Frequency analysis of the amino acid sequences of phage panning positive clones. (d) Determination of the binding activity of the scFv fragments to N protein by ELISA.

Figure 2

The 3-D modelling structures of the 2G4 scFv fragment (a), N protein N-terminus (b), and 2G4 scFv-N protein complex (c); the ribbon denotes the main chain carbon atom orientation of the proteins. (a) The green ribbon denotes the orientation of the 2G4 scFv fragment, the red ribbon denotes HCDR1, the orange denotes HCDR2, the yellow denotes HCDR3, the light blue denotes LCDR1, the white denotes LCDR2, the pink denotes LCDR3, and the deep blue denotes the linker. (b) The pink ribbon denotes the N-terminus of the N protein. (b) The green ribbon denotes the 2G4 scFv fragment, and the blue ribbon denotes the N-terminus of the N protein. (d) The RMSD calculation during the 50 ns MD simulation of the 2G4 scFv fragment and N protein (the X-axis is optimization time and Y-axis is RMSD (angstroms)).

Figure 3

Analysis of important amino acid residues of 2G4 that interact with N protein. (a) The whole binding mode between the 2G4 scFv and N protein. The green ribbon denotes the 2G4 scFv fragment, and the blue ribbon denotes the N-terminus of the N protein. The yellow ball and stick denote the heavy atoms of the key residues in the 2G4 scFv, and red denotes the important residues in N protein. (b) The local binding mode between 2G4 and N protein. The red ball and stick denote the key amino acids in N protein, and the key amino acid residues are marked as name, position and heavy atom orientation.

Figure 4

Conformation comparison of the 2G4 scFv fragment and the three mutants. The orientation of the parent (2G4 scFv) and mutants after structural superposition and translation. (a) The 3-D theoretical ribbon structures of 2G4-M1 and 2G4. (b) The 3-D theoretical ribbon structures of 2G4-M2 and 2G4. (c) The 3-D theoretical ribbon structures of 2G4-M3 and 2G4.

Figure 5

Characterization and comparison of anti-SARS-CoV-2 nucleocapsid protein mutant antibodies. (a) The antibody binding activity to the SARS-CoV-2 N protein was detected by an ELISA. (b–f) The binding kinetics of antibodies to the SARS-CoV-2 N protein were detected by BLI. Experiments were independently repeated at least three times, and one representative experiment is shown.

Figure 6

Confirmation of N protein epitope drift by competitive ELISA. A representative experiment out of two independent experiments is shown.