Design of single-domain VHH antibodies to increase the binding activity in SPR amine coupling

Single-domain antibodies, or VHH, nanobodies, are attractive tools in biotechnology and pharmaceuticals due to their favorable biophysical properties. Single-domain antibodies have potential for use in sensing materials to detect antigens, and in this paper, we propose a generic design strategy of single-domain antibodies for the highly efficient use of immobilized antibodies on a sensing substrate. Amine coupling was used to immobilize the single-domain antibodies on the substrate through a robust co-valent bond. First, for two model single-domain antibodies with lysines at four highly conserved posi-tions (K48, K72, K84, and K95), we mutated the lysines to alanine and measured the binding activity of the mutants (the percentage of immobilized antibodies that can bind antigen) using surface plasmon resonance. The two model single-domain antibodies tended to have higher binding activities when K72, which is close to the antigen binding site, was mutated. Adding a Lys-tag to the C-terminus of single-domain antibodies also increased the binding activity. We also mutated the lysine for another model single-domain antibodies with the lysine in a different position than the four residues mentioned above and measured the binding activity. Thus, single-domain antibodies immobilized in an orientation accessible to the antigen tended to have a high binding activity, provided that the physical properties of the single-domain antibodies themselves (affinity and structural stability) were not significantly reduced. Specifically, the design strategy of single-domain antibodies with high binding activity included mutating the lysine at or near the antigen binding site, adding a Lys-tag to the C-terminus, and mutating a residue away from the antigen binding site to lysine. It is noteworthy that mutating K72 close to the antigen binding site was more effective in increasing the binding activity than Lys-tag addition, and immobilization at the N-terminus close to the antigen binding site did not have such a negative effect on the binding activity compared to immobilization at the K72.(c) 2023 Elsevier Inc. All rights reserved.

Automated summary

Single-domain antibodies, or VHH, nanobodies, are promising tools in biotechnology and pharmaceuticals due to their favorable biophysical properties. In this paper, a generic design strategy of single-domain antibodies for efficient immobilization on a sensing substrate is proposed. The strategy includes mutating the lysine near the antigen binding site, adding a Lys-tag to the C-terminus, and mutating a residue away from the antigen binding site to lysine.