The solution structure of the heavy chain-only C5-Fc nanobody reveals exposed variable regions that are optimal for COVID-19 antigen interactions.

Heavy-chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities compared to conventional antibodies. To address the challenge of SARS-CoV-2, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody, and shows therapeutic efficacy in vivo. Here, we have characterised the solution arrangement of the molecule. Two 1,443 Da N-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys-Cys disulfide bridges within the hinge. The X-ray and neutron Guinier radius of gyration RG values, which provide information about structural elongation, were similar at 4.1-4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modelling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.