Project A03.2

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A3.2

Project Summary

NMR-based profiling of polyclonal antibody mixtures

Small molecules with molecular weights below 300 Da, like ethanol, tryptophan, imidazole, and glucose, interact weakly with many proteins. Such weak interactions can be detected under physiological conditions using Saturation Transfer Difference (STD) NMR experiments. [1] Despite being rather promiscuous, we propose leveraging the detection of weak protein-ligand interactions to classify the binding properties of receptor proteins (e.g. monoclonal antibodies). 

It is hypothesised that monoclonal antibodies recognising different epitopes of a particular viral antigen will interact differently with a mixture of weakly and promiscuously binding small molecules, leading to distinct STD intensity patterns. If the structures of the corresponding monoclonal antibody complexes with the antigen in question are known at atomic resolution, the STD intensity patterns (‚STD-NMR fingerprints‘) could be used as classifiers reflecting the recognition of a certain binding epitope of the viral antigen. Based on a suitable training set of data, it is conjectured that it is possible to delineate the composition of mAbs in polyclonal patient sera (comparable to established neutralisation assay-based approaches [2],[3]) by acquiring STD-NMR fingerprints of the antibody fraction of the sera.In this project, we build on existing anti-HEV ORF2 antibodies [4], which we will characterise for their binding epitopes and create antibody-specific STD fingerprints to delineate polyclonal mixtures by their binding epitopes. 

A3.2

A3.2

References

1. Mayer, M., and Meyer, B. (1999). Characterization of Ligand Binding by Saturation Transfer Difference NMR Spectroscopy. Angew Chem Int Ed Engl 38, 1784–1788. 10.1002/(SICI)1521-3773(19990614)38:12<1784::AID-ANIE1784>3.0.CO;2-Q.
2. Georgiev, I.S., Doria-Rose, N.A., Zhou, T., Kwon, Y.D., Staupe, R.P., Moquin, S., Chuang, G.Y., Louder, M.K., Schmidt, S.D., Altae-Tran, H.R., et al. (2013). Delineating antibody recognition in polyclonal sera from patterns of HIV-1 isolate neutralization. Science 340, 751–756. 10.1126/science.1233989.
3. Kinchen, V.J., Massaccesi, G., Flyak, A.I., Mankowski, M.C., Colbert, M.D., Osburn, W.O., Ray, S.C., Cox, A.L., Crowe, J.E., Jr., and Bailey, J.R. (2019). Plasma deconvolution identifies broadly neutralizing antibodies associated with hepatitis C virus clearance. J Clin Invest 129, 4786–4796. 10.1172/JCI130720.
4. Ssebyatika, G., Dinkelborg, K., Ströh, L.J., Hinte, F., Corneillie, L., Hueffner, L., Guzman, E.M., Nankya, P.L., Plückebaum, N., Fehlau, L., et al. (2025). Broadly neutralizing antibodies isolated from HEV convalescents confer protective effects in human liver-chimeric mice. Nat Commun 16, 1995. 10.1038/s41467-025-57182-1.