A Fast and efficient strategy for the NMR assignment of Fab methyl groups

Henot, F.; Vibert, B.; Giraud, A.; Dbira, S.; Imbert, L.; Favier, A.; Guntert, P.; Clavier, S.; Crublet, E.; Doyen, C.; BOISBOUVIER, J.; Frances, O.

doi:10.1101/2025.10.21.682867

bioRxiv · 10.1101/2025.10.21.682867

A Fast and efficient strategy for the NMR assignment of Fab methyl groups

Henot, F.·Vibert, B.·Giraud, A.·Dbira, S.·Imbert, L.·Favier, A.·Guntert, P.·Clavier, S.·Crublet, E.·Doyen, C.·BOISBOUVIER, J.·Frances, O.

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Abstract

Owing to their high specificity and therapeutic effectiveness, monoclonal antibodies (mAbs) have rapidly become one of the leading classes of biologic drugs used to treat critical illnesses. The antigen-binding fragment (Fab) of mAbs plays a key role in the antigen recognition, so its structural characterization is essential, as even a slight change to its Higher Order Structure (HOS) can impact the antibodys potency. Recently, 2D methyl NMR has been introduced as a powerful method to assess both the structure and integrity of therapeutic Fab fragments. However, the identification of methyl group resonances in NMR spectra remains rare since Fabs are large heterodimers of [~]50 kDa. Here, we present the methyl group assignment of an IgG1 Fab produced in a cell-free system with an optimal isotope labelling. We first assigned 99% of the alanine, isoleucine, leucine, methionine, and valine methyl groups of the therapeutic Fab targeting LAMP1 antigen. Building on this assignment, we propose a "divide and conquer" strategy that exploits sequence identities to rapidly assign methyl groups of other IgG1 Fabs. We demonstrate that the assignment of the Fabs constant region can easily be transferred from one IgG1 to another and that the variable part of a new Fab can be assigned using smaller uniformly 15N,13C-labelled constructs. We applied our strategy to ipilimumabs Fab and, using the assignment of ipilimumabs variable part and Fab anti-LAMP1s constant part, we could transfer the assignment of 89% of the methyl-containing amino acids to the entire ipilimumab Fab without having to produce deuterated samples. This assignment strategy can be generalised to any other IgG1 Fabs provided that their constant regions are identical and the strategy can be adapted to accommodate the expression levels of the different variable domains. This new method drastically facilitates the Fab assignment process, making it suitable for the pharmaceutical timeline.

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Henot, F., Vibert, B., Giraud, A., Dbira, S., Imbert, L., Favier, A., Guntert, P., Clavier, S., Crublet, E., Doyen, C., BOISBOUVIER, J., Frances, O.. 2025-10-21. A Fast and efficient strategy for the NMR assignment of Fab methyl groups. https://doi.org/10.1101/2025.10.21.682867

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