Structure of Nanobody Nb23.

Mathias Percipalle,Federico Fogolari,Gennaro Esposito,Yamanappa Hunashal,Jan Steyaert

doi:10.3390/molecules26123567

Abstract

Background: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of β2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. Methods: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. Results: The solution structure of isolated Nb23 nanobody was determined. Conclusions: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.

Highlights

Single-domain antibodies, or nanobodies, are derived from heavy-chain only antibodies (HcAbs) found in camelids [1]
Molecules 2021, 26, 3567 leading to signal broadening seems confirmed by the fact that neighboring residues in CDR1 and CDR3 (Gly26 and Gly102) exhibit below-average intensities and by the 15N{1H: 4.7 t3 (1H)} NOE data, where residues in conformationally rigid regions show a close-to-average ratio of peak intensity with and without hydrogen saturation (Figure 2)
It is plausible that an unfavorable conformational exchange rate in the CDR regions could affect the detectability of some signal in 15N-1H HSQC and TROSY spectra

Summary

Introduction

Single-domain antibodies, or nanobodies, are derived from heavy-chain only antibodies (HcAbs) found in camelids [1]. The lack of a light chain in HcAbs allows nanobodies to exist as a single domain with less susceptibility to aggregation through hydrophobic interactions, as is the case for scFvs [3,4,5]. Due to their small size and high similarity to the human immunoglobulin variable domain, they provoke little to no immune response [5] which often makes humanization unnecessary. Conclusions: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability

Methods

Results

Conclusion