Antibody Development - Structural Characterization of Antibodies
Structural characterization of antibodies and their interactions with antigens has many applications in antibody development and biologics discovery, including identification of paratopes/epitopes, correlation between structures and activity, identification of cross-specificity, and monoclonal antibody (MAB) optimization. Structural data can also be used in IP protection, and in the formulation and delivery space.
The advantage of using TEM
Owing to their intrinsic flexibility, however, antibodies can be difficult to study structurally by X-ray crystallography and are outside of the acceptable mass range for NMR studies. Transmission electron microscopy imaging of either negative stained or vitrified samples is much more tolerant of flexibility and/or disordered protein termini, making the approach well suited for structural characterization of antibodies, nanobodies and their complexes. Even low-resolution negative stain images can provide a wealth of information not otherwise achievable, particularly when combined with 2D classification.
High Resolution Epitope Mapping by CryoEM
Advancements in single particle analysis workflows have enabled rapid, high resolution epitope mapping by cryoEM. The higher working sample concentrations relative to negative stain workflows and similar tolerance for flexibility make cryoEM ideally suited to return resolutions at the Ab-Ag interface in which clear side change density can be observed. Full length mAbs and multiplex antibody binding can also be readily accommodated when generation the Fab fragments would be challenging or time-consuming.
Epitope mapping by TEM
Negative stain, especially if coupled with 2D class averaging, is a quick way to assess (at low resolution) the epitope/paratope relationship between novel high affinity MABs identified during an antibody campaign and the target antigen. The technique also requires very little sample and is broadly applicable to many sample types.
Antibodies in Vaccine Development
In the case of highly antigenically variable pathogens, isolating pathogen functional antibodies, studying their interaction with their targets and then designing vaccine candidates could be a useful alternative to the traditional vaccines (killed or attenuated pathogens or protein subunits). Negative stain TEM can be used to quickly characterize polyclonal antibodies, identify the different epitopes and correlate structures and activity. High resolution structures of neutralizing antibody fragments (FABs) bound to targets may provide useful information with regard to cross-specificity and potency.
- Pan, J., Peng, H., Chen, B., & Harrison, S. C. (2020). Cryo-EM Structure of Full-length HIV-1 Env Bound with the Fab of Antibody PG16. Journal of Molecular Biology, 432(4), 1158–1168. https://doi.org/10.1016/j.jmb.2019.11.028
- Campbell, M. G., Cormier, A., Ito, S., Seed, R. I., Bondesson, A. J., Lou, J., Marks, J. D., Baron, J. L., Cheng, Y., & Nishimura, S. L. (2020). Cryo-EM Reveals Integrin-Mediated TGF-β Activation without Release from Latent TGF-β. Cell, 180(3), 490-501.e16. https://doi.org/10.1016/j.cell.2019.12.030
- Barnes, C. O. (2020). Structures of Natively-Glycosylated HIV-1 Envelope Trimers Define Antibody-Mediated Neutralization of HIV-1. Biophysical Journal, 118(3), 3a. https://doi.org/10.1016/j.bpj.2019.11.211
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