Nanobodies (Nbs), known as variable antigen-binding (VHH) domain or single-domain antibodies, are small antigen-binding fragments. Nbs typically consist of a single polypeptide chain which contains the antigen-binding site and the effector functions. They are derived from the heavy-chain antibodies found in camelids (such as camels and llamas) and their small size, about one-tenth the size of conventional antibodies, makes them highly versatile and easy to manipulate for various applications1,2.
Nbs are useful tools for dynamic biological systems; they have been used to crystallize flexible membrane proteins, transient multiprotein assemblies, and individual molecules of complex proteins. They have also been used as biosensors to monitor conformational changes of GPCRs in living cells. All the above lightly represent the potential applications of Nbs to facilitate the development of more selective drugs capable of modulating specific signaling pathways, improving therapeutic activity, and minimizing side effects1,2.
Some important characteristics of Nbs are:
Small size and solubility: Their small size and soluble nature make them ideal building blocks for generating multivalent or multispecific constructs.
Tailorable half-life: Nanobodies can extend their half-life through PEGylation or fusion to serum albumin. This allows for tailoring the half-life of nanobodies to increase their therapeutic window depending on the clinical indication.
Binding to cryptic epitopes and conformational selection: Nanobodies have a unique shape that allows them to bind to cavities or clefts on the surfaces of proteins that are inaccessible to conventional antibodies. They can recognize cryptic epitopes often composed of discontinuous amino acid segments and occur only within the fully native protein. Nbs can stabilize specific conformations of proteins, including unstable structural intermediates and substates of conformationally complex proteins.
Generation, selection and functional expression: Nanobodies can be obtained by immunizing a camelid and cloning the variable VHH gene repertoire. Combinatorial biology methods such as phage display, yeast display, and ribosome display can be used to select nanobodies with desired functions. Most Nbs can be functionally expressed as genetically encoded intrabodies within a eukaryotic cell. This allows for correlating structural or dynamic features observed in vitro with functional observations from living cells.
Potential for protein crystallization: Nanobodies can be used to stabilize the protomers of larger protein assemblies and increase the total amount of structured polypeptide. This provides a better starting point for the crystallization of intrinsically unfolded proteins.
Some Nbs that have been use for GPCR research are:
Nb80: This nanobody stabilized an active-state conformation of the β2 adrenergic receptor (β2AR)3.
Nb6B9: This is an affinity-matured nanobody derived from Nb80. It has slower dissociation kinetics at the β2AR and was used to capture the structure of β2AR bound to adrenaline3.
Nb35: This nanobody selectively binds to the β2AR·Gs complex and prevents the dissociation of the nucleotide-free complex by the nonhydrolyzable GTP analog GTPγS4.
Nb37: Similar to Nb35, this nanobody selectively binds to the β2AR·Gs complex and helps reveal the conformational flexibility of the Gαs α-helical domain.
Nb9-8: This nanobody was identified for the M2 muscarinic receptor (M2R) and increased the affinity of agonists.
Nb39: This nanobody was identified for the μ-opioid receptor (μOR) and also increased the affinity of agonists4.
Nb7: This nanobody improves the diffraction quality of the constitutively active GPCR US28·CX3CL1 complex5.
This year Arum Wu et al. reported a library of Nbs to study the allosteric modulation of the rhodopsin receptor activation and investigate the role of specific regions in the switching between different conformational states. This Nbs binds to the extracellular surface of the Rhodopsin receptor and stabilizes the photo-activated receptor in a ground-state-like.
The findings of this study showed that the Nb2 binding to native rhodopsin stabilizes its conformation, protects the Schiff base, and prevents protein degradation, potentially offering therapeutic benefits for retinal diseases6. To know more about this report check this link.
References:
Jin, B. K., Odongo, S., Radwanska, M., & Magez, S. (2023). Nanobodies: A Review of Generation, Diagnostics and Therapeutics. International journal of molecular sciences, 24(6), 5994. https://doi.org/10.3390/ijms24065994
Manglik, A., Kobilka, B. K., & Steyaert, J. (2017). Nanobodies to Study G Protein-Coupled Receptor Structure and Function. Annual review of pharmacology and toxicology, 57, 19–37. https://doi.org/10.1146/annurev-pharmtox-010716-104710
Rasmussen, S. G., Choi, H. J., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., Devree, B. T., Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., Schnapp, A., Konetzki, I., Sunahara, R. K., Gellman, S. H., Pautsch, A., Steyaert, J., Weis, W. I., & Kobilka, B. K. (2011). Structure of a nanobody-stabilized active state of the β(2) adrenoceptor. Nature, 469(7329), 175–180. https://doi.org/10.1038/nature09648
Kruse, A. C., Ring, A. M., Manglik, A., Hu, J., Hu, K., Eitel, K., Hübner, H., Pardon, E., Valant, C., Sexton, P. M., Christopoulos, A., Felder, C. C., Gmeiner, P., Steyaert, J., Weis, W. I., Garcia, K. C., Wess, J., & Kobilka, B. K. (2013). Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature, 504(7478), 101–106. https://doi.org/10.1038/nature12735
Burg, J. S., Ingram, J. R., Venkatakrishnan, A. J., Jude, K. M., Dukkipati, A., Feinberg, E. N., Angelini, A., Waghray, D., Dror, R. O., Ploegh, H. L., & Garcia, K. C. (2015). Structural biology. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor. Science (New York, N.Y.), 347(6226), 1113–1117. https://doi.org/10.1126/science.aaa5026
Wu, A., Salom, D., Hong, J. D., Tworak, A., Watanabe, K., Pardon, E., Steyaert, J., Kandori, H., Katayama, K., Kiser, P. D., & Palczewski, K. (2023). Structural basis for the allosteric modulation of rhodopsin by nanobody binding to its extracellular domain. Nature communications, 14(1), 5209. https://doi.org/10.1038/s41467-023-40911-9