Single-domain antibodies (sdAbs, also known as VHH) can be derived from heavy-chain-only antibodies from members of the camelidae family like llamas and alpacas. These sdAbs are relatively small in size, stable, and have affinities to antigens that are in a similar range as conventional antibodies. Being single-domain, globular recombinant proteins, sdAbs allow for straightforward genetic modifications, fusion of other proteins, and chemical conjugations. At QVQ, we offer off-the-shelf sdAbs against a wide range of targets, as well as services for de novo generation, production, and optimization of sdAbs. In addition, we routinely equip sdAbs with an unpaired thiol for directional conjugation to a wide range of commercially available labels.
Within the European research consortium ONCORNET2.0, Stephanie Anbuhl explored different sdAb formats and types of sdAb functionalization to study the human chemokine receptor CXCR4. Below, a few examples of functionalization of sdAbs targeting human CXCR4 are described.
Due to their convex paratope, sdAbs are well suited to target conformational and cryptic epitopes such as G protein-coupled receptors (GPCRs), which are generally considered challenging targets for biologicals.1 GPCRs are the largest group of cell surface receptors and are crucial for orchestrating cellular responses to external stimuli such as ligands. As such, they are involved in many (patho-)physiological processes. The GPCR CXCR4 is expressed at high levels in many cancers and plays a significant role in cancer progression and metastasis.2 Therefore, CXCR4 is interesting as potential drug target as well as for research on mechanisms of cancer.
A large variety of high affinity sdAbs against CXCR4 was generated by immunizing llamas followed by only a single round of biopanning.3 Some of these molecules acted as GPCR conformational sensors by having different affinities for particular small-molecule-occupied receptor conformations. Furthermore, Stephanie aimed to further improve the binding affinity of one of these monovalent sdAb by constructing a wide range of different multivalent sdAb formats.3 Among the various formats, which reached up to 10-valency, the classical bivalent sdAb-Fc proved to be most potent. Besides improving sdAb binding, Stephanie also added different functional domains to the CXCR4 sdAbs. For example, directional labeling of such sdAbs with fluorescent labels enabled real-time monitoring of ligand binding 4 and super-resolution imaging of CXCR4 (in preparation). Other sdAb formats, such as sdAb-luciferase fusion protein and conjugates for bioluminescence resonance energy transfer (BRET) and sdAb-oligo conjugates for proximity ligation assay (PLA) have been developed for detection of protein complexes. Finally, conjugation of CXCR4 targeting sdAbs to the tubulin stabilizing toxin Aurestatin, enabled the selective killing of CXCR4-overexpressing tumor cells.5
In conclusion, various sdAb formats for a variety of applications have been successfully generated using genetic fusion or site-directed conjugation. The potential and versatility of such reformatted sdAbs are illustrated by the variety of applications.
References
1: De Groof, T.W.M. et al (2019), Nanobodies: New avenues for imaging, stabilizing and modulating GPCRs, Molecular and cellular endocrinology, 484.
2: Scholten, D.J. et al (2012), Pharmacological modulation of chemokine receptor function, British journal of pharmacology, 165(6).
3: Anbuhl, S.M. et al (2024), Multivalent CXCR4-targeting nanobody formats differently affect affinity, receptor clustering, and antagonism, Biochemical Pharmacology, 227.
4: van den Bor, J., Bergkamp, N. D. et al (2023), NanoB2 to monitor interactions of ligands with membrane proteins by combining nanobodies and NanoBRET. Cell reports methods, 3(3).
5: Wakileh, G.A. et al (2023), Molecular characterization of the CXCR4 / CXCR7 axis in germ cell tumors and its targetability using nanobody-drug-conjugates, Experimental hematology & oncology, 12(1).