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VHH antibodies, also known as nanobodies, are the engineered version of antigen-binding domains from heavy-chain-only antibodies found in camelid serum [1]. As the smallest antibody fragments preserving the binding capacity of a whole antibody, they are largely employed for diagnostic and therapeutic applications [2,3]. The small size (~15 kDa) and stability of nanobodies allow their recombinant production in bacterial systems. Lately, a computational-based protocol was developed for the design of VHH sequences against specific epitopes [4].
Here the same algorithm was used to produce nanobodies active against a model protein, the Hen Egg White Lysozyme (HEWL), by starting from a nanobody sequence against an unrelated target. With the aim of improving the in silico design algorithm, complexes between the artificial nanobodies and their target HEWL were studied through structural and biophysical techniques. The VHH protein sequences selected by the algorithm were expressed in Escherichia coli using different protein tags. Their stability was evaluated using a Thermal Shift Assay protocol and results were used to select a suitable buffer for purification and crystallization experiments. An enzymatic inhibition assay was carried out using E. coli cells as substrate of the lysozyme. Despite some predicted overlap between the artificial VHH binding site and the active site of HEWL, no significant inhibition was reported. Nervertheless, for two artificial VHH KD in the nanomolar range were experimentally determined through microscale thermophoresis. The structural characterization of the VHH-HEWL complexes is underway.
[1] Muyldermans S. Nanobodies: Natural Single-Domain Antibodies. Annu Rev Biochem. 2013;82(1):775-797.
[2] Morrison C. Nanobody approval gives domain antibodies a boost. Nature. 2019;18(7):485-487.
[3] Debie P, Van Quathem J, Hansen I, et al. Effect of Dye and Conjugation Chemistry on the Biodistribution Profile of Near-Infrared-Labeled Nanobodies as Tracers for Image-Guided Surgery. Mol Pharmaceutics. 2017;14(4):1145-1153.
[4] Soler MA, Medagli B, Semrau MS, et al. A consensus protocol for the in silico optimisation of antibody fragments. Chem Commun. 2019;55(93):14043-14046.
