Nanobodies and antibody-like proteins as tools in diagnostics and controllable protein-protein interactions

Nanobodies are single-domain antibody fragments derived from heavy-chain-only antibodies. Due to their small size, easier expression in bacterial cells and high stability, compared to regular IgG antibodies, they are attractive for many applications in diagnostics, therapy and basic research.

We and others[1] have designed the first nanobodies that are blocked in binding their antigen until they are activated by irradiation with light. The concept is that a critical tyrosine side chain in the paratope region of the nanobody is substituted by the unnatural amino acid (ncAA) ortho-nitrobenzyl tyrosine (ONBY; Figure 1). Removal of the photolabile protection group reconstitutes the tyrosine side chain and hence the native nanobody. Incorporation of the ncAA is performed by expressing of the protein in E. coli using genetic code expansion.[2] We have shown for several nanobodies that this design concept allows generation of so-called photobodies with changes in dissociation constants of >1,000 to 10,000-fold to control protein interactions on demand within seconds.[3-4] Furthermore, we have transduced these photo-caged nanobodies inside mammalian cells to manipulate endogenous cellular processes with the high spatiotemporal resolution of light.[5]

Fig. 1. Concept of photo-caged nanobodies (photobodies) that are blocked in their antigen binding until a photo-labile group is removed by light.
© Henning Mootz

References

[1]    T. Bridge, S. A. Shaikh, P. Thomas, J. Botta, P. J. McCormick, A. Sachdeva, Angew Chem Int Ed Engl, 2019, 58, 17986-17993. DOI
[2]    A. Deiters, D. Groff, Y. Ryu, J. Xie, P. G. Schultz, Angew Chem Int Ed Engl, 2006, 45, 2728-2731. DOI
[3]    B. Jedlitzke, Z. Yilmaz, W. Dörner, H. D. Mootz, Angew Chem Int Ed Engl, 2020, 59, 1506-1510. DOI
[4]    B. Jedlitzke, H. D. Mootz, Chembiochem, 2022, 23, e202200079. DOI
[5]    B. Jedlitzke, H. D. Mootz, ChemPhotoChem, 2021, 5, 22-25. DOI

In another application we have designed poly-tags of short linear peptide epitopes that can bind multiple anti-tag nanobodies.[6] In collaboration with Prof. Michael Schäfer’s group (University Hospital Münster) these are used for sensitive cell detection and in vivo PET imaging. To this end, the nanobody is equipped with a chelator for a radioisotope like 68Ga. The tumor or immune cells to be imaged are transfected to express the poly-tags before being transplanted or injected into the mouse. Increasing the number of binding nanobodies amplifies the detectable signal

Fig. 2. Concept of poly-tags in combination with radioisotope-labeled nanobodies to amplify the signal in in vivo PET imaging.
© Henning Mootz

References

[6]    K. S. Höffgen, J. Dabel, C. P. Konken, D. A. Depke, S. Hermann, W. Dörner, S. Schelhaas, M. Schäfers, H. D. Mootz, Nucl Med Biol, 2024, 136-137, 108937. DOI

Last modified 05/2026