Non-invasive in vivo imaging of tumour-associated cathepsin B by a highly selective inhibitory DARPin

Lovro Kramer^{1, 2}, Miha Renko¹, Janja Završnik^{1, 2}, Dušan Turk^{1, 3}, Markus A. Seeger⁴, Olga Vasiljeva¹, Markus G. Grütter⁵, Vito Turk¹, Boris Turk^{1, 3✉}

1. Jozef Stefan Institute, Department of Biochemistry and Molecular and Structural Biology, Ljubljana, Slovenia;
2. International Postgraduate School Jozef Stefan, Ljubljana, Slovenia;
3. Center of Excellence CIPKEBIP, Ljubljana, Slovenia;
4. Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland;
5. Department of Biochemistry, University of Zürich, Zürich, Switzerland.

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Citation:
Kramer L, Renko M, Završnik J, Turk D, Seeger MA, Vasiljeva O, Grütter MG, Turk V, Turk B. Non-invasive in vivo imaging of tumour-associated cathepsin B by a highly selective inhibitory DARPin. Theranostics 2017; 7(11):2806-2821. doi:10.7150/thno.19081. Available from https://www.thno.org/v07p2806.htm

Abstract

Cysteine cathepsins often contribute to cancer progression due to their overexpression in the tumour microenvironment and therefore present attractive targets for non-invasive diagnostic imaging. However, the development of highly selective and versatile small molecule probes for cathepsins has been challenging. Here, we targeted tumour-associated cathepsin B using designed ankyrin repeat proteins (DARPins). The selective DARPin 8h6 inhibited cathepsin B with picomolar affinity (K_i = 35 pM) by binding to a site with low structural conservation in cathepsins, as revealed by the X-ray structure of the complex. DARPin 8h6 blocked cathepsin B activity in tumours ex vivo and was successfully applied in in vivo optical imaging in two mouse breast cancer models, in which cathepsin B was bound to the cell membrane or secreted to the extracellular milieu by tumour and stromal cells. Our approach validates cathepsin B as a promising diagnostic and theranostic target in cancer and other inflammation-associated diseases.

Keywords: protease, cathepsin B, designed ankyrin repeat protein (DARPin), non-invasive diagnostic imaging, tumour microenvironment.