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Licensed Unlicensed Requires Authentication Published by De Gruyter November 15, 2017

Selection of an Anticalin® against the membrane form of Hsp70 via bacterial surface display and its theranostic application in tumour models

Lars Friedrich, Petra Kornberger, Claudia T. Mendler, Gabriele Multhoff, Markus Schwaiger and Arne Skerra
From the journal Biological Chemistry

Abstract

We describe the selection of Anticalins against a common tumour surface antigen, human Hsp70, using functional display on live Escherichia coli cells as fusion with a truncated EspP autotransporter. While found intracellularly in normal cells, Hsp70 is frequently exposed in a membrane-bound state on the surface of tumour cells and, even more pronounced, in metastases or after radiochemotherapy. Employing a recombinant Hsp70 fragment comprising residues 383-548 as the target, Anticalins were selected from a naïve bacterial library. The Anticalin with the highest affinity (KD=13 nm), as determined towards recombinant full-length Hsp70 by real-time surface plasmon resonance analysis, was improved to KD=510 pm by doped random mutagenesis and another cycle of E. coli surface display, followed by rational combination of mutations. This Anticalin, which recognises a linear peptide epitope located in the interdomain linker of Hsp70, was demonstrated to specifically bind Hsp70 in its membrane-associated form in immunofluorescence microscopy and via flow cytometry using the FaDu cell line, which is positive for surface Hsp70. The radiolabelled and PASylated Anticalin revealed specific tumour accumulation in xenograft mice using positron emission tomography (PET) imaging. Furthermore, after enzymatic coupling to the protein toxin gelonin, the Anticalin showed potent cytotoxicity on FaDu cells in vitro.

Acknowledgements

The authors wish to thank Alois Bräuer for experimental support and Sybille Reder, Markus Mittelhäuser and Marco Lehmann for performing the imaging measurements. This work was supported by the Bundesministerium für Bildung und Forschung (BMBF), Germany, under grant no. 01EZ0826 and by the Deutsche Forschungsgemeinschaft in frame of the Collaborative Research Centre (SFB) 824. Anticalin® is a registered trademark of Pieris Pharmaceuticals GmbH. PASylation® is a registered trademark of XL-protein GmbH.

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Supplemental Material:

The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2017-0207).


Received: 2017-7-26
Accepted: 2017-10-23
Published Online: 2017-11-15
Published in Print: 2018-2-23

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