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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 EMAIL logo
From the journal Biological Chemistry


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.


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.


Akizawa, H., Uehara, T., and Arano, Y. (2008). Renal uptake and metabolism of radiopharmaceuticals derived from peptides and proteins. Adv. Drug Deliv. Rev. 60, 1319–1328.10.1016/j.addr.2008.04.005Search in Google Scholar

Bertelsen, E.B., Chang, L., Gestwicki, J.E., and Zuiderweg, E.R. (2009). Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate. Proc. Natl. Acad. Sci. USA 106, 8471–8476.10.1073/pnas.0903503106Search in Google Scholar

Bessette, P.H., Rice, J.J., and Daugherty, P.S. (2004). Rapid isolation of high-affinity protein binding peptides using bacterial display. Protein Eng. Des. Sel. 17, 731–739.10.1093/protein/gzh084Search in Google Scholar

Beutling, U., Stading, K., Stradal, T., and Frank, R. (2008). Large-scale analysis of protein-protein interactions using cellulose-bound peptide arrays. Adv. Biochem. Eng. Biotechnol. 110, 115–152.10.1007/10_2008_096Search in Google Scholar

Binder, U., Matschiner, G., Theobald, I., and Skerra, A. (2010). High-throughput sorting of an Anticalin library via EspP-mediated functional display on the Escherichia coli cell surface. J. Mol. Biol. 400, 783–802.10.1016/j.jmb.2010.05.049Search in Google Scholar

Binder, U. and Skerra, A. (2017). PASylation®: a versatile technology to extend drug delivery. Curr. Opin. Colloid Interface Sci. 31, 10–17.10.1016/j.cocis.2017.06.004Search in Google Scholar

Boder, E.T. and Wittrup, K.D. (1998). Optimal screening of surface-displayed polypeptide libraries. Biotechnol. Prog. 14, 55–62.10.1021/bp970144qSearch in Google Scholar

Botzler, C., Li, G., Issels, R.D., and Multhoff, G. (1998). Definition of extracellular localized epitopes of Hsp70 involved in an NK immune response. Cell Stress Chaperones 3, 6–11.10.1379/1466-1268(1998)003<0006:DOELEO>2.3.CO;2Search in Google Scholar

Bratkovič, T. (2010). Progress in phage display: evolution of the technique and its application. Cell. Mol. Life Sci. 67, 749–767.10.1007/s00018-009-0192-2Search in Google Scholar

Dautin, N., Barnard, T.J., Anderson, D.E., and Bernstein, H.D. (2007). Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism. EMBO J. 26, 1942–1952.10.1038/sj.emboj.7601638Search in Google Scholar

De Schutter, K. and Callewaert, N. (2012). Pichia surface display: a tool for screening single domain antibodies. Methods Mol. Biol. 911, 125–134.10.1007/978-1-61779-968-6_8Search in Google Scholar PubMed

English, C.A., Sherman, W., Meng, W., and Gierasch, L.M. (2017). The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains. J. Biol. Chem. 292, 14765–14774.10.1074/jbc.M117.789313Search in Google Scholar PubMed PubMed Central

Epstein, A.L., Herman, M.M., Kim, H., Dorfman, R.F., and Kaplan, H.S. (1976). Biology of the human malignant lymphomas. III. Intracranial heterotransplantation in the nude, athymic mouse. Cancer 37, 2158–2176.10.1007/978-3-642-81246-0_21Search in Google Scholar PubMed

Friedrich, L., Stangl, S., Hahne, H., Küster, B., Köhler, P., Multhoff, G., and Skerra, A. (2010). Bacterial production and functional characterization of the Fab fragment of the murine IgG1/λ monoclonal antibody cmHsp70.1, a reagent for tumour diagnostics. Protein Eng. Des. Sel. 23, 161–168.10.1093/protein/gzp095Search in Google Scholar PubMed

Gebauer, M., Schiefner, A., Matschiner, G., and Skerra, A. (2013). Combinatorial design of an Anticalin directed against the extra-domain B for the specific targeting of oncofetal fibronectin. J. Mol. Biol. 425, 780–802.10.1016/j.jmb.2012.12.004Search in Google Scholar PubMed

Gebauer, M. and Skerra, A. (2009). Engineered protein scaffolds as next-generation antibody therapeutics. Curr. Opin. Chem. Biol. 13, 245–255.10.1016/j.cbpa.2009.04.627Search in Google Scholar PubMed

Gebauer, M. and Skerra, A. (2012). Anticalins: small engineered binding proteins based on the lipocalin scaffold. Methods Enzymol. 503, 157–188.10.1016/B978-0-12-396962-0.00007-0Search in Google Scholar PubMed

Gehrmann, M., Liebisch, G., Schmitz, G., Anderson, R., Steinem, C., De Maio, A., Pockley, G., and Multhoff, G. (2008). Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoS One 3, e1925.10.1371/journal.pone.0001925Search in Google Scholar PubMed PubMed Central

Getz, J.A., Schoep, T.D., and Daugherty, P.S. (2012). Peptide discovery using bacterial display and flow cytometry. Methods Enzymol. 503, 75–97.10.1016/B978-0-12-396962-0.00004-5Search in Google Scholar PubMed

Haas, I.G. (1994). BiP (GRP78), an essential hsp70 resident protein in the endoplasmic reticulum. Experientia 50, 1012–1020.10.1007/BF01923455Search in Google Scholar PubMed

Haas, I.G. and Meo, T. (1988). cDNA cloning of the immunoglobulin heavy chain binding protein. Proc. Natl. Acad. Sci. USA 85, 2250–2254.10.1073/pnas.85.7.2250Search in Google Scholar

Hansel, T.T., Kropshofer, H., Singer, T., Mitchell, J.A., and George, A.J. (2010). The safety and side effects of monoclonal antibodies. Nat. Rev. Drug Discov. 9, 325–338.10.1038/nrd3003Search in Google Scholar

Hosseinimehr, S.J., Tolmachev, V., and Orlova, A. (2012). Liver uptake of radiolabeled targeting proteins and peptides: considerations for targeting peptide conjugate design. Drug Discov. Today 17, 1224–1232.10.1016/j.drudis.2012.07.002Search in Google Scholar

Jäättelä, M. (1999). Escaping cell death: survival proteins in cancer. Exp. Cell Res. 248, 30–43.10.1006/excr.1999.4455Search in Google Scholar

Jiang, J., Prasad, K., Lafer, E.M., and Sousa, R. (2005). Structural basis of interdomain communication in the Hsc70 chaperone. Mol. Cell 20, 513–524.10.1016/j.molcel.2005.09.028Search in Google Scholar

Jose, J., Krämer, J., Klauser, T., Pohlner, J., and Meyer, T.F. (1996). Absence of periplasmic DsbA oxidoreductase facilitates export of cysteine-containing passenger proteins to the Escherichia coli cell surface via the Igaβ autotransporter pathway. Gene 178, 107–110.10.1016/0378-1119(96)00343-5Search in Google Scholar

Katagiri, S., Yonezawa, T., Kuyama, J., Kanayama, Y., Nishida, K., Abe, T., Tamaki, T., Ohnishi, M., and Tarui, S. (1985). Two distinct human myeloma cell lines originating from one patient with myeloma. Int. J. Cancer 36, 241–246.10.1002/ijc.2910360217Search in Google Scholar PubMed

Kim, H.J., Eichinger, A., and Skerra, A. (2009). High-affinity recognition of lanthanide(III) chelate complexes by a reprogrammed human lipocalin 2. J. Am. Chem. Soc. 131, 3565–3576.10.1021/ja806857rSearch in Google Scholar PubMed

Kornberger, P. and Skerra, A. (2014). Sortase-catalyzed in vitro functionalization of a Her2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin. MAbs 6, 25–37.10.4161/mabs.27444Search in Google Scholar PubMed PubMed Central

Kronqvist, N., Löfblom, J., Jonsson, A., Wernerus, H., and Ståhl, S. (2008). A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry. Protein Eng. Des. Sel. 21, 247–255.10.1093/protein/gzm090Search in Google Scholar PubMed

Kuhn, S.M., Rubini, M., Fuhrmann, M., Theobald, I., and Skerra, A. (2010). Engineering of an orthogonal aminoacyl-tRNA synthetase for efficient incorporation of the non-natural amino acid O-methyl-L-tyrosine using fluorescence-based bacterial cell sorting. J. Mol. Biol. 404, 70–87.10.1016/j.jmb.2010.09.001Search in Google Scholar

Liu, Q. and Hendrickson, W.A. (2007). Insights into Hsp70 chaperone activity from a crystal structure of the yeast Hsp110 Sse1. Cell 131, 106–120.10.1016/j.cell.2007.08.039Search in Google Scholar

Mayer, M.P. and Kityk, R. (2015). Insights into the molecular mechanism of allostery in Hsp70s. Front. Mol. Biosci. 2, 58.10.3389/fmolb.2015.00058Search in Google Scholar

Mazor, Y., Van Blarcom, T., Iverson, B.L., and Georgiou, G. (2008). E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display. Nat. Protoc. 3, 1766–1777.10.1038/nprot.2008.176Search in Google Scholar

Mendler, C.T., Friedrich, L., Laitinen, I., Schlapschy, M., Schwaiger, M., Wester, H.J., and Skerra, A. (2015a). High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation. MAbs 7, 96–109.10.4161/19420862.2014.985522Search in Google Scholar

Mendler, C.T., Gehring, T., Wester, H.J., Schwaiger, M., and Skerra, A. (2015b). 89Zr-labeled versus 124I-labeled αHER2 Fab with optimized plasma half-life for high-contrast tumor imaging in vivo. J. Nucl. Med. 56, 1112–1118.10.2967/jnumed.114.149690Search in Google Scholar

Multhoff, G. and Hightower, L.E. (2011). Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies. Cell Stress Chaperones 16, 251–255.10.1007/s12192-010-0247-1Search in Google Scholar

Multhoff, G., Botzler, C., Wiesnet, M., Müller, E., Meier, T., Wilmanns, W., and Issels, R.D. (1995). A stress-inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int. J. Cancer 61, 272–279.10.1002/ijc.2910610222Search in Google Scholar

Multhoff, G., Pockley, A.G., Schmid, T.E., and Schilling, D. (2015). The role of heat shock protein 70 (Hsp70) in radiation-induced immunomodulation. Cancer Lett. 368, 179–184.10.1016/j.canlet.2015.02.013Search in Google Scholar

Myszka, D.G. (1999). Improving biosensor analysis. J. Mol. Recognit. 12, 279–284.10.1002/(SICI)1099-1352(199909/10)12:5<279::AID-JMR473>3.0.CO;2-3Search in Google Scholar

Perk, L.R., Vosjan, M.J., Visser, G.W., Budde, M., Jurek, P., Kiefer, G.E., and van Dongen, G.A. (2010). p-Isothiocyanatobenzyl-desferrioxamine: a new bifunctional chelate for facile radiolabeling of monoclonal antibodies with zirconium-89 for immuno-PET imaging. Eur. J. Nucl. Med. Mol. Imaging 37, 250–259.10.1007/s00259-009-1263-1Search in Google Scholar

Petersen, N.H., Kirkegaard, T., Olsen, O.D., and Jäättelä, M. (2010). Connecting Hsp70, sphingolipid metabolism and lysosomal stability. Cell Cycle 9, 2305–2309.10.4161/cc.9.12.12052Search in Google Scholar

Popp, M.W. and Ploegh, H.L. (2011). Making and breaking peptide bonds: protein engineering using sortase. Angew. Chem. Int. Ed. 50, 5024–5032.10.1002/anie.201008267Search in Google Scholar

Qi, R., Sarbeng, E.B., Liu, Q., Le, K.Q., Xu, X., Xu, H., Yang, J., Wong, J.L., Vorvis, C., Hendrickson, W.A., et al. (2013). Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP. Nat. Struct. Mol. Biol. 20, 900–907.10.1038/nsmb.2583Search in Google Scholar

Rangan, S.R. (1972). A new human cell line (FaDu) from a hypopharyngeal carcinoma. Cancer 29, 117–121.10.1002/1097-0142(197201)29:1<117::AID-CNCR2820290119>3.0.CO;2-RSearch in Google Scholar

Richter, A., Eggenstein, E., and Skerra, A. (2014). Anticalins: exploiting a non-Ig scaffold with hypervariable loops for the engineering of binding proteins. FEBS Lett. 588, 213–218.10.1016/j.febslet.2013.11.006Search in Google Scholar

Salema, V., Manas, C., Cerdan, L., Pinero-Lambea, C., Marin, E., Roovers, R.C., Van Bergen En Henegouwen, P.M., and Fernandez, L.A. (2016). High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display. MAbs 8, 1286–1301.10.1080/19420862.2016.1216742Search in Google Scholar

Schlapschy, M., Gruber, H., Gresch, O., Schäfer, C., Renner, C., Pfreundschuh, M., and Skerra, A. (2004). Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin’s lymphoma using an in vitro evolution approach. Protein Eng. Des. Sel. 17, 847–860.10.1093/protein/gzh098Search in Google Scholar

Schlapschy, M., Binder, U., Börger, C., Theobald, I., Wachinger, K., Kisling, S., Haller, D., and Skerra, A. (2013). PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins. Protein Eng. Des. Sel. 26, 489–501.10.1093/protein/gzt023Search in Google Scholar

Schlehuber, S. and Skerra, A. (2002). Tuning ligand affinity, specificity, and folding stability of an engineered lipocalin variant – a so-called ‘anticalin’ – using a molecular random approach. Biophys. Chem. 96, 213–228.10.1016/S0301-4622(02)00026-1Search in Google Scholar

Schmidt, T.G. and Skerra, A. (2007). The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat. Protoc. 2, 1528–1535.10.1038/nprot.2007.209Search in Google Scholar

Schneider, C.A., Rasband, W.S., and Eliceiri, K.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675.10.1038/nmeth.2089Search in Google Scholar

Schönfeld, D., Matschiner, G., Chatwell, L., Trentmann, S., Gille, H., Hülsmeyer, M., Brown, N., Kaye, P.M., Schlehuber, S., Hohlbaum, A.M., et al. (2009). An engineered lipocalin specific for CTLA-4 reveals a combining site with structural and conformational features similar to antibodies. Proc. Natl. Acad. Sci. USA 106, 8198–8203.10.1073/pnas.0813399106Search in Google Scholar

Schreiber, G. (1988). Ein kombinatorisches Problem aus der Genetik. BioEngineering 2, 32–35.Search in Google Scholar

Siegel, R.W., Coleman, J.R., Miller, K.D., and Feldhaus, M.J. (2004). High efficiency recovery and epitope-specific sorting of an scFv yeast display library. J. Immunol. Methods 286, 141–153.10.1016/j.jim.2004.01.005Search in Google Scholar

Skerra, A. (1994). Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151, 131–135.10.1016/0378-1119(94)90643-2Search in Google Scholar

Skerra, A. (2001). ‘Anticalins’: a new class of engineered ligand-binding proteins with antibody-like properties. J. Biotechnol. 74, 257–275.10.1016/S1389-0352(01)00020-4Search in Google Scholar

Skerra, A. (2003). Imitating the humoral immune response. Curr. Opin. Chem. Biol. 7, 683–693.10.1016/j.cbpa.2003.10.012Search in Google Scholar PubMed

Skerra, A. (2007). Alternative non-antibody scaffolds for molecular recognition. Curr. Opin. Biotechnol. 18, 295–304.10.1016/j.copbio.2007.04.010Search in Google Scholar PubMed

Skerra, A. (2008). Alternative binding proteins: Anticalins – harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities. FEBS J. 275, 2677–2683.10.1111/j.1742-4658.2008.06439.xSearch in Google Scholar PubMed

Skillman, K.M., Barnard, T.J., Peterson, J.H., Ghirlando, R., and Bernstein, H.D. (2005). Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter. Mol. Microbiol. 58, 945–958.10.1111/j.1365-2958.2005.04885.xSearch in Google Scholar PubMed

Stangl, S., Gehrmann, M., Dressel, R., Alves, F., Dullin, C., Themelis, G., Ntziachristos, V., Staeblein, E., Walch, A., Winkelmann, I., et al. (2011a). In vivo imaging of CT26 mouse tumours by using cmHsp70.1 monoclonal antibody. J. Cell. Mol. Med. 15, 874–887.10.1111/j.1582-4934.2010.01067.xSearch in Google Scholar

Stangl, S., Gehrmann, M., Riegger, J., Kuhs, K., Riederer, I., Sievert, W., Hube, K., Mocikat, R., Dressel, R., Kremmer, E., et al. (2011b). Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc. Natl. Acad. Sci. USA 108, 733–738.10.1073/pnas.1016065108Search in Google Scholar

Stangl, S., Themelis, G., Friedrich, L., Ntziachristos, V., Sarantopoulos, A., Molls, M., Skerra, A., and Multhoff, G. (2011c). Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment. Radiother. Oncol. 99, 313–316.10.1016/j.radonc.2011.05.051Search in Google Scholar

Studier, F.W. and Moffatt, B.A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.10.1016/0022-2836(86)90385-2Search in Google Scholar

Tolmachev, V. and Stone-Elander, S. (2010). Radiolabelled proteins for positron emission tomography: pros and cons of labelling methods. Biochim. Biophys. Acta 1800, 487–510.10.1016/j.bbagen.2010.02.002Search in Google Scholar PubMed

van Bloois, E., Winter, R.T., Kolmar, H., and Fraaije, M.W. (2011). Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol. 29, 79–86.10.1016/j.tibtech.2010.11.003Search in Google Scholar PubMed

Voss, S. and Skerra, A. (1997). Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification. Protein Eng. 10, 975–982.10.1093/protein/10.8.975Search in Google Scholar PubMed

Yang, W.P., Green, K., Pinz-Sweeney, S., Briones, A.T., Burton, D.R., and Barbas, C.F., 3rd (1995). CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J. Mol. Biol. 254, 392–403.10.1006/jmbi.1995.0626Search in Google Scholar PubMed

Yeung, Y.A. and Wittrup, K.D. (2002). Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture. Biotechnol. Prog. 18, 212–220.10.1021/bp010186lSearch in Google Scholar PubMed

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

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