Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

12 Issues per year


IMPACT FACTOR 2017: 3.022

CiteScore 2017: 2.81

SCImago Journal Rank (SJR) 2017: 1.562
Source Normalized Impact per Paper (SNIP) 2017: 0.705

Online
ISSN
1437-4315
See all formats and pricing
More options …
Volume 398, Issue 1

Issues

Anticalins directed against vascular endothelial growth factor receptor 3 (VEGFR-3) with picomolar affinities show potential for medical therapy and in vivo imaging

Antonia Richter
  • Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, D-85354 Freising (Weihenstephan), Germany
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Arne Skerra
  • Corresponding author
  • Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, D-85354 Freising (Weihenstephan), Germany
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2016-07-26 | DOI: https://doi.org/10.1515/hsz-2016-0195

Abstract

Members of the vascular endothelial growth factor receptor (VEGFR) family play a central role in angiogenesis as well as lymphangiogenesis and are crucial for tumor growth and metastasis. In particular, VEGFR-3 expression is induced in endothelial cells during tumor angiogenesis. We report the design of anticalins that specifically recognize the ligand-binding domains 1 and 2 of VEGFR-3. To this end, a library of the lipocalin 2 scaffold with 20 randomized positions distributed across its binding site was subjected to phage display selection and enzyme linked immunosorbent assay (ELISA) screening using the VEGF-C binding fragment (D1-2) or the entire extracellular region (D1-7) of VEGFR-3 as target proteins. Promising anticalin candidates were produced in Escherichia coli and biochemically characterized. Three variants with different receptor binding modes were identified, and two of them were optimized with regard to target affinity as well as folding efficiency. The resulting anticalins show dissociation constants down to the single-digit picomolar range. Specific recognition of VEGFR-3 on cells was demonstrated by immunofluorescence microscopy. Competitive binding versus VEGF-C was demonstrated for two of the anticalins with Ki values in the low nanomolar range. Based on these data, VEGFR-3 specific anticalins provide promising reagents for the diagnosis and/or therapeutic intervention of tumor-associated vessel growth.

This article offers supplementary material which is provided at the end of the article.

Keywords: angiogenesis; glioblastoma; in vivo imaging; lipocalin; protein design; protein scaffold

References

  • Baker, N.A., Sept, D., Joseph, S., Holst, M.J., and McCammon, J.A. (2001). Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041.CrossrefGoogle Scholar

  • Bergers, G. and Hanahan, D. (2008). Modes of resistance to anti-angiogenic therapy. Nat. Rev. Cancer 8, 592–603.CrossrefGoogle Scholar

  • Bullock, W.O., Fernandes, J.M., and Short, J.M. (1987). XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. BioTechniques 5, 376–378.Google Scholar

  • Cheng, Y. and Prusoff, W.H. (1973). Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108.Google Scholar

  • Crawford, Y. and Ferrara, N. (2009). VEGF inhibition: insights from preclinical and clinical studies. Cell Tissue Res. 335, 261–269.Google Scholar

  • DuBridge, R.B., Tang, P., Hsia, H.C., Leong, P.M., Miller, J.H., and Calos, M.P. (1987). Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell Biol. 7, 379–387.CrossrefGoogle Scholar

  • Dumont, D.J., Jussila, L., Taipale, J., Lymboussaki, A., Mustonen, T., Pajusola, K., Breitman, M., and Alitalo, K. (1998). Cardiovascular failure in mouse embryos deficient in VEGF receptor-3. Science 282, 946–949.Google Scholar

  • Eggenstein, E., Eichinger, A., Kim, H.J., and Skerra, A. (2014). Structure-guided engineering of anticalins with improved binding behavior and biochemical characteristics for application in radio-immuno imaging and/or therapy. J. Struct. Biol. 185, 203–214.Google Scholar

  • Ferrara, N. (2004). Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 25, 581–611.CrossrefGoogle Scholar

  • Ferrara, N. (2005). VEGF as a therapeutic target in cancer. Oncology 69(Suppl. 3), 11–16.CrossrefGoogle Scholar

  • Ferrara, N. and Kerbel, R.S. (2005). Angiogenesis as a therapeutic target. Nature 438, 967–974.Google Scholar

  • Ferrara, N. and Adamis, A.P. (2016). Ten years of anti-vascular endothelial growth factor therapy. Nat. Rev. Drug. Discov. 15, 385–403.CrossrefGoogle Scholar

  • Folkman, J. (1986). How is blood vessel growth regulated in normal and neoplastic tissue? Cancer Res. 46, 467–473.Google Scholar

  • Fontanella, C., Ongaro, E., Bolzonello, S., Guardascione, M., Fasola, G., and Aprile, G. (2014). Clinical advances in the development of novel VEGFR2 inhibitors. Ann. Transl. Med. 2, 1–12.Google Scholar

  • Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D., and Bairoch, A. (2003). ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31, 3784–3788.CrossrefGoogle Scholar

  • Gebauer, M. and Skerra, A. (2012). Anticalins. Methods Enzymol. 503, 157–188.Google Scholar

  • Gebauer, M. and Skerra, A. (2015). Alternative protein scaffolds as novel biotherapeutics. In: Biobetters: Protein Engineering to Approach the Curative, A. Rosenberg and B. Demeule, eds. (New York, NY: Springer).Google Scholar

  • 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.Google Scholar

  • Gibson, T.J. (1984). Studies on the Epstein-Barr virus genome. PhD Thesis, University of Cambridge.Google Scholar

  • Gille, H., Hülsmeyer, M., Trentmann, S., Matschiner, G., Christian, H.J., Meyer, T., Amirkhosravi, A., Audoly, L.P., Hohlbaum, A.M., and Skerra, A. (2015). Functional characterization of a VEGF-A-targeting anticalin, prototype of a novel therapeutic human protein class. Angiogenesis 19, 79–84.CrossrefGoogle Scholar

  • Goetz, D.H., Holmes, M.A., Borregaard, N., Bluhm, M.E., Raymond, K.N., and Strong, R.K. (2002). The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol. Cell 10, 1033–1043.CrossrefGoogle Scholar

  • Grau, S.J., Trillsch, F., Herms, J., Thon, N., Nelson, P.J., Tonn, J.-C., and Goldbrunner, R. (2007). Expression of VEGFR3 in glioma endothelium correlates with tumor grade. J. Neurooncol. 82, 141–150.CrossrefGoogle Scholar

  • Grau, S., Thorsteinsdottir, J., von Baumgarten, L., Winkler, F., Tonn, J.C., and Schichor, C. (2011). Bevacizumab can induce reactivity to VEGF-C and -D in human brain and tumour derived endothelial cells. J. Neurooncol. 104, 103–112.CrossrefGoogle Scholar

  • Green, N.M. (1965). A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem. J. 94, 23C–24C.CrossrefGoogle Scholar

  • Hirakawa, S., Brown, L.F., Kodama, S., Paavonen, K., Alitalo, K., and Detmar, M. (2007). VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites. Blood 109, 1010–1017.CrossrefGoogle Scholar

  • Jeltsch, M. (2006). Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns. J. Biol. Chem. 281, 12187–12195.Google Scholar

  • Jenny, B., Harrison, J.A., Baetens, D., Tille, J.-C., Burkhardt, K., Mottaz, H., Kiss, J.Z., Dietrich, P.-Y., De Tribolet, N., Pizzolato, G.P., et al. (2006). Expression and localization of VEGF-C and VEGFR-3 in glioblastomas and haemangioblastomas. J. Pathol. 209, 34–43.Google Scholar

  • Jimenez, X., Lu, D., Brennan, L., Persaud, K., Liu, M., Miao, H., Witte, L., and Zhu, Z. (2005). A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3. Mol. Cancer Ther. 4, 427–434.Google Scholar

  • Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V.W., Fang, G.H., Dumont, D., Breitman, M., and Alitalo, K. (1995). Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl. Acad. Sci. USA 92, 3566–3570.CrossrefGoogle Scholar

  • Karpanen, T., Egeblad, M., Karkkainen, M.J., Kubo, H., Ylä-Herttuala, S., Jäättelä, M., and Alitalo, K. (2001). Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res. 61, 1786–1790.Google Scholar

  • Kaur, S., Venktaraman, G., Jain, M., Senapati, S., Garg, P.K., and Batra, S.K. (2012). Recent trends in antibody-based oncologic imaging. Cancer Lett. 315, 97–111.Google Scholar

  • 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.Google Scholar

  • Koch, S., Tugues, S., Li, X., Gualandi, L., and Claesson-Welsh, L. (2011). Signal transduction by vascular endothelial growth factor receptors. Biochem. J. 437, 169–183.Google Scholar

  • Kurenova, E.V., Hunt, D.L., He, D., Fu, A.D., Massoll, N.A., Golubovskaya, V.M., Garces, C.A., and Cance, W.G. (2009). Vascular endothelial growth factor receptor-3 promotes breast cancer cell proliferation, motility and survival in vitro and tumor formation in vivo. Cell Cycle 8, 2266–2280.CrossrefGoogle Scholar

  • Lee, J., Gray, A., Yuan, J., Luoh, S.M., Avraham, H., and Wood, W.I. (1996). Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4. Proc. Natl. Acad. Sci. USA 93, 1988–1992.CrossrefGoogle Scholar

  • Leppänen, V.M., Tvorogov, D., Kisko, K., Prota, A.E., Jeltsch, M., Anisimov, A., Markovic-Mueller, S., Stuttfeld, E., Goldie, K.N., Ballmer-Hofer, K., et al. (2013). Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation. Proc. Natl. Acad. Sci. USA 110, 12960–12965.CrossrefGoogle Scholar

  • Lohela, M., Bry, M., Tammela, T., and Alitalo, K. (2009). VEGFs and receptors involved in angiogenesis versus lymphangiogenesis. Curr. Opin. Cell Biol. 21, 154–165.CrossrefGoogle Scholar

  • Mäkinen, T., Jussila, L., Veikkola, T., Karpanen, T., Kettunen, M.I., Pulkkanen, K.J., Kauppinen, R., Jackson, D.G., Kubo, H., Nishikawa, S., et al. (2001). Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat. Med. 7, 199–205.CrossrefGoogle Scholar

  • Mandriota, S.J., Jussila, L., Jeltsch, M., Compagni, A., Baetens, D., Prevo, R., Banerji, S., Huarte, J., Montesano, R., Jackson, D.G., et al. (2001). Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO J. 20, 672–682.CrossrefGoogle Scholar

  • McDonald, D.M. (2010). New antibody to stop tumor angiogenesis and lymphatic spread by blocking receptor partnering. Cancer Cell 18, 541–543.CrossrefGoogle Scholar

  • Meerman, H.J. and Georgiou, G. (1994). Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins. Bio/Technology 12, 1107–1110.CrossrefGoogle Scholar

  • Mross, K., Richly, H., Fischer, R., Scharr, D., Büchert, M., Stern, A., Gille, H., Audoly, L.P., and Scheulen, M.E. (2013). First-in-human phase I study of PRS-050 (Angiocal), an anticalin targeting and antagonizing VEGF-A, in patients with advanced solid tumors. PLoS One 8, e83232.Google Scholar

  • Myszka, D.G. (1999). Improving biosensor analysis. J. Mol. Recognit. 12, 279–284.CrossrefGoogle Scholar

  • Nasreen, A., Vogt, M., Kim, H., Eichinger, A., and Skerra, A. (2006). Solubility engineering and crystallization of human apolipoprotein D. Protein Sci. 15, 190–199.CrossrefGoogle Scholar

  • Paavonen, K., Puolakkainen, P., Jussila, L., Jahkola, T., and Alitalo, K. (2000). Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing. Am. J. Pathol. 156, 1499–1504.Google Scholar

  • Persaud, K., Tille, J., Liu, M., Zhu, Z., Jimenez, X., Pereira, D., Miao, H., Brennan, L., Witte, L., and Pepper, M. (2004). Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C. J. Cell Sci. 117, 2745–2756.Google Scholar

  • Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612.CrossrefGoogle Scholar

  • Pontén, J. and Macintyre, E.H. (1968). Long term culture of normal and neoplastic human glia. Acta Pathol. Microbiol. Scand. 74, 465–486.CrossrefGoogle 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.Google Scholar

  • Rinderknecht, M., Villa, A., Ballmer-Hofer, K., Neri, D., and Detmar, M. (2010). Phage-derived fully human monoclonal antibody fragments to human vascular endothelial growth factor-C block its interaction with VEGF receptor-2 and 3. PLoS One 5, e11941.Google Scholar

  • Schiweck, W. and Skerra, A. (1995). Fermenter production of an artificial Fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling. Proteins. 23, 561–565.CrossrefGoogle 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.CrossrefGoogle Scholar

  • Schlapschy, M., Grimm, S., and Skerra, A. (2006). A system for concomitant overexpression of four periplasmic folding catalysts to improve secretory protein production in Escherichia coli. Protein Eng. Des. Sel. 19, 385–390.CrossrefGoogle Scholar

  • Schmidt, T.G. and Skerra, A. (1994). One-step affinity purification of bacterially produced proteins by means of the “Strep tag” and immobilized recombinant core streptavidin. J. Chromatogr. A 676, 337–345.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.CrossrefGoogle 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.CrossrefGoogle Scholar

  • Skobe, M., Hawighorst, T., Jackson, D.G., Prevo, R., Janes, L., Velasco, P., Riccardi, L., Alitalo, K., Claffey, K., and Detmar, M. (2001). Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat. Med. 7, 192–198.CrossrefGoogle Scholar

  • Soule, H.D., Vazguez, J., Long, A., Albert, S., and Brennan, M. (1973). A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 51, 1409–1416.CrossrefGoogle Scholar

  • Su, J.L., Chen, P.S., Chien, M.H., Chen, P.B., Chen, Y.H., Lai, C.C., Hung, M.C., and Kuo, M.L. (2008). Further evidence for expression and function of the VEGF-C/VEGFR-3 axis in cancer cells. Cancer Cell 13, 557–560.Google Scholar

  • Tammela, T. and Alitalo, K. (2010). Lymphangiogenesis: molecular mechanisms and future promise. Cell 140, 460–476.Google Scholar

  • Tammela, T., Zarkada, G., Wallgard, E., Murtomäki, A., Suchting, S., Wirzenius, M., Waltari, M., Hellström, M., Schomber, T., Peltonen, R., et al. (2008). Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 454, 656–660.Google Scholar

  • Tvorogov, D., Anisimov, A., Zheng, W., Leppänen, V.-M., Tammela, T., Laurinavicius, S., Holnthoner, W., Heloterä, H., Holopainen, T., Jeltsch, M., et al. (2010). Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization. Cancer Cell 18, 630–640.CrossrefGoogle Scholar

  • Valtola, R., Salven, P., Heikkilä, P., Taipale, J., Joensuu, H., Rehn, M., Pihlajaniemi, T., Weich, H., deWaal, R., and Alitalo, K. (1999). VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer. Am. J. Pathol. 154, 1381–1390.Google Scholar

  • Yanisch-Perron, C., Vieira, J., and Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.CrossrefGoogle Scholar

  • Zhang, D., Li, B., Shi, J., Zhao, L., Zhang, X., Wang, C., Hou, S., Qian, W., Kou, G., and Wang, H. (2010). Suppression of tumor growth and metastasis by simultaneously blocking vascular endothelial growth factor (VEGF)-A and VEGF-C with a receptor-immunoglobulin fusion protein. Cancer Res. 70, 2495–2503.CrossrefGoogle Scholar

About the article

Received: 2016-05-01

Accepted: 2016-07-19

Published Online: 2016-07-26

Published in Print: 2017-01-01


Citation Information: Biological Chemistry, Volume 398, Issue 1, Pages 39–55, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2016-0195.

Export Citation

©2017 Walter de Gruyter GmbH, Berlin/Boston.Get Permission

Supplementary Article Materials

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

[2]
Joanna I. Loch, Piotr Bonarek, Magdalena Tworzydło, Ilona Łazińska, Joanna Szydłowska, Joanna Lipowska, Katarzyna Rzęsikowska, and Krzysztof Lewiński
International Journal of Biological Macromolecules, 2018
[3]
André Schiefner, Michaela Gebauer, Antonia Richter, and Arne Skerra
Structure, 2018
[4]
Stefan Harmansa and Markus Affolter
Development, 2018, Volume 145, Number 2, Page dev148874
[5]
Ben J. Glasgow and Adil R. Abduragimov
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2018
[6]
Xia Xiao, Beng San Yeoh, and Matam Vijay-Kumar
Annual Review of Nutrition, 2017, Volume 37, Number 1, Page 103

Comments (0)

Please log in or register to comment.
Log in