Molecular Biology

, Volume 51, Issue 6, pp 865–873 | Cite as

Bifunctional Toxin DARP-LoPE Based on the Her2-Specific Innovative Module of a Non-Immunoglobulin Scaffold as a Promising Agent for Theranostics

  • G. M. Proshkina
  • D. V. Kiseleva
  • O. N. Shilova
  • A. V. Ryabova
  • E. I. Shramova
  • O. A. Stremovskiy
  • S. M. Deyev
Current Trends in the Application of Monoclonal Antibodies Special Issue


We have generated and characterized HER2-specific targeted toxin based on the low-immunogenic variant of Pseudomonas exotoxin A (LoPE), in which most of the human immunodominant B-cell epitopes have been inactivated. Nonimmunoglobulin DARPin-based HER2-specific protein was used as a targeting module for toxin delivery to the cellular target. Using confocal microscopy, it has been found that both domains in this hybrid toxin retained their functionality, i.e., the specific interaction with HER2 receptor, as well as the internalization and effective transport to ER typical of the wild-type Pseudomonas exotoxin A. The HER2-dependent cytotoxic effect correlated with receptor expression level at the cell surface, as shown in vitro using cell lines with different levels of HER2 expression. Due to the very high selective cytotoxicity against HER2-positive human tumor cells, as well as expected low immunogenicity, we believe that this new targeted toxin may be promising for future in vivo studies as a therapeutic agent for HER2-positive tumors.


DARPin targeted therapy Pseudomonas exotoxin А HER2 



designed ankyrin repeat proteins


human epidermal growth factor receptor 2


fluorescein isothiocyanate


Pseudomonas exotoxin


endoplasmic reticulum.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Deyev S.M., Lebedenko E.N. 2009. Modern technologies for creating synthetic antibodies for clinical application. Acta Naturae. 1, 32–50.PubMedPubMedCentralGoogle Scholar
  2. 2.
    Deyev S.M., Lebedenko E.N. 2008. Multivalency: The hallmark of antibodies used for optimization of tumor targeting by design. Bioessays. 30 (9), 904–918.CrossRefPubMedGoogle Scholar
  3. 3.
    Boersma Y.L., Plückthun A. 2011. DARPins and other repeat protein scaffolds: Advances in engineering and applications. Curr. Opin. Biotechnol. 22 (6), 849–857.CrossRefPubMedGoogle Scholar
  4. 4.
    Löfblom J., Frejd F.Y., Ståhl S. 2011. Non-immunoglobulin based protein scaffolds. Curr. Opin. Biotechnol. 22 (6), 843–848.CrossRefPubMedGoogle Scholar
  5. 5.
    Deyev S.M., Lebedenko E.N., Petrovskaya L.E., et al. 2015. Man-made antibodies and immunoconjugates with desired properties: Function optimization using structural engineering. Russ. Chem. Rev. 84, 1–26.CrossRefGoogle Scholar
  6. 6.
    Binz H.K., Stumpp M.T., Forrer P., et al. 2003. Designing repeat proteins: Well expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol. Biol. 332 (2), 489–503.CrossRefPubMedGoogle Scholar
  7. 7.
    Stumpp M.T., Forrer P., Binz H.K., et al. 2003. Designing repeat proteins: Modular leucine-rich repeat protein libraries based on the mammalian ribonuclease inhibitor family. J. Mol. Biol. 332 (2), 471–487.CrossRefPubMedGoogle Scholar
  8. 8.
    Plückthun A. 2015. Designed ankyrin repeat proteins (DARPins): Binding proteins for research, diagnostics, and therapy. Annu. Rev. Pharmacol. Toxicol. 55, 489–511.CrossRefPubMedGoogle Scholar
  9. 9.
    Steiner D., Forrer P., Plückthun A. 2008. Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. J. Mol. Biol. 382 (5), 1211–1227.CrossRefPubMedGoogle Scholar
  10. 10.
    Jost C., Schilling J., Tamaskovic R., et al. 2013. Structural basis for eliciting a cytotoxic effect in HER2-overexpressing cancer cells via binding to the extracellular domain of HER2. Structure. 21 (11), 1979–1991.CrossRefPubMedGoogle Scholar
  11. 11.
    Simon M., Zangemeister-Wittke U., Plückthun A. 2012. Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries. Bioconjug. Chem. 23 (2), 279–286.CrossRefPubMedGoogle Scholar
  12. 12.
    Martin-Killias P., Stefan N., Rothschild S., et al. 2011. A novel fusion toxin derived from an EpCAM-specific designed ankyrin repeat protein has potent antitumor activity. Clin. Cancer Res. 17 (1), 100–110.CrossRefPubMedGoogle Scholar
  13. 13.
    Zahnd C., Kawe M., Stumpp M.T., et al. 2010. Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: Effects of affinity and molecular size. Cancer Res. 70 (4), 1595–1605.CrossRefPubMedGoogle Scholar
  14. 14.
    Slamon D.J, Clark G.M., Wong S.G., et al. 1987. Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 235 (4785), 177–182.CrossRefPubMedGoogle Scholar
  15. 15.
    Slamon D.J., Godolphin W., Jones L.A., et al. 1989. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 244 (4905), 707–712.CrossRefPubMedGoogle Scholar
  16. 16.
    Holbro T., Hynes N.E. 2004. ErbB receptors: Directing key signaling networks throughout life. Annu. Rev. Pharmacol. Toxicol. 44, 195–217.CrossRefPubMedGoogle Scholar
  17. 17.
    Liu W., Onda M., Lee B., et al. 2012. Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes. Proc. Natl. Acad. Sci. U. S. A. 109 (29), 11782–11787.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mironova K.E., Chernykh O.N., Ryabova A.V., et al. 2014. Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu. Biochemistry (Moscow). 79 (12), 1391–1396.CrossRefPubMedGoogle Scholar
  19. 19.
    Deyev S.M., Waibel R., Lebedenko E.N., et al. 2003. Design of multivalent complexes using the barnase* barstar module. Nat. Biotechnol. 21 (12), 1486–1492.CrossRefPubMedGoogle Scholar
  20. 20.
    Chaudhary V.K., Jinno Y., FitzGerald D., et al. 1990. Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc. Natl. Acad. Sci. U. S. A. 87 (1), 308–312.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Jackson M.E., Simpson J.C., Girod A., et al. 1999. The KDEL retrieval system is exploited by Pseudomonas exotoxin A, but not by Shiga-like toxin-1, during retrograde transport from the Golgi complex to the endoplasmic reticulum. J. Cell Sci. 112 (4), 467–475.PubMedGoogle Scholar
  22. 22.
    Studier F.W. 2014. Stable expression clones and autoinduction for protein production in E. coli. Methods Mol. Biol. 1091, 17–32.CrossRefPubMedGoogle Scholar
  23. 23.
    Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65, 55–63.CrossRefPubMedGoogle Scholar
  24. 24.
    Komatsu N., Oda T., Muramatsu T. 1998. Involvement of both caspase-like proteases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and pseudomonas toxin. J. Biochem. 124 (5), 1038–1044.CrossRefPubMedGoogle Scholar
  25. 25.
    Jenkins C.E., Swiatoniowski A., Issekutz A.C., et al. 2004. Pseudomonas aeruginosa exotoxin A induces human mast cell apoptosis by a caspase-8 and -3-dependent mechanism. J. Biol. Chem. 279 (35), 37201–37207.CrossRefPubMedGoogle Scholar
  26. 26.
    Theurillat J.P., Dreier B., Nagy-Davidescu G., et al. 2010. Designed ankyrin repeat proteins: A novel tool for testing epidermal growth factor receptor 2 expression in breast cancer. Mod. Pathol. 23 (9), 1289–1297.CrossRefPubMedGoogle Scholar
  27. 27.
    Simon M., Stefan N., Borsig L., et al. 2014. Increasing the antitumor effect of an EpCAM-targeting fusion toxin by facile click PEGylation. Mol. Cancer. Ther. 13 (2), 375–385.CrossRefPubMedGoogle Scholar
  28. 28.
    Sokolova E., Proshkina G., Kutova O., et al. 2016. Recombinant targeted toxin based on HER2-specific DARPin possesses a strong selective cytotoxic effect in vitro and a potent antitumor activity in vivo. J. Controlled Release. 10 (233), 48–56.CrossRefGoogle Scholar
  29. 29.
    Friedrich K., Hanauer J.R., Prüfer S., et al. 2013. DARPin-targeting of measles virus: Unique bispecificity, effective oncolysis, and enhanced safety. Mol. Ther. 21 (4), 849–859.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Dreier B., Honegger A., Hess C., et al. 2013. Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters. Proc. Natl. Acad. Sci. U. S. A. 110 (10), E869–E877.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Proshkina G.M., Shilova O.N., Ryabova A.V., et al. 2015. A new anticancer toxin based on HER2/neu-specific DARPin and photoactive flavoprotein miniSOG. Biochimie. 118, 116–122.CrossRefPubMedGoogle Scholar
  32. 32.
    Hommelgaard A.M., Lerdrup M., van Deurs B. 2004. Association with membrane protrusions makes ErbB2 an internalization-resistant receptor. Mol. Biol. Cell. 15 (4), 1557–1567.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Haslekas C., Breen K., Pedersen K.W., et al. 2005. The inhibitory effect of ErbB2 on epidermal growth factorinduced formation of clathrin-coated pits correlates with retention of epidermal growth factor receptor- ErbB2 oligomeric complexes at the plasma membrane. Mol. Biol. Cell. 16 (12), 5832–5842.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Harari D., Yarden Y. 2000. Molecular mechanisms underlying ErbB2/HER2 action in breast cancer. Oncogene. 19 (53), 6102–6114.CrossRefPubMedGoogle Scholar
  35. 35.
    Hendriks B.S., Opresko L.K., Wiley H.S., et al. 2003. Coregulation of epidermal growth factor receptor/ human epidermal growth factor receptor 2 (HER2) levels and locations: Quantitative analysis of HER2 overexpression effects. Cancer Res. 63 (5), 1130–1137.PubMedGoogle Scholar
  36. 36.
    Austin C.D., de Maziere A.M., Pisacane P.I., et al. 2004. Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol. Biol. Cell. 15 (12), 5268–5282.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Shilova O.N., ProshkinaG.M., LebedenkoE.N., Deyev S.M. 2015. Internalization and recycling of the HER2 receptor on human breast adenocarcinoma cells treated with targeted phototoxic protein DARPin-miniSOG. Acta Naturae. 7 (3), 126–132.PubMedPubMedCentralGoogle Scholar
  38. 38.
    Longva K.E., Pedersen N.M., Haslekas C., et al. 2005. Herceptin-induced inhibition of ErbB2 signaling involves reduced phosphorylation of Akt but not endocytic down-regulation of ErbB2. Int. J. Cancer. 116 (3), 359–367.CrossRefPubMedGoogle Scholar
  39. 39.
    Ben-Kasus T., Schechter B., Lavi S., et al. 2009. Persistent elimination of ErbB-2/HER2-overexpressing tumors using combinations of monoclonal antibodies: Relevance of receptor endocytosis. Proc. Natl. Acad. Sci. U. S. A. 106 (9), 3294–3299.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Nahta R., Hung M.C., Esteva F.J. 2004. The HER-2- targeting antibodies trastuzumab and pertuzumab synergistically inhibit the survival of breast cancer cells. Cancer Res. 64 (7), 2343–2346.CrossRefPubMedGoogle Scholar
  41. 41.
    Friedman L.M., Rinon A., Schechter B., et al. 2005. Synergistic down-regulation of receptor tyrosine kinases by combinations of mAbs: Implications for cancer immunotherapy. Proc. Natl. Acad. Sci. U. S. A. 102 (6), 1915–1920.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Weldon J.E., Pastan I. 2011. A guide to taming a toxin: Recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J. 278 (23), 4683–4700.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Munro S., Pelham H.R. 1987. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 48 (5), 899–907.CrossRefPubMedGoogle Scholar
  44. 44.
    Seetharam S., Chaudhary V.K., FitzGerald D., et al. 1991. Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL. J. Biol. Chem. 266 (26), 17376–17381.PubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2017

Authors and Affiliations

  • G. M. Proshkina
    • 1
  • D. V. Kiseleva
    • 1
    • 2
  • O. N. Shilova
    • 1
  • A. V. Ryabova
    • 3
  • E. I. Shramova
    • 1
  • O. A. Stremovskiy
    • 1
  • S. M. Deyev
    • 1
    • 4
  1. 1.Shemyakin–Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Department of BiologyMoscow State UniversityMoscowRussia
  3. 3.Prokhorov General Physics InstituteRussian Academy of SciencesMoscowRussia
  4. 4.National Research Tomsk Polytechnic UniversityTomskRussia

Personalised recommendations