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Intrabody targeting vascular endothelial growth factor receptor-2 mediates downregulation of surface localization

Cancer Gene Therapy volume 24, pages 33–37 (2017)Cite this article

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Abstract

Angiogenesis is among the most important mechanisms that helps cancer cells to survive, grow and undergo metastasis. Therefore, inhibiting angiogenesis will suppress tumor growth. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are believed to be important players of angiogenesis. The goal of this study was to evaluate the success of a novel nanobody against VEGFR2 in tethering its target inside the endoplasmic reticulum and preventing its transport to the cell membrane. Nanobody sequence was cloned in a mammalian vector in fusion with green fluorescent protein and a KDEL retention signal. After transfection of 293KDR cells with this expression vector, surface localization of VEGFR2 was monitored by flow cytometry. This study demonstrates that our intrananobody is effective in targeting VEGFR2 receptor, and therefore, it is a powerful tool to downregulate a surface-exposed target protein, and in this capacity, it has potential to be used as a therapeutic protein to inhibit growth of tumors.

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References

  1. Folkman J Angiogenesis and angiogenesis inhibition: an overview. Regulation of Angiogenesis. Springer: Birkhäuser Basel, 1997, pp 1–8.

  2. Robinson CJ, Stringer SE . The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 2001; 114: 853–865.

    CAS  Google Scholar 

  3. Folkman J, Hanahan D . Switch to the angiogenic phenotype during tumorigenesis. Princess Takamatsu Symp 1990; 22: 339–347.

    Google Scholar 

  4. Folkman J . What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990; 82: 4–7.

    Article  CAS  Google Scholar 

  5. Eichmann A, Simons M . VEGF signaling inside vascular endothelial cells and beyond. Curr Opin Cell Biol 2012; 24: 188–193.

    Article  CAS  Google Scholar 

  6. Carmeliet P, Jain RK . Molecular mechanisms and clinical applications of angiogenesis. Nature 2011; 473: 298–307.

    Article  CAS  Google Scholar 

  7. Sina K, Sonia T, Xiujuan L, Laura G, Lena C-W . Signal transduction by vascular endothelial growth factor receptors. Biochem J 2011; 437: 169–183.

    Article  Google Scholar 

  8. Shibuya M, Yamaguchi S, Yamane A, Ikeda T, Tojo A, Matsushime H et al. Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (flt) closely related to the fms family. Oncogene 1990; 5: 519–524.

    CAS  Google Scholar 

  9. Terman BI, Carrion M, Kovacs E, Rasmussen B, Eddy R, Shows T . Identification of a new endothelial cell growth factor receptor tyrosine kinase. Oncogene 1991; 6: 1677–1683.

    CAS  PubMed  Google Scholar 

  10. Izzedine H . Anti-VEGF cancer therapy in nephrology practice. Int J Nephrol 2014; 2014: 143426.

    Article  Google Scholar 

  11. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z . Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13: 9–22.

    Article  CAS  Google Scholar 

  12. Kazemi-Lomedasht F, Behdani M, Pooshang Bagheri K, Habibi Anbouhi M, Abolhassani M, Khanahmad H et al. Expression and purification of functional human vascular endothelial growth factor-a121; the most important angiogenesis factor. Adv Pharm Bull 2014; 4: 323–328.

    PubMed  PubMed Central  Google Scholar 

  13. Behdani M, Zeinali S, Khanahmad H, Karimipour M, Asadzadeh N, Azadmanesh K et al. Generation and characterization of a functional Nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor. Mol Immunol 2012; 50: 35–41.

    Article  CAS  Google Scholar 

  14. Ferrara N, Hillan KJ, Novotny W . Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys Res Commun 2005; 333: 328–335.

    Article  CAS  Google Scholar 

  15. Sullivan LA, Brekken RA . The VEGF family in cancer and antibody-based strategies for their inhibition. MAbs 2010; 2: 165–175.

    Article  Google Scholar 

  16. Wheeler YY, Kute TE, Willingham MC, Chen S-Y, Sane DC . Intrabody-based strategies for inhibition of vascular endothelial growth factor receptor-2: effects on apoptosis, cell growth, and angiogenesis. FASEB J 2003; 17: 1733–1735.

    Article  CAS  Google Scholar 

  17. Li T, Wang G-d, Tan Y-z, Wang H-j . Inhibition of lymphangiogenesis of endothelial progenitor cells with VEGFR-3 siRNA delivered with PEI-alginate nanoparticles. Int J Biol Sci 2014; 10: 160.

    Article  CAS  Google Scholar 

  18. Wang F, Li H-M, Wang H-P, Ma J-L, Chen X-F, Wei F et al. siRNA-mediated knockdown of VEGF-A, VEGF-C and VEGFR-3 suppresses the growth and metastasis of mouse bladder carcinoma in vivo. Exp Ther Med 2010; 1: 899–904.

    Article  CAS  Google Scholar 

  19. Wang F-q Barfield E, Dutta S, Pua T, Fishman DA . VEGFR-2 silencing by small interference RNA (siRNA) suppresses LPA-induced epithelial ovarian cancer (EOC) invasion. Gynecol Oncol 2009; 115: 414–423.

    Article  Google Scholar 

  20. Cunningham SA, Tran TM, Arrate MP, Brock TA . Characterization of vascular endothelial cell growth factor interactions with the kinase insert domain-containing receptor tyrosine kinase. A real time kinetic study. J Biol Chem 1999; 274: 18421–18427.

    Article  CAS  Google Scholar 

  21. Marasco WA . Intrabodies: turning the humoral immune system outside in for intracellular immunization. Gene Ther 1997; 4: 11–15.

    Article  CAS  Google Scholar 

  22. der Maur AA, Escher D, Barberis A . Antigen-independent selection of stable intracellular single-chain antibodies. FEBS Lett 2001; 508: 407–412.

    Article  Google Scholar 

  23. Carlson JR . A new means of inducibly inactivating a cellular protein. Mol Cell Biol 1988; 8: 2638–2646.

    Article  CAS  Google Scholar 

  24. Lecerf J-M, Shirley TL, Zhu Q, Kazantsev A, Amersdorfer P, Housman DE et al. Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease. Proc Natl Acad Sci 2001; 98: 4764–4769.

    Article  CAS  Google Scholar 

  25. Yu F, Wang Y, Xiao Y, He Y, Luo C, Duan D et al. RP215 single chain fragment variable and single domain recombinant antibodies induce cell cycle arrest at G0/G1 phase in breast cancer. Mol Immunol 2014; 59: 100–109.

    Article  CAS  Google Scholar 

  26. Popkov M, Jendreyko N, McGavern DB, Rader C, Barbas CF . Targeting tumor angiogenesis with adenovirus-delivered anti-Tie-2 intrabody. Cancer Res 2005; 65: 972–981.

    CAS  PubMed  Google Scholar 

  27. Nejatollahi F, Abdi S, Asgharpour M . Antiproliferative and apoptotic effects of a specific antiprostate stem cell single chain antibody on human prostate cancer cells. J Oncol 2013; 2013: 839831.

    Article  Google Scholar 

  28. Sagawa M, Shimizu T, Fukushima N, Kinoshita Y, Ohizumi I, Uno S et al. A new disulfide-linked dimer of a single-chain antibody fragment against human CD47 induces apoptosis in lymphoid malignant cells via the hypoxia inducible factor-1α pathway. Cancer Sci 2011; 102: 1208–1215.

    Article  CAS  Google Scholar 

  29. Intasai N, Tragoolpua K, Pingmuang P, Khunkaewla P, Moonsom S, Kasinrerk W et al. Potent inhibition of OKT3-induced T cell proliferation and suppression of CD147 cell surface expression in HeLa cells by scFv-M6-1B9. Immunobiology 2009; 214: 410–421.

    Article  CAS  Google Scholar 

  30. Zhu X, Yang N, Cai J, Yang G, Liang S, Ren D . The intrabody targeting of hTERT attenuates the immortality of cancer cells. Cell Mol Biol Lett 2010; 15: 32–45.

    Article  CAS  Google Scholar 

  31. Zehner M Sec61 Mediates Antigen Translocation into the Cytosol for Cross-Presentation. Universitäts-und Landesbibliothek Bonn: 2015.

  32. Lippincott-Schwartz J, Bonifacino JS, Yuan LC, Klausner RD . Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Cell 1988; 54: 209–220.

    Article  CAS  Google Scholar 

  33. Fra A, Fagioli C, Finazzi D, Sitia R, Alberini C . Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation. EMBO J 1993; 12: 4755.

    Article  CAS  Google Scholar 

  34. Munro S, Pelham HR . A C-terminal signal prevents secretion of luminal ER proteins. Cell 1987; 48: 899–907.

    Article  CAS  Google Scholar 

  35. Bu G, Rennke S, Geuze HJ . ERD2 proteins mediate ER retention of the HNEL signal of LRP's receptor-associated protein (RAP). J Cell Sci 1997; 110: 65–73.

    CAS  PubMed  Google Scholar 

  36. Behdani M, Zeinali S, Karimipour M, Khanahmad H, Asadzadeh N, Azadmanesh K et al. Expression, purification, and characterization of a diabody against the most important angiogenesis cell receptor: vascular endothelial growth factor receptor 2. Adv Biomed Res 2012; 1: 34.

    Article  Google Scholar 

  37. Gerber H-P, Ferrara N . The role of VEGF in normal and neoplastic hematopoiesis. J Mol Med 2003; 81: 20–31.

    Article  CAS  Google Scholar 

  38. Marasco WA, Haseltine WA, Chen S . Design, intracellular expression, and activity of a human anti-human immunodeficiency virus type 1 gp120 single-chain antibody. Proc Natl Acad Sci 1993; 90: 7889–7893.

    Article  CAS  Google Scholar 

  39. Mhashilkar AM, Lavecchio J, Eberhardt B, Porter-Brooks J, Boisot S, Dove JH et al. Inhibition of human immunodeficiency virus type 1 replication in vitro in acutely and persistently infected human CD4+ mononuclear cells expressing murine and humanized anti-human immunodeficiency virus type 1 Tat single-chain variable fragment intrabodies. Hum Gene Ther 1999; 10: 1453–1467.

    Article  CAS  Google Scholar 

  40. Marasco WA, LaVecchio J, Winkler A . Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS. J Immunol Methods 1999; 231: 223–238.

    Article  CAS  Google Scholar 

  41. Duan L, Bagasra O, Laughlin MA, Oakes JW, Pomerantz RJ . Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody. Proc Natl Acad Sci 1994; 91: 5075–5079.

    Article  CAS  Google Scholar 

  42. Wu Y, Duan L, Zhu M, Hu B, Kubota S, Bagasra O et al. Binding of intracellular anti-Rev single chain variable fragments to different epitopes of human immunodeficiency virus type 1 rev: variations in viral inhibition. J Virol 1996; 70: 3290–3297.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Millauer B, Shawver LK, Plate KH, Risaui W, Ullrich A . Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant. Nature 1994; 367: 576–579.

    Article  CAS  Google Scholar 

  44. Alvarez RD, Barnes MN, Gomez-Navarro J, Wang M, Strong TV, Arafat W et al. A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): a phase I trial. Clin Cancer Res 2000; 6: 3081–3087.

    CAS  PubMed  Google Scholar 

  45. Southwell AL, Ko J, Patterson PH . Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease. J Neurosci 2009; 29: 13589–13602.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the cellular and molecular interaction department-Vrije Universiteit Brussel, Brussels, Belgium. This project was financially supported by the Pasteur Institute of Iran.

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Author notes

  1. E Alirahimi and A Ashkiyan: These authors contributed equally to this work.

Authors and Affiliations

  1. Biotechnology Research Center, Venom & Biotherapeutics Molecules Laboratory, Pasteur Institute of Iran, Tehran, Iran

    E Alirahimi, A Ashkiyan, F Kazemi-Lomedasht, M Hosseininejad-chafi, R Moazami & M Behdani

  2. Virology Department, Pasteur Institute of Iran, Tehran, Iran

    K Azadmanesh

  3. National Cell Bank, Pasteur Institute of Iran, Tehran, Iran

    M Habibi-Anbouhi

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  1. E Alirahimi
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Correspondence to M Behdani.

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Alirahimi, E., Ashkiyan, A., Kazemi-Lomedasht, F. et al. Intrabody targeting vascular endothelial growth factor receptor-2 mediates downregulation of surface localization. Cancer Gene Ther 24, 33–37 (2017). https://doi.org/10.1038/cgt.2016.76

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