Angiogenesis

, Volume 10, Issue 1, pp 23–34 | Cite as

Prophylactic naked DNA vaccination with the human vascular endothelial growth factor induces an anti-tumor response in C57Bl/6 mice

  • Mónica Bequet-Romero
  • Marta Ayala
  • Boris E. Acevedo
  • Ernesto Galbán Rodríguez
  • Omar López Ocejo
  • Isis Torrens
  • Jorge V. Gavilondo
Original Paper

Abstract

Passive immunotherapy against soluble pro-angiogenic factors and/or their receptors in endothelial cells has become a promising approach in cancer therapeutics. There is also experimental evidence indicating that an active immunotherapy strategy directed towards these target molecules could also be effective. In this paper we show that it is possible to reduce tumor growth or increase the survival of tumor-bearing C57Bl/6 mice when animals are vaccinated with the human vascular endothelial growth factor (VEGF) isoform 121 gene (hVEGF121), and later challenged with melanoma or lung carcinoma tumor cells. Immunization was done with 10 μg DNA doses of the hVEGF121 gene, which is highly homologous to its mouse counterpart, administered on a weekly basis using a plasmid bearing 5 CpG bacterial motifs. Histopathology analyses of tumors of hVEGF121 immunized animals showed a decrease in tumor cell density around vessels and in mitotic figures, as well as an increase in apoptotic tumor cells. A statistically significant cell cytotoxic response was found when spleen cells of immunized mice were co-cultured in vitro with mouse tumor VEGF-producing cells. Vaccination with an hVEGF121 gene mutated to make it deficient for VEGF receptor binding, produced similar in vitro and in vivo results, and significantly reduced the number of spontaneous metastases produced by the mouse Lewis lung carcinoma. Our results indicate that human VEGF DNA can be employed for anti-angiogenic active immunotherapy in mice, and that direct cell cytotoxicity is a contributor mechanism to the overall anti-tumor effects seen in immunized animals.

Keywords

Angiogenesis Cancer Immunotherapy VEGF DNA immunization 

References

  1. 1.
    Folkman J (2002) Role of angiogenesis in tumor growth and metastasis. Semin Oncol 29:15–18PubMedGoogle Scholar
  2. 2.
    Folkman J (2003) Angiogenesis and apoptosis. Semin Cancer Biol 13:159–167PubMedCrossRefGoogle Scholar
  3. 3.
    Ohm JE, Carbone DP (2001) VEGF as a mediator of tumor-associated immunodeficiency. Immunol Res 23:263–272PubMedCrossRefGoogle Scholar
  4. 4.
    Ferrara N, Gerber HP, Lecouter J (2003) The biology of VEGF and its receptors. Nat Med 9:669–676PubMedCrossRefGoogle Scholar
  5. 5.
    Ferrara N, Hillan KJ, Novotny W (2005) Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys Res Commun 333:328–335PubMedCrossRefGoogle Scholar
  6. 6.
    Ferrara N, Hillan KJ, Gerber HP, Novotny W (2004) Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 3:391–400PubMedCrossRefGoogle Scholar
  7. 7.
    Midgley R, Kerr D (2005) Bevacizumab – current status and future directions. Ann Oncol 16:999–1004PubMedCrossRefGoogle Scholar
  8. 8.
    Jayson GC, Zweit J, Jackson A et al (2002) Molecular imaging and biological evaluation of HuMV833 anti-VEGF antibody: implications for trial design of antiangiogenic antibodies. J Natl Cancer Inst 94:1484–1493PubMedGoogle Scholar
  9. 9.
    Zhang W, Ran S, Sambade M, Huang X, Thorpe PE (2002) A monoclonal antibody that blocks VEGF binding to VEGFR2 (KDR/Flk-1) inhibits vascular expression of Flk-1 and tumor growth in an orthotopic human breast cancer model. Angiogenesis 5:35–44PubMedCrossRefGoogle Scholar
  10. 10.
    Zhu Z, Hattori K, Zhang H et al (2003) Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity. Leukemia 17:604–611PubMedCrossRefGoogle Scholar
  11. 11.
    Tartour E, Fridman WH (2000) Cancer vaccine 2000. Immunol Lett 74:1–3PubMedCrossRefGoogle Scholar
  12. 12.
    Wei YQ, Huang MJ, Yang L et al (2001) Immunogene therapy of tumors with vaccine based on Xenopus homologous vascular endothelial growth factor as a model antigen. Proc Natl Acad Sci USA 98:11545–11550PubMedCrossRefGoogle Scholar
  13. 13.
    Zagury D, Gallo RC (2004) Anti-cytokine Ab immune therapy: present status and perspectives. Drug Discov Today 9:72–81PubMedCrossRefGoogle Scholar
  14. 14.
    Liu JY, Wei YQ, Yang L et al (2003) Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood 102:1815–1823PubMedCrossRefGoogle Scholar
  15. 15.
    Lu F, Qin ZY, Yang WB, Qi YX, Li YM (2004) A DNA vaccine against extracellular domains 1–3 of flk-1 and its immune preventive and therapeutic effects against H22 tumor cell in vivo. World J Gastroenterol 10:2039–2044PubMedGoogle Scholar
  16. 16.
    Niederman TM, Ghogawala Z, Carter BS, Tompkins HS, Russell MM, Mulligan RC (2002) Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors. Proc Natl Acad Sci USA 99:7009–7014PubMedCrossRefGoogle Scholar
  17. 17.
    Reisfeld RA, Niethammer AG, Luo Y, Xiang R (2004) DNA vaccines designed to inhibit tumor growth by suppression of angiogenesis. Int Arch Allergy Immunol 133:295–304PubMedCrossRefGoogle Scholar
  18. 18.
    Herrera AM, Rodriguez EG, Hernandez T, Sandez B, Duarte CA (2000) A family of compact plasmid vectors for DNA immunization in humans. Biochem Biophys Res Commun 279:548–551PubMedCrossRefGoogle Scholar
  19. 19.
    Lin KY, Guarnieri FG, Staveley-O’Carroll KF et al (1996) Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 56:21–26PubMedGoogle Scholar
  20. 20.
    Eisenbach L, Hollander N, Greenfeld L, Yakor H, Segal S, Feldman M (1984) The differential expression of H-2K versus H-2D antigens, distinguishing high-metastatic from low-metastatic clones, is correlated with the immunogenic properties of the tumor cells. Int J Cancer 34:567–573PubMedCrossRefGoogle Scholar
  21. 21.
    Rodriguez EG, Vazquez DM, Herrera AM, Duarte CA (2003) Enhanced cell-mediated INF-gamma secreting activity against the HIV IIIB V3 peptide of the TAB9 multiepitope after DNA vaccine backbone engineering. Biochem Biophys Res Commun 308:713–718Google Scholar
  22. 22.
    Shen BQ, Lee DY, Gerber HP, Keyt BA, Ferrara N, Zioncheck TF (1998) Homologous up-regulation of KDR/Flk-1 receptor expression by vascular endothelial growth factor in vitro. J Biol Chem 273:29979–29985PubMedCrossRefGoogle Scholar
  23. 23.
    Ojalvo AG, Seralena A, Vázquez R et al (2003) Therapeutic angiogenesis following intramuscular gene transfer of vascular endothelial growth factor 121 in a dog model of hindlimb ischemia. Electronic J Biotechnol 6:208–222Google Scholar
  24. 24.
    Li Y, Wang MN, Li H et al (2002) Active immunization against the vascular endothelial growth factor receptor flk1 inhibits tumor angiogenesis and metastasis. J Exp Med 195:1575–1584PubMedCrossRefGoogle Scholar
  25. 25.
    Wyllie AH (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284:555–556PubMedCrossRefGoogle Scholar
  26. 26.
    Morera Y, Lamdan H, Bequet M et al (2006) Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S-transferase fusion protein. Biotechnol Appl Biochem 44:45–53PubMedCrossRefGoogle Scholar
  27. 27.
    Li Z, Zhang H, Fan X et al (2006) DNA electroporation prime and protein boost strategy enhances humoral immunity of tuberculosis DNA vaccines in mice and non-human primates. Vaccine 24:4565–4568PubMedCrossRefGoogle Scholar
  28. 28.
    Jakob T, Walker PS, Krieg AM, Udey MC, Vogel JC (1998) Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA. J Immunol 161:3042–3049PubMedGoogle Scholar
  29. 29.
    Davis HL, Weeratna R, Waldschmidt TJ, Tygrett L, Schorr J, Krieg AM (1998) CpG DNA is a potent enhancer of specific immunity in mice immunized with recombinant hepatitis B surface antigen. J Immunol 160:870–876PubMedGoogle Scholar
  30. 30.
    Sun S, Kishimoto H, Sprent J (1998) DNA as an adjuvant: capacity of insect DNA and synthetic oligodeoxynucleotides to augment T cell responses to specific antigen. J Exp Med 187:1145–1150PubMedCrossRefGoogle Scholar
  31. 31.
    Plum SM, Fogler WE (2004) Anti-angiogenic vaccines as a treatment modality for cancer. Curr Opin Investig Drugs 5:1243–1246PubMedGoogle Scholar
  32. 32.
    Brand A (2002) Immunological aspects of blood transfusions. Transpl Immunol 10:183–190PubMedCrossRefGoogle Scholar
  33. 33.
    Niethammer AG, Xiang R, Becker JC et al (2002) A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat Med 8:1369–1375PubMedCrossRefGoogle Scholar
  34. 34.
    Ferrara N, Winer J, Burton T et al (1993) Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells. J Clin Invest 91:160–170PubMedCrossRefGoogle Scholar
  35. 35.
    Agnantis NJ, Goussia AC, Batistatou A, Stefanou D (2004) Tumor markers in cancer patients. an update of their prognostic significance. Part II. In Vivo 18:481–488PubMedGoogle Scholar
  36. 36.
    Shinkaruk S, Bayle M, Lain G, Deleris G (2003) Vascular endothelial cell growth factor (VEGF), an emerging target for cancer chemotherapy. Curr Med Chem Anti -Canc Agents 3:95–117CrossRefGoogle Scholar
  37. 37.
    Keyt BA, Nguyen HV, Berleau LT et al (1996) Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis. J Biol Chem 271:5638–5646PubMedCrossRefGoogle Scholar
  38. 38.
    Kim KJ, Li B, Houck K, Winer J, Ferrara N (1992) The vascular endothelial growth factor proteins: identification of biologically relevant regions by neutralizing monoclonal antibodies. Growth Factors 7:53–64PubMedGoogle Scholar
  39. 39.
    Kitamoto Y, Tokunaga H, Miyamoto K, Tomita K (2002) VEGF is an essential molecule for glomerular structuring. Nephrol Dial Transplant 17(Suppl 9):25–27PubMedCrossRefGoogle Scholar
  40. 40.
    Herbst RS, Sandler AB (2004) Non-small cell lung cancer and antiangiogenic therapy: what can be expected of bevacizumab? Oncologist 9 Suppl 9(Suppl 1):19–26CrossRefGoogle Scholar
  41. 41.
    Sandler AB, Johnson DH, Herbst RS (2004) Anti-vascular endothelial growth factor monoclonals in non-small cell lung cancer. Clin Cancer Res 10:4258s–4262sPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media B.V. 2007

Authors and Affiliations

  • Mónica Bequet-Romero
    • 1
  • Marta Ayala
    • 1
  • Boris E. Acevedo
    • 1
  • Ernesto Galbán Rodríguez
    • 2
  • Omar López Ocejo
    • 1
  • Isis Torrens
    • 1
  • Jorge V. Gavilondo
    • 1
  1. 1.Recombinant Antibodies Laboratory, Cancer Research DepartmentCenter for Genetic Engineering and BiotechnologyHavanaCuba
  2. 2.Vaccine DivisionCenter for Genetic Engineering and BiotechnologyHavanaCuba

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