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Generation and characterization of a novel human IgG1 antibody against vascular endothelial growth factor receptor 2

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Abstract

VEGF and its receptors, especially VEGFR2 (KDR), are known to play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies. This study was aimed at developing a fully human IgG1 antibody (mAb-04) constructed from a phage-derived scFv, targeting the VEGF/VEGFR2 pathway. Firstly, an innovative transfection system, containing two recombinant expression vectors (pMH3 and pCApuro), were introduced into CHO-s cells and clones with higher yield selected accordingly. After an optimal fermentation condition was determined, fed-batch fermentation was performed in 5-L bioreactor with a final yield up to 60 mg/L. Further, cell proliferation, wound healing, transwell invasion, tube formation and chick embryo chorioallantoic membrane assays showed significant anti-angiogenic activity of mAb-04 in vitro and in vivo. In addition, the results of Western blotting indicated the ability of mAb-04 to inhibit VEGF-induced VEGFR2 signaling pathway. Finally, ADCC assay demonstrated that mAb-04 is capable of mediating tumor cell killing in presence of effector cells. This study has therefore proved that the full-length antibody targeting human VEGFR2 has potential clinical applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

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Abbreviations

ADCC:

Antibody-dependent cellular cytotoxicity

bFGF:

Basic fibroblast growth factor

CAM:

Chick embryo chorioallantoic membrane

ECGS:

Endothelial cell growth supplement

ECM:

Endothelial culture medium

FBS:

Fetal bovine serum

HAMA:

Human anti-mouse antibody

KDR:

Kinase insert domain-containing receptor

KDR3:

The extracellular domain 3 of human VEGFR2

LDH:

Lactate dehydragenase

scFv:

Single-chain antibody fragment

VEGF:

Vascular endothelial growth factor

VEGFR2:

Vascular endothelial growth factor receptor 2

References

  1. Ozato K, Mayer N, Sachs DH (1980) Hybridoma cell lines screting monoclonal antibodies to mouse H-2 and la antigens. J Immunol 124(2):533–540

    CAS  PubMed  Google Scholar 

  2. Yagami H, Kato H, Tsumoto K, Tomita M (2013) Monoclonal antibodies based on hybridoma technology. Pharm Pat Anal 2(2):249–263

    Article  CAS  PubMed  Google Scholar 

  3. Dubrot J, Portero A, Orive G, Hernández RM, Palazón A, Rouzaut A, Perez-Gracia JL, Hervás-Stubbs S, Pedraz JL, Melero I (2010) Delivery of immunostimulatory monoclonal antibodies by encapsulated hybridoma cells. Cancer Immunol Immunother 59(11):1621–1631

    Article  CAS  PubMed  Google Scholar 

  4. Tjandra JJ, Ramadi L, McKenzie IF (1990) Development of human anti-murine antibody (HAMA) response in patients. Immunol Cell Biol 68(6):367–376

    Article  PubMed  Google Scholar 

  5. Azinovic I, DeNardo GL, Lamborn KR, Mirick G, Goldstein D, Bradt BM, DeNardo SJ (2006) Survival benefit associated with human anti-mouse antibody (HAMA) in patients with B-cell malignancies. Cancer Immunol Immunother 55(12):1451–1458

    Article  CAS  PubMed  Google Scholar 

  6. Morrison SL (1985) Transfectomas provide novel chimeric antibodies. Science 229(4719):1202–1207

    Article  CAS  PubMed  Google Scholar 

  7. Presta LG, Lahr SJ, Shields RL, Porter JP, Gorman CM, Fendly BM, Jardieu PM (1993) Humanization of an antibody directed against IgE. J Immunol 151(5):2623–2632

    CAS  PubMed  Google Scholar 

  8. Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage display technology. Annu Rev Immunol 12:433–455

    Article  CAS  PubMed  Google Scholar 

  9. Cen X, Bi Q, Zhu S (2006) Construction of a large phage display antibody library by in vitro package and in vivo recombination. Appl Microbiol Biotechnol 71(5):767–772

    Article  CAS  PubMed  Google Scholar 

  10. Griffiths AD, Duncan AR (1998) Strategies for selection of selection of antibody phage display. Curr Opin Biotechnol 9(1):102–108

    Article  CAS  PubMed  Google Scholar 

  11. Dübel S, Stoevesandt O, Taussig MJ, Hust M (2010) Generating recombinant antibodies to the complete human proteome. Trends Biotechnol 28(7):333–339

    Article  PubMed  Google Scholar 

  12. Auguste P, Lemiere S, Larrieu-Lahargue F, Bikfalvi A (2005) Molecular mechanisms of tumor vascularization. Crit Rev Oncol Hematol 54(1):53–61

    Article  PubMed  Google Scholar 

  13. Kiselyov A, Balakin KV, Tkachenko SE (2007) VEGF/VEGFR signalling as a target for inhibiting angiogenesis. Expert Opin Investig Drugs 16(1):83–107

    Article  CAS  PubMed  Google Scholar 

  14. Roskoski R Jr (2007) Vascular endothelial growth factor (VEGF) signaling in tumor progression. Crit Rev in Oncol Hematol 62(3):179–213

    Article  Google Scholar 

  15. Miao HQ, Hu K, Jimenez X, Navarro E, Zhang H, Lu D, Ludwig DL, Balderes P, Zhu Z (2006) Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2. Biochem Biophys Res Commun 345(1):438–445

    Article  CAS  PubMed  Google Scholar 

  16. Zhang J, Li H, Wang X, Qi H, Miao X, Zhang T, Chen G, Wang M (2012) Phage-derived fully human antibody scFv fragment directed against human vascular endothelial growth factor receptor 2 blocked its interaction with VEGF. Biotechnol Prog 28(4):981–989

    Article  PubMed  Google Scholar 

  17. Carmeliet P (2003) Angiogenesis in health and disease. Nat Med 9(6):653–660

    Article  CAS  PubMed  Google Scholar 

  18. Folkman J, Shing Y (1992) Angiogenesis. J Biol Chem 267(16):10931–10934

    CAS  PubMed  Google Scholar 

  19. Ziyad S, Iruela-Arispe ML (2011) Molecular mechanisms of tumor angiogenesis. Genes Cancer 2(12):1085–1096

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Shibuya M, Claesson-Welsh L (2006) Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis. Exp Cell Res 312(5):549–560

    Article  CAS  PubMed  Google Scholar 

  21. Li H, Cao W, Chen Z, Acheampong DO, Jin H, Li D, Zhang J, Wang M (2013) The antiangiogenic activity of a soluble fragment of the VEGFR extracellular domain. Biomed Pharmacother 67(7):599–606

    Article  CAS  PubMed  Google Scholar 

  22. Wahl O, Oswald M, Tretzel L, Herres E, Arend J, Efferth T (2011) Inhibition of tumor angiogenesis by antibodies, synthetic small molecules and natural products. Curr Med Chem 18(21):3136–3155

    Article  CAS  PubMed  Google Scholar 

  23. Ferrara N, Kerbel RS (2005) Angiogenesis as a therapeutic target. Nature 438(7070):967–974

    Article  CAS  PubMed  Google Scholar 

  24. Saharinen P, Eklund L, Pulkki K, Bono P, Alitalo K (2011) VEGF and angiopoietin signaling in tumor angiogenesis and metastasis. Trends Mol Med 17(7):347–362

    Article  CAS  PubMed  Google Scholar 

  25. Ferrara N (2010) Pathways mediating VEGF-independent tumor angiogenesis. Cytokine Growth Factor Rev 21(1):21–26

    Article  CAS  PubMed  Google Scholar 

  26. Zhang J, Li H, Chen W, Cao P, Wang M (2009) Preparation of extracellular domain 3 of human VEGF receptor-2 and the monitoring of its real-time binding to VEGF by biosensors. Biotechnol Prog 25(6):1703–1708

    CAS  PubMed  Google Scholar 

  27. Jia Q, Wu H, Zhou X et al (2010) A “GC-rich” method for mammalian gene expression: a dominant role of non-coding DNA GC content in regulation of mammalian gene expression. Sci China Life Sci 53(1):94–100

    CAS  PubMed  Google Scholar 

  28. Zhang F, Zhou Z, Xu X, Wang X, Sullivan C (2008) A bizarre Jurassic maniraptoran from China with elongate ribbon-like feathers. Nature 455(7216):1105–1108

    Article  CAS  PubMed  Google Scholar 

  29. Kilani J, Lebeault JM (2007) Study of the oxygen transfer in a disposable flexible bioreactor with surface aeration in vibrated medium. Appl Microbiol Biotechnol 74(2):324–330

    Article  CAS  PubMed  Google Scholar 

  30. Warnock JN, Al-Rubeai M (2006) Bioreactor systems for the production of biopharmaceuticals from animal cells. Biotechnol Appl Biochem 45(1):1–12

    Article  CAS  PubMed  Google Scholar 

  31. Kaja S, Hilgenberg JD, Everett E, Olitsky SE, Gossage J, Koulen P (2011) Effects of dilution and prolonged storage with preservative in a polyethylene container on Bevacizumab (Avastin™) for topical delivery as a nasal spray in anti-hereditary hemorrhagic telangiectasia and related therapies. Hum Antibodies 20(3–4):95–101

    CAS  PubMed Central  PubMed  Google Scholar 

  32. Chen S 4th, Karnezis T, Davidson TM (2011) Safety of intranasal Bevacizumab (Avastin) treatment in patients with hereditary hemorrhagic telangiectasia-associated epistaxis. Laryngoscope 121(3):644–646

    Article  CAS  PubMed  Google Scholar 

  33. Karnezis TT, Davidson TM (2011) Efficacy of intranasal bevacizumab (Avastin) treatment in patients with hereditary hemorrhagic telangiectasia-associated epistaxis. Laryngoscope 121(3):636–638

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This project was supported by the National Natural Science Foundation of China (NSFC81072561, NSFC81102364 and NSFC81273425). The Project Program of State Key Laboratory of Natural Medicines (China Pharmaceutical University, JKGP201101). Jiangsu Province Qinglan Project (2010). A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Graduate Student Innovation Project Funded by Huahai Pharmaceutical Co. (CX13S-009HH). Revision by Professor Sherie L. Morrison.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, 154#, Tong Jia Xiang 24, Nanjing, 210009, People’s Republic of China

    Wei Xie, Daojuan Li, Juan Zhang, Zhike Li, Desmond Omane Acheampong, Yuan He, Youfu Wang, Zhiguo Chen & Min Wang

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  1. Wei Xie
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  2. Daojuan Li
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  3. Juan Zhang
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  4. Zhike Li
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  5. Desmond Omane Acheampong
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  6. Yuan He
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  7. Youfu Wang
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  8. Zhiguo Chen
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  9. Min Wang
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Corresponding authors

Correspondence to Juan Zhang or Min Wang.

Additional information

The authors Wei Xie and Daojuan Li contributed equally to this work.

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Xie, W., Li, D., Zhang, J. et al. Generation and characterization of a novel human IgG1 antibody against vascular endothelial growth factor receptor 2. Cancer Immunol Immunother 63, 877–888 (2014). https://doi.org/10.1007/s00262-014-1560-9

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  • DOI: https://doi.org/10.1007/s00262-014-1560-9

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