Abstract
In this study, the light chain (κ) and heavy chain (γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies.
Similar content being viewed by others
Abbreviations
- VEGF:
-
Vascular endothelial growth factor
- HR:
-
Homologous recombination
- CHO:
-
Chinese hamster ovary
- PCR:
-
Polymerase chain reaction
- SDS-PAGE:
-
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
- HRP:
-
Horse radish peroxidase
- bp:
-
Base pair
- Hygro:
-
Hygromycin
- Neo:
-
Neomycin
- RPM:
-
Rotation per minute
References
Feldman AL, Libutti SK (2000) Progress in anti-angiogenic gene therapy of cancer. Cancer 89:1181–1194
Daniel TO, Abrahamson D (2000) Endothelial signal integration in vascular assembly. Annu Rev Physiol 62:649–671
Breier G (2000) Angiogenesis in embryonic development. A review. Placenta 21(Suppl A):S11–S15
Iruela-Arispe ML, Dvorak HF (1997) Angiogenesis: a dynamic balance of stimulators and inhibitors. Thromb Haemost 78:672–677
Patz A (1980) Studies on retinal neovascularization. Friedenwald Lecture Invest. Ophthalmol Vis Sci 19:1133–1138
McLaren J, Prentice A, Charmock-Jones DS, Millican SA, Muller KH, Sharkey AM, Smith SK (1996) Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 98:482–489
Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1(1):27–31
Ferrara N (2000) VEGF: an update on biological and therapeutic aspects. Curr Opin Biotechnol 11:617–624
Weidner N, Semple JP, Welch W, Folkman J (1991) Tumor angiogenesis and metastasis. Correlation in invasive breast carcinoma. N Engl J Med 324:1–8
Horak ER, Leek R, Klenk N, Lejeune S, Smith K, Stuart M, Greenall M, Harris AL (1992) Quantitative angiogenesis assessed by anti-PECAM antibodies: correlation with node metastasis and survival in breast cancer. Lancet 340:1120–1124
Nugent MA, Iozzo RV (2000) Fibroblast growth factor-2. Int J Biochem Cell Biol 32:115–120
Gimenez-Gallego G, Odkey RJ, Bennett C, Rios-Candelore M, Disalvo J, Thomas K (1985) Brain-derived acidic fibroblast growth factor: complete amino acid sequence and homologies. Science 230:1385–1388
Schweigerer L, Neufeld G, Friedman J, Abraham JA, Fiddes JC, Gospodarowicz D (1987) Capillary endothelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nature 325:257–259
Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V, Ryan TE, Bruno J, Radziejewski C, Maisonpierre PC, Yancopoulos GD (1996) Isolation of angiopoietin-1, a ligand for theTIE2 receptor, by secretion-trap expression cloning. Cell 87:1161–1169
Eliceiri BP, Cheresh DA (1999) The role of alpha v integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J Clin Invest 103:1227–1230
Roberts DD (1996) Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J 10:1183–1191
O’Reilly MS, Holmgren L, Shin Y, Chen C, Rosenthal RA, Moses M, Lane WS, CAO Y, Sage EH, Folkman J (1994) Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79:315–328
O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88(277):285
Petrova TV, Makinen T, Alitalo K (1999) Signaling via vascular endothelial growth factor receptors. Exp Cell Res 253:117–130
Ferrara N, Davis-Smyth T (1997) The biology of vascular endothelial growth factor. Endocr Rev 18:4–25
Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L (2006) VEGF receptor signalling in the control of vascular function. Nat Rev Mol Cell Biol 7:359–371
Berkman RA, Merrill MJ, Rcinhold WC, Monacci WT, Saxena A, Clark WC, Robertson JT, Ali IU, Oldfield EH (1993) Expression of the vascular permeability/vascular endothelial growth factor gene in central nervous system neoplasms. J Clin Invest 91:153–159
Carmeliet P, Lampugnani MG, Moons L, Breviario F, Compernolle V, Bono F, Balconi G, Spagnuolo R, Oostuyse B, Dewerchin M, Zanetti A, Angellilo A, Mattot V, Nuyens D, Lutgens E, Clotman F, de Ruiter M, Gittenberger-de Groot A, Poelmann R, Lupu F, Herbert J, Collen D, Dejana E (1999) Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98:147–157
Kondo A, Asano M, Matsuo K, Ohmori I, Suzuki H (1994) Vascular endothelial growth factor/vascular permeability factor is detectable in the sera of tumor bearing mice and cancer patients. Biochim Biophys Acta 1221:211–214
Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70
Toi M, Hoshima S, Takayanagi T, Tominaga T (1994) Association of vascular endothelial growth factor expression with tumor angiogenesis and with early relapse in primary breast cancer. Jpn J Cancer Res 85:1045–1049
Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA (1991) The human gene for vascular endothelial growth factor. J Biol Chem 267:11947–11954
Ferrara N, Hillan JK, Novotny W (2005) Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys Res Commun 333:328–335
Chames P, Regenmortel MV, Weiss E, Baty D (2009) Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol 157:220–233
Frentzena A, Yua YA, Chena N, Zhanga Q, Weibelb S, Raabb V, Szalay AA (2009) Anti-VEGF single-chain antibody GLAF-1 encoded by the oncolytic vaccinia virus significantly enhances antitumor therapy. Proc Natl Acad Sci USA 106(31):12915–12920
Fellouse FA, Sidhu SS (2006) Synthetic antibody libraries. In: Sidhu SS (ed) Phage display in biotechnology and drug discovery, 1st edn. Taylor and Francis Group, Boca Raton, FL, pp 709–740
Nissim A, Chernajovsky Y (2008) Historical development of monoclonal antibody therapeutic. Ther Antib Handb Exp pharmacol 181:3–17
Binyamin L, Borghaei H, Weiner L (2006) Cancer therapy engineered monoclonal antibodies. Cancer Ther 1:147–157
Shan D, Ledbetter JA, Ress POW (1998) Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 911:644–1652
Rang HP (2003) Pharmacology. Churchill Livingstone, Edinburgh, p 241, for the examples infliximab, basiliximab, abciximab, daclizumab, palivusamab, gemtuzumab, alemtuzumab and rituximab, and mechanism and mode. ISBN 0-443-07145-4
Trikha M, Yan L, Nakada MT (2002) Monoclonal antibodies as therapeutics in oncology. Curr Opin Biotechnol 13(6):609–614
Goldenberg MM (1999) Trastuzumab, a recombinant DNA-derived humanized monoclonal antibody, a novel agent for the treatment of metastatic breast cancer. Clin Ther 21:309–318
Herbst RS, Langer CJ (2002) Epidermal growth factor receptors as a target for cancer treatment: the emerging role of IMC-C225 in the treatment of lung and head and neck cancers. Semin Oncol 29:27–36
Prewett M, Rockwell PRockwell RF, Giorgio NA, Mendelsohn J, Scher HI, Goldstein NI (1996) The biologic effects of C225, a chimeric monoclonal antibody to the EGFR, on human prostate carcinoma. J Immunother Emphasis Tumor Immunol 19:419–427
Presta LG, Chen H, O’Connor SJ, Chisholm V, Meng YG, Krummen L, Winkler M, Ferrara N (1997) Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 57(20):4593–4599
Kim YGu, Byoungwoo P, Ahn JO, Jung JK, Lee HW, Lee EG (2012) New cell line development of antibody producing Chinese hamster ovary cells using split green fluorescent protein. BMC Biotechnol 12:24
Li F, Vijayasankaran N, Shen A, Kiss R, Amanullah A (2010) Cell culture processes for monoclonal antibody production. MAbs 2(5):466–477
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA 97(11):5978–5983
Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4(3):251–256
Yamamoto T, Moerschell RP, Wakem LP, Ferguson D, Sherman F (1992) Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast. Yeast 8(11):935–948
Liu P, Jenkins NA, Copeland NG (2003) A highly efficient recombineering-based method for generating conditional knockout mutations. J Genome Res 13:476–484
Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. J Ann Rev Gene 36:361–388
Mould R, Pondel MD (2003) Calcitonin receptor gene expression in K562 chronic myelogenous leukemic cells. Cancer Cell Int 3:6
Kim HR, Kang HS, Kim HD (1999) Geldanamycin induces heat shock protein expression through activation of HSF1 in K562 erythroleukemic cells. IUBMB Life 48(4):429–433
Habibi Roudkenar M, Mohammadipour M, Oodi A, Halabian R, Amirizadeh N, Masrouri N, Mohammadi Roshandeh M, parivar K, Rezvan H (2009) Stable expression of recombinant RhD antigen isolated from cord blood in K562 cell line. IJBC 2:69–73
Mould R, Ponde MD (2003) Calcitonin receptor gene expression in K562 chronic myelogenous leukemic cells. Cancer Cell Int 3:6
Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4(2):206–223
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 98:503–517
Thomason L, Court DL, Bubunenko M, Costantino N, Wilson H, Datta S, Oppenheim A (2007) Recombineering: genetic engineering in bacteria using homologous recombination. J Curr Protoc Mol Biol 1.16.1–1.16.24
Eriksson U, Alitalo K (1999) Structure, expression and receptor–binding properties of novel vascular endothelial growth factors. Curr Top Microbiol Immunol 237:41–57
Cheng SY, Huang HJ, Nagane M, Ji XD, Wang D, Shih CC, Arap W, Huang CM, Cavenee WK (1996) Suppression of glioblastoma angiogenicity and tumor igenicity by inhibition of endogenous expression of vascular endothelial growth factor. Proc Natl Acad Sci USA 93:8502–8507
Im SA, Gomez–Manzano C, Fueyo J, Liu TJ, Ke LD, Kim JS, Lee HY, Steck PA, Kyritsis AP, Yung WKA (1999) Antiangiogenesis treatment for gliomas: transfer of antisense–vascular endothelial growth factor inhibits tumor growth in vivo. Cancer Res 59:895–900
Sipkins DA, Cheresh DA, Kazemi MR, Nevin LM, Bednarski MD, Li KC (1998) Detection of tumor angiogenesis in vivo by alphaVbeta3-targeted magnetic resonance imaging. Nat Med 4:623–626
Kohn EC, Felder CC, Jacobs W, Holmes KA, Day A, Freer R, Liotta LA (1994) Structure–function analysis of signal and growth inhibition by carboxyamido–triazole CAI. Cancer Res 54:935–942
Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 93:705–716
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hajirezaei, M., Darbouy, M. & Kazemi, B. Cloning and Expression of the Functional Human Anti-vascular Endothelial Growth Factor (VEGF) Using the pcDNA3.1 Vector and the Human Chronic Myelogenous Leukemia Cell Line K562. Protein J 33, 100–109 (2014). https://doi.org/10.1007/s10930-013-9533-y
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10930-013-9533-y