Abstract
Aptamers are an emerging class of molecules in cancer therapy. They can be easily synthesized and are considered cost-effective drug candidates. In this book chapter we describe the selection and characterization of DNA aptamers specific to the human epidermal growth factor receptor 2 (ERBB2/HER2), an oncogenic tyrosine kinase. First, a DNA aptamer library is applied and ERBB2-specific aptamers are selected using SELEX. Binders are subcloned into a pGEM-T vector, sequenced, and characterized using biochemical and cell biological techniques. By multimerizing the selected ERBB2 aptamers, it might be possible to significantly increase their avidity. For example, we could show that a trimeric ERBB2-specific aptamer could efficiently internalize membranal ERBB2. Furthermore, the receptor assembled in cytoplasmic puncta and was finally degraded by the lysosome. In addition, the selected, trimeric aptamer inhibited proliferation in an XTT assay in comparison to a control sequence. Aptamers selected using the protocol we describe might exert anticancer effect. In our example of a trimeric anti-HER2 aptamer, we could report that a human gastric xenograft mouse model demonstrated pharmacological value of the selected aptamer in vivo. This chapter should enable the interested reader to replicate selection of DNA aptamers specific to oncogenic cell surface. We would like to particularly emphasize some experimental approaches which were used to further characterize selected aptamer sequences, upon SELEX selection. For instance, we included several blotting techniques, antiproliferative assays of aptamers in vitro, and describe the handling of an in vivo human xenograft mouse model.
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References
Köhler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256(5517):495–497
Ben-Kasus T, Schechter B, Sela M, Yarden Y (2007) Cancer therapeutic antibodies come of age: targeting minimal residual disease. Mol Oncol 1(1):42–54
Mahlknecht G, Maron R, Mancini M, Schechter B, Sela M, Yarden Y (2013) Aptamer to ErbB-2/HER2 enhances degradation of the target and inhibits tumorigenic growth. Proc Natl Acad Sci U S A 110(20):8170–8175
Stoltenburg R, Reinemann C, Strehlitz B (2007) SELEX- (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng 24:381–403
Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822
Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–515
Tombelli S, Minunni M, Mascini M (2005) Analytical applications of aptamers. Biosens Bioelectron 20:2424–2434
Hermann T, Patel DJ (2000) Adaptive recognition by nucleic acid aptamers. Biochemistry 287:820–825
Guo KT, Paul A, Schichor C, Ziemer G, Wendel HP (2008) CELL-SELEX: novel perspectives of aptamer-based therapeutics. Int J Mol Sci 9:668–678
Citri A, Yarden Y (2006) EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 7:505–516
Rowinsky EK (2004) The ErbB Family: targets for therapeutic development against cancer and therapeutic Strategies using monoclonal antibodies and tyrosine kinase inhibitors. Annu Rev Med 55:433–457
Chen CH, Chernis GA, Hoang VQ, Landgraf R (2003) Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3. Proc Natl Acad Sci USA 105:15908–15913
Dastjerdi K, Tabar GH, Dehghani H, Haghparast A (2011) Generation of an enriched pool of DNA aptamers for a HER2-overexpressing cell line selected by Cell-SELEX. Biotechnol Appl Biochem 58(4):226–230
Esposito CL, Passaro D, Longobardo I, Condorelli G, Marotta P, Affuso A, De Franciscis V, Cerchia L (2011) A neutralizing RNA aptamer against EGFR causes selective apoptotic cell death. PLoS One 6(9):e24071
Kang HS, Huh YM, Kim S, Lee D (2009) Isolation of RNA aptamers targeting HER-2-overexpressing breast cancer cells using Cell-SELEX. Bull Korean Chem Soc 30(8):1827–1831
Kim MY, Jeong S (2011) In vitro Selection of RNA aptamer and specific targeting of ErbB2 in breast cancer cells. Nucleic Acid Ther 21(3):173–178
Li N, Nguyen HH, Byrom M, Ellington AD (2011) Inhibition of cell proliferation by an anti-EGFR aptamer. PLoS One 6(6):e20299
Thiel KW, Hernandez LI, Dassie JP, Thiel WH, Liu X, Stockdale KR, Rothman AM, Hernandez FJ, McNamara JO 2nd, Giangrande PH (2012) Delivery of chemo-sensitizing siRNAs to HER2+− breast cancer cells using RNA aptamers. Nucleic Acids Res 40(13):6319–6337
Missailidis S (2003) Targeting of antibodies using aptamers. In: Lo BKC (ed) Antibody engineering: methods and protocols, vol 51. Humana Press, New York, pp 547–555
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Mahlknecht, G., Sela, M., Yarden, Y. (2015). Aptamer Targeting the ERBB2 Receptor Tyrosine Kinase for Applications in Tumor Therapy. In: Walther, W., Stein, U. (eds) Gene Therapy of Solid Cancers. Methods in Molecular Biology, vol 1317. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2727-2_1
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DOI: https://doi.org/10.1007/978-1-4939-2727-2_1
Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-2727-2
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