Abstract
This protocol describes the generation of monoclonal antibodies in single-chain variable fragment (scFv)-Fc format. It includes the cloning of the scFv-Fc expression cassette into a mammalian expression vector followed by transient transfection of mammalian cells and purification by protein A affinity chromatography. The protocol is intended for applications in basic and preclinical research that require rapid access to milligram amounts of protein.
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References
Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136
Kontermann RE (2010) Alternative antibody formats. Curr Opin Mol Ther 12:176–183
Shu L, Qi CF, Schlom J et al (1993) Secretion of a single-gene-encoded immunoglobulin from myeloma cells. Proc Natl Acad Sci USA 90:7995–7999
Zhao X, Lapalombella R, Joshi T et al (2007) Targeting CD37-positive lymphoid malignancies with a novel engineered small modular immunopharmaceutical. Blood 11:2569–2577
Robak T, Robak P, Smolewski P (2009) TRU-016, a humanized anti-CD37 IgG fusion protein for the potential treatment of B-cell malignancies. Curr Opin Investig Drugs 10:1383–1390
Hayden-Ledbetter MS, Cerveny CG, Espling E et al (2009) CD20-directed small modular immunopharmaceutical, TRU-015, depletes normal and malignant B cells. Clin Cancer Res 15:2739–2746
Rubbert-Roth A (2010) TRU-015, a fusion protein derived from an anti-CD20 antibody, for the treatment of rheumatoid arthritis. Curr Opin Mol Ther 12:115–123
Brocks B, Rode HJ, Klein M et al (1997) A TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono- and bivalent scFv derivative in insect cells. Immunotechnology 3:173–184
Powers DB, Amersdorfer P, Poul M et al (2001) Expression of single-chain Fv-Fc fusions in Pichia pastoris. J Immunol Methods 251:123–135
Van Droogenbroeck B, Cao J, Stadlmann J et al (2007) Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proc Natl Acad Sci USA 104:1430–1435
Cao P, Zhang S, Gong Z et al (2006) Development of a compact anti-BAFF antibody in Escherichia coli. Appl Microbiol Biotechnol 73:151–157
Acknowledgments
This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health. We thank Marko Modric for comments.
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Yang, J., Rader, C. (2012). Cloning, Expression, and Purification of Monoclonal Antibodies in scFv-Fc Format. In: Proetzel, G., Ebersbach, H. (eds) Antibody Methods and Protocols. Methods in Molecular Biology, vol 901. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-931-0_14
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DOI: https://doi.org/10.1007/978-1-61779-931-0_14
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-930-3
Online ISBN: 978-1-61779-931-0
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