Abstract
The scFv-Fc format allows for rapid characterization of candidate scFvs isolated from phage display libraries before conversion into a full-length IgG. This format offers several advantages over the phage display-derived scFv, including bivalent binding, longer half-life, and Fc-mediated effector functions. Here, a detailed method is presented, which describes the cloning, expression, and purification of an scFv-Fc fragment, starting from scFv fragments obtained from a phage display library. This method facilitates the rapid screening of candidate antibodies, prior to a more time-consuming conversion into a full IgG format. Alternatively, the scFv-Fc format may be used in the clinic for therapeutic applications.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Walsh G (2010) Biopharmaceutical benchmarks 2010. Nat Biotechnol 28:917–924
Villa A, Lovato V, Bujak E et al (2011) A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin. MAbs 3:264–272
Silacci M, Brack S, Schirru G et al (2005) Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics 5:2340–2350
Cuesta ÁM, Sainz-Pastor N, Bonet J et al (2010) Multivalent antibodies: when design surpasses evolution. Trends Biotechnol 28:355–362
Tarli L, Balza E, Viti F et al (1999) A high-affinity human antibody that targets tumoral blood vessels. Blood 94:192–198
Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136
Zuberbühler K, Palumbo A, Bacci C et al (2009) A general method for the selection of high-level scFv and IgG antibody expression by stably transfected mammalian cells. Protein Eng Des Sel 22:169–174
Crothers D, Metzger H (1972) The influence of polyvalency on the binding properties of antibodies. Immunochemistry 9:341–357
Adams GP, Tai M-S, Mccartney JE et al (2006) Avidity-mediated enhancement of in vivo tumor targeting by single-chain Fv dimers. Clin Cancer Res 12:1599–1605
Roopenian DC, Akilesh S (2007) FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol 7:715–725
Weiner LM, Surana R, Wang S (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10:317–327
Clynes RA, Towers TL, Presta LG et al (2000) Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med 6:443–446
Cragg MS, Glennie MJ (2004) Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents. Blood 103:2738–2743
(2012) Study evaluating the SBI-087 in subjects with systemic lupus erythematosus, NCT00714116. http://clinicaltrials.gov/ct2/show/NCT00714116?term=sle&rank=58
(2012) Study evaluating the efficacy and safety of SBI-087 in seropositive subjects with active rheumatoid arthritis, NCT01008852. http://clinicaltrials.gov/ct2/show/NCT01008852?term=SBI-087&rank=4
Hoogenboom HR, Griffiths AD, Johnson KS et al (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19:4133–4137
Borsi L, Balza E, Bestagno M et al (2002) Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin. Int J Cancer 102:75–85
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Bujak, E., Matasci, M., Neri, D., Wulhfard, S. (2014). Reformatting of scFv Antibodies into the scFv-Fc Format and Their Downstream Purification. In: Ossipow, V., Fischer, N. (eds) Monoclonal Antibodies. Methods in Molecular Biology, vol 1131. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-992-5_20
Download citation
DOI: https://doi.org/10.1007/978-1-62703-992-5_20
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-991-8
Online ISBN: 978-1-62703-992-5
eBook Packages: Springer Protocols