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PEDS Advance Access published online on January 25, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzj012
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Received November 8, 2005
Revised December 19, 2005
Accepted December 23, 2005

Article

Construction and optimization of a CC49-Based scFv-{beta}-lactamase fusion protein for ADEPT

Martin Roberge 1, Melodie Estabrook 1, Joshua Basler 1, Regina Chin 1, Pete Gualfetti 1, Amy Liu 1, Stephanie B. Wong 1, M. Harunur Rashid 1, Tom Graycar 1, Lilia Babé 1, and Volker Schellenberger 1 *

1 Genencor International, a Danisco company, 925 Page Mill Road, Palo Alto, CA 94304, USA

* To whom correspondence should be addressed.
Volker Schellenberger, E-mail: vschellenberger{at}mproteins.com


  Abstract

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to {beta}-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5° higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).

Keywords: ADEPT; CC49; consensus mutation; scFv; thermostability.
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