Construction and optimization of a CC49-Based scFv-β-lactamase fusion protein for ADEPT

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 β-lactamase. The first generation fusion protein, TAB2...

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Bibliographic Details
Published in:Protein engineering, design and selection design and selection, 2006-04, Vol.19 (4), p.141-145
Main Authors: Roberge, Martin, Estabrook, Melodie, Basler, Joshua, Chin, Regina, Gualfetti, Pete, Liu, Amy, Wong, Stephanie B., Rashid, M. Harunur, Graycar, Tom, Babé, Lilia, Schellenberger, Volker
Format: Article
Language:English
Subjects:
ADEPT
Amino Acid Sequence
Antibodies, Monoclonal - genetics
Antibodies, Monoclonal - therapeutic use
Antibodies, Neoplasm - genetics
Antibodies, Neoplasm - therapeutic use
Antigens, Neoplasm - immunology
beta-Lactamases - genetics
beta-Lactamases - therapeutic use
CC49
Combinatorial Chemistry Techniques - methods
consensus mutation
Consensus Sequence - genetics
Escherichia coli
Escherichia coli - metabolism
Glycoproteins - immunology
Molecular Sequence Data
Mutagenesis
Peptide Library
Prodrugs - therapeutic use
Protein Engineering - methods
Recombinant Fusion Proteins - chemical synthesis
Recombinant Fusion Proteins - therapeutic use
scFv
thermostability
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Summary: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 β-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).
ISSN:1741-0126
1741-0134
DOI:10.1093/protein/gzj012