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Updated: Aug 13, 2026
The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions
Published on: February 1, 2018
Construction and optimization of a CC49-based scFv-beta-lactamase fusion protein for ADEPT
Martin Roberge1, Melodie Estabrook, Joshua Basler
1Genencor International, a Danisco company, 925 Page Mill Road, Palo Alto, CA 94304, USA.
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 degrees 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).
Insights
Researchers improved a cancer-targeting antibody fragment (scFv) using Combinatorial Consensus Mutagenesis (CCM). The enhanced variant, TAB2.5, shows significantly improved expression and stability for potential cancer therapies like antibody-directed enzyme prodrug therapy (ADEPT).
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- CC49 is a validated antibody targeting TAG-72, a tumor-associated carbohydrate antigen found on many solid tumors.
- Antibody fragments are crucial for targeted therapies, but production challenges like low expression and instability can limit their utility.
- The first-generation fusion protein TAB2.4, based on CC49, exhibited poor expression and degradation in Escherichia coli.
Purpose of the Study:
- To enhance the expression and stability of a CC49-based single-chain variable fragment (scFv) for improved therapeutic applications.
- To evaluate the efficacy of Combinatorial Consensus Mutagenesis (CCM) in optimizing antibody fragment properties.
- To develop a robust fusion protein for antibody-directed enzyme prodrug therapy (ADEPT).
Main Methods:
- Construction of a single-chain fragment (scFv) based on the CC49 antibody and fused to beta-lactamase.
- Optimization of the scFv domain using Combinatorial Consensus Mutagenesis (CCM).
- Expression and characterization of the improved fusion protein variant, TAB2.5, in Escherichia coli.
Main Results:
- The optimized variant TAB2.5 demonstrated a nearly 4-fold increase in expression compared to the parent molecule.
- TAB2.5 exhibited a 2.5°C higher thermostability, indicating improved protein integrity.
- Soluble TAB2.5 could be manufactured at 120 mg/l in low-density E. coli cultures, showcasing efficient production.
Conclusions:
- Combinatorial Consensus Mutagenesis (CCM) is an effective and rapid method for improving antibody fragment stability and expression.
- The enhanced fusion protein TAB2.5 offers significant advantages for developing targeted cancer therapies, particularly ADEPT.
- The improved production efficiency and stability of TAB2.5 pave the way for its clinical application in cancer treatment.
