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Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants
Published on: July 1, 2018
Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems
Patrizia Galeffi1, Alessio Lombardi, Immacolata Pietraforte
1ENEA BIOTEC-GEN, CR Casaccia Via Anguillarese 301, 00060 Rome, Italy. galeffi@casaccia.enea.it
Background:
Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen.
Methods:
Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system.
Results:
An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 x 10(8) M(-1)) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining.
Conclusion:
ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for future in vivo studies.
Insights
Researchers developed a novel single-chain variable fragment (ScFv) targeting ErbB-2, successfully produced in bacteria, plants, and cell-free systems. This advancement offers a scalable solution for producing cancer therapeutics amid manufacturing challenges.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunology
Background:
- Aberrant ErbB-2 signaling drives aggressive carcinomas, especially breast cancer.
- Antibodies targeting ErbB-2 are crucial for advanced breast cancer therapy.
- A global shortage in mammalian cell bioreactor capacity is anticipated.
Purpose of the Study:
- To develop a multi-platform production strategy for ErbB-2 targeting single-chain variable fragments (ScFvs).
- To establish a scalable and versatile method for producing therapeutic antibodies.
Main Methods:
- Functional expression of ErbB-2 ScFvs in bacteria, transgenic tobacco plants (transient and stable), and a cell-free system.
- Selection and optimization of a specific ScFv (ScFv800E6) with various tags, including a novel Strep II tag.
- Characterization of ScFv800E6 binding affinity, stability, and compatibility with diagnostic reagents.
Main Results:
- ScFv800E6 was successfully expressed and functional across all tested platforms, marking a first for non-bacterial/yeast hosts.
- Optimized ScFv800E6 demonstrated high stability, resistance to radiolabeling, and binding affinity comparable to parental antibodies.
- The novel Strep II tag enhanced detection performance over conventional systems.
Conclusions:
- ScFv800E6 is a versatile reagent for in vitro biochemical and immunodiagnostic oncology applications.
- The multi-platform production approach provides a scalable alternative for antibody fragment manufacturing.
- ScFv800E6 shows potential for future in vivo therapeutic studies.
