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Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method
Published on: September 16, 2017
Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay
P-A Löfdahl1, O Nord, L Janzon
1Division of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.
New Biotechnology
|July 7, 2009
Summary
This study developed a novel beta-lactamase protein fragment complementation assay (PCA) for selecting high-affinity affibody molecules that bind human tumor necrosis factor-alpha (TNF-alpha). The assay demonstrated stringent selection and identified potent binders with therapeutic potential.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Protein fragment complementation assays (PCAs) are vital for studying dynamic protein-protein interactions in living cells.
- Existing PCA systems offer powerful tools, but novel applications for specific selection challenges are continuously explored.
Purpose of the Study:
- To construct and validate a novel PCA system utilizing genetic splitting of TEM-1 beta-lactamase.
- To apply this system for the stringent selection of affibody molecules targeting human tumor necrosis factor-alpha (TNF-alpha) from a large combinatorial library.
Main Methods:
- Development of a beta-lactamase split PCA in Escherichia coli periplasm.
- Phage infection to deliver affibody library variants fused to a reporter fragment.
- Selection using ampicillin and tazobactam on plates, followed by binding affinity (K(D)) determination.
- Cross-target PCA and biosensor competition assays using adalimumab.
Main Results:
- The beta-lactamase PCA successfully selected for stringent TNF-alpha binding affibody variants.
- Selected affibody variants exhibited high binding affinities (K(D) in the 14-27 nM range).
- Demonstrated selectivity for TNF-alpha and overlapping epitopes with adalimumab.
Conclusions:
- Beta-lactamase PCA is a promising method for selecting high-affinity and selective binders from complex libraries.
- This approach yields reagents with potential therapeutic applications.
- The developed PCA system offers a robust platform for protein-protein interaction studies and binder selection.
