Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these ZHER2 affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ß-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a ZHER2 affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of ZHER2. This novel anti-idiotypic affibody molecule ZE01 was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of ZHER2 affibody molecules, having a common binding surface for HER2. Eight such ZHER2 affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (99mTc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.
- Affibody molecule
- Affinity column
- Anti-idiotypic affibody ligand
- Molecular imaging
- Protein engineering
- Protein fragment complementation assay
ASJC Scopus subject areas