Selection and characterization of HER2/neu-binding affibody ligands

Abstract

Affibody (affibody) ligands that are specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) have been selected by phage display technology from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. The predominant variants from the phage selection were produced in Escherichia coli, purified by affinity chromatography, and characterized by biosensor analyses. Two affibody variants were shown to selectively bind to the extracellular domain of HER2/neu (HER2-ECD), but not to control proteins. One of the variants, denoted His6-ZHER2/neu:4, was demonstrated to bind with nanomolar affinity (approximately 50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His6-ZHER2/neu:4 affibody showed specific binding to native HER2/neu, overexpressed on the SKBR-3 tumor cell line. Such affibody ligands might be considered in tumor targeting applications for radionuclide diagnostics and therapy of adenocarcinomas such as breast and ovarian cancers.

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

“…It is known that ZH and Herceptin do not bind to the same epitope on the HER2/neu antigen as they cannot block each other. 30 In concordance with this hypothesis, Ad5/FibDCAR-HI-Link-ZHZH and Ad5/EGD/FibDCAR-HI-Link-ZHZH could be shown to poorly infect RD cells but to infect SKOV-3 and SKBR-3 cells well compared to WT ( Figure 9). …”

“…The tandem repeat also has a higher avidity for HER2/neu than the monomer. 32 However, the affinity of the monomeric ZH is already in the nanomolar range, 30 which is comparable to the affinity of the WT knob for CAR (3 nM). 55 By trying to allow for correct folding of the knob sequence and by choosing a ligand that can fold correctly in the cytoplasm of infected cells, we have been able to produce a re-targeted Ad with improved phenotype and a new specificity with clinical relevance.…”

“…Anti-HER2/neu Herceptin (humanized mIgG1) was from Roche (Basel, Switzerland) and HER2-ECD (HER2/neu Extra Cellular Domain) from Fox Chase Cancer Center (Philadelphia). 37 Affinity purified His6-(ZH) 2 Affibody molecule was produced as described in Wikman M et al 30 Ad5 knob and FibDCAR-HI-Link-SpA domain (Zwt) were expressed in Sf9 cells, the cells were lysed and the concentrations of knob and fiber in the soluble fractions were measured by Western blot in comparison with a purified-fiber protein with known concentration.…”

“…19,38 The Affibody molecule used in this study is the ZH, specific for the ECD of HER2/neu. 30 Generation of recombinant truncated knobless fibers Recombinant fibers containing the native fiber tail and the first seven shaft repeats, followed by an external trimerization motif from lung surfactant protein D (neck region peptide (NRP)), 39 a linker from SpA and monomeric or dimeric versions of ZH as a new cellular ligand, were constructed and evaluated as previously described. [10][11][12] A schematic representation of the constructed recombinant Ad5 fibers studied is shown in Figure 1.…”

“…[29][30][31] A dimeric variant of the ZH has been found to bind Her2/neu with 3 nM affinity. 32 HER2/neu is overexpressed in different cancers, above all breast and ovarian.…”