Tumor Imaging Using a Picomolar Affinity HER2 Binding Affibody Molecule

Abstract

The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z HER2:342 showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging. (Cancer Res 2006; 66(8): 4339-48)

Engineering and characterization of a bispecific HER2 × EGFR‐binding affibody molecule

“…When comparing the binding of the bs affibody molecule to the binding of the parental dimeric (Z HER2:342 ) 2 and (Z EGFR:1907 ) 2 proteins used as controls (Figures 2A and 2B), the bs affibody protein showed a similar binding profile as the (Z HER2:342 ) 2 when injected over the HER2 surface (Figure 2A). A retained high association rate and a low dissociation rate was demonstrated, in accordance with previous data for Z HER2:342 [30]. The decrease in binding to HER2 during the first seconds of the dissociation phase is likely to be due to differences in the buffer, but it does not influence the low dissociation rate.…”

“…The dimer format of each affibody molecule in the bs affibody protein will most likely give an additional increase in functional affinity (avidity), as demonstrated previously [38,39]. Furthermore, high selectivity of the Z HER2:342 and Z EGFR:1907 affibody molecules to their respective target protein has been shown in previous studies [30,39]. In the present study, binding to HER2 and EGFR was confirmed for the bs affibody protein (Figure 2).…”

“…The bs affibody molecule was injected at different concentrations over the flow-cell surfaces of a sensor chip containing immobilized HER2 and EGFR respectively. The affinities (equilibrium dissociation constant, K D ) of the monomeric affibody molecules were previously determined to be 22 pM for Z HER2:342 [30] and 5.4 nM for Z EGFR:1907 [31]. The dimer format of each affibody molecule in the bs affibody protein will most likely give an additional increase in functional affinity (avidity), as demonstrated previously [38,39].…”

“…Another flow-cell surface was activated and de-activated as a reference surface. The bs affibody molecule was diluted in a running buffer, HBS (10 mM Hepes, 150 mM NaCl, 3.4 mM EDTA and 0.005 % surfactant P20, pH 7.4), before binding analysis was performed at 25 • C. In a first experiment, the bs (Z HER2:342 ) 2 -(G 4 S) 3 [30] were also injected as controls. After each injection the flow cells were regenerated by the injection of 10 ml of 10 mM HCl.…”

“…Affibody molecules have shown potential in a wide variety of applications, e.g. as detection reagents [23][24][25], in bioseparation [26][27][28], for depletion in proteomics applications [29] and for tumourtargeting applications [30][31][32][33][34]. The small size (∼ 6.5 kDa), generally high stability, efficient production in Eschercihia coli or by chemical synthesis and straightforward thiol-based site-specific coupling or labelling are favourable properties for affinity proteins [35].…”