Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-111

Abstract

Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DOTAGA (1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z₀₈₆₉₈ and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged ¹¹¹In-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

Figure 1

Structural overview of macrocyclic maleimide chelators 1,4,7-triazacyclononane-N,N′,N′′-triacetic acid (NOTA), 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) and charge of the complexes formed with indium-111 when conjugated to Z₀₈₆₉₈.

Figure 2

Binding specificity of ¹¹¹In-Z₀₈₆₉₈-X on BxPC-3 and DU145 cells. HER3 receptors in the blocked group were pre-saturated with excess of unlabeled Z₀₈₆₉₈. Data are presented as an average ± standard deviation from three cell culture dishes.

Figure 3

Cellular processing of ¹¹¹In-Z₀₈₆₉₈-X on BxPC-3 (black) and DU145 cells (grey). Total cell bound activity (solid line) and internalized activity (dashed line) are displayed as percent of cell associated activity. The values were normalized to maximum cell-associated activity for each conjugate. Data are presented as an average ± standard deviation from three cell culture dishes.

Figure 4

SPECT-CT imaging of ¹¹¹In-Z₀₈₆₉₈-X. MIP images coronal and sagittal view at A) 4 h pi and B) 24 h pi. Mice were bearing BxPC-3 xenografts and injected with 2 µg (~1.5 MBq) ¹¹¹In-Z₀₈₆₉₈-X.

Figure 5

Activity uptake in liver (left) and tumor-to-liver ratios for all ¹¹¹In-Z₀₈₆₉₈-X conjugates over time studied in female Balb/c nu/nu mice bearing BxPC-3 xenografts. Significant differences between conjugates are marked with asterisks.