Comparative Preclinical Evaluation of Peptide-Based Chelators for the Labeling of DARPin G3 with 99mTc for Radionuclide Imaging of HER2 Expression in Cancer

Abstract

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [99mTc]Tc-(HE)3-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)3), provided clear imaging of HER2 expressing breast cancer 2–4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E3C), Gly-Gly-Gly-Cys (G3C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)3-linker (designated as G3-(G3S)3C) would further improve the contrast of imaging using 99mTc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9–5 nM. Biodistribution of [99mTc]Tc-G3-G3C, [99mTc]Tc-G3-(G3S)3C, and [99mTc]Tc-G3-E3C in mice was compared with the biodistribution of [99mTc]Tc-(HE)3-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-(HE)3-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.

Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the 99mTc-Based Imaging of HER2-Expressing Malignant Tumors

“…The highest uptake in the liver was observed for [ 99m Tc]­Tc-G3-E 3 C-HYNIC. The high liver uptake of [ 99m Tc]­Tc-G3-E 3 C was also found in our previous study . These findings were somewhat unexpected.…”

“…Overall, the internalized radioactivity for all 99m Tc-labeled variants including [ 99m Tc]­Tc-(HE) 3 -G3 was almost 3-fold lower at 4 h compared to 24 h. Accordingly, the internalization rate of all variants was relatively slow and, as demonstrated earlier, slowly internalized radioconjugates provide better retention of radioactivity in tumors. Interestingly, in our earlier study of G3 variants labeled with [ 99m Tc]­TcO, the internalization patterns were different for glycine-containing and glutamate-containing variants . The glycine-containing variants [ 99m Tc]­Tc-G3-G 3 C and [ 99m Tc]­Tc-G3-(G 3 S) 3 C had a higher internalization rate with ca.…”

“…Comparison of the data from this study with the data for homologous variants labeled using oxotechnetium reveals a strong impact of labeling chemistry biodistribution and targeting features. Thus, [ 99m Tc]­Tc-G3-(G 3 S) 3 C-HYNIC at 4 h after injection had 1.6-fold lower uptake in the liver and more than 2-fold lower uptake in the spleen of Nu/j mice in comparison with [ 99m Tc]­Tc-G3-(G 3 S) 3 C. On the opposite, the renal activity of [ 99m Tc]­Tc-G3-(G 3 S) 3 C-HYNIC was nearly 20-fold higher, and the blood concentration was 4-fold higher compared with [ 99m Tc]­Tc-G3-(G 3 S) 3 C. This is a strong indication that a chelation moiety influences the uptake of DARPin G3 in normal tissues and the retention of the internalized activity.…”

“…It is worth noting that [ 99m Tc]­Tc-G3-(G 3 S) 3 C-HYNIC had a comparable, although slightly higher, hepatic uptake than [ 99m Tc]­Tc-(HE) 3 -G3. Previously, the 99m Tc-labeled analogue without HYNIC had higher liver uptake in CD1 mice than the HYNIC-containing variant …”

“…20 Based on these considerations, a comparative evaluation of [ 99m Tc]Tc-(HE) 3 -G3 and the use of peptide-based cysteinecontaining chelators for labeling of DARPin G3 with 99m Tc has been performed. 21 DARPin G3 variants containing a Glu-Glu-Glu-Cys chelator providing a residualizing label, a Gly-Gly-Gly-Cys chelator providing a nonresidualizing label, and a Gly-Gly-Gly-Ser-Cys chelator connected via a −(Gly-Gly-Gly-Ser)− linker at the C-terminus of G3 were evaluated. The labeled conjugates demonstrated specific and high affinity binding to HER2-expressing human cancer cells in vitro and specific accumulation in HER2-expressing xenografts in vivo.…”