Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for 99mTc-labelling at the C terminus

Abstract

Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide (99m)Tc. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with (99m)Tc. (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, (99m)Tc-Z(HER2:2395)-VDC and (99m)Tc-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of (99m)Tc-Z(HER2:2395)-VDC, but it was substantially higher than uptake of (99m)Tc-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of (99m)Tc was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of (99m)Tc labelling due to a partial loss of site-specificity of nuclide chelation.

Feasibility of imaging of epidermal growth factor receptor expression with ZEGFR:2377 affibody molecule labeled with 99mTc using a peptide-based cysteine-containing chelator

“…In addition, selection of amino acids in the chelator may enable a fine-tuning of residualizing properties of the label (52–54). However, there was a risk of losing site-specificity of the label when amino acid composition of an affibody molecules was modified (43). Indeed, the first experiments demonstrated release of over 30% of the 99m Tc after dilution or under cysteine challenge (Table I).…”

“…Such approach might be considered for anti-EGFR affibody ZEGFR:2377 as well. However, our studies have demonstrated that modification of the amino acid sequence of affibody molecule might create an alternative binding site for technetium-99m, providing lower stability of the complex (43). Since the amino acid composition of ZEGFR:2377 differs profoundly from the composition of ZHER2:2395 we had to consider formation of an alternative chelating site as well.…”

[99mTc(CO)3]+-(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours

“…For a construct containing the HEHEHE-tag, a convenient IMAC purification may be combined with low liver accumulation of radioactivity and the use of a GGGC motif as chelator causes low radioactivity accumulation in kidneys, but requires more complicated purification schemes during tracer production [33]. A combination of these approaches is tempting, but recent data suggest that (Tc=O) 3+ coupling to the GGGC-motif in the presence of a HEHEHEtag in the probe is associated with the loss of site-specificity of labelling and poor control over biodistribution [34]. In this study, we therefore evaluated a labelling approach where [ 99m Tc(CO) 3 ] + was conjugated to the HEHEHE-tag.…”

Imaging of human pancreatic cancer xenografts by single-photon emission computed tomography with 99mTc-Hynic-PEG-AE105

“…Tc is a nuclear isomer of 99 Tc that is detectable within the body using medical equipment such as γ-ray cameras, which emit readily detectable CXL keV γ rays (the same wavelength as emitted by conventional X-ray equipment), and the half-life for γ emission is only 6 h (19). Safe and fast scanning procedures are a result of the relatively short physical half-life of 99 Tc and its biological half-life of 1 day in terms of human activity and metabolism (20). Therefore, Tc-labeled peptides have been used for in vivo targeting of tumors, including pancreatic cancer (21)(22)(23).…”