Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of 99mTc-labeled recombinant Affibody molecules (original) (raw)
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Bioorganic & Medicinal Chemistry Letters, 2009
Affibody molecules represent a novel class of affinity proteins with a high potential as tracers for radionuclide molecular imaging. In this comparative structure-property study, a series of Affibody molecules with the 99m Tc-chelators maGGG, maSSS, or maESE attached to the e-amine of the internally positioned K49 was prepared by peptide synthesis, for comparison to molecules with similar chelators positioned at the N-terminus. The conjugates were labeled with 99m Tc and evaluated in vitro and in vivo. It was found that both composition and position of the chelating moiety influence the label stability, biodistribution and targeting properties of HER2-binding Affibody molecules.
European Journal of Nuclear Medicine and Molecular Imaging, 2008
Purpose Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, 99mTc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. Materials and methods Anti-HER2 ZHER2:342 Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with 99mTc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, 99mTc-maEEE-ZHER2:342, in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, 99mTc-maESE-ZHER2:342, was compared with radioiodinated ZHER2:342. Results All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 ± 5, 68 ± 21 and 71 ± 10%IA/g, for99mTc-maESE-ZHER2:342, 99mTc-maEES-ZHER2:342 and 99mTc-maSEE-ZHER2:342, respectively, were significantly reduced in comparison with 99mTc-maEEE-ZHER2:342 (102 ± 13%IA/g). For 99mTc-maESE-ZHER2:342, a tumour uptake of 9.6 ± 1.8%IA/g and a tumour-to-blood ratio of 58 ± 6 were reached at 4 h p.i. Conclusions A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of 99mTc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area.
Tumor Biology, 2012
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.
Bioconjugate Chemistry, 2007
Affibody molecules present a new class of affinity proteins, which utilizes a scaffold based on a 58-amino acid domain derived from protein A. The small (7 kDa) Affibody molecule can be selected to bind to cell-surface targets with high affinity. An Affibody molecule (Z HER2:342) with a dissociation constant (K d) of 22 pM for binding to the HER2 receptor has been reported earlier. Preclinical and pilot clinical studies have demonstrated the utility of radiolabeled Z HER2:342 in imaging of HER2-expressing tumors. The small size and cysteine-free structure of Affibody molecules enable complete peptide synthesis and direct incorporation of radionuclide chelators. The goal of this study was to evaluate if incorporation of the natural peptide sequences cysteine-diglycine (CGG) and cysteine-triglycine (CGGG) sequences would enable labeling of Affibody molecules with 99m Tc. In a model monomeric form, the chelating sequences were incorporated by peptide synthesis. The HER2-binding affinity was 280 and 250 pM for CGG-Z HER2:342 and CGGG-Z HER2:342, respectively. Conjugates were directly labeled with 99m Tc with 90% efficiency and preserved the capacity to bind specifically to HER2-expressing cells. The biodistribution in normal mice showed a rapid clearance from the blood and the majority of organs (except kidneys). In the mice bearing SKOV-3 xenografts, tumor uptake of 99m Tc-CGG-Z HER2:342 was HER2-specific and a tumorto-blood ratio of 9.2 was obtained at 6 h postinjection. Gamma-camera imaging with 99m Tc-CGG-Z HER2:342 clearly visualized tumors at 6 h postinjection. The results show that the use of a cysteine-based chelator enables 99m Tclabeling of Affibody molecules for imaging.
European journal of medicinal chemistry, 2014
Affibody molecules constitute a class of small (7 kDa) scaffold proteins that can be engineered to have excellent tumor targeting properties. High reabsorption in kidneys complicates development of affibody molecules for radionuclide therapy. In this study, we evaluated the influence of the composition of cysteine-containing C-terminal peptide-based chelators on the biodistribution and renal retention of (188)Re-labeled anti-HER2 affibody molecules. Biodistribution of affibody molecules containing GGXC or GXGC peptide chelators (where X is G, S, E or K) was compared with biodistribution of a parental affibody molecule ZHER2:2395 having a KVDC peptide chelator. All constructs retained low picomolar affinity to HER2-expressing cells after labeling. The biodistribution of all (188)Re-labeled affibody molecules was in general comparable, with the main observed difference found in the uptake and retention of radioactivity in excretory organs. The (188)Re-ZHER2:V2 affibody molecule with a...
Amino Acids, 2018
Epidermal growth factor receptor (EGFR) is overexpressed in a number of cancers and is the molecular target for several anti-cancer therapeutics. Radionuclide molecular imaging of EGFR expression should enable personalization of anti-cancer treatment. Affibody molecule is a promising type of high-affinity imaging probes based on a non-immunoglobulin scaffold. A series of derivatives of the anti-EGFR affibody molecule ZEGFR:2377, having peptide-based cysteine-containing chelators for conjugation of 99m Tc, was designed and evaluated. It was found that glutamate-containing chelators Gly-Gly-Glu-Cys (GGEC), Gly-Glu-Glu-Cys (GEEC) and Glu-Glu-Glu-Cys (EEEC) provide the best labeling stability. The glutamate containing conjugates bound to EGFR-expressing cells specifically and with high affinity. Specific targeting of EGFR-expressing xenografts in mice was demonstrated. The number of glutamate residues in the chelator had strong influence on biodistribution of radiolabeled affibody molecules. Increase of glutamate content was associated with lower uptake in normal tissues. The 99m Tc-labeled variant containing the EEEC chelator provided the highest tumor-to-organ ratios. In conclusion, optimizing the composition of peptide-based chelators enhances contrast of imaging of EGFR-expression using affibody molecules. Keywords Affibody molecules • EGFR • 99m Tc • Peptide-based chelators • Glutamate Abbreviations EGFR Epidermal growth factor receptor HER2 Human epidermal growth factor receptor type 2 HER3 Human epidermal growth factor receptor type 3 IGF-1R Insulin-like growth factor 1 receptor CAIX Carbonic anhydrase IX SPECT Single photon emission computed tomography PET Positron emission tomography ITLC Instant thin-layer chromatography SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Handling Editor: J. Pietzsch.
European Journal of Nuclear Medicine and Molecular Imaging, 2007
Purpose Monitoring HER2 expression is crucial for selection of breast cancer patients amenable to HER2-targeting therapy. The Affibody molecule ZHER2:342 binds to HER2 with picomolar affinity and enables specific imaging of HER2 expression. Previously, ZHER2:342 with the additional N-terminal mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) sequence was labelled with 99mTc and demonstrated specific targeting of HER2-expressing xenografts. However, hepatobiliary excretion caused high radioactivity accumulation in the abdomen. We investigated whether the biodistribution of ZHER2:342 can be improved by substituting glycyl residues in the chelating sequence with more hydrophilic seryl residues. Methods The Affibody molecule ZHER2:342, carrying the chelators mercaptoacetyl-glycyl-seryl-glycyl (maGSG), mercaptoacetyl-glycyl-D-seryl-glycyl [maG(D-S)G] and mercaptoacetyl-seryl-seryl-seryl (maSSS), were prepared by peptide synthesis and labelled with 99mTc. The differences in the excretion pathways were evaluated in normal mice. The tumour targeting capacity of 99mTc-maSSS-ZHER2:342 was studied in nude mice bearing SKOV-3 xenografts and compared with the capacity of radioiodinated ZHER2:342. Results A shift towards renal excretion was obtained when glycine was substituted with serine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced threefold for the maSSS conjugate in comparison with the maGGG conjugate 4 h post injection (p.i.). The tumour uptake of 99mTc-maSSS-ZHER2:342 was 11.5 ± 0.5% IA/g 4 h p.i., and the tumour-to-blood ratio was 76. The pharmacokinetics and uptake characteristics of technetium-labelled ZHER2:342 were better than those of radioiodinated ZHER2:342. Conclusion The introduction of serine residues in the chelator results in better tumour imaging properties of the Affibody molecule ZHER2:342 compared with glycyl-containing chelators and is favourable for imaging of tumours and metastases in the abdominal area.
During the past decade, several peptides containing Arg-Gly-Asp sequence have been conjugated with different chelating agents for labeling with various radionuclides for the diagnosis of tumor development. In this study, we report the synthesis of two tetrapeptides (Asp-Gly-Arg-His and Asp-Gly-Arg-Cys) and one hexapeptide [Asp-Gly-Arg-D-Tyr-Lys-His] by changing the amino acid sequence of the Arg-Gly-Asp motif. Peptide synthesis was initiated from aspartic acid. Aspartic acid placed at C-terminal end of the peptide chain can be conjugated with different drug molecules facilitating their transport to the site of action. The peptides were synthesized in excellent yield and labeled using freshly prepared [ 99m Tc(CO) 3 (H 2 O) 3 ] + intermediate. A complexation yield of over 97% was achieved under mild conditions even at low ligand concentrations of 10 À2 M. Radiolabeled peptides were characterized by HPLC and were found to be substantially stable in saline, in His solution as well as in rat serum and tissue (kidney, liver) homogenates. Internalization studies using Ehrlich ascites carcinoma cell line showed rapid and significant internalization (30-35% at 30 min of incubation attaining maximum value of about 40-60% after 2-4 h incubation). A good percentage of quick internalization was also observed in a v b 3 -receptor-positive B16F10 mouse melanoma cell line (14-16% after 30 min of incubation and 25-30% after 2-4 h incubation). Imaging and biodistribution studies were performed in Swiss albino mice bearing Ehrlich ascites tumor in right thigh. Radiolabeled peptides exhibited fast blood clearance and rapid elimination through the urinary systems. 99m Tc(CO) 3 -tetra-Pep2 exhibited remarkable localization at tumor site (1.15%, 1.17%, and 1.37% ID/g at 2, 4, and 6 h p.i., respectively) which could be due to slow clearance of the radiolabeled peptide from blood in comparison with the other two radiolabeled peptides. However, 99m Tc(CO) 3 -hexa-Pep exhibited the highest tumor to muscle and tumor to blood ratios among the three. The preliminary results with these amino acid-based peptides are encouraging enough to carry out further experiments for targeting tumor.