Three methods for 18F labeling of the HER2-binding affibody molecule Z(HER2:2891) including preclinical assessment.

UNLABELLED: Human epidermal growth factor receptor (HER2)-targeted Affibody molecules radiolabeled with (18)F allow the noninvasive assessment of HER2 status in vivo through PET imaging. Such agents have the potential to improve patient management by selecting individuals for HER2-targeted therapies and allowing therapy monitoring. The aim of this study was to assess different (18)F radiolabeling strategies of the HER2-specific Affibody molecule Z(HER2:2891), preclinically determine the biologic efficacy of the different radiolabel molecules, and select a preferred radiolabeling strategy to progress for automated manufacture. METHODS: Cysteine was added to the C terminus of the Affibody molecule for the coupling of maleimide linkers, and 3 radiolabeling strategies were assessed: silicon-fluoride acceptor approach ((18)F-SiFA), (18)F-AlF-NOTA, and 4-(18)F-fluorobenzaldehyde ((18)F-FBA). The biodistributions of the radiolabeled Affibody molecules were then determined in naïve CD-1 nude mice, and tumor targeting was assessed in CD-1 nude mice bearing high-HER2-expressing NCI-N87 tumors and low-HER2-expressing A431 tumors. The (111)In-ABY-025 compound, which has demonstrable clinical utility, served as a reference tracer. RESULTS: The non-decay-corrected radiochemical yields based on starting (18)F-fluoride using the (18)F-FBA, (18)F-SiFA, and (18)F-AlF-NOTA methods were 13% ± 3% (n = 5), 38% ± 2% (n = 3), and 11% ± 4% (n = 6), respectively. In naïve mice, both the (18)F-AlF-NOTA-Z(HER2:2891) and the (111)In-ABY-025 compounds showed a significant kidney retention (70.3 ± 1.3 and 73.8 ± 3.0 percentage injected dose [%ID], respectively, at 90 min after injection), which was not observed for (18)F-FBA-Z(HER2:2891) or (18)F-SiFA-Z(HER2:2891) (4.8 ± 0.6 and 10.1 ± 0.7 %ID, respectively, at 90 min). The (18)F-SiFA-Z(HER2:2891) conjugate was compromised by increasing bone retention over time (5.3 ± 1.0 %ID/g at 90 min after injection), indicating defluorination. All the radiolabeled Affibody molecules assessed showed significantly higher retention in NCI-N87 tumors than A431 tumors at all time points (P < 0.05), and PET/CT imaging of (18)F-FBA-Z(HER2:2891) in a dual NCI-N87/A431 xenograft model demonstrated high tumor-to-background contrast for NCI-N87 tumors. CONCLUSION: The HER2 Affibody molecule Z(HER2:2891) has been site-selectively radiolabeled by three (18)F conjugation methods. Preliminary biologic data have identified (18)F-FBA-Z(HER2:2891) (also known as GE226) as a favored candidate for further development and radiochemistry automation.

Radiosynthesis and biodistribution of a prosthetic group (¹8F-FENMA) conjugated to cyclic RGD peptides.

We have recently reported a new N-methylaminooxy-based prosthetic group for the site-selective introduction of ¹8F-fluorine under mild acidic aqueous conditions into model peptides functionalized with a Michael acceptor moiety. To further investigate the utility of this methodology, the radiosynthesis of two cyclic RGD peptides was carried out, and in vivo biodistribution and microPET studies were performed in tumor-bearing mice. A cyclic RGD peptide was functionalized with the Michael acceptors trans-ß-nitrostyrene carboxylic acid and 3-vinylsulfonylpropionic acid. Radiolabeling was then performed with the prosthetic group O-(2-(2-[¹8F]fluoroethoxy)ethyl)-N-methylhydroxylamine (¹8F-FENMA) yielding the ¹8F-conjugates in moderate yields (8.5-12%). Biodistribution, blocking, and microPET imaging studies were performed in a mouse xenograft model. The vinylsulfonyl-modified conjugate demonstrated good in vitro plasma stability. Biodistribution and microPET studies revealed excellent tumor uptake with low background in key organs and renal elimination as the predominant route of excretion. Blocking studies with coinjected nonlabeled RGD peptide confirmed the in vivo specificity for the integrin α(v)ß3. On the other hand, ¹8F-FENMA-nitrostyrene-RGD, although stable at conjugation pH 5, was found to rapidly degrade at physiological pH through loss of the ¹8F-prosthetic group.

One step radiosynthesis of 6-[(18)F]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester ([(18)F]F-Py-TFP): a new prosthetic group for efficient labeling of biomolecules with fluorine-18.

The labeling of biomolecules for positron emission tomography (PET) with no-carrier-added fluorine-18 is almost exclusively accomplished using prosthetic groups in a two step procedure. The inherent complexity of the process renders full automation a challenge and leads to protracted synthesis times. Here we describe a new (18)F-labeled prosthetic group based on nicotinic acid tetrafluorophenyl ester. Reaction of [(18)F]fluoride at 40 degrees C with the trimethylammonium precursor afforded 6-[(18)F]fluoronicotinic acid tetrafluorophenyl ester ([(18)F]F-Py-TFP) directly in 60-70% yield. [(18)F]F-Py-TFP was conveniently purified by Sep-Pak cartridge prior to incubation with a peptide containing the RGD sequence. The desired conjugate was formed rapidly and in good yields. An in vitro receptor-binding assay for the integrin alpha(v)beta(3) was established to explore competition with peptide and peptidomimetic prepared from F-Py-TFP with (125)I-echistatin. The nonradioactive conjugates were found to possess high binding affinities with calculated K(i) values in the low nanomolar range.

Methods for 18F-labeling of RGD peptides: comparison of aminooxy [18F]fluorobenzaldehyde condensation with 'click labeling' using 2-[18F]fluoroethylazide, and S-alkylation with [18F]fluoropropanethiol.

Three strategies for chemoselective labeling of RGD peptides with (18)F have been compared. Aminooxy [(18)F]fluorobenzaldehyde conjugation provided 40 +/- 12% decay-corrected radiochemical yield using a fully automated method. An one-pot protocol for 'click labeling' of the RGD scaffold with 2-[(18)F]fluoroethylazide afforded 47 +/- 8% decay-corrected radiochemical yield. Attempted conjugation with 3-[(18)F]fluoropropanethiol led to extensive decomposition and was therefore found unsuitable for labeling of the RGD peptide investigated. The results suggest that 'click labeling' of RGD peptides provides an attractive alternative to aminooxy aldehyde condensation, however, 2-[(18)F]-fluoroethylazide may be too small to allow separation of large (18)F-labeled RGD peptides from their precursors.

A novel prosthetic group for site-selective labeling of peptides for positron emission tomography.

Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.

Radiosynthesis and biodistribution of cyclic RGD peptides conjugated with novel [18F]fluorinated aldehyde-containing prosthetic groups.

Achieving high-yielding, robust, and reproducible chemistry is a prerequisite for the (18)F-labeling of peptides for quantitative receptor imaging using positron emission tomography (PET). In this study, we extend the toolbox of oxime chemistry to include the novel prosthetic groups [(18)F]-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethoxy)acetaldehyde, [(18)F]5, and [(18)F]-4-(3-fluoropropoxy)benzaldehyde, [(18)F]9, in addition to the widely used 4-[(18)F]fluorobenzaldehyde, [(18)F]12. The three (18)F-aldehydes were conjugated to the same aminooxy-bearing RGD peptide and the effect of the prosthetic group on biodistribution and tumor uptake studied in mice. The peptide conjugate [(18)F]7 was found to possess superior in vivo pharmacokinetics with higher tumor to blood, tumor to liver, tumor to muscle, and tumor to lung ratios than either [(18)F]10 or [(18)F]13. The radioactivity from the [(18)F]7 conjugate excreted more extensively through the kidney route with 79%id passing through the urine and bladder at the 2 h time point compared to around 55%id for the more hydrophobic conjugates [(18)F]10 and [(18)F]13. The chemical nature of a prosthetic group can be employed to tailor the overall biodistribution profile of the radiotracer. In this example, the hydrophilic nature of the ethylene glycol containing prosthetic group [(18)F]5 clearly influences the overall excretion pattern for the RGD peptide conjugate.

18F-fluorothiols: a new approach to label peptides chemoselectively as potential tracers for positron emission tomography.

[(18)F]Fluorothiols are a new generation of peptide labeling reagents. This article describes the preparation of suitable methanesulfonyl precursors and their use in no-carrier-added radiosyntheses of (18)F-fluorothiols. The preparations of (3-[(18)F]fluoropropylsulfanyl)triphenylmethane, (2-[2-[2-(2-[(18)F]fluoroethoxy)ethoxy]ethoxy]ethylsulfanyl)triphenylmethane, and 4-[(18)F]fluoromethyl-N-[2-triphenylmethanesulfanyl)ethyl]benzamide starting from the corresponding methanesulfonyl precursors were investigated. Following the removal of the triphenylmethane protecting group, the (18)F-fluorothiols were reacted with the N-terminal chloroacetylated model peptide ClCH(2)C(O)-LysGlyPheGlyLys. The corresponding radiochemical yields of (18)F-labeled isolated model peptide, decay-corrected to (18)F fluoride, were 10%, 32%, and 1%, respectively. These results indicate a considerable potential of (18)F-fluorothiols for the chemoselective labeling of peptides as tracers for positron emission tomography (PET).