FASTlab Radiosynthesis of the 18 F-labelled HER2-binding Affibody molecule [18 F]GE-226.

An 18 F-labelled human epidermal growth factor receptor (HER2) receptor binding radiotracer is a potential tool to non-invasively identify HER2 positive tumour lesions in subjects with recurrent metastatic breast cancer. Having explored the manual radiochemistry to conjugate the Affibody molecule ZHER2:2891 with [18 F]4-fluorobenzaldehyde, we have developed and optimised a full protocol for the automated GE FASTlab synthesiser. Our chemometric model predicted the best radiochemical purity for a short conjugation time (2.8 minutes), a low temperature (65°C), and a medium Affibody molecule precursor amount (5.5 mg). Under these optimised conditions, [18 F]GE-226 was produced after solid-phase extraction purification with activity yield of 30% ± 7 (n = 18) and a radiochemical purity of 94% ± 2 (n = 18). The synthesis and purification was complete after 43 minutes and provided apparent molar activities of 12 to 30 GBq/µmol (n = 12) at the end of synthesis.

Positron emission tomography imaging with 18F-labeled ZHER2:2891 affibody for detection of HER2 expression and pharmacodynamic response to HER2-modulating therapies.

PURPOSE: Expression of HER2 has profound implications on treatment strategies in various types of cancer. We investigated the specificity of radiolabeled HER2-targeting ZHER2:2891 Affibody, [(18)F]GE-226, for positron emission tomography (PET) imaging. EXPERIMENTAL DESIGN: Intrinsic cellular [(18)F]GE-226 uptake and tumor-specific tracer binding were assessed in cells and xenografts with and without drug treatment. Specificity was further determined by comparing tumor localization of a fluorescently labeled analogue with DAKO HercepTest. RESULTS: [(18)F]GE-226 uptake was 11- to 67-fold higher in 10 HER2-positive versus HER2-negative cell lines in vitro independent of lineage. Uptake in HER2-positive xenografts was rapid with net irreversible binding kinetics making possible the distinction of HER2-negative [MCF7 and MCF7-p95HER2: NUV60 (%ID/mL) 6.1 ± 0.7; Ki (mL/cm(3)/min) 0.0069 ± 0.0014] from HER2-positive tumors (NUV60 and Ki: MCF7-HER2, 10.9 ± 1.5 and 0.015 ± 0.0035; MDA-MB-361, 18.2 ± 3.4 and 0.025 ± 0.0052; SKOV-3, 18.7 ± 2.4 and 0.036 ± 0.0065) within 1 hour. Tumor uptake correlated with HER2 expression determined by ELISA (r(2) = 0.78), and a fluorophore-labeled tracer analogue colocalized with HER2 expression. Tracer uptake was not influenced by short-term or continuous treatment with trastuzumab in keeping with differential epitope binding, but reflected HER2 degradation by short-term NVP-AUY922 treatment in SKOV-3 xenografts (NUV60: 13.5 ± 2.1 %ID/mL vs. 9.0 ± 0.9 %ID/mL for vehicle or drug, respectively). CONCLUSIONS: [(18)F]GE-226 binds with high specificity to HER2 independent of cell lineage. The tracer has potential utility for HER2 detection, irrespective of prior trastuzumab treatment, and to discern HSP90 inhibitor-mediated HER2 degradation.

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.

[18F]-FLT positron emission tomography can be used to image the response of sensitive tumors to PI3-kinase inhibition with the novel agent GDC-0941.

The phosphoinositide 3-kinase (PI3K) pathway is deregulated in a range of cancers, and several targeted inhibitors are entering the clinic. This study aimed to investigate whether the positron emission tomography tracer 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]-FLT) is suitable to mark the effect of the novel PI3K inhibitor GDC-0941, which has entered phase II clinical trial. CBA nude mice bearing U87 glioma and HCT116 colorectal xenografts were imaged at baseline with [(18)F]-FLT and at acute (18 hours) and chronic (186 hours) time points after twice-daily administration of GDC-0941 (50 mg/kg) or vehicle. Tumor uptake normalized to blood pool was calculated, and tissue was analyzed at sacrifice for PI3K pathway inhibition and thymidine kinase (TK1) expression. Uptake of [(18)F]-FLT was also assessed in tumors inducibly overexpressing a dominant-negative form of the PI3K p85 subunit p85α, as well as HCT116 liver metastases after GDC-0941 therapy. GDC-0941 treatment induced tumor stasis in U87 xenografts, whereas inhibition of HCT116 tumors was more variable. Tumor uptake of [(18)F]-FLT was significantly reduced following GDC-0941 dosing in responsive tumors at the acute time point and correlated with pharmacodynamic markers of PI3K signaling inhibition and significant reduction in TK1 expression in U87, but not HCT116, tumors. Reduction of PI3K signaling via expression of Δp85α significantly reduced tumor growth and [(18)F]-FLT uptake, as did treatment of HCT116 liver metastases with GDC-0941. These results indicate that [(18)F]-FLT is a strong candidate for the noninvasive measurement of GDC-0941 action.