Peter Iveson

In vivo pH imaging with (99m)Tc-pHLIP.

PurposeA novel molecular imaging agent has been developed recently, which stains tissues of low extracellular pH [pH (low) insertion peptide, pHLIP®]. A pH-dependent process of peptide folding and insertion into cell membranes has been found in vitro. Targeting of acidic solid tumours has been demonstrated in vivo using fluorescence and PET labels. Here, we present proof of feasibility studies of pHLIP with a single-photon emission computed tomography (SPECT) label, 99mTc-AH114567, with focus on preclinical efficacy and imageability.ProceduresLewis lung carcinoma, lymph node carcinoma of the prostate and prostate adenocarcinoma tumour xenografts were grown in mice and characterised by the angiogenesis marker 99mTc-NC100692 and by extracellular pH measurements with 31P-MRS of 3-aminopropyl phosphonate. Biodistribution was assessed and CT/SPECT imaging performed. Oral administration of bicarbonate served as control.Results and ConclusionTc-AH114567 can be obtained via a robust synthesis with good radiolabelling profile and improved formulation. The tracer retains the pH-dependent ability to insert into membranes and to target tumours with similar pharmacokinetics and efficacy that had been demonstrated earlier for pHLIP with optical or 64Cu PET labels. Despite the inherent challenges of SPECT compared to optical and PET imaging, e.g., in terms of lower sensitivity, 99mTc-AH114567 shows adequate image quality and contrast. The main development need for transitioning SPECT labelled pHLIP into the clinic is more rapid background signal reduction, which will be the focus of a subsequent optimisation study.

Three Methods for 18F Labeling of the HER2-Binding Affibody Molecule ZHER2:2891 Including Preclinical Assessment

Human epidermal growth factor receptor (HER2)–targeted Affibody molecules radiolabeled with 18F 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 18F radiolabeling strategies of the HER2-specific Affibody molecule ZHER2:2891, 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 (18F-SiFA), 18F-AlF-NOTA, and 4-18F-fluorobenzaldehyde (18F-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 111In-ABY-025 compound, which has demonstrable clinical utility, served as a reference tracer. Results: The non–decay-corrected radiochemical yields based on starting 18F-fluoride using the 18F-FBA, 18F-SiFA, and 18F-AlF-NOTA methods were 13% ± 3% (n = 5), 38% ± 2% (n = 3), and 11% ± 4% (n = 6), respectively. In naïve mice, both the 18F-AlF-NOTA-ZHER2:2891 and the 111In-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 18F-FBA-ZHER2:2891 or 18F-SiFA-ZHER2:2891 (4.8 ± 0.6 and 10.1 ± 0.7 %ID, respectively, at 90 min). The 18F-SiFA-ZHER2:2891 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 18F-FBA-ZHER2:2891 in a dual NCI-N87/A431 xenograft model demonstrated high tumor-to-background contrast for NCI-N87 tumors. Conclusion: The HER2 Affibody molecule ZHER2:2891 has been site-selectively radiolabeled by three 18F conjugation methods. Preliminary biologic data have identified 18F-FBA-ZHER2:2891 (also known as GE226) as a favored candidate for further development and radiochemistry automation.

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, [18F]GE-226, for positron emission tomography (PET) imaging. Experimental Design: Intrinsic cellular [18F]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: [18F]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/cm3/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 (r2 = 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: [18F]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. Clin Cancer Res; 20(6); 1632–43. ©2014 AACR.

GE-145, a new low-osmolar dimeric radiographic contrast medium

Background: Contrast-induced nephrotoxicity is a significant risk when using radiographic contrast media clinically, especially in high risk patients. Consequently, there is a need for a new contrast agent with improved clinical safety with regards to nephrotoxicity. Purpose: To evaluate the physicochemical properties as well as the preclinical safety and biodistribution parameters of the newly developed radiographic contrast medium GE-145. Material and Methods: Standard methods for radiographic contrast media were used for evaluation of physicochemical properties. The acute toxicity in rats was studied at 8, 10, and 12.5 gI/kg, the clinical chemistry parameters were determined, and histology of the kidneys was performed. Biodistribution was studied in rats using 123I-labeled GE-145. Results: GE-145 is more hydrophilic than iodixanol and has a considerably lower osmolality. The viscosity is similar to that of iodixanol and the protein binding is low. The acute toxicity is similar to that of iodixanol and the biodistribution is similar to that of other radiographic contrast media, showing mainly renal excretion. Kidney histology showed a moderate reversible vacuolization, similar to that of iodixanol. Conclusion: GE-145 exhibits similar preclinical properties to other dimeric radiographic contrast media. In addition, the low osmolality enables an iso-osmolar formulation containing a significantly higher concentration of electrolytes than Visipaque.

Imaging in drug development.

Imaging has played an important part in the diagnosis of disease and development of the understanding of the underlying disease mechanisms and is now poised to make an impact in the development of new pharmaceuticals. This chapter discusses the underlying technologies that make the field ready for this challenge. In particular, the potentials of magnetic resonance imaging and functional magnetic resonance imaging are outlined, including the new methods developed to provide additional information from the scans carried out. The field of nuclear medicine has seen a rapid increase in interest as advances in radiochemistry have enabled a wide range of new radiotracers to be synthesised.

Reply: Al18F Labeling of Affibody Molecules

TO THE EDITOR: Glaser et al. recently described the labeling of F-ZHER2:2891-Cys-NOTA-(COOH)2-AlF (18F-12) (1) and compared it in vivo to the biodistribution of that Affibody (Affibody AB) with 18F attached to carbon and silicon, as well as an 111In-DOTA-Affibody. They reported that the Al18F-labeled Affibody had a similar biodistribution to the 111In-Affibody, as previously noted by Heskamp et al. (2), and also observed that the Al18F-labeled Affibody had high uptake and retention in the kidney (;80 percentage injected dose [%ID], like the 111In-Affibody). This is presumably because the small-sized Affibody is eliminated through the kidneys, where it is rapidly catabolized, with the resulting Al18F complex residualized in the renal tubules in the same manner as the 111In-DOTA complex (3). In contrast, when the carbonand silicon-labeled Affibody molecules are metabolized in the kidney, the 18F-labeled metabolites are eliminated from the kidney cells, greatly reducing renal uptake. Although this clearly serves as an advantage for this product, much like differences between radioiodinated and radiometal-labeled antibody fragments, it is important to emphasize that renal uptake of the Al18F-Affibody product is a property of the Affibody targeting agent and not the Al18F complex. Previous studies with our pretargeting peptide (4) and the Al18F-NOTA-pegylated arginine-glycineaspartic acid dimer (PRGD2) peptide (5) both showed excellent renal clearance in the mouse models, and the Al18F-NOTAPRGD2 peptide also had good renal clearance in humans (6). It should also be noted that the 18F-Affibody labeled through a carbon atom had high hepatobiliary clearance (40–50 %ID in the intestines), whereas the Al18F-labeled Affibody had low uptake in the intestines. The high hepatobiliary accretion might be considered at least as undesirable as the high renal retention, depending on the use of the agent. Glaser et al. also reported a 2-fold lower labeling yield for their Al18F-Affibody than the Al18F-labeling yield of a similar Affibody bearing the same NOTA ligand (11% vs. 21%), and this despite the fact that Heskamp et al. used a lower amount of the Affibody (2). Although we cannot discount the possibility that slight differences in the Affibody structure could have influenced the yields, we strongly suspect the yield differences are attributable to the lack of a co-solvent in the labeling procedure used by Glaser et al. Indeed, we have shown that the use of a co-solvent generally improves yields 2-fold (7). Thus, we believe it is important when comparing labeling technologies to attempt to optimize or normalize each procedure, or if not empirically assessed, to state the conditions that might have affected yields when this information has been published previously. Second, whereas the nonresidualizing 18F-linkage used by Glaser et al. provided lower renal uptake, there likely are other situations, such as in target cells with a more rapid metabolism, in which a residualizing form of 18F afforded by the AlF method would be preferred (8). DISCLOSURE

Abstract B140: Positron emission tomography imaging of HER2 expression and pharmacodynamic response to HSP90 inhibition with the next-generation ZHER2:2891 Affibody molecule [18F]GE-226.

Accurate assessment of HER2 status remains a clinical challenge, with up to 20% of patients being potentially withdrawn from therapy or exposed to unnecessary toxicity. Non-invasive imaging is widely seen as a viable alternative to current methods, in particular in the setting of locoregional and distant recurrences not amenable to biopsy. A next-generation HER2-targeting Affibody-based radiotracer has been developed, [18F]GE-226, with enhanced pharmacokinetic characteristics and improved properties for large-scale and GMP grade synthesis. Kinetic modeling gave insights into Affibody-HER2 interactions. Intrinsic affinity to HER2 (KD = 76 pM) resulted in 11 to 67-fold higher [18F]GE-226 uptake in ten HER2 positive versus negative cell lines in vitro independent of lineage. Uptake correlated with HER2 protein expression but was independent of presence of other targets like EGFR. Blocking with [19F]GE-226 and HER2 siRNA treatment reduced uptake by 96.8 ± 2.6% and 81.7 ± 9.2%, respectively. Uptake in HER2 positive xenografts was rapid with steady state net irreversible binding kinetics making possible the distinction of HER2 negative (MCF7 (n = 6) and MCF7-p95HER2 (n = 3): NUV60 (normalized uptake value at 60 min; %ID/mL) 6.1 ± 0.7; Ki (irreversible uptake rate; mL/cm3/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; all n = 6) within 1 h. Tumor uptake correlated with HER2 expression determined by ELISA (r2=0.78). Specificity was further determined by comparing tumor localization of a fluorescently labeled tracer analogue with DAKO HercepTest. Affibody signal co-localized with HER2 expression at the cellular level independent of spatial heterogeneity. Tracer binding was not influenced by short-term or continuous exposure to trastuzumab in SKOV-3 xenografts (n=6) in keeping with differential epitope binding. Inhibition of the chaperone HSP90– of which HER2 is a client protein– by the therapeutic development candidate NVP-AUY922 caused dose-dependent HER2 degradation and consequently reduced tracer uptake in SKOV-3 cells in vitro and xenografts in vivo (area under the curve, AUC0-60: 618.4±90.1 and 446.7±42.8 %ID/mL*min for vehicle (n=4) and drug (n=5), respectively; P=0.043). In conclusion, [18F]GE-226 differentiates HER2 negative from HER2 expressing tumors. The tracer has potential utility for HER2 detection, irrespective of prior trastuzumab treatment and to monitor response to HSP90 inhibition. Lineage-independence of these results extends application beyond breast cancer. Due to the specific annotation to HER2, enhanced pharmacokinetic properties and completion of initial preclinical toxicology testing, [18F]GE-226 is now transitioning into clinical development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B140. Citation Format: Sebastian Trousil, Susan Hoppmann, Quang-De Nguyen, Maciej Kaliszczak, Giampaolo Tomasi, Peter Iveson, Duncan Hiscock, Eric O. Aboagye. Positron emission tomography imaging of HER2 expression and pharmacodynamic response to HSP90 inhibition with the next-generation ZHER2:2891 Affibody molecule [18F]GE-226. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B140.

Abstract 353: In vivo PET imaging of HER2 expression with GE226: An 18F-labelled affibody molecule

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: The assessment of HER2 expression in biopsies from primary lesions of breast cancer patients is a standard procedure to select patients for HER2-targeted therapies. However, in metastatic disease in which HER2 status can change, determination of HER2 expression is not standard procedure, which complicates patient management. Herein we report evidence for GE226 ([18F]FBA-ZHER2:2891), a highly specific HER2 targeted imaging agent that can determine HER2 expression levels in preclinical tumour models. We propose that GE226 can be progressed to clinical development for non-invasive determination of the HER2 status in recurrent breast cancer patients to improve the clinical management and therapy selection. Methods: The highly selective HER2 targeted 18F-labelled Affibody molecule GE226 was characterized in a murine dual tumour breast cancer model bearing NCI-N87 (high HER2 status) and A431 (low HER2 status) xenografts in separate flanks. Tumour-bearing mice were injected with 3 to 10 MBq of GE226, followed by biodistribution or positron emission tomography (PET) imaging analysis. Results: Biodistribution analysis demonstrated good differentiation of GE226 retention between high and low HER2 expressing tumours (8.4% ID/g and 3.4% ID/g respectively, 30 min post injection). GE226 cleared quickly from background tissue, including kidneys, with excellent ratios for tumour-to-muscle (8.9 for high HER2 status tumours and 3.6 for low HER2 status tumours, 30 min post injection) and tumour-to-blood (2.5 for high HER2 status tumours and 1.0 for low HER2 status tumours, 30 min post injection). PET imaging of GE226 in the dual tumour mouse model showed a marked difference in signal intensity between the two tumour types. Conclusions: The highly selective HER2 targeted Affibody molecule GE226 can image different levels of HER2 expression in a dual-tumour preclinical model of breast cancer with good target-to-background ratios. These data compare favourably with previous patient SPECT and PET studies using the 111In- or 68Ga-labelled HER2-binding Affibody molecule ABY-002 (Baum et al., J Nucl Med. 2010;51(6):892-7) which supports the efficacy of this class of Affibody tracer for visualization of HER2 expressing metastases. We plan to progress GE226 further to assess HER2 status in metastatic breast cancer patients in clinical PET studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 353. doi:1538-7445.AM2012-353