Matthias Glaser

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.

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

Automated synthesis of [11C]-(+)-PHNO from [11C]methyl iodide

Alterations in dopaminergic activity form a fundamental neurobiological process in both schizophrenia and drug abuse and PET imaging offers unrivalled opportunities for investigating the dopamine system in these conditions. Due to competition between dopamine and D2 radiotracers for receptor binding, increases in synaptic dopamine produce reliable reductions in D2 radiotracer binding potential (BP). Herein, we describe a novel method for the synthesis of the potent D2/3 agonist [C]-(+)-PHNO, (+)-4-(3-[C]-propyl)-3,4,4a,5,6,10b-Hexahydro-2HNaphtho[1,2-b][1,4]Oxazin-9-ol. The previously published method for preparation of [C]-(+)-PHNO used multi-step labelling procedure (Brown et al., 1997; Wilson et al., 2005), with the aim of simplifying the radiochemistry and compatibility with other [C]tracer production, an alternative simplified labeling approach from [C]methyl iodide synthesis was examined for the preparation of [C]-(+)-PHNO. In comparison to the previously published [C]-(+)-PHNO synthesis a new [C]methyl iodide route has been developed resulting in carbon-11 labeling at the terminal carbon of the Npropyl chain. Starting from an O-trialkylsilyl-N-acetyl precursor, deprotonation of the N-acetyl group was achieved using LHMDS in THF. Alkylation with [C]methyl iodide afforded the corresponding N-propionyl amide intermediate which was in turn was reduced with borane in THF at 50 °C. Finally, acid deprotection of the O-trialkylsilyl group afforded crude [3-C]-(+)-PHNO which was purified by HPLC before the product was resolubilised in isotonic saline and ascorbic acid was added to prevent radiolysis. The average radiochemical yield of [3-C]-(+)-PHNO achieved from [C]CO2 was ca. 9% (n=13, range 2–30%, non-decay corrected). The radiochemical purity of [3-C]-(+)-PHNO was >95%. For pre-clinical studies the radiochemical purity of [3-C]-(+)-PHNO was >95% with a specific activity at time of injection was 50.58±23.77 GBq/μmol (range 26.80–81.05 GBq/μmol). Pre-clinical evaluation of [3-C]-(+)-PHNO (Egerton et al., 2010) revealed rapid initial brain uptake and accumulation in the D2-rich striatum. The pattern of brain biodistribution was consistent with the distribution of D2/3 receptors and the pharmacokinetic profile observed was similar to that previously reported for [C]-(+)-PHNO, indicating that C-labelling at an alternate position did not markedly alter tracer kinetics or metabolism.

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, ILnnBackground: 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.nnCitation 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

Abstract A222: Assessment of tumor response to therapy with the caspase‐3/7 specific [18F]ICMT‐11 PET imaging tracer

Of the molecular biochemical alterations that occur during apoptosis, activation of caspases, notably caspase‐3, is probably the most attractive for developing specific in vivo molecular imaging probes. We recently designed a library of isatin‐5 sulfonamides and selected [18F]ICMT‐11 for further evaluation on the basis of sub‐nanomolar affinity for activated capsase‐3, high metabolic stability, and facile radiolabeling. We have demonstrated that [18F]ICMT‐11 binds to a range of drug‐induced apoptotic cancer cells in vitro and to 38C13 murine lymphoma xenografts in vivo by up to 2‐fold at 24 h post‐treatment compared to vehicle treatment. We further associated the increased signal intensity in tumors after drug treatment ‐ detected by whole body in vivo microPET imaging ‐ with increased apoptosis detected by immunohistochemistry, and have therefore characterized [18F]ICMT‐11 as a caspase‐3/7 specific PET imaging radiotracer for the assessment of tumor apoptosis that could find utility in anticancer drug development and the monitoring of early responses to therapy. In the present study, we have investigated the sensitivity of [18F]ICMT‐11 compared to [18F]DG in a longitudinal experimental protocol where the same tumor bearing mouse is subjected to pre‐ ,24h and 48h post‐cyclophosphamide treatment microPET imaging. The tumor volumes have been recorded throughout the study for comparison with PET generated data. [18F]ICMT‐11 PET images and imaging variables were characterized by a weak baseline tumor uptake in pretreated animals, which increased after 24h cyclophosphamide treatment, then decreased at 48h posttreatment to levels greater than that of baseline (NUV60 = 0.05±0.01, 0.12±0.03 and 0.09±0.03 at pre‐, 24h and 48h post‐treatment, respectively). In contrast, [18F]DG PET images and imaging variables showed a high tumor uptake in pretreated animals, as expected, that decreased progressively at 24 and 48h posttreatment (NUV60=3.33±0.24, 1.95±0.16 and 1.26±0.10 at pre‐, 24h and 48h post‐treatment, respectively). The changes in [18F]ICMT‐11 and [18F]DG uptake occurred in parallel with small reduction (94.3%±6.0 that of the pretreated mice tumor volumes) of the tumor volume at 24h posttreatment compared to pretreatment, and a more drastic shrinkage (29.9%±3.6 that of the pretreated mice tumor volumes) at 48h posttreatment. We showed that both [18F]ICMT‐11 and [18F]DG PET can be use to monitor response to cyclophosphamide treatment in our longitudinal experimental model. At 24h and 48h posttreatment, [18F]ICMT‐11 PET imaging detects increased tumor apoptosis and [18F]DG PET detects decreased tumor metabolic activity. At 24h posttreatment, the increased [18F]ICMT‐11 and decreased [18F]DG PET tumor intensity signals are associated with only a small decrease in tumor volumes. In contrast, we observed a drastic tumor shrinkage at 48h posttreatment that was, however, associated with less tumor apoptosis detected by [18F]ICMT‐11 and almost no tumor metabolic activity. It is possible that the early increase in tumor apoptosis (24 h) led to tumor shrinkage. Our future studies will investigate [18F]ICMT‐11 PET imaging assessment of tumor response to therapy using a specific apoptosis targeting drug in the same longitudinal experimental model. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A222.