Radiolabelling and preclinical characterization of 89Zr-Df-radiolabelled bispecific anti-PD-L1/TGF-ßRII fusion protein bintrafusp alfa.

PURPOSE: Τhis study aimed to optimize the 89Zr-radiolabelling of bintrafusp alfa investigational drug product and controls, and perform the in vitro and in vivo characterization of 89Zr-Df-bintrafusp alfa and 89Zr-Df-control radioconjugates. METHODS: Bintrafusp alfa (anti-PD-L1 human IgG1 antibody fused to TGF-ß receptor II (TGF-ßRII), avelumab (anti-PD-L1 human IgG1 control antibody), isotype control (mutated inactive anti-PD-L1 IgG1 control antibody), and trap control (mutated inactive anti-PD-L1 human IgG1 fused to active TGF-ßRII) were chelated with p-isothiocyanatobenzyl-desferrioxamine (Df). After radiolabelling with zirconium-89 (89Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. In vivo biodistribution and imaging studies were performed with PET/CT to identify and quantitate 89Zr-Df-bintrafusp alfa tumour uptake in a PD-L1/TGF-ß-positive murine breast cancer model (EMT-6). Specificity of 89Zr-Df-bintrafusp alfa was assessed via a combined biodistribution and imaging experiment in the presence of competing cold bintrafusp alfa (1 mg/kg). RESULTS: Nanomolar affinities for PD-L1 were achieved with 89Zr-Df-bintrafusp alfa and 89Zr-avelumab. Biodistribution and imaging studies in PD-L1- and TGF-ß-positive EMT-6 tumour-bearing BALB/c mice demonstrated the biologic similarity of 89Zr-Df-bintrafusp alfa and 89Zr-avelumab indicating the in vivo distribution pattern of bintrafusp alfa is driven by its PD-L1 binding arm. Competition study with 1 mg of unlabelled bintrafusp alfa or avelumab co-administered with trace dose of 89Zr-labelled bintrafusp alfa demonstrated the impact of dose and specificity of PD-L1 targeting in vivo. CONCLUSION: Molecular imaging of 89Zr-Df-bintrafusp alfa biodistribution was achievable and allows non-invasive quantitation of tumour uptake of 89Zr-Df-bintrafusp alfa, suitable for use in bioimaging clinical trials in cancer patients.

Synthesis and fluorine-18 radiolabeling of a phospholipid as a PET imaging agent for prostate cancer.

INTRODUCTION: Altered lipid metabolism and subsequent changes in cellular lipid composition have been observed in prostate cancer cells, are associated with poor clinical outcome, and are promising targets for metabolic therapies. This study reports for the first time on the synthesis of a phospholipid radiotracer based on the phospholipid 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (PC44:12) to allow tracking of polyunsaturated lipid tumor uptake via PET imaging. This tracer may aid in the development of strategies to modulate response to therapies targeting lipid metabolism in prostate cancer. METHODS: Lipidomics analysis of prostate tumor explants and LNCaP tumor cells were used to identify PC44:12 as a potential phospholipid candidate for radiotracer development. Synthesis of phosphocholine precursor and non-radioactive standard were optimised using click chemistry. The biodistribution of a fluorine-18 labeled analogue, N-{[4-(2-[18F]fluoroethyl)-2,3,4-triazol-1-yl]methyl}-1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine ([18F]2) was determined in LNCaP prostate tumor-bearing NOD SCID gamma mice by ex vivo biodistribution and PET imaging studies and compared to biodistribution of [18F]fluoromethylcholine. RESULTS: [18F]2 was produced with a decay-corrected yield of 17.8 ± 3.7% and an average radiochemical purity of 97.00 ± 0.89% (n = 6). Molar activity was 85.1 ± 3.45 GBq/µmol (2300 ± 93 mCi/µmol) and the total synthesis time was 2 h. Ex vivo biodistribution data demonstrated high liver uptake (41.1 ± 9.2%ID/g) and high splenic uptake (10.9 ± 9.1%ID/g) 50 min post-injection. Ex vivo biodistribution showed low absolute tumor uptake of [18F]2 (0.8 ± 0.3%ID/g). However, dynamic PET imaging demonstrated an increase over time of the relative tumor-to-muscle ratio with a peak of 2.8 ± 0.5 reached 1 h post-injection. In contrast, dynamic PET of [18F]fluoromethylcholine demonstrated no increase in tumor-to-muscle ratios due to an increase in both tumor and muscle over time. Absolute uptake of [18F]fluoromethylcholine was higher and peaked at 60 min post injection (2.25 ± 0.29%ID/g) compared to [18F]2 (1.44 ± 0.06%ID/g) during the 1 h dynamic scan period. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This study demonstrates the ability to radiolabel phospholipids and indicates the potential to monitor the in vivo distribution of phospholipids using fluorine-18 based PET.

Automated synthesis of 18F radiolabelled indole containing Oncrasin-like molecules; a comparison of iodonium salts and boronic ester chemistry.

BACKGROUND: Oncrasin-1 is a small molecule which was identified from a screen of KRAS mutant cancer cells and has shown specificity for KRAS mutant cell killing. We aimed to develop a radiolabelled form of Oncrasin-1 to enable in-vivo imaging of mutant KRAS expression in malignant tumours. This work outlines the synthesis of 3 fluorinated derivatives and development of iodonium salt and boronic ester precursors for radiolabelling with the 18F isotope. RESULTS: In our hands, synthesis of iodonium salts were not easily accessible due to the 3-carbaldehyde indole structure being preferentially oxidized by conditions required for iodonium salt formation, rather than benzyl iodide. Synthesis and radiolabelling of boronic acid pinacol ester precursors were successful, with the products being obtained in yields of 10.76% ± 0.96% (n = 5), 14.7% ±8.58% (n = 3) and 14.92% ±3.9% (n = 3) for 18F KAM001, 18F KAM002 and 18F KAM003 respectively, with radiochemical purity of greater than 99%. CONCLUSIONS: The successful synthesis of these tracers has been undertaken utilizing boronic ester radio-fluorination methods and will allow for investigation of Oncrasin based molecules as potential diagnostics for cancers expressing mutant KRAS protein.

Synthesis and validation of [18F]mBPET-1, a fluorine-18 labelled mTOR inhibitor derivative based on a benzofuran backbone.

BACKGROUND: Targeted therapy of HER2 positive breast cancer has led to clinical success in some cases with primary and secondary resistance being major obstacles. Due to the substantial involvement of mTOR kinase in cell growth and proliferation pathways it is now targeted in combination treatments to counteract HER2 targeted therapy resistance. However, the selection of receptive patient populations for a specific drug combination is crucial. This work aims to develop a molecular probe capable of identifying patients with tumour populations which are receptive to RAD001 combination therapy. Based on the structure of a mTOR inhibitor specific for mTORC1, we designed, synthesised and characterised a novel benzofuran based molecular probe which suits late stage fluorination via Click chemistry. RESULTS: Synthesis of the alkyne precursor 5 proceeded in 27.5% yield over 7 linear steps. Click derivatisation gave the non-radioactive standard in 25% yield. Radiosynthesis of [18F]1-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl) methyl)-4-((5-methoxy-2-phenylbenzofuran-4-yl) methyl) piperazine ([18F]mBPET-1) proceeded over two steps which were automated on an iPhase FlexLab synthesis module. In the first step, 2-[18F]fluoroethylazide ([18F]6) was produced, purified by automated distillation in 60% non-decay-corrected yield and subjected to Click conditions with 5. Semi-preparative RP-HPLC purification and reformulation gave [18F]mBPET-1 in 40% ± 5% (n = 6) overall RCY with a process time of 90 min. Radiochemical purity was ≥99% at end of synthesis (EOS) and ≥ 98% after 4 h at room temperature. Molar activities ranged from typically 24.8 GBq/µmol (EOS) to a maximum of 78.6 GBq/µmol (EOS). Lipophilicity of [18F]mBPET-1 was determined at pH 7.4 (logD7.4 = 0.89). [18F]mBPET-1 showed high metabolic stability when incubated with mouse S9 liver fractions which resulted in a 0.8% drop in radiochemical purity after 3 h. Cell uptake assays showed 1.3-1.9-fold increased uptake of the [18F]mBPET-1 in RAD001 sensitive compared to insensitive cells across a panel of 4 breast cancer cell lines. CONCLUSION: Molecular targeting of mTOR with [18F]mBPET-1 distinguishes mTOR inhibitor sensitive and insensitive cell lines. Future studies will explore the ability of [18F]mBPET-1 to predict response to mTOR inhibitor treatment in in vivo models.

Molecular imaging of T cell co-regulator factor B7-H3 with 89Zr-DS-5573a.

B7-H3 is a transmembrane protein widely expressed in a variety of cancers and has been shown to play a role in anti-tumor immunity. This study aims to develop a molecular imaging probe to identify B7-H3 expression in tumors and to develop 89Zr-DS-5573a as a theranostic that could aid patient selection in clinical Phase I studies. Methods: The anti-B7-H3 humanised monoclonal antibody DS-5573a was labeled with zirconium-89 (89Zr-), and assessed for radiochemical purity, immunoreactivity (Lindmo analysis), antigen binding affinity (Scatchard analysis), and serum stability in vitro. In vivo biodistribution and imaging studies were performed with positron emission tomography and magnetic resonance imaging (PET/MRI) studies to identify and quantitate 89Zr-DS-5573a tumor uptake in a B7-H3-positive breast cancer model (MDA-MB-231) and a B7-H3-negative murine colon cancer model (CT26). Imaging and biodistribution studies were also performed in MDA-MB-231 tumor-bearing SCID mice in the absence and presence of therapeutic DS-5573a antibody dose (3 mg/kg DS-5573a). Results:89Zr-DS-5573a showed high and specific binding to B7-H3-expressing MDA-MB-231 cells (immunoreactivity on day 0, 75.0 ± 2.9%), and low binding to B7-H3-negative CT26 cells (immunoreactivity on day 0, 10.85 ± 0.11%) in vitro. 89Zr-DS-5573a demonstrated good serum stability in vitro with 57.2 ± 2.0% of immunoreactivity remaining on day 7. In vivo biodistribution studies showed high uptake of 89Zr-DS-5573a in B7-H3-expressing MDA-MB-231 tumor-bearing mice, achieving 32.32 ± 6.55 %ID/g on day 7 post injection in BALB/c nu/nu mice and 25.76 ± 1.79 %ID/g in SCID mice, with minimal evidence of non-specific uptake in normal tissues, and excellent tumor localization on PET/MRI. In a combined imaging/therapy study, receptor saturation was demonstrated in tumors responding to therapy. Conclusion:89Zr-DS-5573a demonstrates specific and prolonged targeting of B7-H3-expressing tumors in vivo. Saturation of binding sites was demonstrated in tumors responding to DS-5573a therapy. These results indicate that 89Zr-DS-5573a has potential to target B7-H3-expressing tumors in cancer patients. Furthermore 89Zr-DS-5573a has the potential to provide important insights into T cell biology through its specific binding to B7-H3.

Improved synthesis of [(18)F]FLETT via a fully automated vacuum distillation method for [(18)F]2-fluoroethyl azide purification.

The synthesis of [(18)F]2-fluoroethyl azide and its subsequent click reaction with 5-ethynyl-2'-deoxyuridine (EDU) to form [(18)F]FLETT was performed using an iPhase FlexLab module. The implementation of a vacuum distillation method afforded [(18)F]2-fluoroethyl azide in 87±5.3% radiochemical yield. The use of Cu(CH3CN)4PF6 and TBTA as catalyst enabled us to fully automate the [(18)F]FLETT synthesis without the need for the operator to enter the radiation field. [(18)F]FLETT was produced in higher overall yield (41.3±6.5%) and shorter synthesis time (67min) than with our previously reported manual method (32.5±2.5% in 130min).

Fully automated synthesis and coupling of [(18) F]FBEM to glutathione using the iPHASE FlexLab module.

Site-specific radiolabelling of peptides or antibodies using [(18) F]FBEM is often preferred over non-site-specific radiolabelling with [(18) F]SFB because it does not affect the affinity of the antibody to its target. Unfortunately, the synthesis of [(18) F]FBEM and its conjugation to thiol containing macromolecules requires some manual intervention, which leads to radiation exposure of the radiochemist. In this publication, we report on the complete automation of [(18) F]FBEM production and its subsequent conjugation to glutathione using a slightly modified iPHASE FlexLab module. [(18) F]FBEM was produced in 1.185 ± 0.168 GBq (15-20%; n = 10; 0.75 ± 0.106 GBq non-decay corrected) with a specific activity of 57 ± 10 GBq/µmol. Radiochemical purity was 97 ± 1% and the synthesis time including HPLC purification and reformulation was 70 min. After evaporation to dryness, [(18) F]FBEM was conjugated to glutathione in PBS buffer pH 7.4 in quantitative yields. This fully automated method does not require any manual intervention and therefore reduces the radiation exposure to the operator.

Structural studies on cycloadducts of furan, 2-methoxyfuran, and 5-trimethylsilylcyclopentadiene with maleic anhydride and N-methylmaleimide.

The early stages of the retro-Diels-Alder reaction are clearly apparent in the structures of the cycloadducts formed between furan or 5-trimethylsilylcyclopentadiene with maleic anhydride and N-methylmaleimide. The degree of lengthening of the C-C bonds that break in this reaction is clearly related to the known reactivity of these cycloadducts toward this reaction. In the structures of the cycloadducts 21 and 22 derived from 2-methoxyfuran, the early stages of an alternative fragmentation reaction are apparent, consistent with the reactivity of these compounds in solution.

Structures of oxygenated cyclohexa-1,3-diene-maleic anyhydride cycloadducts. Structural evidence suggests a stepwise retro-Diels-Alder reaction.

The structures of a series of cyclohexadiene-maleic anhydride cycloadducts show structural parameters consistent with the early stages of the retro-Diels-Alder (rDA) reaction in the ground state structure. The symmetrical adduct shows effects consistent with a synchronous rDA reaction. An asynchronous rDA reaction is suggested in the structure of 8, and the first evidence suggesting a two-step retro-Diels-Alder mechanism is provided by the deoxygenated adduct 9.

Manifestations of the Alder-Rickert reaction in the structures of bicyclo[2.2.2]octadiene and bicyclo[2.2.2]octene derivatives.

The Alder-Rickert ethylene extrusion reaction manifests in the ground state structures of compounds 9-12 which contain the bicyclo[2.2.2]octadiene moiety and compounds 13, 14, and 17-20 which contain the bicyclo[2.2.2]octene moiety. A significant decrease of the 13C-13C one-bond coupling constants for the C-C bonds, which break in this fragmentation reaction, suggests lengthening, and hence weakening of these bonds. In the unsymmetrical systems these effects are also shown to be associated with strengthening of the CH2-CH2 bond, which is ultimately lost from the molecule as ethylene. Low-temperature crystal structures of compounds 9-12 and 16 provide evidence for similar crystal packing requirements of the CH2-CH2 and CH=CH moieties.