Radiolabelling and preclinical characterisation of [89Zr]Zr-Df-ATG-101 bispecific to PD-L1/4-1BB.

PURPOSE: ATG-101, a bispecific antibody that simultaneously targets the immune checkpoint PD-L1 and the costimulatory receptor 4-1BB, activates exhausted T cells upon PD-L1 crosslinking. Previous studies demonstrated promising anti-tumour efficacy of ATG-101 in preclinical models. Here, we labelled ATG-101 with 89Zr to confirm its tumour targeting effect and tissue biodistribution in a preclinical model. We also evaluated the use of immuno-PET to study tumour uptake of ATG-101 in vivo. METHODS: ATG-101, anti-PD-L1, and an isotype control were conjugated with p-SCN-Deferoxamine (Df). The Df-conjugated antibodies were radiolabelled with 89Zr, and their radiochemical purity, immunoreactivity, and serum stability were assessed. We conducted PET/MRI and biodistribution studies on [89Zr]Zr-Df-ATG-101 in BALB/c nude mice bearing PD-L1-expressing MDA-MB-231 breast cancer xenografts for up to 10 days after intravenous administration of [89Zr]Zr-labelled antibodies. The specificity of [89Zr]Zr-Df-ATG-101 was evaluated through a competition study with unlabelled ATG-101 and anti-PD-L1 antibodies. RESULTS: The Df-conjugation and [89Zr]Zr -radiolabelling did not affect the target binding of ATG-101. Biodistribution and imaging studies demonstrated biological similarity of [89Zr]Zr-Df-ATG-101 and [89Zr]Zr-Df-anti-PD-L1. Tumour uptake of [89Zr]Zr-Df-ATG-101 was clearly visualised using small-animal PET imaging up to 7 days post-injection. Competition studies confirmed the specificity of PD-L1 targeting in vivo. CONCLUSION: [89Zr]Zr-Df-ATG-101 in vivo distribution is dependent on PD-L1 expression in the MDA-MB-231 xenograft model. Immuno-PET with [89Zr]Zr-Df-ATG-101 provides real-time information about ATG-101 distribution and tumour uptake in vivo. Our data support the use of [89Zr]Zr-Df-ATG-101 to assess tumour and tissue uptake of ATG-101.

An Anti-VEGF-B Antibody Reduces Abnormal Tumor Vasculature and Enhances the Effects of Chemotherapy.

The vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key regulators of blood vessel formation, including in tumors, where their deregulated function can promote the production of aberrant, leaky blood vessels, supporting tumor development. Here we investigated the VEGFR1 ligand VEGF-B, which we demonstrate to be expressed in tumor cells and in tumor stroma and vasculature across a range of tumor types. We examined the anti-VEGF-B-specific monoclonal antibody 2H10 in preclinical xenograft models of breast and colorectal cancer, in comparison with the anti-VEGF-A antibody bevacizumab. Similar to bevacizumab, 2H10 therapy was associated with changes in tumor blood vessels and intra-tumoral diffusion consistent with normalization of the tumor vasculature. Accordingly, treatment resulted in partial inhibition of tumor growth, and significantly improved the response to chemotherapy. Our studies indicate the importance of VEGF-B in tumor growth, and the potential of specific anti-VEGF-B treatment to inhibit tumor development, alone or in combination with established chemotherapies.

Preclinical radiolabeling, in vivo biodistribution and positron emission tomography of a novel pyrrolobenzodiazepine (PBD)-based antibody drug conjugate targeting ASCT2.

INTRODUCTION: Anti-ASCT2 antibody drug conjugate (ADC) MEDI7247 has been under development as a potential anti-cancer therapy for patients with selected relapsed/refractory hematological malignancies and advanced solid tumors by MedImmune. Although promising efficacy was observed in the clinic, pharmacokinetic (PK) analyses observed low exposure of MEDI7247 in phase I hematological patients. To investigate the biodistribution properties of MEDI7247, MEDI7247 and control antibodies were radiolabeled with zirconium-89 and in vitro and in vivo properties characterized. METHODS: MEDI7247 (human anti-ASCT2 antibody conjugated with pyrrolobenzodiazepine (PBD)) and MEDI7519 (MEDI7247 without PBD drug conjugate) and an isotype control antibody drug conjugate construct were conjugated with p-isothiocyanatobenzyl-deferoxamine (Df) and radiolabeled with zirconium-89. In vitro studies included determining the radiochemical purity, protein integrity, immunoreactivity (Lindmo analysis), apparent antigen binding affinity for ASCT2-positive cells by Scatchard analysis and serum stability of the radiolabeled immunoconjugates. In vivo studies included biodistribution and PET/MRI imaging studies of the radiolabeled immunoconjugates in an ASCT2-positive tumor model, HT-29 colorectal carcinoma xenografts. RESULTS: Conditions for the Df-conjugation and radiolabeling of antibody constructs were determined to produce active radioimmunoconjugates. In vivo biodistribution and whole body PET/MRI imaging studies of [89Zr]Zr-Df-MEDI7519 and [89Zr]Zr-Df-MEDI7247 radioimmunoconjugates in HT-29 colon carcinoma xenografts in BALB/c nude mice demonstrated specific tumor localization. However, more rapid blood clearance and non-specific localization in liver was observed for [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 compared to isotype control ADC. Except for liver and bone, other normal tissues demonstrated clearance reflecting the blood clearance for all three constructs and no other abnormal tissue uptake. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: Preclinical biodistribution analyses of [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 showed the biodistribution pattern of anti-ASCT2 ADC MEDI7247 was similar to parental MEDI7519, and both antibodies showed specific tumor uptake compared to an isotype control ADC. This study highlights an important role nuclear medicine imaging techniques can play in early preclinical assessment of drug specificity as part of the drug development pipeline.

Automated radiosynthesis of [89Zr]Zr-DFOSq-Durvalumab for imaging of PD-L1 expressing tumours in vivo.

OBJECTIVES: 89Zr-labelled proteins are gaining importance in clinical research in a variety of diseases. To date, no clinical study has been reported that utilizes an automated approach for radiosynthesis of 89Zr-labelled radiopharmaceuticals. We aim to develop an automated method for the clinical production of 89Zr-labelled proteins and apply this method to Durvalumab, a monoclonal antibody targeting PD-L1 immune-checkpoint protein. PD-L1 expression is poorly understood and can be up-regulated over the course of chemo- and radiotherapy treatment. The ImmunoPET multicentre study aims to examine the dynamics of PD-L1 expression via 89Zr-Durvalumab PET imaging before, during, and after chemoradiotherapy. The developed automated technique will enable reproducible clinical production of [89Zr]Zr-DFOSq-Durvalumab for this study at three different sites. METHODS: Conjugation of Durvalumab to H3DFOSqOEt was optimized for optimal chelator-to-antibody ratio. Automated radiolabelling of H3DFOSq-Durvalumab with zirconium-89 was optimized on the disposable cassette based iPHASE technologies MultiSyn radiosynthesizer using a modified cassette. Activity losses were tracked using a dose calibrator and minimized by optimizing fluid transfers, reaction buffer, antibody formulation additives and pH. The biological profile of the radiolabelled antibody was confirmed in vivo in PD-L1+ (HCC827) and PD-L1- (A549) murine xenografts. Clinical process validation and quality control were performed at three separate study sites to satisfy clinical release criteria. RESULTS: H3DFOSq-Durvalumab with an average CAR of 3.02 was obtained. Radiolabelling kinetics in succinate (20 mM, pH 6) were significantly faster when compared to HEPES (0.5 M, pH 7.2) with >90 % conversion observed after 15 min. Residual radioactivity in the 89Zr isotope vial was reduced from 24 % to 0.44 % ± 0.18 % (n = 7) and losses in the reactor vial were reduced from 36 % ± 6 % (n = 4) to 0.82 % ± 0.75 % (n = 4) by including a surfactant in the reaction and formulation buffers. Overall process yield was 75 % ± 6 % (n = 5) and process time was 40 min. Typically, 165 MBq of [89Zr]Zr-DFOSq-Durvalumab with an apparent specific activity of 315 MBq/mg ± 34 MBq/mg (EOS) was obtained in a volume of 3.0 mL. At end-of-synthesis (EOS), radiochemical purity and protein integrity were always >99 % and >96 %, respectively, and dropped to 98 % and 65 % after incubation in human serum for 7 days at 37 °C. Immunoreactive fraction in HEK293/PD-L1 cells was 83.3 ± 9.0 (EOS). Preclinical in vivo data at 144 h p.i. showed excellent SUVmax in PD-L1+ tumour (8.32 ± 0.59) with a tumour-background ratio of 17.17 ± 3.96. [89Zr]Zr-DFOSq-Durvalumab passed all clinical release criteria at each study site and was deemed suitable for administration in a multicentre imaging trial. CONCLUSION: Fully automated production of [89Zr]Zr-DFOSq-Durvalumab for clinical use was achieved with minimal exposure to the operator. The cassette-based approach allows for consecutive productions on the same day and offers an alternative to currently used manual protocols. The method should be broadly applicable to other proteins and has the potential for clinical impact considering the growing number of clinical trials investigating 89Zr-labelled antibodies.

Bromodomain and extraterminal protein-targeted probe enables tumour visualisation in vivo using positron emission tomography.

Bromodomain and extraterminal (BET) proteins, a family of epigenetic regulators, have emerged as important oncology drug targets. BET proteins have not been targeted for molecular imaging of cancer. Here, we report the development of a novel molecule radiolabelled with positron emitting fluorine-18, [18F]BiPET-2, and its in vitro and preclinical evaluation in glioblastoma models.

18F-labeling and initial in vivo evaluation of a Hitomi peptide for imaging tissue transglutaminase 2.

INTRODUCTION: Tissue transglutaminase 2 (TG2) is a calcium-dependent enzyme which cross-links proteins. It is overexpressed in many diseases and plays a key role in tissue remodeling, including cell adhesion and migration. Overexpression of TG2 in breast cancer is a marker for patients at risk of recurrence. Non-invasive imaging of TG2 can therefore play an important role in patient management. TG2 probes labeled with the positron emitters 11C and 18F have thus far not found widespread application due to purity and metabolism issues. Our approach was to radiolabel a TG2 selective, 13-mer amino acid peptide, which was modified with a 5-azidopentanoic acid group at the N-terminus via a copper free click chemistry approach. METHODS: Radiochemistry was performed and fully automated using an iPhase FlexLab module. We produced the radiolabeling synthon [18F]FBz-DBCO from [18F]SFB and DBCO-amine. After HPLC purification, [18F]FBz-DBCO was reacted with the modified peptide and the putative radiotracer purified by HPLC. In vivo imaging using the radiolabeled amine was performed in mice bearing either TG2 expressing MDA-MB-231 or non-TG2 expressing MCF-7 xenografts as negative control. Expression of the target was confirmed using immunohistochemistry and western blot techniques. RESULTS: We obtained 9 ± 2 GBq of the radiolabeled peptide from 55 ± 5 GBq of fluorine-18 in an overall synthesis time of 160 min from end of bombardment (EOB), including HPLC purification and reformulation. Small animal PET/MR imaging showed that visualization of MDA-MB-231 tumors using the radiolabeled peptide could only be achieved due to differences in clearance between tumor and surrounding tissue. In the MCF-7 xenograft model, radiotracer clearance from tumor and surrounding tissue occurred at a similar rate, thus making it impossible to visualize MCF-7 tumors. The presence of TG2 in MDA-MB-231 tumors and absence in MCF-7 tumors was confirmed by immunohistochemistry staining and western blot analysis. CONCLUSION: A fully automated synthesis of a TG2 selective, 13-amino-acid peptide modified with 5-azido pentynoic acid at the N-terminal was established using [18F]FBzDBCO as a prosthetic group. Although our results show that radiolabeled peptides have potential as imaging agents for TG2, more research needs to be performed to improve radiotracer kinetics.

18F Site-Specific Labelling of a Single-Chain Antibody against Activated Platelets for the Detection of Acute Thrombosis in Positron Emission Tomography.

Positron emission tomography is the imaging modality of choice when it comes to the high sensitivity detection of key markers of thrombosis and inflammation, such as activated platelets. We, previously, generated a fluorine-18 labelled single-chain antibody (scFv) against ligand-induced binding sites (LIBS) on activated platelets, binding it to the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. We used a non-site-specific bio conjugation approach with N-succinimidyl-4-[18F]fluorobenzoate (S[18F]FB), leading to a mixture of products with reduced antigen binding. In the present study, we have developed and characterised a novel fluorine-18 PET radiotracer, based on this antibody, using site-specific bio conjugation to engineer cysteine residues with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). ScFvanti-LIBS and control antibody mut-scFv, with engineered C-terminal cysteine, were reduced, and then, they reacted with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM). Radiolabelled scFv was injected into mice with FeCl3-induced thrombus in the left carotid artery. Clots were imaged in a PET MR imaging system, and the amount of radioactivity in major organs was measured using an ionisation chamber and image analysis. Assessment of vessel injury, as well as the biodistribution of the radiolabelled scFv, was studied. In the in vivo experiments, we found uptake of the targeted tracer in the injured vessel, compared with the non-injured vessel, as well as a high uptake of both tracers in the kidney, lung, and muscle. As expected, both tracers cleared rapidly via the kidney. Surprisingly, a large quantity of both tracers was taken up by organs with a high glutathione content, such as the muscle and lung, due to the instability of the maleimide cysteine bond in vivo, which warrants further investigations. This limits the ability of the novel antibody radiotracer 18F-scFvanti-LIBS to bind to the target in vivo and, therefore, as a useful agent for the sensitive detection of activated platelets. We describe the first fluorine-18 variant of the scFvanti-LIBS against activated platelets using site-specific bio conjugation.

Tumor volumes as a predictor of response to the anti-EGFR antibody drug conjugate depatuxizumab mafadotin.

BACKGROUND: The adverse impact of increasing brain tumor size on the efficacy of antibody-drug conjugates (ADCs) was investigated preclinically then validated with clinical data. METHODS­PRECLINICAL STUDY: The impact of tumor size on ADC tumor delivery and treatment response was evaluated in an EGFR-amplified patient-derived glioblastoma (GBM) model following treatment with Depatuxizumab mafadotin (Depatux-M). Biodistribution and imaging studies correlated drug distribution with starting treatment volume and anti-tumor activity. METHODS­CLINICAL STUDY: M12-356 was a Phase I study of Depatux-M in patients with GBM. Blinded volumetric analysis of baseline tumor volumes of M12-356 patients was undertaken by two reviewers and results correlated with response and survival. RESULTS: Preclinically, imaging and biodistribution studies showed specific and significantly higher tumor uptake of zirconium-89 labeled Depatux-M (89Zr-Depatux-M) in mice with smaller tumor volume (~98 mm3) versus those with larger volumes (~365 mm3); concordantly, mice with tumor volumes ≤100 mm3 at treatment commencement had significantly better growth inhibition by Depatux-M (93% vs 27%, P < .001) and significantly longer overall survival (P < .0001) compared to tumors ≥400 mm3. Clinically, patients with tumor volumes <25 cm3 had significantly higher response rates (17% vs. 0%, P = .009) and longer overall survival (0.5 vs 0.89 years, P = .001) than tumors above 25 cm3. CONCLUSION: Both preclinical and clinical data showed intra-tumoral concentration and efficacy of Depatux-m inversely correlated with tumor size. This finding merit further investigation with pretreatment tumor volume as a predictor for response to ADCs, in both gliomas and other solid tumors.

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.

Targeting and Efficacy of Novel mAb806-Antibody-Drug Conjugates in Malignant Mesothelioma.

Epidermal growth factor receptor (EGFR) is highly overexpressed in malignant mesothelioma (MM). MAb806 is a novel anti-EGFR antibody that selectively targets a tumor-selective epitope. MAb806-derived antibody drug conjugates (ADCs), ABT-414, ABBV-221 and ABBV-322, may represent a novel therapeutic strategy in MM. EGFR and mAb806 epitope expressions in mesothelioma cell lines were evaluated using an array of binding assays, and the in vitro cell effects of ABT-414 and ABBV-322 were determined. In vivo therapy studies were conducted in mesothelioma xenograft and patient-derived xenograft (PDX) tumor models. We also performed biodistribution and imaging studies to allow the quantitative targeting of MM by mAb806 using a 89Zr-labeled immunoconjugate-ch806. A high EGFR expression was present in all mesothelioma cell lines evaluated and mAb806 binding present in all cell lines, except NCIH-2452. ABT-414 and ABBV-322 resulted in significant tumor growth inhibition in MM models with high EGFR and mAb806 epitope expressions. In contrast, in an EGFR-expressing PDX model that was negative for the mAb806 epitope, no growth inhibition was observed. We demonstrated the specific targeting of the mAb806 epitope expressing MM tumors using 89Zr-based PET imaging. Our data suggest that targeting EGFR in MM using specific ADCs is a valid therapeutic strategy and supports further investigation of the mAb806 epitope expression as a predictive biomarker.

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.

Deletion of intestinal Hdac3 remodels the lipidome of enterocytes and protects mice from diet-induced obesity.

Histone deacetylase 3 (Hdac3) regulates the expression of lipid metabolism genes in multiple tissues, however its role in regulating lipid metabolism in the intestinal epithelium is unknown. Here we demonstrate that intestine-specific deletion of Hdac3 (Hdac3IKO) protects mice from diet induced obesity. Intestinal epithelial cells (IECs) from Hdac3IKO mice display co-ordinate induction of genes and proteins involved in mitochondrial and peroxisomal ß-oxidation, have an increased rate of fatty acid oxidation, and undergo marked remodelling of their lipidome, particularly a reduction in long chain triglycerides. Many HDAC3-regulated fatty oxidation genes are transcriptional targets of the PPAR family of nuclear receptors, Hdac3 deletion enhances their induction by PPAR-agonists, and pharmacological HDAC3 inhibition induces their expression in enterocytes. These findings establish a central role for HDAC3 in co-ordinating PPAR-regulated lipid oxidation in the intestinal epithelium, and identify intestinal HDAC3 as a potential therapeutic target for preventing obesity and related diseases.

Smac mimetics LCL161 and GDC-0152 inhibit osteosarcoma growth and metastasis in mice.

BACKGROUND: Current therapies fail to cure over a third of osteosarcoma patients and around three quarters of those with metastatic disease. "Smac mimetics" (also known as "IAP antagonists") are a new class of anti-cancer agents. Previous work revealed that cells from murine osteosarcomas were efficiently sensitized by physiologically achievable concentrations of some Smac mimetics (including GDC-0152 and LCL161) to killing by the inflammatory cytokine TNFα in vitro, but survived exposure to Smac mimetics as sole agents. METHODS: Nude mice were subcutaneously or intramuscularly implanted with luciferase-expressing murine 1029H or human KRIB osteosarcoma cells. The impacts of treatment with GDC-0152, LCL161 and/or doxorubicin were assessed by caliper measurements, bioluminescence, 18FDG-PET and MRI imaging, and by weighing resected tumors at the experimental endpoint. Metastatic burden was examined by quantitative PCR, through amplification of a region of the luciferase gene from lung DNA. ATP levels in treated and untreated osteosarcoma cells were compared to assess in vitro sensitivity. Immunophenotyping of cells within treated and untreated tumors was performed by flow cytometry, and TNFα levels in blood and tumors were measured using cytokine bead arrays. RESULTS: Treatment with GDC-0152 or LCL161 suppressed the growth of subcutaneously or intramuscularly implanted osteosarcomas. In both models, co-treatment with doxorubicin and Smac mimetics impeded average osteosarcoma growth to a greater extent than either drug alone, although these differences were not statistically significant. Co-treatments were also more toxic. Co-treatment with LCL161 and doxorubicin was particularly effective in the KRIB intramuscular model, impeding primary tumor growth and delaying or preventing metastasis. Although the Smac mimetics were effective in vivo, in vitro they only efficiently killed osteosarcoma cells when TNFα was supplied. Implanted tumors contained high levels of TNFα, produced by infiltrating immune cells. Spontaneous osteosarcomas that arose in genetically-engineered immunocompetent mice also contained abundant TNFα. CONCLUSIONS: These data imply that Smac mimetics can cooperate with TNFα secreted by tumor-associated immune cells to kill osteosarcoma cells in vivo. Smac mimetics may therefore benefit osteosarcoma patients whose tumors contain Smac mimetic-responsive cancer cells and TNFα-producing infiltrating cells.

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.

Effect of Structural Modifications to Glyoxal-bis(thiosemicarbazonato)copper(II) Complexes on Cellular Copper Uptake, Copper-Mediated ATP7A Trafficking, and P-Glycoprotein Mediated Efflux.

Bis(thiosemicarbazonato)copper(II) complexes are of interest as potential therapeutics for cancer and neurodegenerative diseases as well as imaging agents for positron emission tomography (PET). The cellular uptake of six bis(thiosemcarbazonato)copper(II)complexes derived from glyoxal, with different functional groups Cu(gtsx) where x = different functional groups, was investigated in SKOV-3, HEK293, and HEK293 P-gp cell lines. Treatment of the cells with the copper complexes increased intracellular copper and increased levels of p-ERK due to activation of the Ras-Raf-MEK-ERK pathway. Treatment of SKOV-3 cells with low concentrations (µM) of two of the copper complexes led to trafficking of the endogenous copper transporter ATP7A from the Golgi network to the cell membrane. Experiments in HEK293 and HEK293-P-gp cells suggest that Cu(gtsm) and Cu(gtse) are substrates for the P-gp efflux protein but the complex with a pyrrolidine functional group, Cu(gtspyr), is not. A PET experiment in mice showed that [64Cu]Cu(gtspyr) has reasonable brain uptake but high liver uptake.

In Vitro and In Vivo Evaluation of 89Zr-DS-8273a as a Theranostic for Anti-Death Receptor 5 Therapy.

Background: DS-8273a, an anti-human death receptor 5 (DR5) agonistic antibody, has cytotoxic activity against human cancer cells and induces apoptosis after specific binding to DR5. DS-8273a is currently being used in clinical Phase I trials. This study evaluated the molecular imaging of DR5 expression in vivo in mouse tumor models using SPECT/CT and PET/MRI, as a tool for drug development and trial design. Methods: DS-8273a was radiolabeled with indium-111 and zirconium-89. Radiochemical purity, immunoreactivity, antigen binding affinity and serum stability were assessed in vitro. In vivo biodistribution and pharmacokinetic studies were performed, including SPECT/CT and PET/MR imaging. A dose-escalation study using a PET/MR imaging quantitative analysis was also performed to determine DR5 receptor saturability in a mouse model. Results:111In-CHX-A″-DTPA-DS-8273a and 89Zr-Df-Bz-NCS-DS-8273a showed high immunoreactivity (100%), high serum stability, and bound to DR5 expressing cells with high affinity (Ka, 1.02-1.22 × 1010 M-1). The number of antibodies bound per cell was 32,000. In vivo biodistribution studies showed high and specific uptake of 111In-CHX-A″-DTPA-DS-8273a and 89Zr-Df-Bz-NCS-DS-8273a in DR5 expressing COLO205 xenografts, with no specific uptake in normal tissues or in DR5-negative CT26 xenografts. DR5 receptor saturation was observed in vivo by biodistribution studies and quantitative PET/MRI analysis. Conclusion:89Zr-Df-Bz-NCS-DS-8273a is a potential novel PET imaging reagent for human bioimaging trials, and can be used for effective dose assessment and patient response evaluation in clinical trials.

Fluorine-18 radiolabeling of a nitrophenyl sulfoxide and its evaluation in an SK-RC-52 model of tumor hypoxia.

The significance of imaging hypoxia with the positron emission tomography ligand [(18) F]FMISO has been demonstrated in a variety of cancers. However, the slow kinetics of [(18) F]FMISO require a 2-h delay between tracer administration and patient scanning. Labeled chloroethyl sulfoxides have shown faster kinetics and higher contrast than [(18) F]FMISO in a rat model of ischemic stroke. However, these nitrogen mustard analogues are unsuitable for routine production and use in humans. Here, we report on the synthesis and in vitro and in vivo evaluation of a novel sulfoxide, which contains an ester moiety for hydrolysis and subsequent trapping in hypoxic cells. Non-decay corrected yields of radioactivity were 1.18 ± 0.24% (n = 27, 2.5 ± 0.5% decay corrected radiochemical yield) based on K[(18) F]F. The radiotracer did not show any defluorination and did not undergo metabolism in an in vitro assay using S9 liver fractions. Imaging studies using an SK-RC-52 tumor model in BALB/c nude mice have revealed that [(18) F]1 is retained in hypoxic tumors and has similar hypoxia selectivity to [(18) F]FMISO. Because of a three times faster clearance rate than [(18) F]FMISO from normoxic tissue, [(18) F]1 has emerged as a promising new radiotracer for hypoxia imaging.

L-Tyrosine Confers Residualizing Properties to a d-Amino Acid-Rich Residualizing Peptide for Radioiodination of Internalizing Antibodies.

PURPOSE: The aims of the study were to develop and evaluate a novel residualizing peptide for labeling internalizing antibodies with (124)I to support clinical development using immuno-positron emission tomography (PET). METHODS: The anti-epidermal growth factor receptor antibody ch806 was radiolabeled directly or indirectly with isotopes and various residualizing peptides. Azido-derivatized radiolabeled peptides were conjugated to dibenzylcyclooctyne-derivatized ch806 antibody via click chemistry. The radiochemical purities, antigen-expressing U87MG.de2-7 human glioblastoma cell-binding properties, and targeting of xenografts at 72 hours post injection of all radioconjugates were compared. Biodistribution of (124)I-PEG4-tptddYddtpt-ch806 and immuno-PET imaging were evaluated in tumor-bearing mice. RESULTS: Biodistribution studies using xenografts at 72 hours post injection showed that (131)I-PEG4-tptddYddtpt-ch806 tumor uptake was similar to (111)In-CHX-A″-DTPA-ch806. (125)I-PEG4-tptddyddtpt-ch806 showed a lower tumor uptake value but higher than directly labeled (125)I-ch806. (124)I-PEG4-tptddYddtpt-ch806 was produced at 23% labeling efficiency, 98% radiochemical purity, 25.9 MBq/mg specific activity, and 64% cell binding in the presence of antigen excess. Tumor uptake for (124)I-PEG4-tptddYddtpt-ch806 was similar to (111)In-CHX-A″-DTPA-ch806. High-resolution immuno-PET/magnetic resonance imaging of tumors showed good correlation with biodistribution data. CONCLUSIONS: The mixed d/l-enantiomeric peptide, dThr-dPro-dThr-dAsp-dAsp-Tyr-dAsp-dAsp-dThr-dPro-dThr, is suitable for radiolabeling antibodies with radiohalogens such as (124)I for high-resolution immuno-PET imaging of tumors and for evaluation in early-phase clinical trials.