Targeting Fn14 as a therapeutic target for cachexia reprograms the glycolytic pathway in tumour and brain in mice.

PURPOSE: Cachexia is a complex syndrome characterized by unintentional weight loss, progressive muscle wasting and loss of appetite. Anti-Fn14 antibody (mAb 002) targets the TWEAK receptor (Fn14) in murine models of cancer cachexia and can extend the lifespan of mice by restoring the body weight of mice. Here, we investigated glucose metabolic changes in murine models of cachexia via [18F]FDG PET imaging, to explore whether Fn14 plays a role in the metabolic changes that occur during cancer cachexia. METHODS: [18F]FDG PET/MRI imaging was performed in cachexia-inducing tumour models versus models that do not induce cachexia. SUVaverage was calculated for all tumours via volume of interest (VOI) analysis of PET/MRI overlay images using PMOD software. RESULTS: [18F]FDG PET imaging demonstrated increased tumour and brain uptake in cachectic versus non-cachectic tumour-bearing mice. Therapy with mAb 002 was able to reduce [18F]FDG uptake in tumours (P < 0.05, n = 3). Fn14 KO tumours did not induce body weight loss and did not show an increase in [18F]FDG tumour and brain uptake over time. In non-cachectic mice bearing Fn14 KO tumours, [18F]FDG tumour uptake was significantly lower (P < 0.01) than in cachectic mice bearing Fn14 WT counterparts. As a by-product of glucose metabolism, l-lactate production was also increased in cachexia-inducing tumours expressing Fn14. CONCLUSION: Our results demonstrate that Fn14 receptor activation is linked to glucose metabolism of cachexia-inducing tumours.

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.

Targeting of immune checkpoint regulator V-domain Ig suppressor of T-cell activation (VISTA) with 89Zr-labelled CI-8993.

BACKGROUND: CI-8993 is a fully human IgG1κ monoclonal antibody (mAb) that binds specifically to immune checkpoint molecule VISTA (V-domain Ig suppressor of T-cell activation). Phase I safety has been established in patients with advanced cancer (NCT02671955). To determine the pharmacokinetics and biodistribution of CI-8993 in patients, we aimed to develop 89Zr-labelled CI-8993 and validate PET imaging and quantitation in preclinical models prior to a planned human bioimaging trial. METHODS: CI-8993 and human isotype IgG1 control were conjugated to the metal ion chelator p-isothiocyanatobenzyl-desferrioxamine (Df). Quality of conjugates were assessed by SE-HPLC, SDS-PAGE, and FACS. After radiolabelling with zirconium-89 (89Zr), radioconjugates were assessed for radiochemical purity, immunoreactivity, antigen binding affinity, and serum stability in vitro. [89Zr]Zr-Df-CI-8993 alone (1 mg/kg, 4.6 MBq) or in combination with 30 mg/kg unlabelled CI-8993, as well as isotype control [89Zr]Zr-Df-IgG1 (1 mg/kg, 4.6 MBq) were assessed in human VISTA knock-in female (C57BL/6 N-Vsirtm1.1(VSIR)Geno, huVISTA KI) or control C57BL/6 mice bearing syngeneic MB49 bladder cancer tumours; and in BALB/c nu/nu mice bearing pancreatic Capan-2 tumours. RESULTS: Stable constructs with an average chelator-to-antibody ratio of 1.81 were achieved. SDS-PAGE and SE-HPLC showed integrity of CI-8993 was maintained after conjugation; and ELISA indicated no impact of conjugation and radiolabelling on binding to human VISTA. PET imaging and biodistribution in MB49 tumour-bearing huVISTA KI female mice showed specific localisation of [89Zr]Zr-Df-CI-8993 to VISTA in spleen and tumour tissues expressing human VISTA. Specific tumour uptake was also demonstrated in Capan-2 xenografted BALB/c nu/nu mice. CONCLUSIONS: We radiolabelled and validated [89Zr]Zr-Df-CI-8993 for specific binding to huVISTA in vivo. Our results demonstrate that 89Zr-labelled CI-8993 is now suitable for targeting and imaging VISTA expression in human trials.

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.

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.

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.

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.

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.

Integral membrane protease fibroblast activation protein sensitizes fibrosarcoma to chemotherapy and alters cell death mechanisms.

Fibroblast activation protein (FAP), an integral membrane serine protease, is found on fibro- and osteo-sarcoma and on myofibroblasts in epithelial carcinoma, but rarely on other adult tissue. FAP has been demonstrated to be an excellent target for tumor imaging in clinical trials, and antibodies and other FAP-targeting drugs are in development. Here we have shown that FAP overexpression increased the growth of HT1080 fibrosarcoma cells in vitro and in vivo, and found that the expression of FAP affects response to chemotherapy. When treated with doxorubicin, expression of FAP increased susceptibility to the drug. In spite of this, FAP-HT1080 cells had fewer markers of classical apoptosis than HT1080 cells and neither necrosis nor necroptosis were enhanced. However, levels of early mitochondrial and lysosomal membrane permeability markers were increased, and autophagy switched from a protective function in HT1080 cells to part of the cell death mechanism with FAP expression. Therefore, FAP may affect how the tumor responds to chemotherapeutic drugs overall, which should be considered in targeted drug development. The overexpression of FAP also alters cell signaling and responses to the environment in this cell line. This includes cell death mechanisms, changing the response of HT1080 cells to doxorubicin from classical apoptosis to an organelle membrane permeability-dependent form of cell death.

Toward hypoxia-selective rhenium and technetium tricarbonyl complexes.

With the aim of preparing hypoxia-selective imaging and therapeutic agents, technetium(I) and rhenium(I) tricarbonyl complexes with pyridylhydrazone, dipyridylamine, and pyridylaminocarboxylate ligands containing nitrobenzyl or nitroimidazole functional groups have been prepared. The rhenium tricarbonyl complexes were synthesized with short reaction times using microwave irradiation. Rhenium tricarbonyl complexes with deprotonated p-nitrophenyl pyridylhydrazone ligands are luminescent, and this has been used to track their uptake in HeLa cells using confocal fluorescent microscopy. Selected rhenium tricarbonyl complexes displayed higher uptake in hypoxic cells when compared to normoxic cells. A (99m)Tc tricarbonyl complex with a dipyridylamine ligand bearing a nitroimidazole functional group is stable in human serum and was shown to localize in a human renal cell carcinoma (RCC; SK-RC-52) tumor in a mouse.

Characterization of an Estrogen Receptor α-Selective 18 F-Estradiol PET Tracer.

Objective Conventional imaging of cancer with modalities such as computed tomography or magnetic resonance imaging provides little information about the underlying biology of the cancer and consequently little guidance for systemic treatment choices. Accurate identification of aggressive cancers or those that are likely to respond to specific treatment regimens would allow more precisely tailored treatments to be used. The expression of the estrogen receptor α subunit is associated with a more aggressive phenotype, with a greater propensity to metastasize. We aimed to characterize the binding properties of an 18 F-estradiol positron emission tomography (PET) tracer in its ability to bind to the α and ß forms of estrogen receptors in vitro and confirmed its binding to estrogen receptor α in vivo. Methods The 18 F-estradiol PET tracer was synthesized and its quality confirmed by high-performance liquid chromatography. Binding of the tracer was assessed in vitro by saturation and competitive binding studies to HEK293T cells transfected with estrogen receptor α ( ESR1 ) and/or estrogen receptor ß ( ESR2 ). Binding of the tracer to estrogen receptor α in vivo was assessed by imaging of uptake of the tracer into MCF7 xenografts in BALB/c nu/nu mice. Results The 18 F-estradiol PET tracer bound with high affinity (94 nM) to estrogen receptor α, with negligible binding to estrogen receptor ß. Uptake of the tracer was observed in MCF7 xenografts, which almost exclusively express estrogen receptor α. Conclusion 18 F-estradiol PET tracer binds in vitro with high specificity to the estrogen receptor α isoform, with minimal binding to estrogen receptor ß. This may help distinguish human cancers with biological dependence on estrogen receptor subtypes.

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.

Global protein profiling reveals anti-EGFR monoclonal antibody 806-modulated proteins in A431 tumor xenografts.

An important mediator of tumorigenesis, the epidermal growth factor receptor (EGFR) is expressed in almost all non-transformed cell types, associated with tumor progression, angiogenesis and metastasis. The significance of the EGFR as a cancer therapeutic target is underscored by the clinical development of several different classes of EGFR antagonists, including monoclonal antibodies (mAb) and tyrosine kinase inhibitors. Extensive preclinical studies have demonstrated the anti-tumor effects of mAb806 against tumor xenografts overexpressing EGFR. EGF stimulation of A431 cells induces rapid tyrosine phosphorylation of intracellular signalling proteins which regulate cell proliferation and apoptosis. Detailed understanding of the intracellular signalling pathways and components modulated by mAbs (such as mAb806) to EGFR, and other growth factor receptors, remain limited. The use of fluorescence 2D difference gel electrophoresis (2D DIGE), coupled with sensitive MS-based protein profiling in A431 tumor (epidermoid carcinoma) xenografts, in combination with mAb806, revealed proteins modulating endocytosis, cell architecture, apoptosis, cell signalling pathways and cell cycle regulation, including Dynamin-1-like protein, cofilin-1 protein, and 14-3-3 protein zeta/delta. Further, we report various proteins, including Interferon-induced protein 53 (IFI53), and Oncogene EMS1 (EMS1) which have roles in the tumor microenvironment, regulating cancer cell invasiveness, angiogenesis and formation of metastases. These findings contribute to understanding the underlying biological processes associated with mAb806 therapy of EGFR-positive tumors, and identifying further potential protein markers that may contribute in assessment of the treatment response.

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.

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.

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.

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.

18F-THK523: a novel in vivo tau imaging ligand for Alzheimer's disease.

While considerable effort has focused on developing positron emission tomography ß-amyloid imaging radiotracers for the early diagnosis of Alzheimer's disease, no radiotracer is available for the non-invasive quantification of tau. In this study, we detail the characterization of (18)F-THK523 as a novel tau imaging radiotracer. In vitro binding studies demonstrated that (18)F-THK523 binds with higher affinity to a greater number of binding sites on recombinant tau (K18Δ280K) compared with ß-amyloid(1-42) fibrils. Autoradiographic and histofluorescence analysis of human hippocampal serial sections with Alzheimer's disease exhibited positive THK523 binding that co-localized with immunoreactive tau pathology, but failed to highlight ß-amyloid plaques. Micro-positron emission tomography analysis demonstrated significantly higher retention of (18)F-THK523 (48%; P < 0.007) in tau transgenic mice brains compared with their wild-type littermates or APP/PS1 mice. The preclinical examination of THK523 has demonstrated its high affinity and selectivity for tau pathology both in vitro and in vivo, indicating that (18)F-THK523 fulfils ligand criteria for human imaging trials.

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.