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.

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.

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.

Monte Carlo calculated calibration settings for commercially available nuclear medicine ionization chambers.

PURPOSE: A method for calculating nuclear medicine ionization chamber (NMIC) calibration settings with a Monte Carlo model is presented and validated against physical measurements. This work provides Monte Carlo-calculated calibration settings for select isotopes with no current manufacturer recommendations and a method by which NMIC manufacturers or standards laboratories can utilize highly detailed specifications to calculate comprehensive lists of calibration settings for general isotopes. METHODS: A Monte Carlo model of a Capintec PET series NMIC was developed and used to calculate the chamber response to relevant radioactive decay products over an energy range relevant to nuclear medicine. The photon detection efficiency (PDE) of a high purity germanium (HPGe) detector was modeled and physically validated to facilitate measurements of NMIC calibration settings with HPGe detector spectroscopy. Modeled NMIC response to various isotopes was compared against spectroscopic measurements and National Institute of Standards and Technology (NIST)-validated calibration settings to validate the Monte Carlo-calculated NMIC calibration settings. RESULTS: HPGe detector PDE was validated against the physical measurements to within 3.3 % $3.3\%$ at 95 % $95\%$ confidence and used to measure calibration settings, which produced activity readings 0.7 % $0.7\%$ , 1.6 % $1.6\%$ , 0.8 % $0.8\%$ , and 1.0 % $1.0\%$ different than those validated by NIST for 11 $^{11}$ C, 18 $^{18}$ F, 68 $^{68}$ Ga, and 64 $^{64}$ Cu respectively. The Monte Carlo model of the NMIC reproduced measured calibration settings to within 7 % $7\%$ at 95 % $95\%$ confidence for isotopes with a sufficiently small yield of low energy photons. CONCLUSIONS: A method of calculating NMIC calibration settings with Monte Carlo modeling has been developed and validated against HPGe detector spectroscopy. NMIC manufacturers or standards laboratories can use more detailed specifications of the chamber geometries to extend the applicability of this method to a wider range of isotopes.

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.

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.