Customizable Porphyrin Platform Enables Folate Receptor PET Imaging Using Copper-64.

Folate receptors including folate receptor α (FRα) are overexpressed in up to 90% of ovarian cancers. Ovarian cancers overexpressing FRα often exhibit high degrees of drug resistance and poor outcomes. A porphyrin chassis has been developed that is readily customizable according to the desired targeting properties. Thus, compound O5 includes a free base porphyrin, two water-solubilizing groups that project above and below the macrocycle plane, and a folate targeting moiety. Compound O5 was synthesized (>95% purity) and exhibited aqueous solubility of at least 0.48 mM (1 mg/mL). Radiolabeling of O5 with 64Cu in HEPES buffer at 37 °C gave a molar activity of 1000 µCi/µg (88 MBq/nmol). [64Cu]Cu-O5 was stable in human serum for 24 h. Cell uptake studies showed 535 ± 12% bound/mg [64Cu]Cu-O5 in FRα-positive IGROV1 cells when incubated at 0.04 nM. Subcellular fractionation showed that most radioactivity was associated with the cytoplasmic (39.4 ± 2.7%) and chromatin-bound nuclear (53.0 ± 4.2%) fractions. In mice bearing IGROV1 xenografts, PET imaging studies showed clear tumor uptake of [64Cu]Cu-O5 from 1 to 24 h post injection with a low degree of liver uptake. The tumor standardized uptake value at 24 h post injection was 0.34 ± 0.16 versus 0.06 ± 0.07 in the blocking group. In summary, [64Cu]Cu-O5 was synthesized at high molar activity, was stable in serum, exhibited high binding to FRα-overexpressing cells with high nuclear translocation, and gave uptake that was clearly visible in mouse tumor xenografts.

Optimizing scan time and bayesian penalized likelihood reconstruction algorithm in copper-64 PET/CT imaging: a phantom study.

Objectives: The aim of this study was to evaluate Cu-64 PET phantom image quality using Bayesian Penalized Likelihood (BPL) and Ordered Subset Expectation Maximum with point-spread function modeling (OSEM-PSF) reconstruction algorithms. In the BPL, the regularization parameterßwas varied to identify the optimum value for image quality. In the OSEM-PSF, the effect of acquisition time was evaluated to assess the feasibility of shortened scan duration.Methods: A NEMA IEC PET body phantom was filled with known activities of water soluble Cu-64. The phantom was imaged on a PET/CT scanner and was reconstructed using BPL and OSEM-PSF algorithms. For the BPL reconstruction, variousßvalues (150, 250, 350, 450, and 550) were evaluated. For the OSEM-PSF algorithm, reconstructions were performed using list-mode data intervals ranging from 7.5 to 240 s. Image quality was assessed by evaluating the signal to noise ratio (SNR), contrast to noise ratio (CNR), and background variability (BV).Results: The SNR and CNR were higher in images reconstructed with BPL compared to OSEM-PSF. Both the SNR and CNR increased with increasingß, peaking atß= 550. The CNR for allß, sphere sizes and tumor-to-background ratios (TBRs) satisfied the Rose criterion for image detectability (CNR > 5). BPL reconstructed images withß= 550 demonstrated the highest improvement in image quality. For OSEM-PSF reconstructed images with list-mode data duration ≥ 120 s, the noise level and CNR were not significantly different from the baseline 240 s list-mode data duration.Conclusions: BPL reconstruction improved Cu-64 PET phantom image quality by increasing SNR and CNR relative to OSEM-PSF reconstruction. Additionally, this study demonstrated scan time can be reduced from 240 to 120 s when using OSEM-PSF reconstruction while maintaining similar image quality. This study provides baseline data that may guide future studies aimed to improve clinical Cu-64 imaging.

Neurotensin (8-13) and Neuromedin N Neuropeptides Radiolabelling with Copper-64 Produced on Solid or Liquid Targets.

On the verge of a theranostic approach to personalised medicine, copper-64 is one of the emerging radioisotopes in nuclear medicine due to its exploitable nuclear and biochemical characteristics. The increased demand for copper-64 for preclinical and clinical studies has prompted the development of production routes. This research aims to compare the (p,n) reaction on nickel-64 solid versus liquid targets and evaluate the effectiveness of [64Cu]CuCl2 solutions prepared by the two routes. As new treatments for neurotensin receptor-overexpressing tumours have developed, copper-64 was used to radiolabel Neurotensin (8-13) and Neuromedin N. High-quality [64Cu]CuCl2 solutions were prepared using ACSI TR-19 and IBA Cyclone Kiube cyclotrons. The radiochemical purity after post-irradiation processing reached 99% (LT) and 99.99% (ST), respectively. The irradiation of a solid target with 11.8 MeV protons and 150 µAh led to 704 ± 84 MBq/µA (17.6 ± 2.1 GBq/batch at EOB). At the end of the purification process (1 h, 90.90% activity yield), the solution for peptide radiolabelling had a radioactive concentration of 1340.4 ± 70.1 MBq/mL (n.d.c.). The irradiation of a liquid target with 16.9 MeV protons and 230 µAh resulted in 3.7 ± 0.2 GBq/batch at EOB, which corresponds to an experimental production yield of 6.89 GBq.cm3/(g.µA)sat. Benefiting from a shorter purification process (40 min), the activity yielded 90.87%, while the radioactive concentration of the radiolabelling solution was lower (492 MBq/mL, n.d.c.). The [64Cu]CuCl2 solutions were successfully used for the radiolabelling of DOTA-NT(8-13) and DOTA-NN neuropeptides, resulting in a high RCP (>99%) and high molar activity (27.2 and 26.4 GBq/µmol for LT route compared to 45 and 52 GBq/µmol for ST route, respectively). The strong interaction between the [64Cu]Cu-DOTA-NT(8-13) and the colon cancerous cell lines HT29 and HCT116 proved that the specificity for NTR had not been altered, as shown by the uptake and retention data.

Development of a CD163-Targeted PET Radiotracer That Images Resident Macrophages in Atherosclerosis.

Tissue-resident macrophages are complementary to proinflammatory macrophages to promote the progression of atherosclerosis. The noninvasive detection of their presence and dynamic variation will be important to the understanding of their role in the pathogenesis of atherosclerosis. The goal of this study was to develop a targeted PET radiotracer for imaging CD163-positive (CD163+) macrophages in multiple mouse atherosclerosis models and assess the potential of CD163 as a biomarker for atherosclerosis in humans. Methods: CD163-binding peptide was identified using phage display and conjugated with a NODAGA chelator for 64Cu radiolabeling ([64Cu]Cu-ICT-01). CD163-overexpressing U87 cells were used to measure the binding affinity of [64Cu]Cu-ICT-01. Biodistribution studies were performed on wild-type C57BL/6 mice at multiple time points after tail vein injection. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages upregulated on the surface of atherosclerotic plaques were assessed in multiple mouse atherosclerosis models. Immunostaining, flow cytometry, and single-cell RNA sequencing were performed to characterize the expression of CD163 on tissue-resident macrophages. Human carotid atherosclerotic plaques were used to measure the expression of CD163+ resident macrophages and test the binding specificity of [64Cu]Cu-ICT-01. Results: [64Cu]Cu-ICT-01 showed high binding affinity to U87 cells. The biodistribution study showed rapid blood and renal clearance with low retention in all major organs at 1, 2, and 4 h after injection. In an ApoE-/- mouse model, [64Cu]Cu-ICT-01 demonstrated sensitive and specific detection of CD163+ macrophages and capability for tracking the progression of atherosclerotic lesions; these findings were further confirmed in Ldlr-/- and PCSK9 mouse models. Immunostaining showed elevated expression of CD163+ macrophages across the plaques. Flow cytometry and single-cell RNA sequencing confirmed the specific expression of CD163 on tissue-resident macrophages. Human tissue characterization demonstrated high expression of CD163+ macrophages on atherosclerotic lesions, and ex vivo autoradiography revealed specific binding of [64Cu]Cu-ICT-01 to human CD163. Conclusion: This work reported the development of a PET radiotracer binding CD163+ macrophages. The elevated expression of CD163+ resident macrophages on human plaques indicated the potential of CD163 as a biomarker for vulnerable plaques. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages warrant further investigation in translational settings.

Investigation of imaging the somatostatin receptor by opening the blood-brain barrier with melittin - A feasibility study using positron emission tomography and [64Cu]Cu-DOTATATE.

DOTATATE is a somatostatin peptide analog used in the clinic to detect somatostatin receptors which are highly expressed on neuroendocrine tumors. Somatostatin receptors are found naturally in the intestines, pancreas, lungs, and brain (mainly cortex). In vivo measurement of the somatostatin receptors in the cortex has been challenging because available tracers cannot cross the blood-brain barrier (BBB) due to their intrinsic polarity. A peptide called melittin, a main component of honeybee venom, has been shown to disrupt plasma membranes and increase the permeability of biological membranes. In this study, we assessed the feasibility of using melittin to facilitate the passage of [64Cu]Cu-DOTATATE through the BBB and its binding to somatostatin receptors in the cortex. Evaluation included in vitro autoradiography on Long Evans rat brains to estimate the binding affinity of [64Cu]Cu-DOTATATE to the somatostatin receptors in the cortex and an in vivo evaluation of [64Cu]Cu-DOTATATE binding in NMRI mice after injection of melittin. This study found an in vitro Bmax = 89 ± 4 nM and KD = 4.5 ± 0.6 nM in the cortex, resulting in a theoretical binding potential (BP) calculated as Bmax/KD ≈ 20, which is believed suitable for in vivo brain PET imaging. However, the in vivo results showed no significant difference between the control and melittin injected mice, indicating that the honeybee venom failed to open the BBB. Additional experiments, potentially involving faster injection rates are required to verify that melittin can increase brain uptake of non-BBB permeable PET tracers. Furthermore, an evaluation of whether a venom with a narrow therapeutic range can be used for clinical purposes needs to be considered.

64Cu tumor labeling with hexadentate picolinic acid-based bispidine immunoconjugates.

Discussed are two picolinate appended bispidine ligands (3,7-diazabicyclo[3.3.1]nonane derivatives) in comparison with an earlier described bis-pyridine derivative, which are all known to strongly bind CuII. The radiopharmacological characterization of the two isomeric bispidine complexes includes quantitative labeling with 64CuII at ambient conditions with high radiochemical purities and yields (molar activities >200 MBq/nmol). Challenge experiments in presence of EDTA, cyclam, human serum and SOD demonstrate high stability and inertness of the 64Cu-bispidine complexes. Biodistribution studies performed in Wistar rats indicate a rapid renal elimination for both 64Cu-labeled chelates. The bispidine ligand with the picolinate group in N7 position was selected for further biological experiments, and its backbone was therefore substituted with a benzyl-NCS group at C9. Two tumor target modules (TMs), targeting prostate stem cell antigen (PSCA), overexpressed in prostate cancer, and the fibroblast activation protein (FAP) in fibrosarcoma, were selected for thiourea coupling with the NCS-functionalized ligand and lysine residues of TMs. Small animal PET experiments on tumor-bearing mice showed specific accumulation of the 64Cu-labeled TMs in PSCA- and FAP-overexpressing tumors (standardized uptake value (SUV) for PC3: 2.7±0.6 and HT1080: 7.2±1.25) with almost no uptake in wild type tumors.

Radiolabeling Diaminosarcophagine with Cyclotron-Produced Cobalt-55 and [55Co]Co-NT-Sarcage as a Proof of Concept in a Murine Xenograft Model.

Cobalt-sarcophagine complexes exhibit high kinetic inertness under various stringent conditions, but there is limited literature on radiolabeling and in vivo positron emission tomography (PET) imaging using no carrier added 55Co. To fill this gap, this study first investigates the radiolabeling of DiAmSar (DSar) with 55Co, followed by stability evaluation in human serum and EDTA, pharmacokinetics in mice, and a direct comparison with [55Co]CoCl2 to assess differences in pharmacokinetics. Furthermore, the radiolabeling process was successfully used to generate the NTSR1-targeted PET agent [55Co]Co-NT-Sarcage (a DSar-functionalized SR142948 derivative) and administered to HT29 tumor xenografted mice. The [55Co]Co-DSar complex can be formed at 37 °C with purity and stability suitable for preclinical in vivo radiopharmaceutical applications, and [55Co]Co-NT-Sarcage demonstrated prominent tumor uptake with a low background signal. In a direct comparison with [64Cu]Cu-NT-Sarcage, [55Co]Co-NT-Sarcage achieved a higher tumor-to-liver ratio but with overall similar biodistribution profile. These results demonstrate that Sar would be a promising chelator for constructing Co-based radiopharmaceuticals including 55Co for PET and 58mCo for therapeutic applications.

Effectiveness of [67Cu]Cu-trastuzumab as a theranostic against HER2-positive breast cancer.

PURPOSE: To evaluate the imaging and therapeutic properties (theranostic) of 67Cu-labeled anti-human epidermal growth factor receptor II (HER2) monoclonal antibody trastuzumab against HER2-positive breast cancer (BC). METHODS: We conjugated trastuzumab with p-SCN-Bn-NOTA, 3p-C-NETA-NCS, or p-SCN-Bn-DOTA, and radiolabeled with [67Cu]CuCl2. Immunoconjugate internalization was evaluated in BT-474, JIMT-1 and MCF-7 BC cells. In vitro stability was studied in human serum (HS) and Phosphate Buffered Saline (PBS). Flow cytometry, radioligand binding and immunoreactive fraction assays were carried out. ImmunoSPECT imaging of [67Cu]Cu-NOTA-trastuzumab was done in mice bearing BT-474, JIMT-1 and MCF-7 xenografts. Pharmacokinetic was studied in healthy Balb/c mice while dosimetry was done in both healthy Balb/c and in athymic nude mice bearing JIMT-1 xenograft. The therapeutic effectiveness of [67Cu]Cu-NOTA-trastuzumab was evaluated in mice bearing BT-474 and JIMT-1 xenografts after a single intravenous (i.v.) injection of ~ 16.8 MBq. RESULTS: Pure immunoconjugates and radioimmunoconjugates (> 95%) were obtained. Internalization was HER2 density-dependent with highest internalization observed with NOTA-trastuzumab. After 5 days, in vitro stabilities were 97 ± 1.7%, 31 ± 6.2%, and 28 ± 4% in HS, and 79 ± 3.5%, 94 ± 1.2%, and 86 ± 2.3% in PBS for [67Cu]Cu-NOTA-trastuzumab, [67Cu]Cu-3p-C-NETA-trastuzumab and [67Cu]Cu-DOTA-trastuzumab, respectively. [67Cu]Cu-NOTA-trastuzumab was chosen for further evaluation. BT-474 flow cytometry showed low KD, 8.2 ± 0.2 nM for trastuzumab vs 26.5 ± 1.6 nM for NOTA-trastuzumab. There were 2.9 NOTA molecules per trastuzumab molecule. Radioligand binding assay showed a low KD of 2.1 ± 0.4 nM and immunoreactive fraction of 69.3 ± 0.9. Highest uptake of [67Cu]Cu-NOTA-trastuzumab was observed in JIMT-1 (33.9 ± 5.5% IA/g) and BT-474 (33.1 ± 10.6% IA/g) xenograft at 120 h post injection (p.i.). Effectiveness of the radioimmunoconjugate was also expressed as percent tumor growth inhibition (%TGI). [67Cu]Cu-NOTA-trastuzumab was more effective than trastuzumab against BT-474 xenografts (78% vs 54% TGI after 28 days), and JIMT-1 xenografts (90% vs 23% TGI after 19 days). Mean survival of [67Cu]Cu-NOTA-trastuzumab, trastuzumab and saline treated groups were > 90, 77 and 72 days for BT-474 xenografts, while that of JIMT-1 were 78, 24, and 20 days, respectively. CONCLUSION: [67Cu]Cu-NOTA-trastuzumab is a promising theranostic agent against HER2-positive BC.

Development and characterization of DIA 12.3, a fully human intact anti-CEACAM1 monoclonal antibody.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), a homotypic cell adhesion molecule glycoprotein with apical expression on normal epithelial cells and activated lymphocytes, is overexpressed on many tumors and acts as an inhibitory receptor on NK cells, preventing their killing of CEACAM1 positive tumors. Production of humanized anti-CEACAM1 antibodies to block the inhibitory activity of CEACAM1 for immunotherapy and immunoimaging. Starting from a scFv, a fully human intact anti-CEACAM1 (DIA 12.3) that recognizes the N-terminal domain of CEACAM1 was developed and shown to bind CEACAM1 positive tumor cells and enhanced NK cell killing of CEACAM1 positive targets. DIA 12.3 bound to human neutrophils without activation, indicating they would be safe for human use. DIA 12.3 exhibited some cross-reactivity to CEACAM5, a tumor marker with high sequence homology to the N-terminal domain of CEACAM1. CEACAM1 PET imaging with 64Cu-COTA-DIA 12.3 showed excellent imaging of CEACAM1 positive tumors with reduced binding to CEACAM5 tumors. Based on its immunoinhibitory an immunoimaging activities, DIA 12.3 shows promise for therapeutic studies in man.

Development of PET Radioisotope Copper-64-Labeled Theranostic Immunoliposomes for EGFR Overexpressing Cancer-Targeted Therapy and Imaging.

Combining standard surgical procedures with personalized chemotherapy and the continuous monitoring of cancer progression is necessary for effective NSCLC treatment. In this study, we developed liposomal nanoparticles as theranostic agents capable of simultaneous therapy for and imaging of target cancer cells. Copper-64 (64Cu), with a clinically practical half-life (t1/2 = 12.7 h) and decay properties, was selected as the radioisotope for molecular PET imaging. An anti-epidermal growth factor receptor (anti-EGFR) antibody was used to achieve target-specific delivery. Simultaneously, the chemotherapeutic agent doxorubicin (Dox) was encapsulated within the liposomes using a pH-gradient method. The conjugates of 64Cu-labeled and anti-EGFR antibody-conjugated micelles were inserted into the doxorubicin-encapsulating liposomes via a post-insertion procedure (64Cu-Dox-immunoliposomes). We evaluated the size and zeta-potential of the liposomes and analyzed target-specific cell binding and cytotoxicity in EGFR-positive cell lines. Then, we analyzed the specific therapeutic effect and PET imaging of the 64Cu-Dox-immunoliposomes with the A549 xenograft mouse model. In vivo therapeutic experiments on the mouse models demonstrated that the doxorubicin-containing 64Cu-immunoliposomes effectively inhibited tumor growth. Moreover, the 64Cu-immunoliposomes provided superior in vivo PET images of the tumors compared to the untargeted liposomes. We suggest that nanoparticles will be the potential platform for cancer treatment as a widely applicable theranostic system.

Theranostic Nuclear Medicine with Gallium-68, Lutetium-177, Copper-64/67, Actinium-225, and Lead-212/203 Radionuclides.

Molecular changes in malignant tissue can lead to an increase in the expression levels of various proteins or receptors that can be used to target the disease. In oncology, diagnostic imaging and radiotherapy of tumors is possible by attaching an appropriate radionuclide to molecules that selectively bind to these target proteins. The term "theranostics" describes the use of a diagnostic tool to predict the efficacy of a therapeutic option. Molecules radiolabeled with γ-emitting or ß+-emitting radionuclides can be used for diagnostic imaging using single photon emission computed tomography or positron emission tomography. Radionuclide therapy of disease sites is possible with either α-, ß-, or Auger-emitting radionuclides that induce irreversible damage to DNA. This Focus Review centers on the chemistry of theranostic approaches using metal radionuclides for imaging and therapy. The use of tracers that contain ß+-emitting gallium-68 and ß-emitting lutetium-177 will be discussed in the context of agents in clinical use for the diagnostic imaging and therapy of neuroendocrine tumors and prostate cancer. A particular emphasis is then placed on the chemistry involved in the development of theranostic approaches that use copper-64 for imaging and copper-67 for therapy with functionalized sarcophagine cage amine ligands. Targeted therapy with radionuclides that emit α particles has potential to be of particular use in late-stage disease where there are limited options, and the role of actinium-225 and lead-212 in this area is also discussed. Finally, we highlight the challenges that impede further adoption of radiotheranostic concepts while highlighting exciting opportunities and prospects.

Porphyrin-Based Brain Tumor-Targeting Agents: [64Cu]Cu-porphyrin and [64Cu]Cu-TDAP.

The aim of this study is to evaluate a radioactive metal complex platform for brain tumor targeting. Herein, we introduce a new porphyrin derivative, 5,10,15,20-(tetra-N,N-dimethyl-4-aminophenyl)porphyrin (TDAP), in which four N,N-dimethyl-4-p-phenylenediamine (DMPD) moieties are conjugated to the porphyrin labeled with the radiometal 64Cu. DMPD affected the pharmacokinetics of porphyrin in terms of retention time in vivo and tumor-targeting ability relative to those of unmodified porphyrin. [64Cu]Cu-TDAP showed stronger enhancement than [64Cu]Cu-porphyrin in U87MG glioblastoma cells, especially in the cytoplasm and nucleus, indicating its tumor-targeting properties and potential use as a therapeutic agent. In the subcutaneous and orthotopic models of brain-tumor-bearing mice, [64Cu]Cu-TDAP was clearly visualized in the tumor site via positron emission tomography imaging and showed a tumor-to-brain ratio as high as 13. [64Cu]Cu-TDAP deserves attention as a new diagnostic agent that is suitable for the early diagnosis and treatment of brain tumors.

The design of functional proteins using tensorized energy calculations.

In protein design, the energy associated with a huge number of sequence-conformer perturbations has to be routinely estimated. Hence, enhancing the throughput and accuracy of these energy calculations can profoundly improve design success rates and enable tackling more complex design problems. In this work, we explore the possibility of tensorizing the energy calculations and apply them in a protein design framework. We use this framework to design enhanced proteins with anti-cancer and radio-tracing functions. Particularly, we designed multispecific binders against ligands of the epidermal growth factor receptor (EGFR), where the tested design could inhibit EGFR activity in vitro and in vivo. We also used this method to design high-affinity Cu2+ binders that were stable in serum and could be readily loaded with copper-64 radionuclide. The resulting molecules show superior functional properties for their respective applications and demonstrate the generalizable potential of the described protein design approach.

Impact of human serum albumin on CuII and ZnII complexation by ATSM (diacetyl-bis(N4-methylthiosemicarbazone)) and a water soluble analogue.

The chelator diacetyl-bis(N4-methylthiosemicarbazone) (ATSM) and its complexes with CuII and ZnII are becoming increasingly investigated for medical applications such as PET imaging for anti-tumour therapy and the treatment of amyotrophic lateral sclerosis. However, the solubility in water of both the ligand and the complexes presents certain limitations for in vitro studies. Moreover, the stability of the CuII and ZnII complexes and their metal exchange reaction against the potential biological competitor human serum albumin (HSA) has not been studied in depth. In this work it was observed that the ATSM with an added carboxylic group into the structure increases its solubility in aqueous solutions without altering the coordination mode and the conjugated system of the ligand. The poorly water-soluble CuII- and ZnII-ATSM complexes were prevented from precipitating due to the binding to HSA. Both HSA and ATSM show a similar thermodynamic affinity for ZnII. Finally, the CuII-competition experiments with EDTA and the water-soluble ATSM ligands yielded an apparent log Kd at pH 7.4 of about -19. When ATSM was added to CuII- and ZnII-loaded HSA, withdrawing of ZnII was kinetically favoured, but this metal is slowly substituted by the CuII afterwards taken from HSA so that this protein could be considered as a source of CuII for ATSM.

Urokinase-Type Plasminogen Activator Receptor (uPAR) Expression and [64Cu]Cu-DOTA-AE105 uPAR-PET/CT in Patient-Derived Xenograft Models of Oral Squamous Cell Carcinoma.

PURPOSE: [64Cu]Cu-DOTA-AE105 urokinase-type plasminogen activator receptor (uPAR)-PET/CT is a novel and promising imaging modality for cancer visualization, although it has not been tested in head and neck cancer patients nor in preclinical models that closely resemble these heterogenous tumors, i.e., patient-derived xenograft (PDX) models. The aim of the present study was to establish and validate oral squamous cell carcinoma (OSCC) PDX models and to evaluate [64Cu]Cu-uPAR-PET/CT for tumor imaging in these models. PROCEDURES: PDX flank tumor models were established by engrafting tumor tissue from three patients with locally advanced OSCC into immunodeficient mice. [64Cu]Cu-DOTA-AE105 was injected in passage 2 (P2) mice, and [64Cu]Cu-uPAR-PET/CT was performed 1 h and 24 h after injection. After the last PET scan, all animals were euthanized, and tumors dissected for autoradiography and immunohistochemical (IHC) staining. RESULTS: Three PDX models were established, and all of them showed histological stability and unchanged heterogenicity, uPAR expression, and Ki67 expression through passages. A significant correlation between uPAR expression and tumor growth was found. All tumors of all models (n=29) showed tumor uptake of [64Cu]Cu-DOTA-AE105. There was a clear visual concordance between the distribution of uPAR expression (IHC) and [64Cu]Cu-DOTA-AE105 uptake pattern in tumor tissue (autoradiography). No significant correlation was found between IHC (H-score) and PET-signal (SUVmax) (r=0.34; p=0.07). CONCLUSIONS: OSCC PDX models in early passages histologically mimic donor tumors and could serve as a valuable platform for the development of uPAR-targeted imaging and therapeutic modalities. Furthermore, [64Cu]Cu-uPAR-PET/CT showed target- and tumor-specific uptake in OSCC PDX models demonstrating the diagnostic potential of this modality for OSCC patients.

Positron Emission Tomography Using 64-Copper as a Tracer for the Study of Copper-Related Disorders.

Copper is an essential trace element, functioning in catalysis and signaling in biological systems. Radiolabeled copper has been used for decades in studying basic human and animal copper metabolism and copper-related disorders, such as Wilson disease (WD) and Menke's disease. A recent addition to this toolkit is 64-copper (64Cu) positron emission tomography (PET), combining the accurate anatomical imaging of modern computed tomography (CT) or magnetic resonance imaging (MRI) scanners with the biodistribution of the 64Cu PET tracer signal. This allows the in vivo tracking of copper fluxes and kinetics, thereby directly visualizing human and animal copper organ traffic and metabolism. Consequently, 64Cu PET is well-suited for evaluating clinical and preclinical treatment effects and has already demonstrated the ability to diagnose WD accurately. Furthermore, 64Cu PET/CT studies have proven valuable in other scientific areas like cancer and stroke research. The present article shows how to perform 64Cu PET/CT or PET/MR in humans. Procedures for 64Cu handling, patient preparation, and scanner setup are demonstrated here.

Two-Chain Mature Hepatocyte Growth Factor-Specific Positron Emission Tomography Imaging in Tumors Using 64Cu-Labeled HiP-8, a Nonstandard Macrocyclic Peptide Probe.

Two-chain hepatocyte growth factor (tcHGF), the mature form of HGF, is associated with malignancy and anticancer drug resistance; therefore, its quantification is an important indicator for cancer diagnosis. In tumors, activated tcHGF hardly discharges into the systemic circulation, indicating that tcHGF is an excellent target for molecular imaging using positron emission tomography (PET). We recently discovered HGF-inhibitory peptide-8 (HiP-8) that binds specifically to human tcHGF with nanomolar affinity. The purpose of this study was to investigate the usefulness of HiP-8-based PET probes in human HGF knock-in humanized mice. 64Cu-labeled HiP-8 molecules were synthesized using a cross-bridged cyclam chelator, CB-TE1K1P. Radio-high-performance liquid chromatography-based metabolic stability analyses showed that more than 90% of the probes existed in intact form in blood at least for 15 min. In PET studies, significantly selective visualization of hHGF-overexpressing tumors versus hHGF-negative tumors was observed in double-tumor-bearing mice. The accumulation of labeled HiP-8 into the hHGF-overexpressing tumors was significantly reduced by competitive inhibition. In addition, the radioactivity and distribution of phosphorylated MET/HGF receptor were colocalized in tissues. These results demonstrate that the 64Cu-labeled HiP-8 probes are suitable for tcHGF imaging in vivo, and secretory proteins like tcHGF can be a target for PET imaging.

An Engineered Probiotic Platform for Cancer Epitope-Independent Targeted Radionuclide Therapy of Solid Tumors.

Targeted radionuclide therapy (TRT) is an emerging therapeutic modality for the treatment of various solid cancers. Current approaches rely on the presence of cancer-specific epitopes and receptors against which a radiolabeled ligand is systemically administered to specifically deliver cytotoxic doses of α and ß particles to tumors. In this proof-of-concept study, tumor-colonizing Escherichia coli Nissle 1917 (EcN) is utilized to deliver a bacteria-specific radiopharmaceutical to solid tumors in a cancer-epitope independent manner. In this microbe-based pretargeted approach, the siderophore-mediated metal uptake pathway is leveraged to selectively concentrate copper radioisotopes, 64 Cu and 67 Cu, complexed to yersiniabactin (YbT) in the genetically modified bacteria. 64 Cu-YbT facilitates positron emission tomography (PET) imaging of the intratumoral bacteria, whereas 67 Cu-YbT delivers a cytotoxic dose to the surrounding cancer cells. PET imaging with 64 Cu-YbT reveals persistence and sustained growth of the bioengineered microbes in the tumor microenvironment. Survival studies with 67 Cu-YbT reveals significant attenuation of tumor growth and extends survival of both MC38 and 4T1  tumor-bearing mice harboring the microbes. Tumor response to this pretargeted approach correlates with promising anti-tumor immunity, with noticeable CD8+ T:Treg cell ratio. Their strategy offers a pathway to target and ablate multiple solid tumors independent of their epitope and receptor phenotype.

Pre-targeting amyloid-ß with antibodies for potential molecular imaging of Alzheimer's disease.

With the aim of developing the concept of pretargeted click chemistry for the diagnosis of Alzheimer's disease two antibodies specific for amyloid-ß were modified to incorporate trans-cyclooctene functional groups. Two bis(thiosemicarbazone) compounds with pendant 1,2,4,5-tetrazine functional groups were prepared and radiolabelled with positron emitting copper-64. The new copper-64 complexes rapidly react with the trans-cyclooctene functionalized antibodies in a bioorthogonal click reaction and cross the blood-brain barrier in mice.

Gastro-Entero-Pancreatic Tumors: FDG Positron Emission Tomography/Computed Tomography.

Gastro-entero-pancreatic tumors comprise a group of heterogenous neoplasms, with medical imaging being paramount in the diagnosis, staging, and treatment planning of these tumors. Moreover, with the advent of newer radiopharmaceuticals, such as 68 Ga-labeled and 64 Cu-labeled somatostatin analogs (eg, 68 Ga-DOTATOC, 68 Ga-DOTATATE, 68 Ga-DOTANOC, and 64Cu-DOTATATE) that bind to the somatostatin receptor (SSTR), molecular imaging plays an increasing and critical role in the diagnosis, staging, and treatment planning of these neoplasms. Dual-tracer imaging with 18F-FDG PET/CT and SSTR agents may play a significant role in treatment planning and predicting patient outcomes in the setting of high-grade or poorly differentiated neuroendocrine tumors.