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Harnessing 64Cu/67Cu for a theranostic approach to pretargeted radioimmunotherapy.

ABSTRACT: Over the past decade, theranostic imaging has emerged as a powerful clinical tool in oncology for identifying patients likely to respond to targeted therapies and for monitoring the response of patients to treatment. Herein, we report a theranostic approach to pretargeted radioimmunotherapy (PRIT) based on a pair of radioisotopes of copper: positron-emitting copper-64 (64Cu, t 1/2 = 12.7 h) and beta particle-emitting copper-67 (67Cu, t 1/2 = 61.8 h). This strategy is predicated on the in vivo ligation between a trans-cyclooctene (TCO)-bearing antibody and a tetrazine (Tz)-based radioligand via the rapid and bioorthogonal inverse electron-demand Diels-Alder reaction. Longitudinal therapy studies were conducted in a murine model of human colorectal carcinoma using an immunoconjugate of the huA33 antibody modified with TCO (huA33-TCO) and a 67Cu-labeled Tz radioligand ([67Cu]Cu-MeCOSar-Tz). The injection of huA33-TCO followed 72 h later by the administration of 18.5, 37.0, or 55.5 MBq of [67Cu]Cu-MeCOSar-Tz produced a dose-dependent therapeutic response, with the median survival time increasing from 68 d for the lowest dose to >200 d for the highest. Furthermore, we observed that mice that received the highest dose of [67Cu]Cu-MeCOSar-Tz in a fractionated manner exhibited improved hematological values without sacrificing therapeutic efficacy. Dual radionuclide experiments in which a single administration of huA33-TCO was followed by separate injections of [64Cu]Cu-MeCOSar-Tz and [67Cu]Cu-MeCOSar-Tz revealed that the positron emission tomography images produced by the former accurately predicted the efficacy of the latter. In these experiments, a correlation was observed between the tumoral uptake of [64Cu]Cu-MeCOSar-Tz and the subsequent therapeutic response to [67Cu]Cu-MeCOSar-Tz.

SUBMITTER: Keinanen O

PROVIDER: S-EPMC7668034 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Harnessing 64Cu/67Cu for a theranostic approach to pretargeted radioimmunotherapy.

Keinänen Outi O Fung Kimberly K Brennan James M JM Zia Nicholas N Harris Matt M van Dam Ellen E Biggin Colin C Hedt Amos A Stoner Jon J Donnelly Paul S PS Lewis Jason S JS Zeglis Brian M BM

Proceedings of the National Academy of Sciences of the United States of America 20201026 45

Over the past decade, theranostic imaging has emerged as a powerful clinical tool in oncology for identifying patients likely to respond to targeted therapies and for monitoring the response of patients to treatment. Herein, we report a theranostic approach to pretargeted radioimmunotherapy (PRIT) based on a pair of radioisotopes of copper: positron-emitting copper-64 (64Cu, t1/2 = 12.7 h) and beta particle-emitting copper-67 (67Cu, t1/2 ...[more]

PMID: 33106429

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Project description:BackgroundA major challenge to the long-term success of neuroblastoma therapy is widespread metastases that survive initial therapy as minimal residual disease (MRD). The SSTR2 receptor is expressed by most neuroblastoma tumors making it an attractive target for molecularly targeted radionuclide therapy. SARTATE consists of octreotate, which targets the SSTR2 receptor, conjugated to MeCOSar, a bifunctional chelator with high affinity for copper. Cu-SARTATE offers the potential to both detect and treat neuroblastoma MRD by using [64Cu]Cu-SARTATE to detect and monitor the disease and [67Cu]Cu-SARTATE as the companion therapeutic agent. In the present study, we tested this theranostic pair in a preclinical model of neuroblastoma MRD. An intrahepatic model of metastatic neuroblastoma was established using IMR32 cells in nude mice. The biodistribution of [64Cu]Cu-SARTATE was measured using small-animal PET and ex vivo tissue analysis. Survival studies were carried out using the same model: mice (6-8 mice/group) were given single doses of saline, or 9.25 MBq (250 µCi), or 18.5 MBq (500 µCi) of [67Cu]Cu-SARTATE at either 2 or 4 weeks after tumor cell inoculation.ResultsPET imaging and ex vivo biodistribution confirmed tumor uptake of [64Cu]Cu-SARTATE and rapid clearance from other tissues. The major clearance tissues were the kidneys (15.6 ± 5.8% IA/g at 24 h post-injection, 11.5 ± 2.8% IA/g at 48 h, n = 3/4). Autoradiography and histological analysis confirmed [64Cu]Cu-SARTATE uptake in viable, SSTR2-positive tumor regions with mean tumor uptakes of 14.1-25.0% IA/g at 24 h. [67Cu]Cu-SARTATE therapy was effective when started 2 weeks after tumor cell inoculation, extending survival by an average of 13 days (30%) compared with the untreated group (mean survival of control group 43.0 ± 8.1 days vs. 55.6 ± 9.1 days for the treated group; p = 0.012). No significant therapeutic effect was observed when [67Cu]Cu-SARTATE was started 4 weeks after tumor cell inoculation, when the tumors would have been larger (control group 14.6 ± 8.5 days; 9.25 MBq group 9.5 ± 1.6 days; 18.5 MBq group 15.6 ± 4.1 days; p = 0.064).ConclusionsClinical experiences of peptide-receptor radionuclide therapy for metastatic disease have been encouraging. This study demonstrates the potential for a theranostic approach using [64/67Cu]Cu-SARTATE for the detection and treatment of SSTR2-positive neuroblastoma MRD.

Project description:BackgroundDiacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia. However, various controversial studies highlighted potential pitfalls that may disable its use as a selective hypoxic marker. They also highlighted that the results may be tumor location dependent. Here, we first analyzed uptake of Cu-ATSM and its less lipophilic counterpart Cu-Cl2 in the tumor over time in an orthotopic glioblastoma model. An in vitro study was also conducted to investigate the hypoxia-dependent copper uptake in tumor cells. We then further performed a comprehensive ex vivo study to compare 64Cu uptake to hypoxic markers, specific cellular reactions, and also transporter expression.MethodsμPET was performed 14 days (18F-FMISO), 15 days (64Cu-ATSM and 64Cu-Cl2), and 16 days (64Cu-ATSM and 64Cu-Cl2) after C6 cell inoculation. Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry. C6 cells were also grown in hypoxic workstation to analyze cellular uptake of Cu complexes in different oxygen levels.ResultsIn vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors. Cu-ATSM also stained, to a lesser extent, non-hypoxic regions, such as regions of astrogliosis, with high expression of copper transporters and in particular DMT-1 and CTR1, and also characterized by the expression of elevated astrogliosis. In vitro results show that 64Cu-ATSM showed an increase in the uptake only in severe hypoxia at 0.5 and 0.2% of oxygen while for 64Cu-Cl2, the cell retention was significantly increased at 5% and 1% of oxygen with no significant rise at lower oxygen percentages.ConclusionIn the present study, we show that Cu-complexes undoubtedly accumulate in hypoxic areas of the tumors. This uptake may be the reflection of a direct dependency to a redox metabolism and also a reflection of hypoxic-induced overexpression of transporters. We also show that Cu-ATSM also stained non-hypoxic regions such as astrogliosis.

Project description:The Cu2+ complexes formed by a series of cyclen derivatives bearing sulfur pendant arms, 1,4,7,10-tetrakis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO4S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO3S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-10-acetamido-1,4,7,10-tetraazacyclododecane (DO3SAm), and 1,7-bis[2-(methylsulfanyl)ethyl]-4,10-diacetic acid-1,4,7,10-tetraazacyclododecane (DO2A2S), were studied in aqueous solution at 25 °C from thermodynamic and structural points of view to evaluate their potential as chelators for copper radioisotopes. UV-vis spectrophotometric out-of-cell titrations under strongly acidic conditions, direct in-cell UV-vis titrations, potentiometric measurements at pH >4, and spectrophotometric Ag+-Cu2+ competition experiments were performed to evaluate the stoichiometry and stability constants of the Cu2+ complexes. A highly stable 1:1 metal-to-ligand complex (CuL) was found in solution at all pH values for all chelators, and for DO2A2S, protonated species were also detected under acidic conditions. The structures of the Cu2+ complexes in aqueous solution were investigated by UV-vis and electron paramagnetic resonance (EPR), and the results were supported by relativistic density functional theory (DFT) calculations. Isomers were detected that differed from their coordination modes. Crystals of [Cu(DO4S)(NO3)]·NO3 and [Cu(DO2A2S)] suitable for X-ray diffraction were obtained. Cyclic voltammetry (CV) experiments highlighted the remarkable stability of the copper complexes with reference to dissociation upon reduction from Cu2+ to Cu+ on the CV time scale. The Cu+ complexes were generated in situ by electrolysis and examined by NMR spectroscopy. DFT calculations gave further structural insights. These results demonstrate that the investigated sulfur-containing chelators are promising candidates for application in copper-based radiopharmaceuticals. In this connection, the high stability of both Cu2+ and Cu+ complexes can represent a key parameter for avoiding in vivo demetalation after bioinduced reduction to Cu+, often observed for other well-known chelators that can stabilize only Cu2+.

Project description:Purpose64 Cu and 67 Cu radioisotopes have nuclear characteristics suitable for nuclear medicine applications. The production of 64 Cu is already well established. However, the production of 67 Cu in quantities suitable to conduct clinical trials is more challenging as it leads to the coproduction of other Cu isotopes, in particular 64 Cu. The aim of this study is to investigate the possibility of using a CuCl2 solution with a mixture of 67/64 Cu radioisotopes for therapeutic purposes, providing an alternative solution for the cyclotron production problem.MethodsCopper radioisotopes activities were calculated by considering proton beam irradiation of the following targets: (i) 70 Zn in the energy range 70-45 MeV; (ii) 68 Zn in the energy range 70-35 MeV; (iii) a combination of 70 Zn (70-55 MeV) and 68 Zn (55-35 MeV). The contribution of each copper radioisotope to the human-absorbed dose was estimated with OLINDA/EXM software using the biokinetic model for CuCl2 published by ICRP 53. The total absorbed dose generated by the 67/64 CuCl2 mixture, obtained through different production routes, was calculated at different times after the end of the bombardment (EOB). A simple spherical model was used to simulate tumors of different sizes containing uniformly distributed 67/64 Cu mixture and to calculate the absorbed dose of self-irradiation. The biological damage produced by 67 Cu and 64 Cu was also evaluated through cellular dosimetry and cell surviving fraction assessment using the MIRDcell code, considering two prostate cancer cell lines with different radiosensitivity.ResultsThe absorbed dose to healthy organs and the effective dose (ED) per unit of administered activity of 67 CuCl2 are higher than those of 64 CuCl2 . Absorbed dose values per unit of administered activity of 67/64 CuCl2 mixture increase with time after the EOB because the amount of 67 Cu in the mixture increases. Survival data showed that the biological damage caused per each decay of 67 Cu is greater than that of 64 Cu, assuming that radionuclides remain accumulated in the cell cytoplasm. Sphere model calculations demonstrated that 64 Cu administered activity must be about five times higher than that of 67 Cu to obtain the same absorbed dose for tumor mass between 0.01 and 10 g and about 10 times higher for very small spheres. Consequently, the 64 CuCl2 -absorbed dose to healthy organs will reach higher values than those of 67 CuCl2 . The supplemental activity of the 67/64 CuCl2 mixture, required to get the same tumor-absorbed dose produced by 67 CuCl2 , triggers a dose increment (DI) in healthy organs. The waiting time post-EOB necessary to keep this DI below 10% (t10% ) depends on the irradiation methods employed for the production of the 67/64 CuCl2 mixture.ConclusionsA mixture of cyclotron produced 67/64 Cu radioisotopes proved to be an alternative solution for the therapeutic use of CuCl2 with minimal DI to healthy organs compared with pure 67 Cu. Irradiation of a 70 Zn+68 Zn target in the 70-35 MeV proton energy range for 185 h appears to be the best option from among all the production routes investigated, as it gives the maximum amount of activity, the shortest t10% (10 h), and less than 1% of 61 Cu and 60 Cu impurities.

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Harnessing 64Cu/67Cu for a theranostic approach to pretargeted radioimmunotherapy.

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Harnessing <sup>64</sup>Cu/<sup>67</sup>Cu for a theranostic approach to pretargeted radioimmunotherapy.

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