Alpha-particle emitting 213Bi-anti-EGFR immunoconjugates eradicate tumor cells independent of oxygenation

Hypoxia is a central problem in tumor treatment because hypoxic cells are less sensitive to chemo- and radiotherapy than normoxic cells. Radioresistance of hypoxic tumor cells is due to reduced sensitivity towards low Linear Energy Transfer (LET) radiation. High LET α-emitters are thought to eradicate tumor cells independent of cellular oxygenation. Therefore, the aim of this study was to demonstrate that the cell-bound α-particle emitting 213Bi immunoconjugates efficiently kill hypoxic just like normoxic CAL33 tumor cells. For that purpose CAL33 cells were incubated with 213Bianti- EGFR-MAb or irradiated with photons with a nominal energy of 6 MeV both under hypoxic and normoxic conditions. Oxygenation of cells was checked via the hypoxia-associated marker HIF-1α. Survival of cells was analysed using the clonogenic assay. Cell viability was monitored with the WST colorimetric assay. Results were evaluated statistically using a t-test and a Generalized Linear Mixed Model (GLMM). Survival and viability of CAL33 cells decreased both after incubation with increasing 213Bi-anti-EGFR-MAb activity concentrations (9.25 kBq/ml – 1.48 MBq/ml) and irradiation with increasing doses of photons (0.5 – 12 Gy). Following photon irradiation survival and viability of normoxic cells were significantly lower than those of hypoxic cells at all doses analysed. In contrast, cell death induced by 213Bianti- EGFR-MAb turned out to be independent of cellular oxygenation. These results demonstrate for the first time that α-particle emitting 213Bi-immunoconjugates eradicate hypoxic tumor cells as effective as normoxic cells. Therefore, 213Biradioimmunotherapy seems to be an appropriate strategy for treatment of hypoxic tumors.JRC.E.5-Nuclear chemistr

Development of [²²⁵ac]ac-psma-i&t for targeted alpha therapy according to gmp guidelines for treatment of mcrpc

Recently, promising results of the antitumor effects were observed in patients with metastatic castration-resistant prostate cancer treated with177Lu-labeled PSMA-ligands. Radionu-clide therapy efficacy may even be improved by using the alpha emitter Ac-225. Higher efficacy is claimed due to high linear energy transfer specifically towards PSMA positive cells, causing more double-strand breaks. This study aims to manufacture [225Ac]Ac-PSMA-I&T according to good manufacturing practice guidelines for the translation of [225Ac]Ac-PSMA-I&T into a clinical phase 1 dose escalation study. Quencher addition during labeling was investigated. Quality control of [225Ac]Ac-PSMA-I&T was based on measurement of Fr-221 (218 keV), in equilibrium with Ac-225 in approximately six half-lives of Fr-221 (T12 = 4.8 min). Radio-(i)TLC methods were utilized for identification of the different radiochemical forms, gamma counter for concentration determination, and HPGe-detector for the detection of the radiochemical yield. Radiochemical purity was determined by HPLC. The final patient dose was prepared and diluted with an optimized concentration of quenchers as during labeling, with an activity of 8–12 MBq (±5%), pH > 5.5, 100 ± 20 µg/dose, PSMA-I&T, radiochemical yield >95%, radiochemical purity >90% (up to 3 h), endotoxin levels of <5 EU/mL, osmolarity of 2100 mOsmol, and is produced according to current guidelines. The start of the phase I dose escalation study is planned in the near future

Influence of tumour size on the efficacy of targeted alpha therapy with 213Bi-[DOTA0,Tyr3]-octreotate

BACKGROUND: Targeted alpha therapy has been postulated to have great potential for the treatment of small clusters of tumour cells as well as small metastases. (213)Bismuth, an α-emitter with a half-life of 46 min, has shown to be effective in preclinical as well as in clinical applications. In this study, we evaluated whether (213)Bi-[DOTA(0), Tyr(3)]-octreotate ((213)Bi-DOTATATE), a (213)Bi-labelled somatostatin analogue with high affinity for somatostatin receptor subtype 2 (SSTR(2)), is suitable for the treatment of larger neuroendocrine tumours overexpressing SSTR(2) in comparison to its effectiveness for smaller tumours. We performed a preclinical targeted radionuclide therapy study with (213)Bi-DOTATATE in animals bearing tumours of different sizes (50 and 200 mm(3)) using two tumour models: H69 (human small cell lung carcinoma) and CA20948 (rat pancreatic tumour). METHODS: Pharmacokinetics was determined for calculation of dosimetry in organs and tumours. H69- or CA20948-xenografted mice with tumour volumes of approximately 120 mm(3) were euthanized at 10, 30, 60 and 120 min post injection of a single dose of (213)Bi-DOTATATE (1.5–4.8 MBq). To investigate the therapeutic efficacy of (213)Bi-DOTATATE, xenografted H69 and CA20948 tumour-bearing mice with tumour sizes of 50 and 200 mm(3) were administered daily with a therapeutic dose of (213)Bi-DOTATATE (0.3 nmol, 2–4 MBq) for three consecutive days. The animals were followed for 90 days after treatment. At day 90, mice were injected with 25 MBq (99m)Tc-DMSA and imaged by SPECT/CT to investigate possible renal dysfunction due to (213)Bi-DOTATATE treatment. RESULTS: Higher tumour uptakes were found in CA20948 tumour-bearing animals compared to those in H69 tumour-bearing mice with the highest tumour uptake of 19.6 ± 6.6 %IA/g in CA20948 tumour-bearing animals, while for H69 tumour-bearing mice, the highest tumour uptake was found to be 9.8 ± 2.4 %IA/g. Nevertheless, as the anti-tumour effect was more pronounced in H69 tumour-bearing mice, the survival rate was higher. Furthermore, in the small tumour groups, no regrowth of tumour was found in two H69 tumour-bearing mice and in one of the CA20948 tumour-bearing mice. No renal dysfunction was observed in (213)Bi-DOTATATE-treated mice after the doses were applied. CONCLUSIONS: (213)Bi-DOTATATE demonstrated a great therapeutic effect in both small and larger tumour lesions. Higher probability for stable disease was found in animals with small tumours. (213)Bi-DOTATATE was effective in different neuroendocrine (H69 and CA20948) tumour models with overexpression of SSTR(2) in mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13550-016-0162-2) contains supplementary material, which is available to authorized users

Uptake and subcellular distribution of radiolabeled polymersomes for radiotherapy

Polymersomes have the potential to be applied in targeted alpha radionuclide therapy, while in addition preventing release of recoiling daughter isotopes. In this study, we investigated the cellular uptake, post uptake processing and intracellular localization of polymersomes. Methods: High-content microscopy was used to validate polymersome uptake kinetics. Confocal (live cell) microscopy was used to elucidate the uptake mechanism and DNA damage induction. Intracellular distribution of polymersomes in 3-D was determined using super-resolution microscopy. Results: We found that altering polymersome size and concentration affects the initial uptake and overall uptake capacity; uptake efficiency and eventual plateau levels varied between cell lines;

Spät-Neurotoxizität der diffusiblen Beta-Radiopeptid-Brachytherapie im Vergleich zur Alphatherapie

Das entscheidende Kriterium der Malignität von glialen Hirntumoren der WHO-Grade II-IV ist die diffuse Tumorzell-Infiltration von gesundem Hirngewebe. Das weitere Kriterium der Knotenbildung – mit Ausnahme der Gliomatosis cerebri, einer rein infiltrativen seltenen Unterform – ist bedingt durch die noch ungenügende Darstellung der Tumorinfiltrationszone durch die diversen Bildgebungsverfahren. Maligne Gliome bilden keine Tumorkapsel und deshalb auch keine abgrenzbaren Knoten. Vielmehr erstreckt sich der Infiltrationsgradient über die gesamte ipsi- und kontralaterale Hirnhälfte. Frühere Beschreibungen dieses Phänomens, im Handbuch für Neurochirurgie durch den Kölner Neuropathologen K.J. Zülch (1) zusammengefasst, wurden in den 1970ern durch den amerikanische Neuropathologen Peter Burger vertieft anhand von Ganzhirn-Gewebeschnitten von malignen Gliomen. Diese Infiltration von gesundem Hirngewebe durch Gliomzellen ist aus therapeutischer Sicht der Schlüsselfaktor, welcher die Malignität der Gliome der WHO Grade II-IV definiert. Die zunehmende Infiltrationsdichte vom niedriggradigen Gliom bis hin zum Glioblastom erklärt auch den viel rascheren Verlauf der Erkrankung beim hochmalignen Gliom. Dieses infiltrative Verhalten der Gliomzellen ist der eigentliche Grund, warum alle Gliome mit Ausnahme der seltenen pilozytischen Grad I Astrozytome nicht heilbar sind, selbst wenn es gelingt, die nodulären Anteile gut zu kontrollieren. Bei der Brachytherapie kommen therapeutische Radionuklide zum Einsatz, die entweder als eingekapselte ruhende Strahlenquellen stereotaktisch implantiert oder in ihrer modernisierten Form als radioaktive Injektionslösung in ein Kapsel-Kathetersystem injiziert werden. Dabei werden radioaktiv markierte kleinmolekulare Peptidemoleküle lokoregional in die Resektionshöhle oder direkt in die Tumormasse als gelöstes Radiopharmazeutikum injiziert (2). Die Peptidvektoren mit einem Molekulargewicht unter 2000 Dalton können bei optimaler Bioverteilung weite Teile des Gehirns durchdringen und an spezifische Rezeptoren auf der Tumorzelloberfläche andocken. Der Radiopeptid-Rezeptor-Komplex wird daraufhin internalisiert (2). Die metallischen Radioisotope sind stabil an einen DOTA-Chelator gekoppelt, welcher an das nicht bindende N-terminale Ende des Peptids konjugiert ist. Im Gegensatz zur alten Seeds-Technik werden nicht stationäre Strahlenquellen stereotaktisch implantiert, sondern es wird ein in einem Volumen von ca. 2 ml gelöstes Radiopharmazeutikum direkt in den Extrazellulärraum injiziert, in jenes Kompartiment also, in welchem die Tumorzellen entstehen, wachsen und wandern. Die Radiopeptidvektoren verteilen sich nach intratumoraler Injektion vor allem entlang der axonalen Bahnen durch Diffusion und Konvektion infolge des erhöhten intratumoralen Druckes. Sie durchdringen also das Gewebekompartiment, in welchem sich die malignen Gliome ausbreiten, unter Umgehung der Blut-Hirnschranke. Als therapeutische Nuklide kommen traditionellerweise Betastrahler, sog. Elektronenstrahler, zur Anwendung (2,3). In den letzten Jahren wurden aber auch erste klinische Studien mit Alphastrahlern durchgeführt (4,5). Das Nebenwirkungsprofil dieser Chelator-gebundenen metallischen Radionuklide hängt von den physikalischen Eigenschaften ab, genauer von der mittleren Reichweite, der Energie und der Halbwertszeit des jeweiligen Isotops. Der häufig verwendete Betastrahler Yttrium-90 hat eine mittlere Reichweite von 5mm und eine sehr hohe Energie von 2,1 MeV. Lutetium-177 hingegen hat nur eine Reichweite von 1mm eine viel niedrigere Energie von 0,13 MeV, hat also einen viel steileren Dosisabfall als Yttrium-90 und damit ein deutlich günstigeres Nebenwirkungsprofil, wenn die therapeutischen Elektronen in funktionell wichtigen Arealen freigesetzt werden. Die Schädigung von wichtigen Hirnzentren durch das hochenergetische Yttrium-90 kann auch als späte Fernwirkung auftreten, weil es sich um eine diffus infiltrative Krankheit handelt, deren Infiltrationsmuster bei der Diagnosestellung aufgrund fehlender diagnostischer Methoden gar nicht getreu abgebildet werden kann. Deswegen kann bei der Therapieplanung über mögliche Nebenwirkungen auch keine genaue Voraussage gemacht werden. Das erste Fallbeispiel zeigt, dass repetitiv applizierte, mit Yttrium-90 markierte Vektoren (z.B. Y-90 DOTAGA-Substanz P) noch viele Jahre nach Abschluss der Behandlung zu Nebenwirkungen führen können, und zwar an Orten , die ursprünglich gar nicht befallen schienen.JRC.G.I.5-Advanced Nuclear Knowledg

Reducing renal uptake of free 213Bi associated with the decay of 225Ac-labeled radiopharmaceuticals

Alpha-emitting radionuclides provide cytotoxic agents that are considered impervious to conventional cellular resistance mechanisms such as effusion pumps, signaling pathway redundancy, and cell cycle modulation (e.g., cell dormancy, G1/G0 or G2/M block). Actinium-225 is a promising α-emitting radionuclide due to its net decay of four α-particles. The longest-lived progeny of 225Ac, bismuth-213 concentrates in the kidneys. Depending upon tumor burden, and the accessibility of tumor cells the percent tumor uptake of IV-administered radiolabeled antibody in humans is typically of the order of a few percent or less. This means that the majority of antibody-bound 225Ac decays would occur in the circulation and lead to eventual renal toxicity as has been observed in pre-clinical studies The objective of this work is to evaluate the potential to reduce renal uptake of free 213Bi by exploiting the mechanism associated with bismuth uptake.JRC.G.I.5-Advanced Nuclear Knowledg

The impact of tumor burden on the absorbed dose to the kidneys from Actinium-225 labeled antibody therapy in a murine model and predicted dosimetric impact for human clinical use

There has been increasing interest in the use alpha-particle emitting radionuclides to combat cancer, especially in metastasized disease. The short range of the emitted alpha-particles combined with the high LET makes them effective against single cells, cell clusters and solid tumors. Studies have shown that the main part of the absorbed dose to the kidneys when using 225Ac labeled antibodies originates from the free daughter 213Bi, which is generated from 225Ac in the rest of the body. Furthermore, the dosimetry has been well qualitatively characterized using the macro-to-micro (M2M) small scale methodology. In this study we examine how the tumor burden affects the absorbed dose to the kidneys from both the 225Ac labeled antibody and the free daughter 213Bi in a transgenic immunocompetent mouse model applying M2M and extrapolate to human use.JRC.G.I.5-Advanced Nuclear Knowledg

Targeting aberrant DNA double strand break repair in triple negative breast cancer with alpha particle emitter radiolabeled anti-EGFR antibody

The greater potential efficacy of alpha-particle emitter radiopharmaceutical therapy lies in the 3 to 8-fold greater biological effectiveness (RBE) of alpha particles relative to photon or beta-particle radiation. The greater RBE, however, generally applies to both tumor and normal tissue, thereby reducing efficacy. Since alpha particles typically cause DNA double strand breaks (DSBs), targeting tumors that are defective in DNA DSB repair would effectively increase the RBE, yielding a secondary, RBE-based differentiation between tumor and normal tissue that is complementary to conventional, delivery-based tumor targeting. In some triple negative breast cancer (ER-/PR-/HER-2-, TNBC) patients, both germline predisposed mutation or sporadic gene silencing in BRCA-1, a key gene in homologous recombination (HR) DSB repair, are well established. Such patients have few treatment options once the cancer has metastasized. In this study, we investigated the efficacy of alpha particle emitter, 213Bi labeled anti-EGFR antibody, Cetuximab, in TNBC cells that are defective in DNA DSB repair. 213Bi-Cetuximab was found to be significantly more effective in BRCA-1 mutated TNBC cell HCC1937. siRNA knockdown of BRCA-1 or DNA-PKcs, a key gene in non-homologous end joining (NHEJ) DSB repair, sensitized TNBC cells to 213Bi-Cetuximab. Furthermore, the small molecule inhibitor of DNA-PKcs, NU7441, also sensitized TNBC cells to alpha radiation. Both immunofluorescent staining of H2AX foci and Comet assay confirmed that enhanced RBE is caused by impaired DNA DSB repair. These data suggest a strategy for enhancing conventional receptor-mediated targeting with an additional, potentially synergistic radiobiological alpha-emitter targeting that could be applied to TNBC metastases.JRC.E.5-Nuclear chemistr

Pharmacokinetic variability of 225Ac and 213Bi from vector labeled 225Ac cancer therapy as a function of vector type (antibody vs. small molecule)

Objectives Actinium-225 (T1/2 = 9.9 days) labeled targeted radiopharmaceuticals’ pharmacokinetics are determined by the choice of targeting vector used (i.e. antibody, small molecule, etc.). The decay of 225Ac results in the release of the decay daughters from the targeting vector. For example, decay of 225Ac-labeled agent in the blood results with the longest lived daughter 213Bi (T1/2 = 46 minutes) accumulating in the cortex region of the kidneys and is associated with the majority of the radiation dose to the kidneys. Depending on the targeting vector accumulation within different normal tissues the dominant supplier of unbound 213Bi to the kidneys may be altered. In this study we used two different targeting vectors (antibody and a small molecule peptidomimetic) labeled with 225Ac against breast cancer (BC) and castrate resistant prostate cancer (CRPC), respectively, to determine the pharmacokinetics of unbound 213Bi to the kidneys. Methods To compare pharmacokinetics differences between targeting vectors, we selected the 7.16.4 antibody, which is targeted to Erbb2, and a PSMA-targeted small molecule peptidomimetic. The 7.16.4 antibody was labeling with 225Ac and the resulting agent, 225Ac-7.16.4, was injected into neu-N mice bearing Erbb2+ tumors. The 225Ac-labeled PSMA-targeted peptidomimetic was injected into mice bearing PSMA+ tumors. For both models at select time points the mice were sacrificed, organs removed, and measured for activity. Select organs were measured directly after harvesting, and continuously measured over a period of several hours to determine the amount of unbound 213Bi compared to the intact 225Ac-labeled targeting vector present in the selected organ. Differentiation in the detection of these two isotopes is delicate as only the 213Bi emits photons captured in the gamma well counter; therefore fits to the time activity data from the gamma well counter using the half-lives of the two isotopes can distinguish between the intact 225Ac-labeled agent and unbound 213Bi. Results The use of the small molecule vector for the CRPC murine model showed uptake of unbound 213Bi in the kidneys at all time points and the main supplying organ of unbound 213Bi was the liver. In the BC murine model using an antibody the blood was the main supplier of unbound 213Bi to the kidneys. These results were combined with the different micro-scale distribution in the kidneys for 213Bi and the respective vector-225Ac to determine the radiation dose to kidneys and other normal organs. Conclusions This study demonstrated that the use of different targeting vectors for delivery of 225Ac alters the pharmacokinetics and suppliers of unbound 213Bi, to the kidneys. These properties are important for translation to clinical studies as in vivo imaging cannot distinguish between the intact 225Ac-labeled agent and the unbound 213Bi, resulting in an overestimation of the average radiation dose to kidneys and an underestimation of the average radiation dose to the supplying organ (blood and liver, respectively).JRC.G.I.5-Advanced Nuclear Knowledg