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Die vorliegende Arbeit untersuchte 62 Patienten/-innen mit metastasierten neuroendokrinen Tumoren des Gastrointestinaltrakt, die im Zeitraum von 2014 bis 2019 in der Klinik für Nuklearmedizin im Universitätsklinikum Marburg, eine Ga-68-DOTATOC-PET-Untersuchung und darauffolgend, eine Peptid-Radio-Rezeptor-Therapie mit Lutetium-177-DOTATOC, erhielten. Die Analyse fokussierte sich auf die möglichen Prädiktoren des progressionsfreien Überlebens: Ki67, SUVmax, WHO-Grading, Chromogranin A, Primariuslokalisation, Alter bei Erstdiagnose und Geschlecht. Zusammenfassend konnte diese Arbeit die Rolle von Ki67 als Prognoseparameter für das progressionsfreie Überleben von Patienten/-innen mit metastasierten GEP-NET nach Lu-177-PRRT demonstrieren. Eine Überarbeitung der Abgrenzung zwischen G1 und G2 Tumoren sollte von Ki67=2% auf Ki67=5% zur Erhöhung der prognostischen Aussagekraft erwogen werden. Der SUVmax konnte für das bereits zur PRRT-selektierte Kollektiv keine prognostischen Rückschlüsse bieten. Chromogranin A behält weiterhin seine Berechtigung als Tumormarker und Verlaufsparameter, ist aber zur Formulierung einer Prognose hinsichtlich des PFS nicht geeignet. Das Alter bei Erstdiagnose konnte das PFS nicht vorhersagen. Für p-NET konnte eine schlechtere Prognose als für NET des Dünndarms gezeigt werden. Signifikante Unterschiede im Ga-68-Speicherverhalten konnte nur für die Variable „Primarius“ festgestellt werden, nicht jedoch für das WHO-Grading oder das Auftreten eines Progresses. Überraschend war der deutliche Unterschied im PFS zwischen Männern und Frauen des Kollektivs – dessen Genese jedoch nicht abschließend aufzuklären war. Außerdem kam es bei den Frauen häufiger zu einem Tumorprogress. Prospektive Studien in der Zukunft sollten weiterhin die möglichen Unterschiede zwischen den Geschlechtern untersuchen um Ergebnisse wie diese einordnen zu können., In this paper 62 patients were investigated who were diagnosed and treated with Ga-68-DOTATOC-PET/CT and consecutive Lu-177-PRRT therapy for metastatic neuroendocrine neoplasms of the digestive system at the University Hospital of Marburg between 2014 and 2019. The analysis focused mainly on potential predictors of progression free survival: Ki67 proliferation index, WHO-Grading, Chromogranin A, localization of the primary, age at diagnosis and sex. To conclude, this paper showed Ki67 proliferation index as a feasible prognostic marker of progression free survival in patients with metastatic neuroendocrine tumors of the digestive system after Lu-177-PRRT treatment. The differenciation between G1 and G2 neoplasms should be revised from Ki67=2% to a threshold of Ki67=5% in order to provide a more reliable prognosis. SUVmax was proven to be an unfeasible prognostic marker for progression free survival in patients who had already been selected for PRRT by SUVmax measurement. Chromogranin A can be used as a tumormarker and representative of disease progression. Age at diagnosis and Chromogranin A were did not present as suitable prognostic values of progression free survival. Pancreatic neoendocrine neoplasms were associated with a poorer prognosis than NET with an ileal or jejunal primary. There was a significant difference in the tracer accumulation in metastases of different primary sites but none with regard to WHO-Grading or status progressive disease. Surprisingly, there was a clear difference in progression free survival of men and women, which could not finally be clarified. Furthermore, women suffered from progressive disease more often than men. Future prospective studies should continue to examine differences with regard to gender in order to offer more insight as to integrate such results.

The high energy demand of fast and aggressively growing tumor cells is covered by both anaer-obic and aerobic glycolysis. This results in an intracellular accumulation of mostly acidic metabol-ic products. Thus, balancing the intracellular pH is demanding for tumor cells, yet crucial for their optimal proliferation and survival. The carbonic anhydrases (CA) constitute a family of enzymes that catalyse the hydration of carbon dioxide, yielding the bicarbonate ion, which serves as the main buffer system in most organisms. CA XII is a membrane-tethered isozyme on several types of tumors and an excellent target for nuclear medicine diagnosis and therapy. The specific CA XII binding 6A10 Fab (fragment antigen binding) was shown to be of great interest as a new carrier molecule for a radioactive nuclide. A suitable radionuclide for therapeutic applications accompa-nied with single photon emission computed tomography (SPECT) imaging techniques is lutetium-177. In this study, the 177Lu-labelled 6A10 Fab was for the first time evaluated in vitro and in vivo as an agent for local intracavitary radioimmunotherapy (RIT) of glioblastomas. To extend the 6A10 Fab´s application beyond the local treatment in the brain, a systemic injection for the detec-tion of CA XII expressing tumors was investigated. For this purpose, this study evaluated the automated production of copper-64 for radiolabelling purposes to provide 64Cu-labelled 6A10 Fab for positron emission tomography (PET) studies. For therapeutic applications, the protein was conjugated with p-SCN-Bn-CHX-A´´-DTPA at a mo-lar chelator-to-protein ratio of 1:0.96 and radiolabelled with lutetium-177 with a specific activity of 1.5 GBq/mg. Radio-TLC revealed a radiochemical purity of greater than 96%. The radioconjugate was analyzed via flow cytometry, confirming the persisting CA XII binding capacity after suc-cessful modification. The radiochemical stability of the compound was determined to be greater than 90% after 72 h of incubation in human cerebrospinal fluid (37 °C), human plasma (37 °C) and labelling buffer (room temperature). A mouse xenograft model with CA XII-positive glioma cells was used for biodistribution studies, autoradiographic examinations and first in vivo imaging experiments. The tumor revealed a moderate uptake of up to 3.1 ± 0.9 %ID/g 6 h post injection (p.i.) while significantly high and long-lasting kidney retention (55.6 ± 13.0 %ID/g 48 h p.i.) was found. Tumor slices were analyzed autoradiographically and microscopically to confirm that re-gions of high activity uptake also show high cell density and vice versa. Tumors were success-fully visualized using planar scintigraphy or SPECT/CT techniques and a tumor-to-contralateral ratio of 2.4 (5 h p.i.) was measured. Copper-64 was produced using an automated setup and characterized with respect to its use as a radiolabelling nuclide. Nickel-64, which was used as target material, was electrochemically de-posited with yields of up to 96%, automatically send to a cyclotron, and irradiated with protons (2 - 5 h, 11 – 14.5 MeV, 20 - 30 µA), yielding a maximum activity of approx. 5 GBq. In addition to copper-64, irradiation by-products were quantified by gamma spectroscopy as 0.03 ± 0.10 At% 55Co, 0.004 ± 0.014 At% 57Co, and 1.4 ± 3.4 At% 61Co at the end of irradiation. Ion chromatog-raphy of the dissolved target led to a radiochemical purity of >99% in the final product fraction. The copper-64, dissolved in 9 ml HCl, was evaporated to dryness and taken up in 400 µl of 0.1 M HCl. Optimization of the module rinsing methods lead to molar activities of up to 133 GBq/µmol. The efficiency of rinsing methods was evaluated by determination of non-radioactive metal con-taminations via ICP-OES while the molar activity was calculated by titration of the copper-64 with 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). For diagnostic purposes, the 6A10 Fab was conjugated with p-NCS-Bn-NODA-GA, radiolabelled with copper-64 and used for PET imaging and a biodistribution study in mice. First in vivo experi-ments of the 64Cu-labelled 6A10 Fab showed a high tumor uptake of up to 6.9 ± 1.6 %ID/g. PET analysis revealed a tumor-to-contralateral ratio of 11.8 ± 0.3 (n = 2) 4 h p.i.. The results of this study showed the high potential of the radiolabelled 6A10 Fab fragment for possible therapeutic and diagnostic applications. Radiolabelled with lutetium-177, the protein is suitable for the local therapeutic application in the brain since it showed a moderate tumor uptake, and a good radiochemical stability under physiological conditions. Additionally, it does not cross the blood-brain barrier (BBB). Further investigations on an orthotopic animal model to generate data closer to the final clinical application would be beneficial to estimate the compound´s distribu-tion after a local application. By radiolabelling the Fab with copper-64, high tumor uptake enabling high resolution PET images was achieved, which encourages further investigations concerning a future systemic application for diagnostic purposes. The automated production of copper-64 re-sulted in good radioactive yields, moderate molar activities and high radiochemical purity. Repro-ducibility of the production process still needs to be improved further for routine supply of copper-64 with high quality for radiolabelling procedures., Aufgrund ihres schnellen und aggressiven Wachstums benötigen Tumorzellen überdurchschnitt-lich viel Energie. Dieser Bedarf wird größtenteils über aerobe und anaerobe Glykolyse gedeckt, was intrazellulär zur Entstehung von sauren Stoffwechselprodukten führt. Um einer Übersäue-rung entgegenzuwirken sind Puffermoleküle wie Hydrogencarbonate notwendig, die u.a. von der Membran-assoziierten Carboanhydrase XII (CA XII) zur Verfügung gestellt werden. Aus diesem Grund ist dieses Enzym auf vielen Arten von Tumorzellen überexprimiert und stellt somit ein viel-versprechendes Zielmolekül für nuklearmedizinische Ansätze in Diagnostik und Therapie dar. Das 6A10 Fab Fragment bindet spezifisch an CA XII, weshalb es als Trägermolekül eines radio-aktiven Nuklides prädestiniert ist. In dieser Arbeit wurde das Protein mit Lutetium-177 markiert und durch diverse in-vitro und in-vivo Charakterisierungen auf seine mögliche Eignung für die lokale Therapie von Glioblastomen untersucht. Darüber hinaus ist eine diagnostische Anwendung erstrebenswert, weshalb die automatisierte Produktion des PET-Nuklids Kupfer-64 etabliert und dieses charakterisiert wurde. Schlussendlich wurde das Fab Fragment mit Kupfer-64 markiert und damit erste in-vivo Daten mittels PET-Bildgebung generiert. Das Protein wurde zunächst mit dem Chelator p-SCN-Bn-CHX-A´´-DTPA in einem Verhältnis von 1:0,96 gekoppelt und anschließend mit 1,5 GBq 177Lu/mg Fab radiomarkiert. Die radiochemische Reinheit von mehr als 96% wurde mittels Dünnschichtchromatographie ermittelt. Der Erhalt der Bindungsaffinität des modifizierten Fab Fragments am Zielmolekül CA XII wurde mittels Durch-flusszytometrie bestätigt und eine radiochemische Stabilität von über 90% nach 72 h Inkubation in verschiedenen Medien (Liquor, Plasma und Markierungspuffer) gefunden. Autoradiographie-, Biodistributions- und erste Bildgebungsexperimente wurden an Mäusen mit subkutanen Tumor-transplantaten durchgeführt. Der Tumor zeigte 6 h p.i. eine moderate Aktivitätsaufnahme von 3,1 ± 0,9 %ID/g, während signifikant hohe Aktivitätswerte in der Niere gefunden wurden, die auch 48 h p.i. noch bei 55,6 ± 13,0 %ID/g lagen. Durch autoradiographische Untersuchungen an Tu-morschnitten, gefolgt von HE-Färbungen und entsprechender mikroskopischer Charakterisie-rung konnte eine Korrelation zwischen Bereichen mit hoher Aktivitätsakkumulation und hoher Zelldichte und vice versa festgestellt werden. Die Verteilung des radioaktiven Konjugats konnte außerdem in ersten Bildgebungsexperimenten (planare Szintigraphie und SPECT/CT) visualisiert und ein Tumor-zu-kontralateral Verhältnis von 2,4 (5 h p.i.) bestätigt werden. Im zweiten Teil dieser Arbeit wurde die Konjugation und Radiomarkierung des 6A10 Fabs mit p-NCS-Bn-NODA-GA und Kupfer-64 angestrebt, um erste Information zum in-vivo Verhalten der Verbindung mittels PET-Bildgebung und Biodistribution zu erhalten. Dazu wurde die Herstellung des Nuklids unter Verwendung der automatisierten Alceo-Module etabliert und das Produkt hin-sichtlich seiner Eignung zur Radiomarkierung von Proteinen charakterisiert. Elementares Nickel-64 wurde mit Ausbeuten über 96% abgeschieden, automatisch im Strahlengang des Zyklotrons positioniert und bis zu 5 h lang bei 11,0 – 14,5 MeV und 20 - 30 µA mit Protonen bestrahlt. Aus-beuten von maximal 5 GBq konnten erzielt werden. Entstandene Nebenprodukte wurden mittels Gammaspektroskopie quantifiziert und auf das Ende der Bestrahlung zerfallskorrigiert (0,03 ± 0,10 At% 55Co, 0,004 ± 0,014 At% 57Co und 1,4 ± 3,4 At% 61Co). Das Nuklidgemisch wurde mit-tels Ionenchromatographie aufgetrennt, was zu einer radiochemischen Reinheit der 64Cu-Fraktion von über 99% führte. Nach Volumenreduktion lag das Produkt in 400 µl einer 0,1 M HCl vor. Die molare Aktivität des Kupfers, ermittelt durch Titration mit TETA, konnte auf 133 GBq/µmol angehoben werden, nachdem durch Optimierung der Reinigungsmethodik der Anteil an nicht-radioaktiven Metallkontaminationen, quantifiziert mittels ICP-OES Messungen, sig-nifikant reduziert wurde. Erste Biodistributionsergebnisse mit dem 64Cu-markiertem 6A10 Fab zeigten eine hohe Tumoranreicherung von bis zu 6,9 ± 1,6 %ID/g 5 h p.i.. Die PET-Quantifizierung ergab 4 h p.i. ein Tumor-zu-kontralateral Verhältnis von 11,8 ± 0,3 (n = 2). Die ersten in-vivo und in-vitro Charakterisierungen des 6A10 Fab in Hinblick auf zukünftige the-rapeutische und diagnostische Anwendungen zeigten vielversprechende Ergebnisse. Durch die Radiomarkierung mit Lutetium-177 in Hinblick auf die lokale Applikation in das Gehirn, konnte eine moderate Tumoranreicherung und eine ausreichende radiochemische Stabilität unter physiologi-schen Bedingungen im systemisch applizierten Xenograft-Modell nachgewiesen werden. Zusätz-lich dazu zeigte die mangelnde Anreicherung im Gehirn, dass das Radiokonjugat die Blut-Hirn-Schranke nicht passiert. Weitere Informationen über das in-vivo Verhalten des Moleküls nach der lokalen Applikation können zudem durch die Etablierung eines anwendungsnäheren Tiermodells generiert werden. Die systemische Gabe des 64Cu-markierten Proteins führte zu einer guten Tumoranreicherung und ermöglichte hochauflösende PET-Bildgebung, was zu weiteren Studien in Hinblick auf die Anwendung des Konjugats im Bereich der Krebsdiagnostik motiviert. Die wissenschaftliche Rele-vanz dieser ersten Ergebnisse sollte in ausführlichen Biodistributions- und Bildgebungsstudien bestätigt werden um das weitere Vorgehen in Richtung der humanen Anwendung zu rechtferti-gen. Die genutzten Alceo-Module ermöglichen die automatische Produktion von Kupfer-64 in gu-ter radioaktiver Ausbeute, moderaten molaren Aktivitäten und hoher radiochemischer Reinheit. Die Reproduzierbarkeit des Herstellungsprozesses sollte durch weitere Modifikationen weiter verbessert werden, um die regelmäßige Bereitstellung von Kupfer-64 in hoher Qualität für Radi-omarkierungszwecke gewährleisten zu können.

Single-domain antibody fragments (sdAbs) are the smallest functional antigen-binding fragments, derived from heavy chain-only camelid antibodies. When designed as radiolabeled monomeric probes for imaging and therapy of cancer, their fast and specific targeting results in high tumor-to-background ratios early after injection. However, their moderate absolute uptake into tumors might not always be sufficient to treat cancerous lesions. We have evaluated the pharmacokinetics of seven constructs derived from a CD20-targeting monomeric sdAb (αCD20). The constructs differed in affinity or avidity towards CD20 (dimeric αCD20-αCD20 and αCD20 fused to a non-targeting control sdAb, referred to as αCD20-ctrl) and blood half-lives (αCD20 fused to an albumin-targeting sdAb (αAlb) = αCD20-αAlb). The constructs were radiolabeled with 111In (imaging) and 177Lu (therapy) using the bifunctional chelator CHX-A”-DTPA and evaluated in vitro and in vivo. In mice, tumor uptake of 177Lu-DTPA-αCD20 decreased from 4.82 ± 1.80 to 0.13 ± 0.05% IA/g over 72 h. Due to its rapid blood clearance, tumor-to-blood (T/B) ratios of >100 were obtained within 24 h. Although in vitro internalization indicated that dimeric 177Lu-DTPA-αCD20-αCD20 was superior in terms of total cell-associated radioactivity, this was not confirmed in vivo. Blood clearance was slower and absolute tumor uptake became significantly higher for αCD20-αAlb. Blood levels of 177Lu-DTPA-αCD20-αAlb decreased from 68.30 ± 10.53 to 3.58 ± 0.66% IA/g over 120 h, while tumor uptake increased from 6.21 ± 0.94 to 24.90 ± 2.83% IA/g, resulting in lower T/B ratios. Taken together, these results indicate that the increased size of dimeric αCD20-αCD20 or the fusion of monomeric αCD20 to an albumin-targeting moiety (αAlb) counterbalance their improved tumor targeting capacity compared to monomeric αCD20.

Background Recently, 177Lu-dotatate therapy for neuroendocrine tumours has received regulatory approval. Dosimetry can be used to optimize treatment on an individual basis, but there is no international consensus as to how it should be done. The aim of this study is to determine a feasible and accurate dosimetry method to guide individualized peptide receptor radionuclide therapy (PRRT) for patients with neuroendocrine tumours. As part of a clinical trial on 177Lu-dotatate therapy, renal dosimetry was performed for all patients in each treatment cycle, using a hybrid planar-SPECT/CT method. In the present study, we use the image data acquired from 22 patients and 119 cycles and define a set of alternative treatment planning strategies, each representing a simplification in terms of image acquisition and dosimetric calculations. The results from the simplified strategies are compared to the results from the protocol-prescribed hybrid planar-SPECT/CT-based method by analysing differences both in per-cycle and total cumulative absorbed dose (AD) analyses. Results In general, the SPECT-based methods gave results that were largely consistent with the protocol-specified hybrid method, both in the per-cycle and cumulative AD analyses. Notably, performing one SPECT/CT per cycle at 96 h yielded ADs that were very similar to the protocol method. The methods using planar dosimetry resulted in larger variations, as expected, while giving 4 cycles to all patients resulted in the largest inter-individual differences in cumulative AD. Conclusions Performing one SPECT/CT at 96 h in every treatment cycle gives sufficiently reliable dosimetric results to base individualized treatment planning on, with a reasonable demand on resources.

Background Recently, 177Lu-dotatate therapy for neuroendocrine tumours has received regulatory approval. Dosimetry can be used to optimize treatment on an individual basis, but there is no international consensus as to how it should be done. The aim of this study is to determine a feasible and accurate dosimetry method to guide individualized peptide receptor radionuclide therapy (PRRT) for patients with neuroendocrine tumours. As part of a clinical trial on 177Lu-dotatate therapy, renal dosimetry was performed for all patients in each treatment cycle, using a hybrid planar-SPECT/CT method. In the present study, we use the image data acquired from 22 patients and 119 cycles and define a set of alternative treatment planning strategies, each representing a simplification in terms of image acquisition and dosimetric calculations. The results from the simplified strategies are compared to the results from the protocol-prescribed hybrid planar-SPECT/CT-based method by analysing differences both in per-cycle and total cumulative absorbed dose (AD) analyses. Results In general, the SPECT-based methods gave results that were largely consistent with the protocol-specified hybrid method, both in the per-cycle and cumulative AD analyses. Notably, performing one SPECT/CT per cycle at 96 h yielded ADs that were very similar to the protocol method. The methods using planar dosimetry resulted in larger variations, as expected, while giving 4 cycles to all patients resulted in the largest inter-individual differences in cumulative AD. Conclusions Performing one SPECT/CT at 96 h in every treatment cycle gives sufficiently reliable dosimetric results to base individualized treatment planning on, with a reasonable demand on resources. Electronic supplementary material The online version of this article (10.1186/s40658-018-0210-2) contains supplementary material, which is available to authorized users.

RIT has become an attractive strategy in cancer treatment, but still faces important drawbacks due to poor tumor penetration and undesirable pharmacokinetics of the targeting vehicles. Smaller radiolabeled antibody fragments and peptides feature highly specific target accumulation, resulting in low accumulation in healthy tissue, except for the kidneys. Nanobodies are the smallest (MW < 15 kDa) functional antigen-binding fragments that are derived from heavy chain-only camelid antibodies. Here, we show that the extend of kidney retention of nanobodies is predominantly dictated by the number of polar residues in the C-terminal amino acid tag. Three nanobodies were produced with different C-terminal amino-acid tag sequences (Myc-His-tagged, His-tagged, and untagged). Dynamic planar imaging of Wistar rats with 111In-DTPA-nanobodies revealed that untagged nanobodies showed a 70 % drop in kidney accumulation compared to Myc-His-tagged nanobodies at 50 min p.i.. In addition, coinfusion of untagged nanobodies with the plasma expander Gelofusin led to a final reduction of 90 %. Similar findings were obtained with different 177Lu-DTPA-2Rs15d nanobody constructs in HER2pos tumor xenografted mice at 1 h p.i.. Kidney accumulation decreased 88 % when comparing Myc-His-tagged to untagged 2Rs15d nanobody, and 95 % with a coinfusion of Gelofusin, without affecting the tumor targeting capacity. Consequently, we identified a generic method to reduce kidney retention of radiolabeled nanobodies. Dosimetry calculations of Gelofusin-coinfused, untagged 177Lu-DTPA-2Rs15d revealed a dose of 0.90 Gy/MBq that was delivered to both tumor and kidneys and extremely low doses to healthy tissues. In a comparative study, 177Lu-DTPA-Trastuzumab supplied 6 times more radiation to the tumor than untagged 177Lu-DTPA-2Rs15d, but concomitantly also a 155, 34, 80, 26 and 4180 fold higher radioactivity burden to lung, liver, spleen, bone and blood. Most importantly, nanobody-based targeted radionuclide therapy in mice bearing small estiblashed HER2pos tumors led to an almost complete blockade of tumor growth and a significant difference in event-free survival between the treated and the control groups (P < 0.0001). Based on histology analyses, no evidence of renal inflammation, apoptosis or necrosis was obtained. In conclusion, these data highlight the importance of the amino acid composition of the nanobody's C-terminus, as it has a predominant effect on kidney retention. Moreover, we show successful nanobody-based targeted radionuclide therapy in a xenograft model and highlight the potential of radiolabeled nanobodies as a valuable adjuvant therapy candidate for treatment of minimal residual and metastatic disease.