Publications by authors named "Stefanie Pektor"

22 Publications

  • Page 1 of 1

Iodine-124 PET quantification of organ-specific delivery and expression of NIS-encoding RNA.

EJNMMI Res 2021 Feb 10;11(1):14. Epub 2021 Feb 10.

TRON - Translational Oncology at the University Medical Center, Johannes Gutenberg University Mainz gGmbH, Mainz, Germany.

Background: RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. Here, we have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein. For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was in vitro transcribed in cell lines and evaluated for expression. RNA-lipoplex nanoparticles were then assembled by complexing RNA with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice.

Results: NIS expression was detected on the membrane of two cell lines as early as 6 h after transfection and gradually decreased over 48 h. In vivo and ex vivo PET/MRI of anionic spleen-targeting or cationic lung-targeting NIS-RNA lipoplexes revealed a visually detectable rapid increase of Iodine-124 uptake in the spleen or lung compared to control-RNA-lipoplexes, respectively, with minimal background in other organs except from thyroid, stomach and salivary gland.

Conclusions: The strong organ selectivity and high target-to-background acquisition of NIS-RNA lipoplexes indicate the feasibility of small animal PET/MRI to quantify organ-specific delivery of RNA.
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http://dx.doi.org/10.1186/s13550-021-00753-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876195PMC
February 2021

Characterization of activation induced [18]F-FDG uptake in Dendritic Cells.

Nuklearmedizin 2021 Apr 16;60(2):90-98. Epub 2020 Dec 16.

Department of Nuclear Medicine, University Medical Center Mainz, Germany.

Aim:  Activation of immune cells leads to enhanced glucose uptake that can be visualized by []F-Fluorodeoxyglucose ([]F-FDG) positron emission tomography/computed tomography (PET/CT). Dendritic cells (DC) are essential for the function of the adaptive immune system. In contrast to other immune cells metabolic changes leading to an increase of []F-FDG uptake are poorly investigated. Here, we analysed the impact of different DC activation pathways on their []F-FDG uptake. This effect was then used to radiolabel DC with []F-FDG and track their migration in vivo.

Methods:  DC were generated from bone marrow progenitors (BMDC) or isolated from spleens (SPDC) of C57BL/6 mice. After stimulation with the TLR ligands LPS and CpG or anti-CD40 antibody for up to 72 hours activation markers and glucose transporters (GLUTs) were measured by flow cytometry. Uptake of []F-FDG was measured by gamma-counting. DC lysates were analysed for expression of glycolysis relevant proteins by mass spectrometry (MS). []F-FDG-labeled DC were injected into footpads of mice to image DC migration.

Results:  BMDC and SPDC showed strong upregulation of activation markers predominantly 24 hours after TLR stimulation followed by higher uptake of []F-FDG. In line with this, the expression of GLUTs was upregulated during the course of activation. Furthermore, MS analyses of DC lysates revealed differential regulation of glycolysis relevant proteins according to the stimulatory pathway. As a proof of principle, DC were labeled with []F-FDG upon activation to follow their migration in vivo via PET/MRI.

Conclusion:  Immune stimulation of DC leads to enhanced []F-FDG uptake into DC, representing the typical shift to aerobic glycolysis in immune cells after activation.
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http://dx.doi.org/10.1055/a-1308-0589DOI Listing
April 2021

PSA and PSA Kinetics Thresholds for the Presence of Ga-PSMA-11 PET/CT-Detectable Lesions in Patients With Biochemical Recurrent Prostate Cancer.

Cancers (Basel) 2020 Feb 8;12(2). Epub 2020 Feb 8.

Clinic of Nuclear Medicine, Johannes Gutenberg-University, 55101 Mainz, Germany.

Ga-PSMA-11 positron-emission tomography/computed tomography (PET/CT) is commonly used for restaging recurrent prostate cancer (PC) in European clinical practice. The goal of this study is to determine the optimum time for performing these PET/CT scans in a large cohort of patients by identifying the prostate-specific-antigen (PSA) and PSA kinetics thresholds for detecting and localizing recurrent PC. This retrospective analysis includes 581 patients with biochemical recurrence (BC) by definition. The performance of Ga-PSMA-11 PET/CT in relation to the PSA value at the scan time as well as PSA kinetics was assessed by the receiver-operating-characteristic-curve (ROC) generated by plotting sensitivity versus 1-specificity. Malignant prostatic lesions were identified in 77%. For patients that were treated with radical prostatectomy (RP) a PSA value of 1.24 ng/mL was found to be the optimal cutoff level for predicting positive and negative scans, while for patients previously treated with radiotherapy (RT) it was 5.75 ng/mL. In RP-patients with PSA value <1.24 ng/mL, 52% scans were positive, whereas patients with PSA ≥1.24 ng/mL had positive scan results in 87%. RT-patients with PSA <5.75 ng/mL had positive scans in 86% and and for those with PSA ≥5.75 ng/mL 94% had positive scans. This study identifies the PSA and PSA kinetics threshold levels for the presence of Ga-PSMA-11 PET/CT-detectable PC-lesions in BC patients.
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http://dx.doi.org/10.3390/cancers12020398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072299PMC
February 2020

Evaluation of a novel monoclonal antibody against tumor-associated MUC1 for diagnosis and prognosis of breast cancer.

Int J Med Sci 2019 14;16(9):1188-1198. Epub 2019 Aug 14.

Institute for Immunology, University Medical Center.

There is still a great unmet medical need concerning diagnosis and treatment of breast cancer which could be addressed by utilizing specific molecular targets. Tumor-associated MUC1 is expressed on over 90 % of all breast cancer entities and differs strongly from its physiological form on epithelial cells, therefore presenting a unique target for breast cancer diagnosis and antibody-mediated immune therapy. Utilizing an anti-tumor vaccine based on a synthetically prepared glycopeptide, we generated a monoclonal antibody (mAb) GGSK-1/30, selectively recognizing human tumor-associated MUC1. This antibody targets exclusively tumor-associated MUC1 in the absence of any binding to MUC1 on healthy epithelial cells thus enabling the generation of breast tumor-specific radiolabeled immune therapeutic tools. MAb GGSK-1/30 was used for immunohistochemical analysis of human breast cancer tissue. Its desferrioxamine (Df')-conjugate was synthesized and labelled with Zr. [Zr]Zr-Df'-GGSK-1/30 was evaluated as a potential PET tracer. Binding and pharmacokinetic properties of [Zr]Zr-Df'-GGSK-1/30 were analyzed using human and murine cell lines that express tumor-associated MUC1. Self-generated primary murine breast cancer cells expressing human tumor-associated MUC1 were transplanted subcutaneously in wild type and human MUC1-transgenic mice. The pharmacology of [Zr]Zr-Df'-GGSK-1/30 was investigated using breast tumor-bearing mice by PET/MRT imaging as well as by organ biodistribution analysis. The mAb GGSK-1/30 stained specifically human breast tumor tissue and can be possibly used to predict the severity of disease progression based on the expression of the tumor-associated MUC1. For imaging, the Df'-conjugated mAb was radiolabeled with a radiochemical yield of 60 %, a radiochemical purity of 95 % and an apparent specific activity of 6.1 GBq/µmol. After 7 d, stabilities of 84 % in human serum and of 93 % in saline were observed. cell studies showed strong binding to human tumor-associated MUC1 expressing breast cancer cells. The breast tumor-bearing mice showed an tumor uptake of >50 %ID/g and clearly visible specific enrichment of the radioconjugate PET/MRT. Tumor-associated MUC1 is a very important biomarker for breast cancer next to the traditional markers estrogen receptor (ER), progesterone receptor (PR) and HER/2-neu. The mAb GGSK-1/30 can be used for the diagnosis of over 90% of breast cancers, including triple negative breast cancer based on biopsy staining. Its radioimmunoconjugate represents a promising PET-tracer for breast cancer imaging selectively targeting breast cancer cells.
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http://dx.doi.org/10.7150/ijms.35452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775261PMC
April 2020

Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy.

Cell Rep 2019 10;29(1):135-150.e9

Department of Chemistry, The Scripps Research Institute, Scripps-Florida, Jupiter, FL, USA.

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.
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http://dx.doi.org/10.1016/j.celrep.2019.08.068DOI Listing
October 2019

Using immuno-PET imaging to monitor kinetics of T cell-mediated inflammation and treatment efficiency in a humanized mouse model for GvHD.

Eur J Nucl Med Mol Imaging 2020 05 31;47(5):1314-1325. Epub 2019 Aug 31.

Department of Nuclear Medicine, University Medical Center Mainz, Langenbeckstr. 1, 55131, Mainz, Germany.

Purpose: Hematopoietic stem cell transplantation is the only curative treatment for several hematological malignancies and immune deficiency syndromes. Nevertheless, the development of graft-versus-host disease (GvHD) after transplantation is a severe complication with high morbidity and mortality. The aim of this study was to image human T cells during GvHD development and their migration into GvHD-related organs. By using a radiolabeled anti-human CD3 monoclonal antibody (mAb), we were able to visualize GvHD progression in a humanized mouse model.

Methods: Human peripheral blood mononuclear cells (PBMC) were transferred into immunodeficient mice (initially n = 11 mice/group) to induce GvHD. One group additionally received regulatory T cells (Treg) for prevention of GvHD. T cell migration was visualized by sequential small animal PET/MRI using Zr-labeled anti-human CD3 mAb. Flow cytometry and immunohistochemistry were used to measure T cell frequencies in relevant organs at different time points after engraftment.

Results: Using radiolabeled anti-CD3 mAb, we successfully visualized human T cells in inflamed organs of mice by Zr-anti-CD3-PET/MRI. Upon GvHD progression, we observed increased numbers of CD3 T cells in the liver (22.9% on day 3; 94.2% on day 10) and the spleen (4.4% on day 3; 58.8% on day 10) which correlated with clinical symptoms. The liver showed distinct spot-like lesions representing a strong focal accumulation of T cells. Administration of Treg prior GvHD induction reduced T cell accumulation in the liver from 857 ± 177 CD3 cells/mm to 261 ± 82 CD3 cells/mm and thus prevented GvHD.

Conclusion: Zr-labeled anti-human CD3 mAb can be used as a proof of concept to detect the exact spatio-temporal distribution of GvHD-mediating T cells. In the future, radiolabeled T cell-specific mAb could be employed as a predictive early biomarker during the course of GvHD maybe even before clinical signs of the disease become evident. Furthermore, monitoring T cell migration and proliferation might improve tailored GvHD therapy.
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http://dx.doi.org/10.1007/s00259-019-04507-0DOI Listing
May 2020

Evaluation of the inverse electron demand Diels-Alder reaction in rats using a scandium-44-labelled tetrazine for pretargeted PET imaging.

EJNMMI Res 2019 May 28;9(1):49. Epub 2019 May 28.

Department of Clinical Physiology, Nuclear Medicine & PET, Rigshospitalet, Blegdamsvej 9, 2100, Copenhagen, Denmark.

Background: Pretargeted imaging allows the use of short-lived radionuclides when imaging the accumulation of slow clearing targeting agents such as antibodies. The biotin-(strept)avidin and the bispecific antibody-hapten interactions have been applied in clinical pretargeting studies; unfortunately, these systems led to immunogenic responses in patients. The inverse electron demand Diels-Alder (IEDDA) reaction between a radiolabelled tetrazine (Tz) and a trans-cyclooctene (TCO)-functionalized targeting vector is a promising alternative for clinical pretargeted imaging due to its fast reaction kinetics. This strategy was first applied in nuclear medicine using an In-labelled Tz to image TCO-functionalized antibodies in tumour-bearing mice. Since then, the IEDDA has been used extensively in pretargeted nuclear imaging and radiotherapy; however, these studies have only been performed in mice. Herein, we report the Sc labelling of a Tz and evaluate it in pretargeted imaging in Wistar rats.

Results: Sc was obtained from an in house Ti/Sc generator. A 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized tetrazine was radiolabelled with Sc resulting in radiochemical yields of 85-95%, a radiochemical purity > 99% at an apparent molar activity of 1 GBq/mmol. The Sc-labelled Tz maintained stability in solution for up to 24 h. A TCO-functionalized bisphosphonate, which accumulates in skeletal tissue, was used as a targeting vector to evaluate the Sc-labelled Tz. Biodistribution data of the Sc-labelled Tz showed specific uptake (0.9 ± 0.3% ID/g) in the bones (humerus and femur) of rats pre-treated with the TCO-functionalized bisphosphonate. This uptake was not present in rats not receiving pre-treatment (< 0.03% ID/g).

Conclusions: We have prepared a Sc-labelled Tz and used it in pretargeted PET imaging with rats treated with TCO-functionalized bisphosponates. This allowed for the evaluation of the IEDDA reaction in animals larger than a typical mouse. Non-target accumulation was low, and there was a 30-fold higher bone uptake in the pre-treated rats compared to the non-treated controls. Given its convenient half-life and the ability to perform positron emission tomography with a previously studied DOTA-functionalized Tz, scandium-44 (t = 3.97 h) proved to be a suitable radioisotope for this study.
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http://dx.doi.org/10.1186/s13550-019-0520-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538705PMC
May 2019

Ga[Ga]-, In[In]-oxine: a novel strategy of radiolabeling of HPMA-based micelles.

Am J Nucl Med Mol Imaging 2019 15;9(1):67-83. Epub 2019 Feb 15.

Institute of Nuclear Chemistry, Johannes Gutenberg-University Fritz-Straßmann-Weg 2, Mainz 55128, Germany.

Polymeric micelles are of increasing interest as drug delivery vehicles since they can accumulate in tumor tissue through EPR effect and deliver their hydrophobic cargo. The pharmacology can be visualized and quantified noninvasively by molecular imaging techniques. Here, a novel, fast and efficient technique for radiolabeling various HPMA-LMA based micellar aggregates with hydrophobic oxine-complexes of the trivalent radiometals Ga and In was investigated. The radiometal-oxine complexes resemble the hydrophobic drug In[In]-oxine considered for the diagnosis of infection and inflammation. Promising stability lead to evaluation in healthy mice in terms of quantitative organ distribution. The results show that while the hydrophobic radiometal-oxine complexes were safely encapsulated in aqueous saline, they left the polymeric micelles slowly in contact with blood serum and more rapidly . Due to the similarity between the radiometal complexes and hydrophobic drugs transported in the polymeric micelles this has significant implications for further strategies on transport mechanisms of hydrophobically encapsulated drugs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420711PMC
February 2019

Evaluation of Radiolabeled Girentuximab In Vitro and In Vivo.

Pharmaceuticals (Basel) 2018 Nov 28;11(4). Epub 2018 Nov 28.

Clinic for Nuclear Medicine, University Medical Center Mainz, 55131 Mainz, Germany.

Girentuximab (cG250) targets carbonic anhydrase IX (CAIX), a protein which is expressed on the surface of most renal cancer cells (RCCs). cG250 labeled with Lu has been used in clinical trials for radioimmunotherapy (RIT) of RCCs. In this work, an extensive characterization of the immunoconjugates allowed optimization of the labeling conditions with Lu while maintaining immunoreactivity of cG250, which was then investigated in in vitro and in vivo experiments. cG250 was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA(SCN)) by using incubation times between 30 and 90 min and characterized by mass spectrometry. Immunoconjugates with five to ten DOTA(SCN) molecules per cG250 molecule were obtained. Conjugates with ratios less than six DOTA(SCN)/cG250 had higher in vitro antigen affinity, both pre- and postlabeling with Lu. Radiochemical stability increased, in the presence of sodium ascorbate, which prevents radiolysis. The immunoreactivity of the radiolabeled cG250 tested by specific binding to SK-RC-52 cells decreased when the DOTA content per conjugate increased. The in vivo tumor uptake was < 10% ID/g and independent of the total amount of protein in the range between 5 and 100 µg cG250 per animal. Low tumor uptake was found to be due to significant necrotic areas and heterogeneous CAIX expression. In addition, low vascularity indicated relatively poor accessibility of the CAIX target.
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http://dx.doi.org/10.3390/ph11040132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316122PMC
November 2018

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages.

Nat Immunol 2018 12 5;19(12):1319-1329. Epub 2018 Nov 5.

Institute for Immunology, University Medical Center, Johannes Gutenberg University Mainz, Mainz, Germany.

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.
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http://dx.doi.org/10.1038/s41590-018-0226-8DOI Listing
December 2018

In vivo imaging of the immune response upon systemic RNA cancer vaccination by FDG-PET.

EJNMMI Res 2018 Aug 15;8(1):80. Epub 2018 Aug 15.

Department of Nuclear Medicine, University Medical Center Mainz of Johannes Gutenberg University Mainz, Mainz, Germany.

Background: [F]Fluoro-2-deoxy-2-D-glucose positron emission tomography (FDG-PET) is commonly used in the clinic for diagnosis of cancer and for follow-up of therapy outcome. Additional to the well-established value in tumor imaging, it bears potential to depict immune processes in modern immunotherapies. T cells enhance their glucose consumption upon activation and are crucial effectors for the success of such novel therapies. In this study, we analyzed the T cell immunity in spleen after antigen-specific stimulation of T cells via highly innovative RNA-based vaccines using FDG-PET/MRI. For this purpose, we employed systemic administration of RNA-lipoplexes encoding the endogenous antigen of Moloney murine leukemia virus (gp70) which have been previously shown to induce potent innate as well as adaptive immune mechanisms for cancer immunotherapy. Feasibility of clinical imaging of increased splenic FDG uptake was demonstrated in a melanoma patient participating in a clinical phase 1 trial of a tetravalent RNA-lipoplex cancer vaccine.

Results: We observed exclusive increase of glucose uptake in spleen compared to other organs thanks to liposome-mediated RNA targeting to this immune-relevant organ. In vivo and ex vivo FDG uptake analysis in the spleen of vaccinated mice correlated well with antigen-specific T cell activation. Moreover, the use of an irrelevant (antigen non-specific) RNA also resulted in enhanced FDG uptake early after vaccination through the activation of several other splenic cell populations. The glucose uptake was also dependent on the dose of RNA administered in line with the activation and frequencies of proliferating antigen-specific T cells as well as the general activation pattern of splenic cell populations.

Conclusions: Our preclinical results show rapid and transient vaccination-induced increase of FDG uptake within the spleen reflecting immune activation preceding T cell proliferation. FDG-PET/CT in patients is also capable to image this immune activation resulting in a new potential application of FDG-PET/CT to image immune processes in new immunological therapies.
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http://dx.doi.org/10.1186/s13550-018-0435-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093825PMC
August 2018

Evaluation of [Ac]Ac-DOTA for α-Therapy of Bone Metastases.

Curr Radiopharm 2018 ;11(3):223-230

Institute of Nuclear Chemistry, Johannes Gutenberg University, Mainz, Germany.

Background: Conjugates of bisphosphonates with macrocyclic chelators possess high potential in bone targeted radionuclide imaging and therapy. DOTAZOL, zoledronic acid conjugated to DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), demonstrated promising results in vivo in small animals as well as in first patient applications using 68Ga for diagnosis via PET and the lowenergy β-emitter 177Lu for therapy of painful bone metastases. In consideration of the fact that targeted α-therapy probably offers various advantages over the use of β--emitters, the 225Ac-labelled derivative [225Ac]Ac-DOTAZOL was synthesized and evaluated in vivo. Here, we report on radiolabelling and biodistribution of [225Ac]Ac-DOTAZOL in healthy Wistar rats.

Methods: DOTAZOL was labelled with 225Ac and injected without further purification into the tail vein with activities of 404 ± 47 kBq per animal. Ex vivo biodistribution studies were performed in healthy Wistar rats at 1 hour, 24 hours, 5 days and 10 days post injection. The accumulation of [225Ac]Ac- DOTAZOL on healthy bone and soft tissue organs was determined in terms of SUV. The results were compared to those of other radiolabelled bisphosphonates such as [68Ga]Ga-DOTAZOL and [177Lu]Lu- DOTAZOL. A group of 7 animals was observed over a period of 3 month after application of 394 kBq ± 10 kBq of [225Ac]Ac-DOTAZOL for signs of toxicity. After 3 months, kidneys were microscopically analysed for signs of chronic kidney damage.

Results: Radiolabelling of DOTAZOL with 225Ac at 98 °C provided radiochemical yields ≥98 % within 30 minutes. [225Ac]Ac-DOTAZOL showed high femur uptake (SUVfemur = 4.99 ± 0.97, 10 d p.i.), which was comparable to that of other Me(III)-DOTAZOL derivatives. Ratios between bone uptake and blood pool activity reached levels of 5, 940, 2181 and 2409 at 1 hour, 24 hours, 5 days and 10 days post injection. During the observation period of the first two month no toxicity was observed clinically. Histopathology of kidneys after 3 month revealed significant tubular damage in most of the animals.

Conclusion: [225Ac]Ac-DOTAZOL repeats the well-known pharmacology of DOTAZOL derivatives in preclinical evaluations. It thus may be considered for translational application together with strategies to reduce renal toxicity.
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http://dx.doi.org/10.2174/1874471011666180604083911DOI Listing
January 2019

Comparison of Linear and Hyperbranched Polyether Lipids for Liposome Shielding by F-Radiolabeling and Positron Emission Tomography.

Biomacromolecules 2018 07 27;19(7):2506-2516. Epub 2018 Apr 27.

Institute of Nuclear Chemistry , Johannes Gutenberg University , Fritz-Strassmann-Weg 2 , D-55128 Mainz , Germany.

Multifunctional and highly biocompatible polyether structures play a key role in shielding liposomes from degradation in the bloodstream, providing also multiple functional groups for further attachment of targeting moieties. In this work hyperbranched polyglycerol ( hbPG) bearing lipids with long alkyl chain anchor are evaluated with respect to steric stabilization of liposomes. The branched polyether lipids possess a hydrophobic bis(hexadecyl)glycerol membrane anchor for the liposomal membrane. hbPG was chosen as a multifunctional alternative to PEG, enabling the eventual linkage of multiple targeting vectors. Different hbPG lipids ( M = 2900 and 5200 g mol) were examined. A linear bis(hexadecyl)glycerol-PEG lipid ( M = 3000 g mol) was investigated as well, comparing hbPG and PEG with respect to shielding properties. Radiolabeling of the polymers was carried out using 1-azido-2-(2-(2-[F]fluoroethoxy)ethoxy)ethane ([F]F-TEG-N) via copper-catalyzed alkyne-azide cycloaddition with excellent radiochemical yields exceeding 95%. Liposomes were prepared by the thin-film hydration method followed by repeated extrusion. Use of a custom automatic extrusion device gave access to reproducible sizes of the liposomes (hydrodynamic radius of 60-94 nm). The in vivo fate of the bis(hexadecyl)glycerol polyethers and their corresponding assembled liposome structures were evaluated via noninvasive small animal positron emission tomography (PET) imaging and biodistribution studies (1 h after injection and 4 h after injection) in mice. Whereas the main uptake of the nonliposomal polyether lipids was observed in the kidneys and in the bladder after 1 h due to rapid renal clearance, in contrast, the corresponding liposomes showed uptake in the blood pool as well as in organs with good blood supply, that is, heart and lung over the whole observation period of 4 h. The in vivo behavior of all three liposomal formulations was comparable, albeit with remarkable differences in splenic uptake. Overall, liposomes shielded by the branched polyglycerol lipids show a favorable biodistribution with greatly prolonged blood circulation times, rendering them promising novel nanovesicles for drug transport and targeting.
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http://dx.doi.org/10.1021/acs.biomac.8b00115DOI Listing
July 2018

Comparison Study of Two Differently Clicked F-Folates-Lipophilicity Plays a Key Role.

Pharmaceuticals (Basel) 2018 Mar 17;11(1). Epub 2018 Mar 17.

Hannover Medical School, Department of Nuclear Medicine, Radiopharmaceutical Chemistry, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

Within the last decade, several folate-based radiopharmaceuticals for Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) have been evaluated; however, there is still a lack of suitable F-folates for clinical PET imaging. Herein, we report the synthesis and evaluation of two novel F-folates employing strain-promoted and copper-catalyzed click chemistry. Furthermore, the influence of both click-methods on lipophilicity and pharmacokinetics of the F-folates was investigated. F-Ala-folate and F-DBCO-folate were both stable in human serum albumin. In vitro studies proved their high affinity to the folate receptor (FR). The lipophilic character of the strain-promoted clicked F-DBCO-folate (logD = 0.6) contributed to a higher non-specific binding in cell internalization studies. In the following in vivo PET imaging studies, FR-positive tumors could not be visualized in a maximum intensity projection images. Compared with F-DBCO-folate, F-Ala-folate (logD = -1.4), synthesized by the copper-catalyzed click reaction, exhibited reduced lipophilicity, and as a result an improved in vivo performance and a clear-cut visualization of FR-positive tumors. In view of high radiochemical yield, radiochemical purity and favorable pharmacokinetics, F-Ala-folate is expected to be a promising candidate for FR-PET imaging.
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http://dx.doi.org/10.3390/ph11010030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874726PMC
March 2018

Labeling of DOTA-conjugated HPMA-based polymers with trivalent metallic radionuclides for molecular imaging.

EJNMMI Res 2018 Feb 27;8(1):16. Epub 2018 Feb 27.

Institute of Nuclear Chemistry, Johannes Gutenberg University Mainz, Mainz, Germany.

Background: In this work, the in vitro and in vivo stabilities and the pharmacology of HPMA-made homopolymers were studied by means of radiometal-labeled derivatives. Aiming to identify the fewer amount and the optimal DOTA-linker structure that provides quantitative labeling yields, diverse DOTA-linker systems were conjugated in different amounts to HPMA homopolymers to coordinate trivalent radiometals Me(III)* = gallium-68, scandium-44, and lutetium-177.

Results: Short linkers and as low as 1.6% DOTA were enough to obtain labeling yields > 90%. Alkoxy linkers generally exhibited lower labeling yields than alkane analogues despite of similar chain length and DOTA incorporation rate. High stability of the radiolabel in all examined solutions was observed for all conjugates. Labeling with scandium-44 allowed for in vivo PET imaging and ex vivo measurements of organ distribution for up to 24 h.

Conclusions: This study confirms the principle applicability of DOTA-HPMA conjugates for labeling with different trivalent metallic radionuclides allowing for diagnosis and therapy.
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http://dx.doi.org/10.1186/s13550-018-0372-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829281PMC
February 2018

Highly Loaded Semipermeable Nanocapsules for Magnetic Resonance Imaging.

Macromol Biosci 2018 04 2;18(4):e1700387. Epub 2018 Feb 2.

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128, Mainz, Germany.

Magnetic resonance imaging has become an essential tool in medicine for the investigation of physiological processes. The key issues related to contrast agents, i.e., substances that are injected in the body for imaging, are the efficient enhancement of contrast, their low toxicity, and their defined biodistribution. Polyurea nanocapsules containing the gadolinium complex Gadobutrol as a contrast agent in high local concentration and high relaxivity up to 40 s mmol L are described. A high concentration of the contrast agent inside the nanocapsules can be ensured by increasing the crystallinity in the shell of the nanocapsules. Nanocapsules from aliphatic polyurea are found to display higher crystallinity and higher relaxivity at an initial Gadobutrol concentration of 0.1 m than aromatic polyurea nanocapsules. The nanocapsules and the contrast agent are clearly identified in cells. After injection, the nanocarriers containing the contrast agent are mostly found in the liver and in the spleen, which allow for a significant contrast enhancement in magnetic resonance imaging.
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http://dx.doi.org/10.1002/mabi.201700387DOI Listing
April 2018

Toll like receptor mediated immune stimulation can be visualized in vivo by [F]FDG-PET.

Nucl Med Biol 2016 Nov 14;43(11):651-660. Epub 2016 Jul 14.

Department of Nuclear Medicine, University Medical Center Mainz, Germany.

Introduction: High uptake of [F]-2-fluorodeoxyglucose ([F]FDG) by inflammatory cells is a frequent cause of false positive results in [F]FDG-positron-emission tomography (PET) for cancer diagnostics. Similar to cancer cells, immune cells undergo significant increases in glucose utilization following activation, e.g., in infectious diseases or after vaccination during cancer therapy. The aim of this study was to quantify certain immune effects in vitro and in vivo by [F]FDG-PET after stimulation with TLR ligands and specific antibodies.

Methods: In vivo [F]FDG-PET/magnetic resonance imaging (MRI) and biodistribution was performed with C57BL/6 mice immunized with CpG or LPS. Cellular [F]FDG-uptake assays were performed with B cells and T cells or with whole spleen cells after stimulation with CpG, LPS and anti-CD3/CD28. In vitro and in vivo activation of B and T cells was examined by concomitant FACS analysis to correlate immune cell activation with the strength of [F]FDG accumulation.

Results: We could show that TLR mediated activation of B cells increases [F]FDG uptake, and that B cells show faster kinetics and greater effect than T cells stimulated by the CD3/CD28 pathway. In the whole spleen cell population the [F]FDG signal was triggered mainly by the activation of B cells, corresponding closely to expression of typical stimulation markers. This finding could also been seen in vivo in [F]FDG-PET/MRI, where the spleen was clearly visible after TLR stimulation and B cells showed upregulation of CD80 and CD86.

Conclusion: In vivo TLR stimulation can be visualized by increased [F]FDG uptake in lymphoid organs. The signal generated in the spleen after immunization might be mainly attributed to the activation of B cells within.

Advances In Knowledge And Implications For Patient Care: Knowledge of the composition of cells that take up [F]FDG during vaccination or in response to therapy may improve successful treatment of cancer patients in the future.
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http://dx.doi.org/10.1016/j.nucmedbio.2016.07.004DOI Listing
November 2016

Harnessing the potential of noninvasive in vivo preclinical imaging of the immune system: challenges and prospects.

Nanomedicine (Lond) 2016 Oct 15;11(20):2711-2722. Epub 2016 Sep 15.

Department of Nuclear Medicine, University Medical Center Mainz, Mainz, Germany.

Preclinical imaging has become a powerful method for investigation of in vivo processes such as pharmacokinetics of therapeutic substances and visualization of physiologic and pathophysiological mechanisms. These are important aspects to understand diseases and develop strategies to modify their progression with pharmacologic interventions. One promising intervention is the application of specifically tailored nanoscale particles that modulate the immune system to generate a tumor targeting immune response. In this complex interaction between immunomodulatory therapies, the immune system and malignant disease, imaging methods are expected to play a key role on the way to generate new therapeutic strategies. Here, we summarize examples which demonstrate the current potential of imaging methods and develop a perspective on the future value of preclinical imaging of the immune system.
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http://dx.doi.org/10.2217/nnm-2016-0187DOI Listing
October 2016

Fate of linear and branched polyether-lipids in vivo in comparison to their liposomal formulations by 18F-radiolabeling and positron emission tomography.

Biomacromolecules 2015 Mar 24;16(3):842-51. Epub 2015 Feb 24.

Institute of Nuclear Chemistry and §Institute of Organic Chemistry, Johannes Gutenberg University , Mainz, Germany.

In this study, linear poly(ethylene glycol) (PEG) and novel linear-hyperbranched, amphiphilic polyglycerol (hbPG) polymers with cholesterol (Ch) as a lipid anchor moiety were radiolabeled with fluorine-18 via copper-catalyzed click chemistry. In vivo investigations via positron emission tomography (PET) and ex vivo biodistribution in mice were conducted. A systematic comparison to the liposomal formulations with and without the polymers with respect to their initial pharmacokinetic properties during the first hour was carried out, revealing remarkable differences. Additionally, cholesterol was directly labeled with fluorine-18 and examined likewise. Both polymers, Ch-PEG27-CH2-triazole-TEG-(18)F and Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F (TEG: triethylene glycol), showed rapid renal excretion, whereas the (18)F-cholesten displayed retention in lung, liver, and spleen. Liposomes containing Ch-PEG27-CH2-triazole-TEG-(18)F revealed a hydrodynamic radius of 46 nm, liposomal Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F showed a radius of 84 nm and conventional liposomes with (18)F-cholesten 204 nm, respectively. The results revealed fast uptake of the conventional liposomes by liver, spleen, and lung. Most importantly, the novel hbPG-polymer stabilized liposomes showed similar behavior to the PEG-shielded vesicles. Thus, an advantage of multifunctionality is achieved with retained pharmacokinetic properties. The approach expands the scope of polymer tracking in vivo and liposome tracing in mice via PET.
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http://dx.doi.org/10.1021/bm5017332DOI Listing
March 2015

Dendritic cell motility and T cell activation requires regulation of Rho-cofilin signaling by the Rho-GTPase activating protein myosin IXb.

J Immunol 2014 Apr 19;192(8):3559-68. Epub 2014 Mar 19.

Institute of Molecular Cell Biology, Westfalian Wilhelms University-Münster, Münster 48149, Germany.

Directed migration of stimulated dendritic cells (DCs) to secondary lymphoid organs and their interaction with Ag-specific T cells is a prerequisite for the induction of primary immune responses. In this article, we show that murine DCs that lack myosin IXB (Myo9b), a motorized negative regulator of RhoA signaling, exhibit increased Rho signaling activity and downstream acto-myosin contractility, and inactivation of the Rho target protein cofilin, an actin-depolymerizing factor. On a functional level, Myo9b(-/-) DCs showed impaired directed migratory activity both in vitro and in vivo. Moreover, despite unaltered Ag presentation and costimulatory capabilities, Myo9b(-/-) DCs were poor T cell stimulators in vitro in a three-dimensional collagen matrix and in vivo, associated with altered DC-T cell contact dynamics and T cell polarization. Accordingly, Myo9b(-/-) mice showed an attenuated ear-swelling response in a model of contact hypersensitivity. The impaired migratory and T cell stimulatory capacity of Myo9b(-/-) DCs was restored in large part by pharmacological activation of cofilin. Taken together, these results identify Myo9b as a negative key regulator of the Rho/RhoA effector Rho-kinase [Rho-associated coiled-coil-forming kinase (ROCK)]/LIM domain kinase signaling pathway in DCs, which controls cofilin inactivation and myosin II activation and, therefore may control, in part, the induction of adaptive immune responses.
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http://dx.doi.org/10.4049/jimmunol.1300695DOI Listing
April 2014

A trifunctional dextran-based nanovaccine targets and activates murine dendritic cells, and induces potent cellular and humoral immune responses in vivo.

PLoS One 2013 5;8(12):e80904. Epub 2013 Dec 5.

Department of Dermatology, University Medical Center Mainz, Mainz, Germany.

Dendritic cells (DCs) constitute an attractive target for specific delivery of nanovaccines for immunotherapeutic applications. Here we tested nano-sized dextran (DEX) particles to serve as a DC-addressing nanocarrier platform. Non-functionalized DEX particles had no immunomodulatory effect on bone marrow (BM)-derived murine DCs in vitro. However, when adsorbed with ovalbumine (OVA), DEX particles were efficiently engulfed by BM-DCs in a mannose receptor-dependent manner. A DEX-based nanovaccine containing OVA and lipopolysaccharide (LPS) as a DC stimulus induced strong OVA peptide-specific CD4(+) and CD8(+) T cell proliferation both in vitro and upon systemic application in mice, as well as a robust OVA-specific humoral immune response (IgG1>IgG2a) in vivo. Accordingly, this nanovaccine also raised both a more pronounced delayed-type hypersensitivity response and a stronger induction of cytotoxic CD8(+) T cells than obtained upon administration of OVA and LPS in soluble form. Therefore, DEX-based nanoparticles constitute a potent, versatile and easy to prepare nanovaccine platform for immunotherapeutic approaches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080904PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855172PMC
March 2015

The chemotherapeutic agent topotecan differentially modulates the phenotype and function of dendritic cells.

Cancer Immunol Immunother 2013 Aug 11;62(8):1315-26. Epub 2013 May 11.

Department of Dermatology, Clinical Research Unit Allergology, Medical Center of the Johannes Gutenberg-University, Obere Zahlbacher-Str. 63, 55131, Mainz, Germany.

The camptothecin analogue topotecan (TPT) induces tumor cell apoptosis due to interference with topoisomerase I and is clinically used as a second-line chemotherapeutic in the treatment for metastasizing ovarian and small cell lung carcinoma. Based on the more recent finding of TPT-mediated inhibition of the transcription factor hypoxia-induced factor-1α, a hallmark of solid tumors, TPT, is currently tested in clinical trials for its suitability as a first-line chemotherapeutic for the treatment for various types of tumors. Due to the gained clinical interest in TPT and in light of its modulatory effect on signaling pathways, which are also of importance for immune cell functions, we asked for potential effects of TPT on dendritic cells (DCs), the main antigen-presenting cell population of the immune system. Here, we show that TPT at a therapeutically relevant dose partially activated monocyte-derived DCs as reflected by enhanced migratory activity, elevated expression of HLA-DR and costimulatory/maturation markers, and accordingly an increased allogenic CD4(+) T cell stimulation. In marked contrast, TPT prevented full maturation of DCs stimulated with a cocktail of proinflammatory mediators, accompanied by somewhat lower upregulation of NF-κB factors p65 and RelB.
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http://dx.doi.org/10.1007/s00262-013-1431-9DOI Listing
August 2013