The Danish Chinese Center for Proteases and Cancer

Chinese, Denmark

The Danish Chinese Center for Proteases and Cancer

Chinese, Denmark
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Persson M.,The Danish Chinese Center for Proteases and Cancer | Persson M.,Rigshospitalet | Persson M.,Copenhagen University | Persson M.,The BRIC | And 7 more authors.
Nuclear Medicine and Biology | Year: 2012

The proposition of uPAR as a potential target in cancer therapy is advanced by its predominant expression at the invasive front of colorectal cancer (CRC) and its value as prognostic biomarker for poor survival in this disease. In this study, we provide the first in vivo proof-of-concept for a theranostic approach as treatment modality in a human xenograft colorectal cancer model. Methods: A DOTA-conjugated 9-mer high affinity uPAR binding peptide (DOTA-AE105) was radiolabeled with 64Cu and 177Lu, for PET imaging and targeted radionuclide therapy study, respectively. Human uPAR-positive CRC HT-29 cells were inoculated in Nude mice and treated with 177Lu-DOTA-AE105 once a visible tumor had formed. To evaluate the true effect of the targeted radiotherapy, two controls groups were included in this study, one receiving a 177Lu-labeled non-binding control peptide and one receiving vehicle. All animals were treated day 0 and 7. A parallel 18F-FLT PET/CT study was performed on day 0, 1, 3 and 6. Dosimetry calculations were based on a biodistribution study, where organs and tissue of interest were collected 0.5, 1.0, 2.0, 4.0 and 24h post injection of 177Lu-DOTA-AE105. Toxicity was assessed by recording mouse weight and by H&E staining of kidneys in each treatment group. Results: uPAR-positive HT-29 xenograft was clearly visualized by PET/CT imaging using 64Cu-DOTA-AE105. Subsequently, these xenograft transplants were locally irradiated using 177Lu-DOTA-AE105, where a significant effect on tumor size and the number of uPAR-positive cells in the tumor was found (p<0.05). Evaluations of biodistribution and dosimetry revealed highest accumulation of radioactivity in kidneys and tumor tissue. 18F-FLT PET/CT imaging study revealed a significant correlation between 18F-FLT tumor uptake and efficacy of the radionuclide therapy. A histological examination of the kidneys from one animal in each treatment group did not reveal any gross abnormalities and the general performance of all treated animals also showed no indications of radioactivity-induced toxicity. Conclusion: These findings document for the first time the in vivo efficacy of an uPAR-targeted radionuclide therapeutic intervention on both tumor size and its content of uPAR expressing cells thus setting the stage for future translation into clinical use. © 2012 Elsevier Inc.


Persson M.,The Danish Chinese Center for Proteases and Cancer | Persson M.,The BRIC | Persson M.,Copenhagen University | Persson M.,Rigshospitalet | And 11 more authors.
Theranostics | Year: 2013

The correlation between uPAR expression, cancer cell invasion and metastases is now well-established and has prompted the development of a number of uPAR PET imaging agents, which could potentially identify cancer patients with invasive and metastatic lesions. In the present study, we synthesized and characterized two new cross-bridged 64Cu-labeled peptide conjugates for PET imaging of uPAR and performed a head-to-head comparison with the corresponding and more conventionally used DOTA conjugate. Based on in-source laser-induced reduction of chelated Cu(II) to Cu(I), we now demonstrate the following ranking with respect to the chemical inertness of their complexed Cu ions: DOTA-AE105 « CB-TE2A-AE105 < CB-TE2A-PA-AE105, which is correlated to their corresponding demetallation rate. No penalty in the uPAR receptor binding affinity of the targeting peptide was encountered by conjugation to either of the macrobicyclic chelators (IC50 ~ 5-10 nM) and high yields and radiochemical purities (>95%) were achieved in all cases by incubation at 95°C. In vivo, they display identical tumor uptake after 1 h, but differ significantly after 22 hrs, where the DOTA-AE105 uptake remains surprisingly high. Importantly, the more stable of the new uPAR PET tracers, 64Cu-CB-TE2A-PA-AE105, exhibits a significantly reduced liver uptake compared to 64Cu-DOTA-AE105 as well as 64Cu-CB-TE2A-AE105, (p<0.0001), emphasizing that our new in vitro stability measurements by mass spectrometry predicts in vivo stability in mice. Specificity of the best performing ligand, 64Cu-CB-TE2A-PA-AE105 was finally confirmed in vivo using a non-binding 64Cu-labeled peptide as control (64Cu-CB-TE2A-PA-AE105mut). This control PET-tracer revealed significantly reduced tumor uptake (p<0.0001), but identical hepatic uptake compared to its active counterpart (64Cu-CB-TE2A-PA-AE105) after 1h. In conclusion, our new approach using in-source laser-induced reduction of Cu(II)-chelated PET-ligands provides useful information, which are predictive for the tracer stability in vivo in mice. Furthermore, the increased stability of our new macrobicyclic 64Cu-CB-TE2A-PA-AE105 PET ligand is paralleled by an excellent imaging contrast during non-invasive PET scanning of uPAR expression in preclinical mouse cancer models. The translational promises displayed by this PET-tracer for future clinical cancer patient management remains, however, to be investigated. © Ivyspring International Publisher.


Persson M.,The Danish Chinese Center for Proteases and Cancer | Persson M.,Rigshospitalet | Persson M.,Copenhagen University | Madsen J.,Rigshospitalet | And 6 more authors.
Nuclear Medicine and Biology | Year: 2012

Introduction: The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH 2 labeled with 64Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce 68Ga-DOTA-AE105-NH 2 and 68Ga-NODAGA-AE105-NH 2 and evaluate their imaging properties using small-animal PET. Methods: Synthesis of DOTA-AE105-NH 2 and NODAGA-AE105-NH 2 was based on solid-phase peptide synthesis protocols using the Fmoc strategy. 68GaCl 3 was eluted from a 68Ge/ 68Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. Results: In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH 2 and NODAGA-AE105-NH 2 with 68Ga was done at 95°C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH 2 was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for 68Ga-NODAGA-AE105-NH 2 and 68Ga-DOTA-AE105-NH 2, respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for 68Ga-NODAGA-AE105-NH 2 60 min postinjection. Conclusions: The use of 68Ga-DOTA-AE105-NH 2 and 68Ga-NODAGA-AE105-NH 2 as the first gallium-68 labeled uPAR radiotracers for noninvasive PET imaging is reported, which combine versatility with good imaging properties. These new tracers thus constitute an interesting alternative to the 64Cu-labeled version ( 64Cu-DOTA-AE105 and 64Cu-DOTA-AE105-NH 2) for detecting uPAR expression in tumor tissue. In our hands, the fractionated elution approach was superior for labeling of peptides, and 68Ga-NODAGA-AE105-NH 2 is the favored tracer as it provides the highest tumor-to-background ratio. © 2012 Elsevier Inc.


Persson M.,The Danish Chinese Center for Proteases and Cancer | Persson M.,Rigshospitalet | Persson M.,Copenhagen University | El Ali H.H.,Rigshospitalet | And 10 more authors.
Nuclear Medicine and Biology | Year: 2014

64Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of 64Cu-DOTA-AE105. Methods: Five mice received iv tail injection of 64Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung, liver, kidney, spleen, intestine, muscle, bone and bladder. The activity concentrations in the mentioned organs [%ID/g] were used for the dosimetry calculation. The %ID/g of each organ at 1, 4.5 and 22h was scaled to human value based on a difference between organ and body weights. The scaled values were then exported to OLINDA software for computation of the human absorbed doses. The residence times as well as effective dose equivalent for male and female could be obtained for each organ. To validate this approach, of human projection using mouse data, five mice received iv tail injection of another 64Cu-DOTA peptide-based tracer, 64Cu-DOTA-TATE, and underwent same procedure as just described. The human dosimetry estimates were then compared with observed human dosimetry estimate recently found in a first-in-man study using 64Cu-DOTA-TATE. Results: Human estimates of 64Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918mSv/MBq) followed by the liver (0.0815mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02-0.04mSv/MBq. The mean effective whole-body dose of 64Cu-DOTA-AE105 was estimated to be 0.0317mSv/MBq. Relatively good correlation between human predicted and observed dosimetry estimates for 64Cu-DOTA-TATE was found. Importantly, the effective whole body dose was predicted with very high precision (predicted value: 0.0252mSv/Mbq, Observed value: 0.0315mSv/MBq) thus validating our approach for human dosimetry estimation. Conclusion: Favorable dosimetry estimates together with previously reported uPAR PET data fully support human testing of 64Cu-DOTA-AE105. © 2014 Elsevier Inc.


Persson M.,The Danish Chinese Center for Proteases and Cancer | Persson M.,Rigshospitalet | Persson M.,Copenhagen University | Persson M.,Stanford University | And 6 more authors.
Nuclear Medicine and Biology | Year: 2013

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed in human prostate cancer and uPAR has been found to be associated with metastatic disease and poor prognosis. AE105 is a small linear peptide with high binding affinity to uPAR. We synthesized an N-terminal NOTA-conjugated version (NOTA-AE105) for development of the first 18F-labeled uPAR positron-emission-tomography PET ligand using the Al18F radiolabeling method. In this study, the potential of 18F-AlF-NOTA-AE105 to specifically target uPAR-positive prostate tumors was investigated. Methods: NOTA-conjugated AE105 was synthesized and radiolabeled with 18F-AlF according to a recently published optimized protocol. The labeled product was purified by reverse phase high performance liquid chromatography RP-HPLC. The tumor targeting properties were evaluated in mice with subcutaneously inoculated PC-3 xenografts using small animal PET and ex vivo biodistribution studies. uPAR-binding specificity was studied by coinjection of an excess of a uPAR antagonist peptide AE105 analogue (AE152). Results: NOTA-AE105 was labeled with 18F-AlF in high radiochemical purity (>92%) and yield (92.7%) and resulted in a specific activity of greater than 20GBq/μmol. A high and specific tumor uptake was found. At 1h post injection, the uptake of 18F-AlF-NOTA-AE105 in PC-3 tumors was 4.22±0.13%ID/g. uPAR-binding specificity was demonstrated by a reduced uptake of 18F-AlF-NOTA-AE105 after coinjection of a blocking dose of uPAR antagonist at all three time points investigated. Good tumor-to-background ratio was observed with small animal PET and confirmed in the biodistribution analysis. Ex vivo uPAR expression analysis on extracted tumors confirmed human uPAR expression that correlated close with tumor uptake of 18F-AlF-NOTA-AE105. Conclusion: The first 18F-labeled uPAR PET ligand, 18F-AlF-NOTA-AE105, has successfully been prepared and effectively visualized noninvasively uPAR positive prostate cancer. The favorable in vivo kinetics and easy production method facilitate its future clinical translation for identification of prostate cancer patients with an invasive phenotype and poor prognosis. © 2013 Elsevier Inc.


PubMed | The Danish Chinese Center for Proteases and Cancer
Type: Journal Article | Journal: Molecular pharmaceutics | Year: 2014

The urokinase-type plasminogen activator receptor (uPAR) is implicated in cancer invasion and metastatic development in prostate cancer and provides therefore an attractive molecular target for both imaging and therapy. In this study, we provide the first in vivo data on an antimetastatic effect of uPAR radionuclide targeted therapy in such lesions and show the potential of uPAR positron emission tomography (PET) imaging for identifying small foci of metastatic cells in a mouse model of disseminating human prostate cancer. Two radiolabeled ligands were generated in high purity and specific activity: a uPAR-targeting probe ((177)Lu-DOTA-AE105) and a nonbinding control ((177)Lu-DOTA-AE105mut). Both uPAR flow cytometry and ELISA confirmed high expression levels of the target uPAR in PC-3M-LUC2.luc cells, and cell binding studies using (177)Lu-DOTA-AE105 resulted in a specific binding with an IC50 value of 100 nM in a competitive binding experiment. In vivo, uPAR targeted radionuclide therapy significantly reduced the number of metastatic lesions in the disseminated metastatic prostate cancer model, when compared to vehicle and nontargeted (177)Lu groups (p < 0.05) using bioluminescence imaging. Moreover, we found a significantly longer metastatic-free survival, with 65% of all mice without any disseminated metastatic lesions present at 65 days after first treatment dose (p = 0.047). In contrast, only 30% of all mice in the combined control groups treated with (177)Lu-DOTA-AE105mut or vehicle were without metastatic lesions. No treatment-induced toxicity was observed during the study as evaluated by observing animal weight and H&E staining of kidney tissue (dose-limiting organ). Finally, uPAR PET imaging using (64)Cu-DOTA-AE105 detected all small, disseminated metastatic foci when compared with bioluminescence imaging in a cohort of animals during the treatment study. In conclusion, uPAR targeted radiotherapy resulted in a significant reduction in the number of metastatic lesions in a human metastatic prostate cancer model. Furthermore, we have provided the first evidence of the potential for identification of small metastatic lesions using uPAR PET imaging in disseminated prostate cancer, illustrating the promising strategy of uPAR theranostics in prostate cancer.


PubMed | The Danish Chinese Center for Proteases and Cancer
Type: Journal Article | Journal: Nuclear medicine and biology | Year: 2012

The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH(2) labeled with (64)Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) and evaluate their imaging properties using small-animal PET.Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide synthesis protocols using the Fmoc strategy. (68)GaCl(3) was eluted from a (68)Ge/(68)Ga generator. The eluate was either concentrated on a cation-exchange column or fractionated and used directly for labeling. For in vitro characterization of both tracers, partition coefficient, buffer and plasma stability, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft.In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated peptides and identical to AE105. Labeling of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) with (68)Ga was done at 95C and room temperature, respectively. The highest radiochemical yield and purity were obtained using fractionated elution, whereas a negative effect of acetone on labeling efficiency for NODAGA-AE105-NH(2) was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min postinjection and 2.0% ID/g and 1.1% ID/g 60 min postinjection for (68)Ga-NODAGA-AE105-NH(2) and (68)Ga-DOTA-AE105-NH(2), respectively. A significantly higher tumor-to-muscle ratio (P<.05) was found for (68)Ga-NODAGA-AE105-NH(2) 60 min postinjection.The use of (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) as the first gallium-68 labeled uPAR radiotracers for noninvasive PET imaging is reported, which combine versatility with good imaging properties. These new tracers thus constitute an interesting alternative to the (64)Cu-labeled version ((64)Cu-DOTA-AE105 and 64Cu-DOTA-AE105-NH(2)) for detecting uPAR expression in tumor tissue. In our hands, the fractionated elution approach was superior for labeling of peptides, and (68)Ga-NODAGA-AE105-NH(2) is the favored tracer as it provides the highest tumor-to-background ratio.


PubMed | The Danish Chinese Center for Proteases and Cancer
Type: Journal Article | Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine | Year: 2012

Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expression levels of uPAR across different human cancer xenograft models in mice and to illustrate the clinical potential of uPAR PET in future settings for individualized therapy.To accomplish our objective, a linear, high-affinity uPAR peptide antagonist, AE105, was conjugated with DOTA and labeled with (64)Cu ((64)Cu-DOTA-AE105). Small-animal PET was performed in 3 human cancer xenograft mice models, expressing different levels of human uPAR, and the tumor uptake was correlated with the uPAR expression level determined by uPAR enzyme-linked immunosorbent assay. The tumor uptake pattern of this tracer was furthermore compared with (18)F-FDG uptake, and finally the correlation between sensitivity toward 5-fluorouracil therapy and uPAR expression level was investigated.The uPAR-targeting PET tracer was produced in high purity and with high specific radioactivity. A significant correlation between tumor uptake of (64)Cu-DOTA-AE105 and uPAR expression was found (R(2) = 0.73; P < 0.0001) across 3 cancer xenografts, thus providing a strong argument for specificity. A significantly different uptake pattern of (64)Cu-DOTA-AE105, compared with that of (18)F-FDG, was observed, thus emphasizing the additional information that can be obtained on tumor biology using (64)Cu-DOTA-AE105 PET. Furthermore, a significant correlation between baseline uPAR expression and sensitivity toward 5-fluorouracil was revealed, thus illustrating the possible potentials of uPAR PET in a clinical setting.Our results clearly demonstrate that the peptide-based PET tracer (64)Cu-DOTA-AE105 enables the noninvasive quantification of uPAR expression in tumors in vivo, thus emphasizing its potential use in a clinical setting to detect invasive cancer foci and for individualized cancer therapy.

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