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Purpose Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. Methods We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. Results Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). Conclusion We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

Contains fulltext : 218875.pdf (Publisher’s version ) (Open Access) Myxofibrosarcoma(MFS) is the most common soft tissue sarcoma(STS) in elderly patients. Surgical resection remains the main treatment modality but tumor borders can be difficult to delineate with conventional clinical methods. Incomplete resections are a common problem and local recurrence remains a clinical issue. A technique that has shown great potential in improving surgical treatment of solid tumors is tumor targeted imaging and image-guided surgery with near-infrared fluorescence. To facilitate this technique, it is essential to identify a biomarker that is highly and homogenously expressed on tumor cells, while being absent on healthy non-malignant tissue. The purpose of this study was to identify suitable molecular targets for tumor-targeted imaging of myxofibrosarcoma. Ten potential molecular targets for tumor targeted imaging were investigated with immunohistochemical analysis in myxofibrosarcoma tissue (n = 34). Results were quantified according to the immunoreactive score(IRS). Moderate expression rates were found for uPAR, PDGFRa and EMA/MUC1. High expression rates of VEGF and TEM1 were seen. Strong expression was most common for TEM1 (88.2%). These results confirms that TEM1 is a suitable target for tumor-targeted imaging of myxofibrosarcoma. Keywords Image-guided surgery; Immunohistochemistry; Molecular imaging; Myxofibrosarcoma; Soft tissue sarcoma; Tumor endothelial marker 1(TEM1), Vascular endothelial growth factor (VEGF).

GLP-1R imaging with radiolabelled exendin has proven to be a powerful tool to quantify beta-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose levels on GLP-1R-mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and beta-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of blood glucose levels restored absolute tracer uptake and GLP-1R expression in beta-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation non-invasively. Our findings have significant implications for clinical practice, indicating that blood glucose levels should be near-normalized for at least three weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis.

The treatment of choice for insulinomas and focal lesions in congenital hyperinsulinism (CHI) is surgery. However, intraoperative detection can be challenging. This challenge could be overcome with intraoperative fluorescence imaging, which provides real-time lesion detection with a high spatial resolution. Here, a novel method for targeted near-infrared (NIR) fluorescence imaging of glucagonlike peptide 1 receptor (GLP-1R)-positive lesions, using the GLP-1 agonist exendin-4 labeled with IRDye 800CW, was examined in vitro and in vivo. Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with GLP-1R. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. In vivo NIR fluorescence imaging of CHL-GLP-1R xenografts was performed. Localization of the tracer in the pancreatic islets of BALB/c nude mice was examined using fluorescence microscopy. Laparoscopic imaging was performed to detect the fluorescent signal of the tracer in the pancreas of mini pigs. Results: Exendin-4-IRDye 800CW binds GLP-1R with a half-maximal inhibitory concentration of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice, and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These data provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1R-positive lesions. Intraoperative lesion delineation using exendin-4-IRDye 800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI.

Glucagon-like peptide 1 receptor (GLP-1R) imaging with radiolabeled exendin has proven to be a powerful tool to quantify β-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose (BG) levels on GLP-1R–mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and β-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of BG levels restored absolute tracer uptake and GLP-1R expression in β-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation noninvasively. Our findings have significant implications for clinical practice, indicating that BG levels should be near-normalized for at least 3 weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis.

Rationale: Prostate cancer (PCa) recurrences after surgery frequently occur. To improve the outcome after surgical resection of the tumor, the theranostic multimodal anti-PSMA targeting agent 111In-DTPA-D2B-IRDye700DX was developed and characterized for both pre- and intra-operative tumor localization and eradication of (residual) tumor tissue by PSMA-targeted photodynamic therapy (tPDT), which is a highly selective cancer treatment based on targeting molecules conjugated to photosensitizers that can induce cell destruction upon exposure to near-infrared (NIR) light. Methods: The anti-PSMA monoclonal antibody D2B was conjugated with IRDye700DX and DTPA and subsequently radiolabeled with 111In. To determine the optimal dose and time point for tPDT, BALB/c nude mice with PSMA-expressing (PSMA+) s.c. LS174T-PSMA xenografts received the conjugate (24-240 µg/mouse) intravenously (8 MBq/mouse) followed by µSPECT/CT, near-infrared fluorescence imaging, and ex vivo biodistribution at 24, 48, 72 and 168 h p.i. Tumor growth of LS174T-PSMA xenografts and overall survival of mice treated with 1-3 times of NIR light irradiation (50, 100, 150 J/cm2) 24 h after injection of 80 µg of DTPA-D2B-IRDye700DX was compared to control conditions. Results: Highest specific tumor uptake was observed at conjugate doses of 80 µg/mouse. Biodistribution revealed no significant difference in tumor uptake in mice at 24, 48, 72 and 168 h p.i. PSMA+ tumors were clearly visualized with both µSPECT/CT and NIR fluorescence imaging. Overall survival in mice treated with 80 µg of DTPA-D2B-IRDye700DX and 1x 150 J/cm2 of NIR light at 24 h p.i. was significantly improved compared to the control group receiving neither conjugate nor NIR light (73 days vs. 16 days, respectively, p=0.0453). Treatment with 3x 150 J/cm2 resulted in significantly prolonged survival compared to treatment with 3x 100 J/cm2 (p = 0.0067) and 3x 50 J/cm2 (p = 0.0338). Principal conclusions: 111In-DTPA-D2B-IRDye700DX can be used for pre- and intra-operative detection of PSMA+ tumors with radionuclide and NIR fluorescence imaging and PSMA-targeted PDT. PSMA-tPDT using this multimodal agent resulted in significant prolongation of survival and shows great potential for treatment of (metastasized) prostate cancer.

The changes in β-cell mass (BCM) during the development and progression of diabetes could potentially be measured by radionuclide imaging using radiolabeled exendin. In this study, we investigated the potential of

Pancreatic islet transplantation is a promising therapy for patients with type 1 diabetes. However, the duration of long-term graft survival is limited due to inflammatory as well as non-inflammatory processes and routine clinical tests are not suitable to monitor islet survival. 111In-exendin-SPECT (single photon emission computed tomography) is a promising method to non-invasively image islets after transplantation and has the potential to help improve the clinical outcome. Whether 111In-exendin-SPECT allows detecting small differences in beta-cell mass (BCM) and measuring the actual volume of islets that were successfully engrafted has yet to be demonstrated. Here, we evaluated the performance of 111In-exendin-SPECT using an intramuscular islet transplantation model in C3H mice. In vivo imaging of animals transplanted with 50, 100, 200, 400 and 800 islets revealed an excellent linear correlation between SPECT quantification of 111In-exendin uptake and insulin-positive area of islet transplants, demonstrating that 111In-exendin-SPECT specifically and accurately measures BCM. The high sensitivity of the method allowed measuring small differences in graft volumes, including grafts that contained less than 50 islets. The presented method is reliable, convenient and holds great potential for non-invasive monitoring of BCM after islet transplantation in humans.

Contains fulltext : 153713.pdf (Publisher’s version ) (Closed access) PURPOSE: Pretargeting with bispecific antibodies and radiolabeled hapten-peptides could be used to specifically target tumors with high target-to-background ratios. TF12 is a trivalent bispecific antibody that consists of two anti-TROP-2 Fab fragments and one anti-HSG (histamine-succinyl-glycine) Fab fragment. The TROP-2 antigen is expressed in many epithelial cancers, including prostate cancer (PC), and therefore, this bispecific antibody can be used for pretargeting of PC. In this study, the potential for pretargeted radioimmunoPET with TF12 and the (68)Ga-labeled di-HSG peptide IMP288 in mice with human PC xenografts was investigated using 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) as a reference. PROCEDURES: The potential of pretargeted immunoPET with TF12 and the (68)Ga-labeled di-HSG hapten-peptide, IMP288, was studied in mice with subcutaneous PC3 tumors using [(18)F]FDG as a reference. Furthermore, the use of this pretargeting system for imaging PC lesions was evaluated in mice with intraperitoneally growing tumors with [(18)F]FDG as a reference. RESULTS: [(68)Ga]lMP288 showed rapid accumulation in the TF12 pretargeted subcutaneous tumor (7.2 +/- 1.1 % ID/g) with low uptake in the kidneys (1.8 +/- 0.5 % ID/g) and high tumor-to-blood ratios (17.4 +/- 11.2) at 1 h p.i. Accumulation of [(18)F]FDG in the s.c. tumors was significantly lower (3.4 +/- 0.9 % ID/g, P = 0.008), with lower tumor-to-blood ratios (3.0 +/- 1.9, P = 0.011). ImmunoPET/CT images clearly visualized both subcutaneous and intraperitoneal tumors as small as 5 mm(3) with low blood levels and kidney uptake as early as 1 h p.i. CONCLUSION: Pretargeted immunoPET with TF12 in combination with a (68)Ga-labeled hapten-peptide is an efficient system for rapid, sensitive, and specific imaging of prostate cancer.

TF12 is a trivalent bispecific antibody that consists of two anti-TROP-2 Fab fragments and one anti-histamine-succinyl-glycine (HSG) Fab fragment. The TROP-2 antigen is found in many epithelial cancers, including prostate cancer (PC), and therefore this bispecific antibody could be suitable for pretargeting in this cancer. In this study, the characteristics and the potential for pretargeted radioimmunoimaging and radioimmunotherapy with TF12 and the radiolabeled di-HSG peptide IMP288 in mice with human PC were investigated.The optimal TF12 protein dose, IMP288 peptide dose, and dose interval for PC targeting were assessed in nude mice with s.c. PC3 xenografts. Immuno-positron emission tomography (PET)/CT was performed using TF12/⁶⁸Ga-IMP288 at optimized conditions. The potential of pretargeted radioimmunotherapy (PRIT) using the TF12 pretargeted ¹⁷⁷Lu-IMP288 was determined.TF12 and ¹¹¹In-IMP288 showed high and fast accumulation in the tumor [20.4 ± 0.6%ID/g at 1 h post-injection (p.i.)] at optimized conditions, despite the internalizing properties of TF12. The potential for PRIT was shown by retention of 50% of the ¹¹¹In-IMP288 in the tumor at 48 h p.i. One cycle of treatment with TF12 and ¹⁷⁷Lu-IMP288 showed significant improvement of survival compared to treatment with ¹⁷⁷Lu-IMP288 alone (90 vs. 67 days, p0.0001) with no renal or hematological toxicity.TROP-2-expressing PC can be pretargeted efficiently with TF12, with very rapid uptake of the radiolabeled hapten-peptide, IMP288, sensitive immuno-PET, and effective therapy.

Radiolabeled exendin is used for non-invasive quantification of beta cells in the islets of Langerhans in vivo. High accumulation of radiolabeled exendin in the islets raised concerns about possible radiation-induced damage to these islets in man. In this work, islet absorbed doses resulting from exendin-imaging were calculated by combining whole organ dosimetry with small scale dosimetry for the islets. Our model contains the tissues with high accumulation of radiolabeled exendin: kidneys, pancreas and islets. As input for the model, data from a clinical study (radiolabeled exendin distribution in the human body) and from a preclinical study with Biobreeding Diabetes Prone (BBDP) rats (islet-to-exocrine uptake ratio, beta cell mass) were used. We simulated 111In-exendin and 68Ga-exendin absorbed doses in patients with differences in gender, islet size, beta cell mass and radiopharmaceutical uptake in the kidneys. In all simulated cases the islet absorbed dose was small, maximum 1.38 mGy for 68Ga and 66.0 mGy for 111In. The two sources mainly contributing to the islet absorbed dose are the kidneys (33–61%) and the islet self-dose (7.5–57%). In conclusion, all islet absorbed doses are low (

Single Photon Emission Computed Tomography (SPECT) has become a promising experimental approach to monitor changes in β-cell mass (BCM) during diabetes progression. SPECT imaging of pancreatic islets is most commonly cross-validated by stereological analysis of histological pancreatic sections after insulin staining. Typically, stereological methods do not accurately determine the total β-cell volume, which is inconvenient when correlating total pancreatic tracer uptake with BCM. Alternative methods are therefore warranted to cross-validate β-cell imaging using radiotracers. In this study, we introduce multimodal SPECT - optical projection tomography (OPT) imaging as an accurate approach to cross-validate radionuclide-based imaging of β-cells. Uptake of a promising radiotracer for β-cell imaging by SPECT, 111In-exendin-3, was measured by ex vivo-SPECT and cross evaluated by 3D quantitative OPT imaging as well as with histology within healthy and alloxan-treated Brown Norway rat pancreata. SPECT signal was in excellent linear correlation with OPT data as compared to histology. While histological determination of islet spatial distribution was challenging, SPECT and OPT revealed similar distribution patterns of 111In-exendin-3 and insulin positive β-cell volumes between different pancreatic lobes, both visually and quantitatively. We propose ex vivo SPECT-OPT multimodal imaging as a highly accurate strategy for validating the performance of β-cell radiotracers.

Item does not contain fulltext RS7 is an internalizing anti-Trop-2 pancarcinoma antibody capable of targeting most epithelial cancers. Because pretargeting strategies could improve the tumor localization of radionuclides, a new anti-Trop-2 x antihapten bispecific antibody for pretargeting, based on humanized RS7, was prepared and evaluated with a radiolabeled hapten-peptide in vitro and in vivo to determine whether its internalization properties would interfere with pretargeting. METHODS: The anti-Trop-2 x antihapten bispecific antibody, TF12, was prepared using the modular dock-and-lock method. TF12 and humanized RS7 binding was assessed by cell binding assays and fluorescence-activated cell sorting analysis in a variety of human carcinoma cell lines. The internalization of TF12 was evaluated in vitro using a fluorescent TF12 conjugate or hapten-peptide and (111)In-labeled TF12 and RS7. The biodistribution of TF12 and its use as a pretargeting agent with an (111)In-labeled hapten-peptide were assessed in several human epithelial cancer xenografts. Dose optimization was examined in 2 tumor models. RESULTS: TF12 internalizes, but a substantial fraction remained accessible on the tumor surface. Fluorescence-activated cell sorting analysis showed only a minor change in fluorescent signal when the tumor was probed with a fluorescent hapten-peptide over 4 h, and microscopy showed substantial membrane staining when reassessed at 24 h after TF12 exposure. Only 40.1% of (111)In-TF12 was internalized after 24 h. In vivo, excellent tumor localization of the (111)In-labeled peptide was observed in several tumor models. CONCLUSION: TF12 was retained sufficiently on the cell surface in several epithelial cancers, thereby making it suitable for pretargeted imaging and therapy of various Trop-2-expressing carcinomas.

Islet transplantation is a novel promising strategy to cure type 1 diabetes. However, the long-term outcome is still poor, because both function and survival of the transplant decline over-time. Non-invasive imaging methods have the potential to enable monitoring of islet survival after transplantation and the effects of immunosuppressive drugs on transplantation outcome. 111In-labeled exendin-3 is a promising tracer to visualize native and transplanted islets by SPECT (Single Photon Emission Computed Tomography). In the present study, we hypothesized that islet microvasculature plays an important role determining the uptake of exendin-3 in islets when monitoring transplant survival. We observed 111In-exendin-3 accumulation in the transplant as early as three days after transplantation and an increase in the uptake up to three weeks post-transplantation. Islet-revascularization correlated with the increase in 111In-exendin-3 uptake, whereas fully re-established islet vasculature coincided with a stabilized uptake of the radiotracer in the transplant. Here, we demonstrate the importance of islet vasculature for in vivo delivery of radiotracers to transplanted islets and we demonstrate that optimal and stable uptake of exendin four weeks after transplantation opens the possibility for long-term monitoring of islet survival by SPECT imaging.

Item does not contain fulltext Targeting of the Glucagon-like peptide 1 receptor with (111)In-labeled exendin is an attractive approach to determine the beta cell mass (BCM). Preclinical studies as well as a proof-of-concept study in type 1 diabetic patients and healthy subjects showed a direct correlation between BCM and radiotracer uptake. Despite these promising initial results, the influence of alpha cells on the uptake of the radiotracer remains a matter of debate. In this study we determined the correlation between pancreatic tracer uptake and beta and alpha cell mass in a rat model for beta cell loss. The uptake of (111)In-exendin (\%ID/g) showed a strong positive linear correlation with the BCM (Pearson r = 0.82). The fraction of glucagon positive cells in the total endocrine mass was increased after alloxan treatment (26\% ± 4\%, 43\% ± 8\%, and 69\% ±21\% for 0, 45 and 60 mg/kg alloxan, respectively). The uptake of (111)In-exendin showed a negative linear correlation with the alpha cell fraction (Pearson r = -0.76). These data clearly indicate towards specificity of (111)In-exendin for beta cells and that the influence of the alpha cells on (111)In-exendin uptake is negligible.

Radical removal of malignant lesions may be improved using tumor-targeted dual-modality probes that contain both a radiotracer and a fluorescent label to allow for enhanced intraoperative delineation of tumor resection margins. Because pretargeting strategies yield high signal-to-background ratios, we evaluated the feasibility of a pretargeting strategy for intraoperative imaging in prostate cancer using an anti–TROP-2 x anti-HSG bispecific antibody (TF12) in conjunction with the dual-labeled diHSG peptide (RDC018) equipped with both a DOTA chelate for radiolabeling purposes and a fluorophore (IRdye800CW) to allow near-infrared optical imaging. Nude mice implanted s.c. with TROP-2–expressing PC3 human prostate tumor cells or with PC3 metastases in the scapular and suprarenal region were injected i.v. with 1 mg of TF12 and, after 16 hours of tumor accumulation and blood clearance, were subsequently injected with 10 MBq, 0.2 nmol/mouse of either 111In-RDC018 or 111In-IMP288 as a control. Two hours after injection, both microSPECT/CT and fluorescence images were acquired, both before and after resection of the tumor nodules. After image acquisition, the biodistribution of 111In-RDC018 and 111In-IMP288 was determined and tumors were analyzed immunohistochemically. The biodistribution of the dual-label RDC018 showed specific accumulation in the TROP-2–expressing PC3 tumors (12.4 ± 3.7% ID/g at 2 hours postinjection), comparable with 111In-IMP288 (9.1 ± 2.8% ID/g at 2 hours postinjection). MicroSPECT/CT and near-infrared fluorescence (NIRF) imaging confirmed this TROP-2–specific uptake of the dual-label 111In-RDC018 in both the s.c. and metastatic growing tumor model. In addition, PC3 metastases could be visualized preoperatively with SPECT/CT and could subsequently be resected by image-guided surgery using intraoperative NIRF imaging, showing the preclinical feasibility of pretargeted dual-modality imaging approach in prostate cancer. Cancer Res; 74(21); 6216–23. ©2014 AACR.

Item does not contain fulltext Arginine-glycine-aspartic acid (RGD)-based imaging tracers allow specific imaging of integrin alphavbeta3, a protein overexpressed during angiogenesis, leading to the possibility that it might serve as a tool to stratify patients for antiangiogenic treatment. However, these tracers have generally been characterized in xenograft models in which integrin alphavbeta3 was constitutively expressed by the tumor cells themselves. In the studies presented here, the use of (111)In-RGD2 as a tracer to image only integrin alphavbeta3 expression on blood vessels in the tumor was determined using tumor xenografts in which tumor cells were integrin alphavbeta3-negative. METHODS: DOTA-E-[c(RGDfK)]2 was radiolabeled with (111)In ((111)In-RGD2), and biodistribution studies were performed in squamous cell carcinoma of the head and neck (HNSCC) xenograft mouse models to determine the optimal peptide dose to image angiogenesis. Next, biodistribution and imaging studies were performed at the optimal peptide dose in 3 HNSCC mouse models, FaDu, SCCNij3, and SCCNij202. Immunohistochemical analysis of tumor vascular and cell surface expression of integrin alphavbeta3 and correlation analysis of vascular integrin alphavbeta3 and autoradiography were completed. RESULTS: All 3 HNSCC xenografts expressed integrin alphavbeta3 on the vessels only. The optimal peptide dose of (111)In-RGD2 was 1 mug or less for specific integrin alphavbeta3-mediated uptake of the tracer. SPECT/CT imaging showed clear uptake of the tracer in the periphery of the tumors, corresponding with well-vascularized areas of the tumor. Within the tumor, (111)In-RGD2 autoradiography coincided with vascular integrin alphavbeta3 expression, as determined immunohistochemically. Integrin alphavbeta3-mediated uptake was also detected in nontumor tissues, which, through immunohistochemical analysis, proved positive for integrin alphavbeta3. CONCLUSION: (111)In-RGD2 allows the visualization of integrin alphavbeta3 in xenograft models in which integrin alphavbeta3 is expressed only on the neovasculature, such as in the HNSCC tumors. Thus, (111)In-RGD2 allows specific visualization of angiogenesis in tumor models lacking constitutive tumoral integrin alphavbeta3 expression but may be less useful for this purpose in many tumors in which tumor cells express integrin alphavbeta3.

Contains fulltext : 136392.pdf (Publisher’s version ) (Closed access) AIMS/HYPOTHESIS: A reliable method for in vivo quantification of pancreatic beta cell mass (BCM) could lead to further insight into the pathophysiology of diabetes. The glucagon-like peptide 1 receptor, abundantly expressed on beta cells, may be a suitable target for imaging. We investigated the potential of radiotracer imaging with the GLP-1 analogue exendin labelled with indium-111 for determination of BCM in vivo in a rodent model of beta cell loss and in patients with type 1 diabetes and healthy individuals. METHODS: The targeting of (111)In-labelled exendin was examined in a rat model of alloxan-induced beta cell loss. Rats were injected with 15 MBq (111)In-labelled exendin and single photon emission computed tomography (SPECT) acquisition was performed 1 h post injection, followed by dissection, biodistribution and ex vivo autoradiography studies of pancreatic sections. BCM was determined by morphometric analysis after staining with an anti-insulin antibody. For clinical evaluation SPECT was acquired 4, 24 and 48 h after injection of 150 MBq (111)In-labelled exendin in five patients with type 1 diabetes and five healthy individuals. The tracer uptake was determined by quantitative analysis of the SPECT images. RESULTS: In rats, (111)In-labelled exendin specifically targets the beta cells and pancreatic uptake is highly correlated with BCM. In humans, the pancreas was visible in SPECT images and the pancreatic uptake showed high interindividual variation with a substantially lower uptake in patients with type 1 diabetes. CONCLUSIONS/INTERPRETATION: These studies indicate that (111)In-labelled exendin may be suitable for non-invasive quantification of BCM. TRIAL REGISTRATION: ClinicalTrials.gov NCT01825148, EudraCT: 2012-000619-10.

A reliable method for in vivo quantification of pancreatic beta cell mass (BCM) could lead to further insight into the pathophysiology of diabetes. The glucagon-like peptide 1 receptor, abundantly expressed on beta cells, may be a suitable target for imaging. We investigated the potential of radiotracer imaging with the GLP-1 analogue exendin labelled with indium-111 for determination of BCM in vivo in a rodent model of beta cell loss and in patients with type 1 diabetes and healthy individuals.The targeting of (111)In-labelled exendin was examined in a rat model of alloxan-induced beta cell loss. Rats were injected with 15 MBq (111)In-labelled exendin and single photon emission computed tomography (SPECT) acquisition was performed 1 h post injection, followed by dissection, biodistribution and ex vivo autoradiography studies of pancreatic sections. BCM was determined by morphometric analysis after staining with an anti-insulin antibody. For clinical evaluation SPECT was acquired 4, 24 and 48 h after injection of 150 MBq (111)In-labelled exendin in five patients with type 1 diabetes and five healthy individuals. The tracer uptake was determined by quantitative analysis of the SPECT images.In rats, (111)In-labelled exendin specifically targets the beta cells and pancreatic uptake is highly correlated with BCM. In humans, the pancreas was visible in SPECT images and the pancreatic uptake showed high interindividual variation with a substantially lower uptake in patients with type 1 diabetes.These studies indicate that (111)In-labelled exendin may be suitable for non-invasive quantification of BCM.ClinicalTrials.gov NCT01825148, EudraCT: 2012-000619-10.

Item does not contain fulltext Numerous studies have shown that the physicochemical properties of biomaterials can control cell activity. Cell adhesion, proliferation, differentiation as well as tissue formation in vivo can be tuned by properties such as the porosity, surface micro- and nanoscale topography and chemical composition of biomaterials. This concept is very appealing for tissue engineering since instructive properties in bioactive materials can be more economical and time efficient than traditional strategies of cell pre-differentiation in vitro prior to implantation. The biomaterial surface, which is easy to modify due to its accessibility, may provide the necessary signals to elicit a certain cellular behavior. Here, we used gas plasma technology at atmospheric pressure to modify the physicochemical properties of polylactic acid and analyzed how this influenced pre-osteoblast proliferation and differentiation. Tetramethylsilane and 3-aminopropyl-trimethoxysilane with helium as a carrier gas or a mixture of nitrogen and hydrogen were discharged to polylactic acid discs to create different surface chemical compositions, hydrophobicity and microscale topographies. Such modifications influenced protein adsorption and pre-osteoblast cell adhesion, proliferation and osteogenic differentiation. Furthermore polylactic acid treated with tetramethylsilane enhanced osteogenic differentiation compared to the other surfaces. This promising surface modification could be further explored for potential development of bone graft substitutes. 01 augustus 2012

Contains fulltext : 97008.pdf (Publisher’s version ) (Closed access) hRS7 is a humanized IgG1 monoclonal antibody directed against the epithelial glycoprotein-1 (EGP-1; also known as TROP2). This antigen is found in many epithelial cancers, including prostate cancer, and therefore this antibody could be suitable for targeting this cancer. In this study, the characteristics of hRS7 for targeting prostate cancer were examined. The potential for immuno-PET with (89)Zr-hRS7 and immuno-SPECT with (111)In-hRS7 was assessed using nude mice with human prostate cancer xenografts. METHODS: EGP-1 expression was assessed by immunohistology in human primary and metastatic prostate cancer samples and in PC3 xenografts. The optimal antibody protein dose for prostate cancer targeting was examined in nude mice with subcutaneous PC3 xenografts, and then the biodistribution of (111)In-, (125)I-, and (89)Zr-labeled hRS7 was determined in subcutaneous PC3 xenografts at 1, 3, and 7 d after injection. Immuno-PET and immuno-SPECT were performed with (89)Zr-hRS7 and (111)In-hRS7 in mice with subcutaneous and intraprostatic PC3 xenografts, respectively. RESULTS: Immunohistochemical analysis showed abundant EGP-1 expression in human primary and metastatic prostate cancers and in PC3 xenografts. (111)In-hRS7 and (89)Zr-hRS7 preferentially and specifically accumulated in PC3 xenografts, with tumor uptake as high as 60% injected dose per gram at a protein dose of 0.1 mug per mouse. PC3 tumors in nude mice were clearly visualized with both tracers with immuno-PET and immuno-SPECT. CONCLUSION: hRS7 shows excellent in vivo tumor targeting in human prostate cancer xenografts. Therefore, hRS7 is a potential vehicle for targeting prostate cancer.

Contains fulltext : 51419.pdf (Publisher’s version ) (Closed access) Ovarian cancer is the fourth most common cause of cancer deaths among females in the western world after cancer of the breast, colon and lung. The inability to control the disease within the peritoneal cavity is the major cause of treatment failure in patients with ovarian cancer. The majority of ovarian carcinomas express the alpha(v)beta(3) integrin. Here we studied the tumor targeting potential of an (111)In-labeled cyclic RGD peptide in athymic BALB/c mice with intraperitoneally (i.p.) growing NIH:OVCAR-3 human ovarian carcinoma tumors. The cyclic RGD peptide, c(RGDfK)E, was synthesized, conjugated with DOTA and radiolabeled with (111)In. The targeting potential of (111)In-DOTA-E-c(RGDfK) was studied in athymic mice with i.p. growing NIH:OVCAR-3 xenografts and the optimal dose of this compound was determined (0.01 microg up to 10 microg). The biodistribution at optimal peptide dose was determined at various time points (0.5 up to 72 hr). Furthermore, the therapeutic potential of (177)Lu-DOTA-E-c(RGDfK) was studied in this model. Two hours after i.p. administration, (111)In-DOTA-E-c(RGDfK) showed high and specific uptake in the i.p. growing tumors. Optimal uptake in the i.p. growing tumors was observed at a 0.03-0.1 microg dose range. Tumor uptake of (111)In-DOTA-E-c(RGDfK) peaked 4 hr p.i. [(38.8 +/- 2.7)% ID/g], gradually decreasing at later time points [(24.0 +/- 4.1)% ID/g at 48 hr p.i.]. Intraperitoneal growth of OVCAR-3 could be significantly delayed by injecting 37 MBq (177)Lu-labeled peptide i.p. Radiolabeled DOTA-E-c(RGDfK) is suitable for targeting of i.p. growing tumors and potentially can be used for peptide receptor radionuclide therapy of these tumors.

Contains fulltext : 49400.pdf (Publisher’s version ) (Closed access) Dendritic cells (DC) are professional antigen-presenting cells that play a pivotal role in the induction of immunity. Ex vivo-generated, tumour antigen-loaded mature DC are currently exploited as cancer vaccines in clinical studies. However, antigen loading and maturation of DC directly in vivo would greatly facilitate the application of DC-based vaccines. We formerly showed in murine models that radiofrequency-mediated tumour destruction can provide an antigen source for the in vivo induction of anti-tumour immunity, and we explored the role of DC herein. In this paper we evaluate radiofrequency and cryo ablation for their ability to provide an antigen source for DC and compare this with an ex vivo-loaded DC vaccine. The data obtained with model antigens demonstrate that upon tumour destruction by radiofrequency ablation, up to 7% of the total draining lymph node (LN) DC contained antigen, whereas only few DC from the conventional vaccine reached the LN. Interestingly, following cryo ablation the amount of antigen-loaded DC is almost doubled. Analysis of surface markers revealed that both destruction methods were able to induce DC maturation. Finally, we show that in situ tumour ablation can be efficiently combined with immune modulation by anti-CTLA-4 antibodies or regulatory T-cell depletion. These combination treatments protected mice from the outgrowth of tumour challenges, and led to in vivo enhancement of tumour-specific T-cell numbers, which produced more IFN-gamma upon activation. Therefore, in situ tumour destruction in combination with immune modulation creates a unique, 'in situ DC-vaccine' that is readily applicable in the clinic without prior knowledge of tumour antigens.

Purpose: The aim of these studies was to develop a pretargeting strategy for CEA-expressing cancers using biologically produced bispecific monoclonal antibodies (bsMAb). The bsMAbs used in this system have affinity for the carcinoembryonic antigen on the one hand, and for indium-labeled diethylenetriaminepentaacetic acid (DTPA), on the other. Experimental Design: Stable quadroma clones producing bsMAb MN-14xDTIn-1 were isolated. LS174T tumor–bearing mice were injected with 1 to 100 μg of bsMAb followed by 1 to 60 ng of an 111In-labeled bivalent peptide [Ac-Phe-Lys(DTPA)-Tyr-Lys(DTPA)-NH2]. Mice were killed at 24 hours postinjection and the biodistribution of the radiolabel was determined. The biodistribution of diDTPA labeled with four different radionuclides (111In, 99mTc, nonresidualizing 125I, and residualizing 125I) was determined at various time points postinjection following pretargeting of LS174T tumors with bsMAb MN-14xDTIn-1. Results: Optimal tumor targeting was observed when tumors were pretargeted with 10 μg of bsMAb MN-14xDTIn-1 and when 6 ng of a radiolabeled peptide was given 72 hours later. The uptake of the four radiolabels in LS174T tumors at 4 hours postinjection was similar. However, at later time points, the 111In-label and residualizing 125I-label were better retained in the tumor than the nonresidualizing 125I label. Although the absolute uptake in the tumor (in terms of percentage of injected dose per gram of tissue) was 5-fold lower than the uptake obtained with directly labeled MN-14, the pretargeting strategy revealed much higher tumor-to-blood ratios due to the rapid clearance of the radiolabel from the circulation as compared with 111In-MN-14 (445 ± 90 and 5.3 ± 1.1, respectively, at 72 hours postinjection). Conclusions: Effective targeting of carcinoembryonic antigen-expressing tumors was achieved with a newly produced bispecific antibody. The 111In-labeled l-amino acid peptide and 125I-d-amino acid peptide were better retained in the tumor than the 99mTc- and 125I-l-amino acid peptide. Very high tumor-to-blood ratios were obtained due to rapid background clearance.

Pancreatic islet transplantation is a promising experimental treatment for type I diabetic patients. A non-invasive method to accurately determine beta cell volume in the transplant could give insight in the faith of the islets after transplantation. Such a method will also provide the possibility to monitor the effects of therapeutic interventions on beta cell volume, which could improve the treatment of diabetic patients.

The CXC chemokines are a family of closely related chemoattractant cytokines that bind to, attract, and activate neutrophils to variable degrees. In this study, the relationship between neutrophil-binding affinity and suitability for infection imaging was investigated in a selected group of CXC chemokines. Neutrophil-activating peptide-2 (NAP-2, 70 residues; also called CXCL7) binds with high affinity to the CXCR2 receptor on neutrophils. Recently, C-terminally truncated NAP-2-variants have been described that have enhanced neutrophil-binding affinity and neutrophil-stimulating capacity. Here, NAP-2 and its C-terminal shortened variants NAP-2(1-68), NAP-2(1-66), and NAP-2(1-63) were labeled with (99m)Tc via the hydrazinonicotinamide (HYNIC) chelator and their potential for imaging of infection was investigated in a rabbit model of infection. The CXC chemokine interleukin-8 (IL-8) was used for comparison. In addition, a series of (99m)Tc-labeled CXC chemokines were screened for their potential to image infection, including CTAP-III, GCP-2, ENA-78, PF-4, and IP-10.The receptor-binding affinity of HYNIC-conjugated NAP-2 and its analogs was compared in competitive binding assays on Jurkat cells transfected with the CXCR2 receptor gene. Biodistribution of labeled NAP-2 (analogs) and other CXC chemokines in rabbits with intramuscular Escherichia coli infections was determined both by gamma-camera imaging and by counting dissected tissues at 6 h after injection.The CXCR2-binding affinity of the HYNIC-conjugated NAP-2 analogs relative to NAP-2 was as follows: NAP-2(1-68), 2.5-fold; NAP-2(1-66), 10-fold; and NAP-2(1-63), 3-fold. In the rabbit model, uptake in the abscess (in percentage injected dose per gram +/- SEM) was 0.084 +/- 0.015 for NAP-2, 0.098 +/- 0.010 for NAP-2(1-68), 0.189 +/- 0.044 for NAP-2(1-66), and 0.114 +/- 0.017 for NAP-2(1-63) at 6 h after injection. In comparison, higher uptake in the abscess was found for labeled IL-8, a modest uptake was found for GCP-2 and ENA-78, and a low uptake was found for CTAP-III, PF-4, and IP-10.This study showed a clear relationship between affinity to receptors on neutrophils and suitability for infection imaging. Of the NAP-2 variants, NAP-2(1-66) combined highest affinity to CXCR2 with the best characteristics for imaging. IL-8 binds to both CXCR1 and CXCR2 with high affinity and showed a superior imaging quality. The other CXC chemokines tested bind to neutrophils with lower affinity and were shown to be less suitable for infection imaging in this study.

Radioimmunotherapy (RIT) can be performed with various radionuclides. We tested the stability, biodistribution, and therapeutic efficacy of various radioimmunoconjugates ((131)I, (88/90)Y, (177)Lu, and (186)Re) of chimeric antirenal cell cancer monoclonal antibody G250 (mAb cG250) in nude mice with subcutaneous renal cell cancer (RCC) tumors.The (88/90)Y and (177)Lu labeling procedures of cG250 conjugated with cyclic diethylenetriaminepentaacetic acid anhydride (cDTPA), isothiocyanatobenzyl-DTPA (SCN-Bz-DTPA), or 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) were characterized. Stability of the labeled conjugates in plasma at 37 degrees C was assessed. Biodistribution and therapeutic efficacy of labeled cG250 were compared in nude mice with SK-RC-52 human RCC xenografts.Both SCN-Bz-DTPA and DOTA were stable in vitro (5% release of the radiolabel during 14 and 21 d of incubation) and in vivo (uptake in bone/= 1.5 percentage injected dose per gram [%ID/g] at 7 d after injection) when used to label (88)Y or (177)Lu to cG250. The DOTA conjugate was slightly but significantly more stable than SCN-Bz-DTPA at 7 d after injection. In vivo, these cG250 preparations showed high tumor uptake (70 +/- 15 %ID/g +/- SD at 7 d after injection). Maximum tumor uptake for (125)I-cG250 and (186)Re-mercaptoacetyltriglycine-(MAG3)-cG250 (20 +/- 3 %ID/g +/- SD) was reached at 3 d after injection and was much lower in comparison with cG250 labeled with the residualizing radionuclides. Because the highest specific activities could be prepared using SCN-Bz-DTPA, and relatively low protein doses of cG250 could be administered without saturating the tumor, cG250-SCN-Bz-DTPA conjugates were used in RIT studies. In RIT experiments at maximum tolerated dose, tumor growth was delayed most effectively by cG250 labeled with (177)Lu, next most effectively by (90)Y and (186)Re (which were approximately equal), and least by (131)I (delayed by approximately 185, 125, 90, and 25 d, respectively). The best median survival (300 d) was observed for (177)Lu-SCN-Bz-DTPA-cG250. Median survival for control groups was150 d.DOTA-conjugated radiolabeled antibodies were the most stable radioimmunoconjugates in vitro and in vivo as manifested by the lowest bone uptake. However, specific activity was higher for SCN-Bz-DTPA. The RIT studies clearly showed that the therapeutic efficacy of mAb cG250 labeled with (177)Lu, (90)Y, or (186)Re was superior to that of (131)I-cG250. The residualizing radionuclides (177)Lu and (90)Y led to higher radiation doses to the tumor and most likely are better candidates than conventionally radiolabeled (131)I for RIT with cG250 in patients with RCC.

Contains fulltext : 58153.pdf (Publisher’s version ) (Open Access) Arginine-glycine-aspartic acid (RGD) peptides preferentially bind to alphavbeta3 integrin, an integrin expressed on newly formed endothelial cells and on various tumor cells. When labeled with beta-emitting radionuclides, these peptides can be used for peptide-receptor radionuclide therapy of malignant tumors. These studies aimed to investigate whether tumor targeting and tumor therapy could be optimized by dose fractionation. The RGD-peptide DOTA-E-[c(RGDfK)]2 was labeled with 111In for biodistribution experiments and with 90Y for therapy experiments. In mice with NIH:OVCAR-3 ovarian carcinoma xenografts, optimal tumor uptake was obtained at peptide doses up to 1.0 microg (4.8 %ID/g). A peptide dose of 5 microg, required to administer the maximum tolerable dose (MTD) 90Y-DOTA-E-[c(RGDfK)]2, was administered as 5 portions of 1.0 microg. Tumor uptake of the fifth portion was significantly higher than that of the single 5.0 microg portion (3.3 %ID/g versus 2.1 %ID/g). The therapeutic efficacy of 37 MBq 90Y-DOTA-E-[c(RGDfK)]2 (1 x 5.0 microg) was compared with that of 37 MBq administered in five equal portions (5 x 1.0 microg). No difference in tumor growth between the fractionated and the nonfractionated therapy was observed. In conclusion, dose fractionation resulted in higher radiation doses. However, therapeutic efficacy of the radiolabeled peptide was not significantly improved by dose fractionation.

The alpha(v)beta(3) integrin is expressed on proliferating endothelial cells such as those present in growing tumors, as well as on tumor cells of various origin. Tumor-induced angiogenesis can be blocked in vivo by antagonizing the alpha(v)beta(3) integrin with small peptides containing the Arg-Gly-Asp (RGD) amino acid sequence. This tripeptidic sequence, naturally present in extracellular matrix proteins, is the primary binding site of the alpha(v)beta(3) integrin. Because of selective expression of alpha(v)beta(3) integrin in tumors, radiolabeled RGD peptides are attractive candidates for alpha(v)beta(3) integrin targeting in tumors. We studied the in vivo behavior of the radiolabeled dimeric RGD peptide E-[c(RGDfK)](2) in the NIH:OVCAR-3 s.c. ovarian carcinoma xenograft model in BALB/c nude mice. Conjugation of the 1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) and hydrazinonicotinamide (HYNIC) chelators enabled efficient radiolabeling with (111)In/(90)Y and (99m)Tc, respectively. The radiolabeled peptide was rapidly excreted renally. Uptake in nontarget organs such as liver and spleen was considerable. Tumor uptake peaked at 7.5% injected dose (ID)/g ((111)In-DOTA-E-[c(RGDfK)](2)) or 6.0%ID/g ((99m)Tc-HYNIC-E-[c(RGDfK)](2)) at 2 and 1 h postinjection, respectively. Integrin alpha(v)beta(3) receptor binding specificity was demonstrated by reduced tumor uptake after injection of the scrambled control peptide (111)In-DOTA-E-[c(RDKfD)](2) (0.28%ID/g at 2 h p.i.) and after coinjection of excess nonradioactive (115)In-DOTA-E-[c(RGDfK)](2) (0.22%ID/g at 2 h p.i.). A single injection of (90)Y-DOTA-E-[c(RGDfK)](2) at the maximum-tolerated dose (37 MBq) in mice with small s.c. tumors caused a significant growth delay as compared with mice treated with 37 MBq (90)Y-labeled scrambled peptide or untreated mice (median survival of 54 versus 33.5 versus 19 days, respectively). In conclusion, the radiolabeled RGD peptides (111)In-DOTA-E-[c(RGDfK)](2) and (99m)Tc-HYNIC-E-[c(RGDfK)](2) demonstrated high and specific tumor uptake in a human tumor xenograft. Injection of (90)Y-DOTA-E-[c(RGDfK)](2) induced a significant delay in tumor growth. Potentially, these peptides can be used for peptide receptor radionuclide imaging as well as therapy.

Contains fulltext : 186517.pdf (Publisher’s version ) (Closed access) The alpha v beta 3 integrin, a transmembrane heterodimeric protein expressed on sprouting endothelial cells, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin. Growing malignant tumors continuously require angiogenesis. As a result, alpha v beta 3 is preferentially expressed in growing tumors and is a potential target for radiolabeled RGD-peptides. In this study we compared the tumor targeting characteristics of a monomeric radiolabeled RGD-peptide with those of a dimeric analogue. Both peptides were radiolabeled with 99mTc via the hydrazinoni-cotinamid (= HYNIC) moiety to form 99mTc-HYNIC-c(RGDfK) and 99mTc-HYNIC-E-[c(RGDfK)]2. In vitro, the IC50 showed a 10-fold higher affinity of the dimer for the alpha v beta 3 integrin as compared to the monomer (0.1 vs. 1.0 nM). In athymic female BALB/c mice with subcutaneously growing OVCAR-3 ovarian carcinoma xenografts, tumor uptake peaked at 5.8 +/- 0.7% ID/g and 5.2 +/- 0.6% ID/g for the dimer and the monomer, respectively. At 1, 2, and 4 h postinjection (p.i.) uptake of the dimer in the tumor was significantly higher than that of the monomeric analogue. Tumor-to-blood ratios were highest at 24 h p.i. at a value of 63 for both compounds. At all timepoints kidney retention of the dimer was significantly higher as compared to kidney retention of the monomer. In conclusion, in this mouse model the dimeric RGD-peptide showed better retention in the tumor than the monomeric analogue, most likely due to the bivalent interaction with the target cell. Furthermore, kidney retention of the dimeric peptide was higher than that of the monomeric peptide.

The beta cell mass (BCM) plays a key role in the development and progression of diabetes mellitus. A reliable, non-invasive method for quantification of the pancreatic (BCM) in vivo would give further insight in the pathophysiology of type 1 and 2 diabetes. The glucagonlike peptide 1 receptor (GLP-1R) is specifically expressed on beta cells.

Currently, transplantation of islets of Langerhans is a promising experimental treatment for diabetes type 1 patients. Despite the ability of the transplanted islets to normalize blood glucose, the rate of insulin-independency drops to less than 15% after 5 years. The visualization of transplanted islets could gain important insights in the processes underlying islet transplantation and might improve the outcome of islet transplantation.

Contains fulltext : 95807.pdf (Publisher’s version ) (Open Access) Renal ischemia/reperfusion injury (IRI) frequently complicates shock, renal transplantation and cardiac and aortic surgery, and has prognostic significance. The translocation of phosphatidylserines to cell surfaces is an important pro-inflammatory signal for cell-stress after IRI. We hypothesized that shielding of exposed phosphatidylserines by the annexin A5 (ANXA5) homodimer Diannexin protects against renal IRI. Protective effects of Diannexin on the kidney were studied in a mouse model of mild renal IRI. Diannexin treatment before renal IRI decreased proximal tubule damage and leukocyte influx, decreased transcription and expression of renal injury markers Neutrophil Gelatinase Associated Lipocalin and Kidney Injury Molecule-1 and improved renal function. A mouse model of ischemic hind limb exercise was used to assess Diannexin biodistribution and targeting. When comparing its biodistribution and elimination to ANXA5, Diannexin was found to have a distinct distribution pattern and longer blood half-life. Diannexin targeted specifically to the ischemic muscle and its affinity exceeded that of ANXA5. Targeting of both proteins was inhibited by pre-treatment with unlabeled ANXA5, suggesting that Diannexin targets specifically to ischemic tissues via phosphatidylserine-binding. This study emphasizes the importance of phosphatidylserine translocation in the pathophysiology of IRI. We show for the first time that Diannexin protects against renal IRI, making it a promising therapeutic tool to prevent IRI in a clinical setting. Our results indicate that Diannexin is a potential new imaging agent for the study of phosphatidylserine-exposing organs in vivo.