Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging

IntroductionAffibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL).MethodsThe HER2-targeting NCL-cyclized Affibody molecule Z_HER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-Z_HER2:342min was labeled with ¹¹¹In and ⁶⁸ Ga. The binding affinity of DOTA-Z_HER2:342min was evaluated in vitro. The targeting properties of ¹¹¹In- and ⁶⁸ Ga-DOTA-Z_HER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of ¹¹¹In- and ⁶⁸ Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge.ResultsThe dissociation constant (K_D) for DOTA-Z_HER2:342min binding to HER2 was 18 nM according to SPR measurements. DOTA-Z_HER2:342min was labeled with ¹¹¹In and ⁶⁸ Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with K_D1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 ± 1.3 for ¹¹¹In- DOTA-Z_HER2:342min and 4.6 ± 0.7 for ⁶⁸ Ga-DOTA-Z_HER2:342min. However, the uptake of DOTA-Z_HER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts.ConclusionsNative chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (Z_HER2:342min) with low nanomolar target affinity and specific tumor uptake.     

Targeting breast carcinoma with radioiodinated anti-HER2 Nanobody

IntroductionWith a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors.Methods5F7GGC Nb was radioiodinated with ¹²⁵I using Iodogen and with ¹³¹I using the residualizing agent N^?-(3-[¹³¹I]iodobenzoyl)-Lys⁵-N^α-maleimido-Gly¹-GEEEK ([¹³¹I]IB-Mal-d-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations.ResultsThe radiochemical yields for Iodogen and [¹³¹I]IB-Mal-d-GEEEK labeling were 83.6 ± 5.0% (n = 10) and 59.6 ± 9.4% (n = 15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [¹³¹I]IB-Mal-d-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65 ± 0.61% ID/g vs. 2.92 ± 0.24% ID/g at 8 h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios.ConclusionsTaken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [¹³¹I]IB-Mal-d-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT (¹²⁹I) and PET imaging (¹²⁴I) of patients with HER2-expressing tumors.     

Phase I trial of intraoperative detection of tumor margins in patients with HER2-positive carcinoma of the breast following administration of 111In-DTPA-trastuzumab Fab fragments

IntroductionOur aim was to conduct a Phase I clinical trial to determine the feasibility of intraoperative detection of tumor margins in HER2 positive breast carcinoma using a hand-held γ-probe following administration of ¹¹¹In-DTPA-trastuzumab Fab fragments. Accurate delineation of tumor margins is important for preventing local recurrence.MethodsSix patients with HER2-positive in situ or invasive ductal carcinoma were administered 74 MBq (0.5 mg) of ¹¹¹In-DTPA-trastuzumab Fab fragments and counts in the tumor, surgical cavity wall and en face margins were measured intraoperatively at 72 h post-injection using the Navigator or C-Trak γ-probes. Margins were evaluated histologically. Quantitative whole body planar imaging was performed to estimate radiation absorbed doses using OLINDA/EXM software. SPECT imaging of the thorax was performed to evaluate tumor uptake. The pharmacokinetics of elimination from the blood and plasma were determined over 72 h.ResultsThere were no acute adverse reactions from ¹¹¹In-DTPA-trastuzumab Fab fragments and no changes in hematological or biochemical indices were found over a 3 month period. ¹¹¹In-DTPA-trastuzumab Fab fragments exhibited a biphasic elimination from the blood and plasma with t_1/2α = 11.9 h and 7.5 h, respectively, and t_1/2β = 26.6 and 20.7 h, respectively. The radiopharmaceutical accumulated in the liver, spleen and kidneys. SPECT imaging did not reveal tumor in any patient. The mean effective dose was 0.146 mSv/MBq (10.8 mSv for 74 MBq). Counts in excised tumors were low but were higher than in margins. Margins in two patients harboured tumor but this was not correlated with counts obtained using the γ-probes. Surgical cavity counts were high and likely due to detection of γ-photons outside the surgical field.ConclusionWe conclude that it was not feasible, at least at the administered amount of radioactivity used in this study, to reliably detect the margins of disease in patients with in situ or invasive ductal carcinoma intraoperatively using a hand-held γ-probe and ¹¹¹In-DTPA-trastuzumab Fab fragments due to low uptake in the tumor and involved margins.     

Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical

¹¹¹In-DTPA-anti-γH2AX-Tat, which combines an anti-γH2AX antibody with a cell-penetrating peptide, Tat, and the Auger electron-emitting radioisotope, ¹¹¹In, targets the DNA damage signalling protein, γH2AX, and has potential as a probe for imaging DNA damage in vivo. The goal of this study was to investigate whether ¹¹¹In-DTPA-anti-γH2AX-Tat labelled to high specific activity (6 MBq/μg) can amplify treatment-related DNA damage for therapeutic gain.MethodsMDA-MB-468 and MDA-MB-231/H2N (231-H2N) breast cancer cells were incubated with ¹¹¹In-DTPA-anti-γH2AX-Tat (3 MBq, 6 MBq/μg) or a control radioimmunoconjugate, ¹¹¹In-DTPA-mIgG-Tat, and exposed to IR or bleomycin. DNA damage was studied by counting γH2AX foci and by neutral comet assay. Cytotoxicity was evaluated using clonogenic assays. ¹¹¹In-DTPA-anti-γH2AX-Tat was administered intravenously to 231-H2N-xenograft-bearing Balb/c nu/nu mice in tumor growth inhibition studies.ResultsThe number of γH2AX foci was greater after exposure of cells to IR (10 Gy) plus ¹¹¹In-DTPA-anti-γH2AX-Tat compared to IR alone (20.6 ± 2.5 versus 10.4 ± 2.3 foci/cell; P < .001).¹¹¹In-DTPA-anti-γH2AX-Tat resulted in a reduced surviving fraction in cells co-treated with IR (4 Gy) versus IR alone (5.2% ± 0.9% versus 47.8% ± 2.8%; P < .001). Similarly, bleomycin (25–200 μg/mL) plus ¹¹¹In-DTPA-anti-γH2AX-Tat resulted in a lower SF compared to bleomycin alone. The combination of a single exposure to IR (10 Gy) plus ¹¹¹In-DTPA-anti-γH2AX-Tat significantly decreased the growth rate of 231-H2N xenografts in vivo compared to either ¹¹¹In-DTPA-anti-γH2AX-Tat or IR alone (? 0.002 ± 0.004 versus 0.036 ± 0.011 and 0.031 ± 0.014 mm³/day, respectively, P < .001).Conclusion¹¹¹In-DTPA-anti-γH2AX-Tat amplifies anticancer treatment-related DNA damage in vitro and has a potent anti-tumor effect when combined with IR in vivo.     

[11C]Sorafenib: Radiosynthesis and preclinical evaluation in tumor-bearing mice of a new TKI-PET tracer

IntroductionTyrosine kinase inhibitors (TKIs) like sorafenib are important anticancer therapeutics with thus far limited treatment response rates in cancer patients. Positron emission tomography (PET) could provide the means for selection of patients who might benefit from TKI treatment, if suitable PET tracers would be available. The aim of this study was to radiolabel sorafenib (1) with carbon-11 and to evaluate its potential as TKI-PET tracer in vivo.MethodsSynthetic methods were developed in which sorafenib was labeled at two different positions, followed by a metabolite analysis in rats and a PET imaging study in tumor-bearing mice.Results[methyl-¹¹C]-1 and [urea-¹¹C]-1 were synthesized in yields of 59% and 53%, respectively, with a purity of > 99%. The identity of the products was confirmed by coinjection on HPLC with reference sorafenib. In an in vivo metabolite analysis [¹¹C]sorafenib proved to be stable. The percentage of intact product in blood–plasma after 45 min was 90% for [methyl-¹¹C]-1 and 96% for [urea-¹¹C]-1, respectively. Due to the more reliable synthesis, further research regarding PET imaging was performed with [methyl-¹¹C]-1 in nude mice bearing FaDu (head and neck cancer), MDA-MB-231 (breast cancer) or RXF393 (renal cancer) xenografts. Highest tracer accumulation at a level of 2.52 ± 0.33 %ID/g was observed in RXF393, a xenograft line extensively expressing the sorafenib target antigen Raf-1 as assessed by immunohistochemistry.ConclusionIn conclusion, we have synthesized [¹¹C]sorafenib as PET tracer, which is stable in vivo and has the capability to be used as PET tracer for imaging in tumor-bearing mice.     

Synthesis and in vivo preclinical evaluation of an 18F labeled uPA inhibitor as a potential PET imaging agent

IntroductionThe urokinase plasminogen activator (uPA) system is a proteolytic cascade involved in tumor invasion and metastasis. uPA and its inhibitor PAI-1 are described as biomarkers for breast cancer with the highest level of evidence. The present study describes the synthesis and first in vivo application of an activity based uPA PET probe.MethodsBased on the design of a small irreversible and selective uPA inhibitor we developed an ¹⁸F-labeled activity based probe for uPA imaging. Human uPA expressing MDA-MB-231-luc2-GFP cells were inoculated in the mammary fat pads of nude mice and treated with the probe once tumors reached a volume of 150 mm³. Scans were performed at 0.25, 0.75, 1.5, 4 and 6 h post injection. To evaluate tumor uptake in vivo and ex vivo data were gathered. Biodistribution data of the organs and tissues of interest were collected at all time points. Due to a relatively low tumor uptake, probe stability was further evaluated.ResultsThe uPA targeting PET tracer was produced in high purity and with good specific radioactivity. In vivo PET data showed a maximum tumor uptake of 2,51 ± 0,32 %ID/g at 4 h p.i. A significant correlation between in vivo and ex vivo tumor uptake calculation was found (R = 0.75; p < 0.01). Due to a high blood signal at all time points, probe stability was further examined revealing high plasma protein binding and low plasma stability.ConclusionsIn vivo and ex vivo results clearly demonstrate that uPA expressing tumors can be detected with non-invasive PET imaging. Stability tests suggest that further optimization is needed to provide a better tumor-to-background contrast.     

[99mTc]O2-AMD3100 as a SPECT tracer for CXCR4 receptor imaging

PurposeCXCR4 plays an important role in HIV infection, tumor progression, neurogenesis, and inflammation. In-vivo imaging of CXCR4 could provide more insight in the role of this receptor in health and disease. The aim of this study was to investigate [^99mTc]O₂-AMD3100 as a potential SPECT tracer for imaging of CXCR4.MethodAMD3100 was labelled with [^99mTc]pertechnetate. A cysteine challenge assay was performed to test the tracer stability. Heterologous and homologous receptor binding assay and internalization assay were performed in CXCR4 expressing Jurkat-T cells. Ex vivo biodistribution was studied in healthy mice at 30, 60, and 120 min after tracer injection. Tumor uptake of the tracer was determined by microSPECT imaging in nude mice xenografted with human PC-3 prostate tumor. Specificity of tracer uptake was determined by blocking studies using an excess of unlabelled AMD3100.ResultsAMD3100 was labelled with technetium-99 m with a radiochemical yield of > 98%. The tracer was stable in PBS and mouse plasma for at least 6 h at 37 °C. Heterologous and homologous binding assays with AMD3100 showed IC₅₀ values of 240 ± 10 μM, and 92 ± 5 μM for [¹²⁵I]SDF-1α and [^99mTc]O₂-AMD3100 respectively, with negligible receptor internalisation. The tracer showed high uptake in liver, lungs, spleen, thymus, intestine and bone. Blocking dose of AMD3100.8HCl (20 mg/kg) decreased the uptake in these organs (p < 0.05). [^99mTc]O₂-AMD3100 showed specific tumor accumulation in mice bearing PC-3 xenografts model. Time activity curves (TAC) in AMD3100 pre-treated animals tracer showed 1.7 times less tumor uptake as compared to control animals (p < 0.05).Conclusion[^99mTc]O₂-AMD3100 is readily labelled, is stable in plasma and displays a favourable binding affinity for the CXCR4 receptors. [^99mTc O₂-AMD3100 shows specific binding in organs with high CXCR4 expression and in CXCR4 positive tumors. These results justify further evaluation of this radiopharmaceutical as a potential biomarker for the non-invasive imaging of CXCR4 receptors.     

Dosimetry of 64Cu-DOTA-AE105, a PET tracer for uPAR imaging

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.MethodsFive mice received iv tail injection of ⁶⁴Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22 h 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 22 h 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.ResultsHuman estimates of ⁶⁴Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918 mSv/MBq) followed by the liver (0.0815 mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02–0.04 mSv/MBq. The mean effective whole-body dose of ⁶⁴Cu-DOTA-AE105 was estimated to be 0.0317 mSv/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: 0.0252 mSv/Mbq, Observed value: 0.0315 mSv/MBq) thus validating our approach for human dosimetry estimation.ConclusionFavorable dosimetry estimates together with previously reported uPAR PET data fully support human testing of ⁶⁴Cu-DOTA-AE105.     

Antilipolytic drug boosts glucose metabolism in prostate cancer

IntroductionThe antilipolytic drug Acipimox reduces free fatty acid (FFA) levels in the blood stream. We examined the effect of reduced FFAs on glucose metabolism in androgen-dependent (CWR22Rv1) and androgen-independent (PC3) prostate cancer (PCa) xenografts.MethodsSubcutaneous tumors were produced in nude mice by injection of PC3 and CWR22Rv1 PCa cells. The mice were divided into two groups (Acipimox vs. controls). Acipimox (50 mg/kg) was administered by oral gavage 1 h before injection of tracers. 1 h after i.v. co-injection of 8.2 MBq (222 ± 6.0 μCi) ¹⁸ F-FDG and ~ 0.0037 MBq (0.1 μCi) ¹⁴C-acetate, ¹⁸ F-FDG imaging was performed using a small-animal PET scanner. Counting rates in reconstructed images were converted to activity concentrations. Quantification was obtained by region-of-interest analysis using dedicated software. The mice were euthanized, and blood samples and organs were harvested. ¹⁸ F radioactivity was measured in a calibrated γ-counter using a dynamic counting window and decay correction. ¹⁴C radioactivity was determined by liquid scintillation counting using external standard quench corrections. Counts were converted into activity, and percentage of the injected dose per gram (%ID/g) tissue was calculated.ResultsFDG biodistribution data in mice with PC3 xenografts demonstrated doubled average %ID/g tumor tissue after administration of Acipimox compared to controls (7.21 ± 1.93 vs. 3.59 ± 1.35, P = 0.02). Tumor-to-organ ratios were generally higher in mice treated with Acipimox. This was supported by PET imaging data, both semi-quantitatively (mean tumor FDG uptake) and visually (tumor-to-background ratios). In mice with CWR22Rv1 xenografts there was no effect of Acipimox on FDG uptake, either in biodistribution or PET imaging. ¹⁴C-acetate uptake was unaffected in PC3 and CWR22Rv1 xenografts.ConclusionsIn mice with PC3 PCa xenografts, acute administration of Acipimox increases tumor uptake of ¹⁸ F-FDG with general improvements in tumor-to-background ratios. Data indicate that administration of Acipimox prior to ¹⁸ F-FDG PET scans has potential to improve sensitivity and specificity in patients with castration-resistant advanced PCa.     

18F-FDG-PET/CT in patients with breast cancer and rising Ca 15-3 with negative conventional imaging: A multicentre study

ObjectivesBreast cancer is the second cause of death in women in Europe and North America. The mortality of this disease can be reduced with effective therapy and regular follow up to detect early recurrence. Tumor markers are sensitive in detecting recurrent or residual disease but imaging is required to customize the therapeutic option. Rising tumor markers and negative conventional imaging (US, X-mammography, CT and MR) poses a management problem. Our aim is to assess the role of ¹⁸F-FDG-PET/CT in the management of post-therapy patients with rising markers but negative conventional imaging.Materials and methodsIn the period from January 2008 to September 2009, 89 female patients with breast cancer who developed post-therapy rising markers (serum Ca 15-3 levels = 64.8 ± 16.3 U/mL) but negative clinical examination and conventional imaging were investigated with ¹⁸F-FDG-PET/CT.ResultsTumor deposits were detected in 40/89 patients in chest wall, internal mammary nodes, lungs, liver and skeleton. The mean SUVmax value calculated in these lesions was 6.6 ± 1.7 (range 3.1–12.8). In 23/40 patients solitary small lesion were amenable to radical therapy. In 7 out of these 23 patients a complete disease remission lasting more than 1 year was observed.Conclusions¹⁸F-FDG-PET/CT may have a potential role in asymptomatic patients with rising markers and negative conventional imaging. Our findings agree with other studies in promoting regular investigations such as tumor markers and ¹⁸F-FDG-PET/CT rather than awaiting the developments of physical symptoms as suggested by current guidelines since the timely detection of early recurrence may have a major impact on therapy and survival.     

Specific uptake of 99mTc-NC100692, an αvβ3-targeted imaging probe,in subcutaneous and orthotopic tumors

IntroductionThe α_vβ₃ integrin, which is expressed by angiogenic epithelium and some tumor cells, is an attractive target for the development of both imaging agents and therapeutics. While optimal implementation of α_vβ₃-targeted therapeutics will require a priori identification of the presence of the target, the clinical evaluation of these compounds has typically not included parallel studies with α_vβ₃-targeted diagnostics. This is at least partly due to the relatively limited availability of PET radiopharmaceuticals in comparison to those labeled with ^99mTc. In an effort to begin to address this limitation, we evaluated the tumor uptake of ^99mTc-NC100692, a cyclic RGD peptide that binds to α_vβ₃ with ~ 1-nM affinity, in an α_vβ₃-positive tumor model as well as its in vivo specificity.MethodsMicroSPECT imaging was used to assess the ability of cilengitide, a therapeutic with high affinity for α_vβ₃, to block and displace ^99mTc-NC100692 in an orthotopic U87 glioma tumor. The specificity of ^99mTc-NC100692 was quantitatively evaluated in mice bearing subcutaneous U87MG tumors, by comparison of the biodistribution of ^99mTc-NC100692 with that of the non-specific structural analogue ^99mTc-AH-111744 and by blocking uptake of ^99mTc-NC100692 with excess unlabeled NC100692.ResultsMicroSPECT imaging studies demonstrated that uptake of ^99mTc-NC100692 in the intracranial tumor model was both blocked and displaced by the α_vβ₃-targeted therapeutic cilengitide. Biodistribution studies provided quantitative confirmation of these imaging results. Tumor uptake of ^99mTc-NC100692 at 1 h post-injection was 2.8 ± 0.7% ID/g compared to 0.38 ± 0.1% ID/g for ^99mTc-AH-111744 (p < 0.001). Blocking ^99mTc-NC100692 uptake by pre-injecting the mice with excess unlabeled NC100692 reduced tumor uptake by approximately five-fold, to 0.68 ± 0.3% ID/g (p = 0.01).ConclusionThese results confirm that ^99mTc-NC100692 does, in fact, target the α_vβ₃ integrin and may, therefore, be useful in identifying patients prior to anti-α_vβ₃ therapy as well as monitoring the response of these patients to therapy.     

64Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis

IntroductionWe synthesized and evaluated ⁶⁴Cu-labeled tetraiodothyroacetic acid (tetrac)-conjugated liposomes for PET imaging of tumor angiogenesis, because tetrac inhibits angiogenesis via integrin α_Vβ₃.MethodsTetrac-PEG-DSPE and DOTA-PEG-DSPE were synthesized and formulated with other lipids into liposomes. The resulting tetrac/DOTA-liposomes were labeled with ⁶⁴Cu at 40 °C for 1 h and purified using a PD-10 column. ⁶⁴Cu-DOTA-liposomes were also prepared for comparison. Human aortic endothelial cell (HAEC) binding studies were performed by incubating the liposomes with the cells at 37 °C. MicroPET imaging followed by tissue distribution study was carried out using U87MG tumor-bearing mice injected with tetrac/⁶⁴Cu-DOTA-liposomes or ⁶⁴Cu-DOTA-liposomes.ResultsHAEC binding studies exhibited that tetrac/⁶⁴Cu-DOTA-liposomes were avidly taken up by the cells from 1.02 %ID at 1 h to 11.89 %ID at 24 h, while ⁶⁴Cu-DOTA-liposomes had low uptake from 0.47 %ID at 1 h to 1.57 %ID at 24 h. MicroPET imaging of mice injected with tetrac/⁶⁴Cu-DOTA-liposomes showed high radioactivity accumulation in the liver and spleen. ROI analysis of the tumor images revealed 1.93 ± 0.12 %ID/g at 1 h and 2.70 ± 0.36 %ID/g at 22 h. In contrast, tumor ROI analysis of ⁶⁴Cu-DOTA-liposomes revealed 0.54 ± 0.08 %ID/g at 1 h and 0.52 ± 0.09 %ID/g at 22 h. Tissue distribution studies confirmed that the tumor uptakes of tetrac/⁶⁴Cu-DOTA-liposomes and ⁶⁴Cu-DOTA-liposomes were 1.75 ± 0.03 %ID/g and 0.36 ± 0.01 %ID/g at 22 h, respectively.ConclusionThese results demonstrate that tetrac/⁶⁴Cu-DOTA-liposomes have significantly enhanced tumor uptake compared to ⁶⁴Cu-DOTA-liposomes due to tetrac conjugation. Further studies are warranted to reduce the liver and spleen uptake of tetrac/⁶⁴Cu-DOTA-liposomes.     

68Ga-NODAGA-VEGF121 for in vivo imaging of VEGF receptor expression

PurposeVascular endothelial growth factor (VEGF) is a crucial regulator of angiogenesis. In this study, we labeled VEGF₁₂₁ with ⁶⁸Ga using a hydrophilic chelating agent, NODAGA and evaluated the resulting ⁶⁸Ga-NODAGA-VEGF₁₂₁ for in vivo imaging of VEGF receptor (VEGFR) expression.MethodsNODAGA-VEGF₁₂₁ was prepared and its binding affinity for VEGFR2 was measured using ¹²⁵I-VEGF₁₂₁. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was prepared by labeling NODAGA-VEGF₁₂₁ with ⁶⁸GaCl₃ followed by purification using a PD-10 column. Human aortic endothelial cell (HAEC) binding studies of ⁶⁸Ga-NODAGA-VEGF₁₂₁ were performed at 37 °C for 4 h. MicroPET imaging followed by biodistribution studies were performed in U87MG tumor-bearing mice injected with ⁶⁸Ga-NODAGA-VEGF₁₂₁. Immunofluorescence staining of the tumor tissues was performed to verify VEGFR2 expression.ResultsBinding affinity of NODAGA-VEGF₁₂₁ for VEGFR2 was found to be comparable to that of VEGF₁₂₁. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was prepared in 47.8% yield with specific activity of 3.4 GBq/mg. ⁶⁸Ga-NODAGA-VEGF₁₂₁ was avidly taken up by HAECs with a time-dependent increase from 9.88 %ID at 1 h to 20.86 %ID at 4 h. MicroPET imaging of mice demonstrated high liver and spleen uptake with clear visualization of tumor at 1 h after injection. ROI analysis of tumors revealed 2.53 ± 0.11 %ID/g at 4 h after injection. In the blocking study, tumor uptake was inhibited by 29% at 4 h. Subsequent biodistribution studies demonstrated tumor uptake of 2.38 ± 0.15 %ID/g. Immunofluorescence staining of the tumor tissues displayed high level of VEGFR2 expression.ConclusionsThese results demonstrate that ⁶⁸Ga-NODAGA-VEGF₁₂₁ led to VEGFR-specific distribution in U87MG tumor-bearing mice. This study also suggests that altered physicochemical properties of VEGF₁₂₁ after radiolabeling may affect biodistribution of the radiolabeled VEGF₁₂₁.     

99mTc-labeled SWL specific peptide for targeting EphA2 receptor

IntroductionEphA2, one member of the Eph receptor family, is widely expressed in multiple aggressive cancers. SWL, a small peptide identified by phage display, has high binding affinity to EphA2, suggesting that it could be exploited for targeted molecular imaging. Therefore, a novel peptide-based probe, ^99mTc-HYNIC-SWL, was developed and its potential to specifically target EphA2-positive tumors was investigated.MethodsThe SWL peptide was labeled with hydrazinonicotinic acid (HYNIC), followed by ^99mTc labeling. Immunofluorescence staining was carried out to detect the expression of EphA2 in A549 lung cancer cells and OCM-1 melanoma cells. Saturation binding experiments were performed by incubating A549 cells with increasing concentrations of radiolabeled peptide in vitro. To test the probe in vivo, nude mice bearing either A549 or OCM-1 derived tumors were established, injected with ^99mTc-HYNIC-SWL, and subjected to SPECT imaging. Mice injected with excess unlabeled SWL were used as a specific control. Ex vivo γ-counting of dissected tissues from the mice was also performed to evaluate biodistribution.ResultsImmunofluorescence staining showed that A549 cells intensively expressed EphA2, while OCM-1 cells had little expression. ^99mTc-HYNIC-SWL displayed high binding affinity with A549 cells (K_D = 2.6 ± 0.7 nM). From the SPECT images and the results of the biodistribution study, significantly higher uptake of the tracer was seen in A549 tumors (1.44 ± 0.12 %ID/g) than in OCM-1 tumors (0.43 ± 0.20 %ID/g) at 1 h after injection. Pre-injection with excess unlabeled peptide in A549-bearing nude mice, significantly reduced tumor uptake of the radiolabeled probe (0.58 ± 0.20 %ID/g) was seen. These data suggest that ^99mTc-HYNIC-SWL specifically targets EphA2 in tumors.ConclusionsThe expression of EphA2 can be noninvasively investigated using ^99mTc-HYNIC-SWL by SPECT imaging. The in vitro and in vivo characteristics of ^99mTc-HYNIC-SWL make it a promising probe for EphA2-positive tumor imaging.     

Synthesis and biological evaluation of copper-64 radiolabeled [DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2], a novel bivalent targeting vector having affinity for two distinct biomarkers (GRPr/PSMA) of prostate cancer

Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂] radioligand for prostate cancer imaging, where DUPA = (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH₂ = bombesin, a GRPr-specific peptide targeting probe.MethodsThe PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH₂], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with ⁶⁴CuCl₂ and ^natCuCl₂. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.).ResultsCompetitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-(^natCu-NODAGA)-5-Ava-BBN(7-14)NH₂] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18 h p.i. with collateral, background radiation also being observed in non-target tissue.ConclusionsDUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular of imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-(⁶⁴Cu-NODAGA)-5-Ava-BBN(7-14)NH₂ in tumor-bearing mice indicate that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues.     

The human polynucleotide kinase/phosphatase (hPNKP) inhibitor A12B4C3 radiosensitizes human myeloid leukemia cells to Auger electron-emitting anti-CD123 111In-NLS-7G3 radioimmunoconjugates

IntroductionLeukemia stem cells (LSCs) are believed to be responsible for initiating and propagating acute myeloid leukemia (AML) and for causing relapse after treatment. Radioimmunotherapy (RIT) targeting these cells may improve the treatment of AML, but is limited by the low density of target epitopes. Our objective was to study a human polynucleotide kinase/phosphatase (hPNKP) inhibitor that interferes with DNA repair as a radiosensitizer for the Auger electron RIT agent, ¹¹¹In-NLS-7G3, which recognizes the CD123⁺/CD131^- phenotype uniquely displayed by LSCs.MethodsThe surviving fraction (SF) of CD123⁺/CD131^- AML-5 cells exposed to ¹¹¹In-NLS-7G3 (33–266 nmols/L; 0.74 MBq/μg) or to γ-radiation (0.25-5 Gy) was determined by clonogenic assays. The effect of A12B4C3 (25 μmols/L) combined with ¹¹¹In-NLS-7G3 (16–66 nmols/L) or with γ-radiation (0.25–2 Gy) on the SF of AML-5 cells was assessed. The density of DNA double-strand breaks (DSBs) in the nucleus was measured using the γ-H2AX assay. Cellular dosimetry was estimated based on the subcellular distribution of ¹¹¹In-NLS-7G3 measured by cell fractionation.ResultsBinding of ¹¹¹In-NLS-7G3 to AML-5 cells was reduced by 2.2-fold in the presence of an excess (1 μM) of unlabeled NLS-7G3, demonstrating specific binding to the CD123⁺/CD131^- epitope. ¹¹¹In-NLS-7G3 reduced the SF of AML-5 cells from 86.1 ± 11.0% at 33 nmols/L to 10.5 ± 3.6% at 266 nmols/L. Unlabeled NLS-7G3 had no significant effect on the SF. Treatment of AML-5 cells with γ-radiation reduced the SF from 98.9 ± 14.9% at 0.25 Gy to 0.03 ± 0.1% at 5 Gy. A12B4C3 combined with ¹¹¹In-NLS-7G3 (16–66 nmols/L) enhanced the cytotoxicity up to 1.7-fold compared to treatment with radioimmunoconjugates alone and was associated with a 1.6-fold increase in DNA DSBs in the nucleus. A12B4C3 enhanced the cytotoxicity of γ-radiation (0.25–0.5 Gy) on AML-5 cells by up to 1.5-fold, and DNA DSBs were increased by 1.7-fold. Exposure to ¹¹¹In-NLS-7G3 (66 nmols/L) delivered up to 0.6 Gy to AML-5 cells.ConclusionsWe conclude that A12B4C3 radiosensitized AML cells to the DNA damaging effects of ¹¹¹In-NLS-7G3. Combination treatment may increase the effectiveness for Auger electron RIT of AML targeting the LSC subpopulation.     

Radiosynthesis and initial evaluation of 18F labeled nanocarrier composed of poly(L-lactic acid)-block-poly(sarcosine) amphiphilic polydepsipeptide

IntroductionWith the aim of developing radiotracers for in vivo positron emission tomography (PET) imaging of solid tumors based on the enhanced permeability and retention effect of nanocarriers, we have developed a polymer micelle named “Lactosome”, which is composed of the amphiphilic polydepsipeptide, poly(L-lactic acid)-block-poly(sarcosine). This paper describes and evaluates the initial evaluation of the ¹⁸F-labeled Lactosome as a novel contrast agent for the tumor PET imaging technique carried out.Methods¹⁸F-labeled Lactosomes were prepared by a film hydration method under sonication in water at 50 °C from a mixture of 4-[¹⁸F]fluoro-benzoyl poly-L-lactic acid (¹⁸F-BzPLLA₃₀) and the amphiphilic polydepsipeptide. For biodistribution studies, BALB/cA Jcl-nu/nu mice bearing HeLa cells in the femur region were used. We took both PET and near-infrared fluorescence (NIRF) images of tumor bearing mice after co-injection of ¹⁸F-labeled Lactosome and NIRF-labeled Lactosome.Results¹⁸F-labeled Lactosomes were prepared at good yields (222–420 MBq) and more than 99% of ¹⁸F-BzPLLA₃₀ was incorporated into ¹⁸F-labeled Lactosome. The radioactivity of ¹⁸F-labeled Lactosome was found to be stable and maintained at high level for up to 6 h after injection into the blood stream. Tumor uptake increased gradually after the injection. The uptake ratio of tumor/muscle was 2.7 at 6 h from the time of injection. Tumor PET imaging with ¹⁸F-labeled Lactosome was as capable as tumor NIRF imaging with NIRF-labeled Lactosome.ConclusionTumor PET imaging using Lactosome as a nanocarrier may be therefore a potential candidate for a facile and general solid tumor imaging technique.     

Development of [18F]afatinib as new TKI-PET tracer for EGFR positive tumors

IntroductionAfatinib is an irreversible ErbB family blocker that was approved for the treatment of EGFR mutated non-small cell lung cancer in 2013. Positron emission tomography (PET) with fluorine-18 labeled afatinib provides a means to obtain improved understanding of afatinib tumor disposition in vivo. PET imaging with [¹⁸F]afatinib may also provide a method to select treatment responsive patients. The aim of this study was to label afatinib with fluorine-18 and evaluate its potential as TKI-PET tracer in tumor bearing mice.MethodsA radiochemically novel coupling, using peptide coupling reagent BOP, was explored and optimized to synthesize [¹⁸F]afatinib, followed by a metabolite analysis and biodistribution studies in two clinically relevant lung cancer cell lines, xenografted in nude mice.ResultsA reliable [¹⁸F]afatinib radiosynthesis was developed and the tracer could be produced in yields of 17.0 ± 2.5% calculated from [¹⁸F]F⁻ and >98% purity. The identity of the product was confirmed by co-injection on HPLC with non-labeled afatinib. Metabolite analysis revealed a moderate rate of metabolism, with >80% intact tracer in plasma at 45 min p.i. Biodistribution studies revealed rapid tumor accumulation and good retention for a period of at least 2 hours, while background tissues showed rapid clearance of the tracer.ConclusionWe have developed a method to synthesize [¹⁸F]afatinib and related fluorine-18 labeled 4-anilinoquinazolines. [¹⁸F]Afatinib showed good stability in vivo, justifying further evaluation as a TKI-PET tracer.     

Inflammation imaging of atherosclerosis in Apo-E-deficient mice using a 99mTc-labeled dual-domain cytokine ligand

Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) play a critical role in initiating and accelerating atherosclerosis. This study evaluated the imaging properties of ^99mTc-TNFR2-Fc-IL-1RA (^99mTc-TFI), a dual-domain cytokine radioligand that targets TNF-α and IL-1β pathways, in assessing atherosclerosis development in apolipoprotein-E-deficient (ApoE^−/−) mice.MethodsThe feasibility and specificity of detecting atherosclerosis with ^99mTc-TFI SPECT imaging were investigated in ApoE^−/− and ApoE^+/+ mice. Fifty-four ApoE^−/− mice were fed either an atherogenic diet (AGD) or a normal diet (ND) beginning at 5 weeks of age. Eighteen Apo-E wild-type (ApoE^+/+) mice were fed an ND. Two groups of ApoE^−/− mice (n = 12 each group) on AGD and ND were imaged three times with ^99mTc-TFI and a high-resolution SPECT system at 20–25, 30–40, and 48–52 weeks to study the evolution of atherosclerotic plaque.ResultsFocal radioactive accumulations in the aortic arch region were observed in the ApoE^−/− mice (n = 12) on AGD but not in the ApoE^+/+ mice on ND (n = 10). Apo-E^−/− mice on ND (n = 11) exhibited lower radioactive uptake than ApoE^−/− mice on AGD (P < 0.05). Co-injection of an excess of cold ligand with ^99mTc-TFI resulted in significant reduction of ^99mTc-TFI uptake in the ApoE^−/− mice on AGD. Longitudinal studies showed that ^99mTc-TFI uptake in the aortas of ApoE^−/− mice progressively increased from 20 to 48 weeks. Real-time PCR assays demonstrated that atherosclerotic aortas expressed significantly higher IL-1β and TNF-α than the aortas from wild-type controls.ConclusionsAtherosclerotic plaques were detected by ^99mTc-TFI imaging in ApoE^−/− mice. ^99mTc-TFI is promising for specific detection of inflammatory response in atherosclerotic plaques.