Background: Molecular targeting remains to be a promising approach in oncology. Overexpression of G protein-coupled receptors (GPCRs) in human cancer is offering a powerful opportunity for tumor-selective imaging and treatment employing nuclear medicine. We utilized novel chemerin-based peptide conjugates for chemokine-like receptor 1 (CMKLR1) targeting in a breast cancer xenograft model.Methods: By conjugation with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of five highly specific, high-affinity tracers for hybrid positron emission tomography/magnetic resonance (PET/MR) imaging. A xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. We acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments.Results: Five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be validated by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers [68Ga]Ga-DOTA-AHX-CG34, [68Ga]Ga-DOTA-KCap-CG34 and [68Ga]Ga-DOTA-ADX-CG34 with best tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 3.3% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. In addition, tumor uptake was blocked by excess of unlabeled peptide (6.4-fold, 5.5-fold and 3.4-fold 1 h post-injection), further confirming CMKLR1 specificity. Out of five tracers, we identified these three tracers with moderate, balanced hydrophilicity to be the most potent in receptor-mediated tumor targeting.Conclusion: We demonstrated the applicability of 68Ga-labeled peptide tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging, paving the way for developing them into theranostics for tumor treatment.

Surgery with curative intent can be offered to Congenital Hyperinsulinism (CHI) patients, provided that the lesion is focal. Radiolabeled Exendin-4 specifically binds the glucagon-like peptide 1 receptor (GLP-1R) on pancreatic beta cells. In this study we compared the performance of 18 F-DOPA positron emission tomography/computed tomography (DOPA PET), the current standard imaging method for CHI, and PET/CT with the new tracer 68 Ga-NODAGA-exendin-4 (Exendin PET) in the preoperative detection of focal CHI. MethodsNineteen CHI patients underwent both DOPA PET and Exendin PET prior to surgery. The images were evaluated in three settings a) standard clinical reading b) blinded expert reading and c) joined reading. Target (lesion) / non target (normal pancreas) ratio were determined using maximum standard uptake value (SUV max ). Image quality was rated by pediatric surgeons in a questionnaire. ResultsFourteen/nineteen patients having focal lesions underwent surgery. Based on clinical readings, the sensitivity of Exendin PET (100% (confidence interval 77-100%)) was higher than that of DOPA PET (71% (confidence interval 42-92%)). Interobserver agreement between readings was higher for Exendin than for DOPA PET (Fleiss' kappa 0.91 vs. 0.56). Exendin PET provided significantly (p=0.021) higher target / non target ratios (2.02 ± 0.65 ) than DOPA PET (1.40 ± 0.40). On a five point scale, Pediatric surgeons rated Exendin PET superior to DOPA PET. ConclusionExendin PET has higher clinical sensitivity and better interobserver correlation for the detection of focal CHI than DOPA PET. Better contrast and image quality makes Exendin PET superior to DOPA PET in surgeons' intra-operative quest for lesion localization.

BackgroundProstate-specific membrane antigen (PSMA) targeting radioligands have transformed treatment of prostate cancer. Radioligand therapy (RLT) with 177Lu-PSMA in metastasized castration resistant prostate cancer (mCRPC) achieves objective response and disease stabilization in roughly two third of patients, whereas one third of patients progress. This study was performed to assess the role of interim PSMA PET/CT after the 2nd cycle of RLT for early prediction of overall survival in patients undergoing RLT with 177Lu-PSMA.Methods38 mCRPC patients (68.9 ± 8.1 y) treated with at least two cycles of RLT at 8 week intervals and interim 68Ga-PSMA PET/CT (PET) at 8–10 weeks after the 2nd cycle of RLT were included in this study. Prostate-specific antigen (PSA) response was evaluated according to the Prostate Cancer Working Group 3 criteria. Radiographic response assessment of soft tissue, lymph node, and bone lesions was performed according to RECIST 1.1 including the PET component. Patients’ data were collected for follow-up from the local Comprehensive Cancer Center Register.ResultsMedian follow-up was 19.7 months (4.7–45.3). PSA response after the 2nd therapy cycle showed partial remission (PR) in 23.7%, stable disease (SD) in 50%, and progressive disease (PD) in 26.3% of patients. In comparison, 52.6, 23.7, and 23.7% of patients showed PR, SD, and PD respectively on PET/CT. The strength of agreement between PSA response and PET/CT response criteria was only fair (kappa 0.346). Median overall survival (OS) was 22.5 months (95% CI: 15.8–29.2). Median OS stratified to PSA/PET response was 25.6/25.6 months for PR, 21.7/30.6 months for SD and 19.4/13.1 months for PD (p = 0.496 for PSA and 0.013 for PET/CT response).ConclusionsInterim PSMA PET/CT based response evaluation at 8–10 weeks after the 2nd cycle of RLT is predictive of overall survival and PD in patients treated with 177Lu-PSMA. On the contrary, PSA appears to have only limited predictive value.

Peptide receptor radionuclide therapy (PRRT) using radiolabeled somatostatin receptor (SSTR) analogs is a common approach in advanced neuroendocrine neoplasms. Recently, SSTR antagonists have shown promising results for imaging and therapy due to a higher number of binding sites than in commonly used agonists. We evaluated PRRT with SSTR agonist 177 Lu-DOTATOC and antagonist 177 Lu-DOTA-JR11 longitudinally in an orthotopic murine pancreatic neuroendocrine neoplasm model expressing human SSTR2. Morphologic and metabolic changes during treatment were assessed using multimodal imaging, including hybrid PET/MRI and SPECT/ CT. Methods: In vitro radioligand binding and internalization assays and cell-cycle analysis were performed. SSTR2-transfected BON cells (BON-SSTR2) were used for in vivo experiments. Tumor-bearing mice received 2 intravenous injections of 100 μL of saline, 30 MBq of 177 Lu-DOTATOC, or 20 MBq of 177 Lu-DOTA-JR11 with an interval of 3 wk. Weekly T2-weighted MRI was performed for tumor monitoring. Viability of the tumor tissue was assessed by 18 F-FDG PET/MRI once after PRRT. Tumor and kidney uptake of the respective radiopharmaceuticals was measured 24 h after injection by SPECT/CT. Results: Compared with 177 Lu-DOTATOC, 177 Lu-DOTA-JR11 treatment resulted in an increased accumulation of cells in G2/M phase. Animals treated with the SSTR antagonist showed a significant reduction in tumor size (P , 0.001) and an increased median survival (207 d; interquartile range [IQR], 132-228) compared with 177 Lu-DOTATOC (126 d; IQR,(118)(119)(120)(121)(122)(123)(124)(125)(126)(127)(128)(129). SPECT/CT revealed a 4-fold higher median tumor uptake for the antagonist and a 3-fold higher tumor-to-kidney ratio in the first treatment cycle. During the second therapy cycle, tumor uptake of 177 Lu-DOTATOC was significantly lower (P 5 0.01) whereas 177 Lu-DOTA-JR11 uptake remained stable. Imaging of tumor morphology indicated comparatively larger necrotic fractions for 177 Lu-DOTA-JR11 despite further tumor growth. These results were confirmed by 18 F-FDG PET, revealing the least amount of viable tumor tissue in 177 Lu-DOTA-JR11-treated animals, at 6.2% (IQR, 2%-23%). Conclusion: 177 Lu-DOTA-JR11 showed a higher tumorto-kidney ratio and a more pronounced cytotoxic effect than did 177 Lu-DOTATOC. Additionally, tumor uptake was more stable over the course of 2 treatment cycles.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin αvβ6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin αvβ6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin αvβ6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC.MethodsThe expression of integrin αvβ6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model.ResultsRT-qPCR and immunofluorescence results showed high expression of integrin αvβ6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin αvβ6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 ± 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 ± 2.5 %IA/g, declining much more rapidly than in tumors.Conclusion177Lu-DOTA-integrin αvβ6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

Multimodal imaging probes have attracted the interest of ongoing research, for example, for the surgical removal of tumors. Modular synthesis approaches allow the construction of hybrid probes consisting of a radiotracer, a fluorophore and a targeting unit. We present the synthesis of a new asymmetric bifunctional cyanine dye that can be used as a structural and functional linker for the construction of such hybrid probes. 68Ga‐DOTATATE, a well‐characterized radiopeptide targeting the overexpressed somatostatin receptor subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our cyanine dye, thus providing additional information along with the data obtained from the radiotracer. We tested the SSTR2‐targeting and imaging properties of the resulting probe 68Ga‐DOTA‐ICC‐TATE in vitro and in a tumor xenograft mouse model. Despite the close proximity between dye and pharmacophore, we observed a high binding affinity towards SSTR2 as well as elevated uptake in SSTR2‐overexpressing tumors in the positron emission tomography (PET) scan and histological examination.

Purpose Melanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68 Gallium ( 68 Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the 68 Ga-DOTA peptide purified from the excess of unlabeled peptide. Procedures The MC1R ligand DOTA-NAPamide was labeled with 68 Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68 Ga (95°C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. Results In biodistribution studies, non-purified 68 Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified 68 Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. Conclusions We demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68 Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.

REPLY: Science needs differences in opinions to ensure that facts are presented in a truly reproducible way. That is why we welcome the comments by Banerjee et al. ( 1), and we have to admit that we actually agree with most of them.Needless to say, the results of our study must be tested for replicability-as every set of novel data must be tested. Having said that, we would like to highlight that we have never claimed that this study was a registration trial. In fact, we clearly state that these are the first results of clinical imaging of congenital hyperinsulinism with 68 Ga NODAGA-exendin-4, and we did raise this issue in the discussion and conclusion sections (2).Pertaining to the comments on retrospectively including 11 patients outside the prospective study, we do agree that there is a possibility of extending the bias. However, as the nature of the study was proof of concept and considering the expertise of the Charit e Berlin Centre in treating congenital hyperinsulinism patients from all around the world, we decided to include all available patient data.