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Yang Liu,1 Zhengyuan Zhou,2 Yutian Feng,2 Xiao-Guang Zhao,2 Ganesan Vaidyanathan,2 Michael R Zalutsky,1,2 Tuan Vo-Dinh1,3,4 1Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA; 2Department of Radiology, Duke University Medical Center, Durham, NC, 27710, USA; 3Department of Chemistry, Duke University, Durham, NC, 27708, USA; 4Fitzpatrick Institute for Photonics, Duke University, Durham, NC, 27708, USACorrespondence: Michael R ZalutskyDepartment of Radiology, Duke University Medical Center, 311 Research Drive, 161H Bryan Research Building, Durham, NC, 27710, USAEmail michael.zalutsky@duke.eduTuan Vo-DinhDepartment of Biomedical Engineering, Duke University, 1427 CIEMAS, Box 90281, Durham, NC, 27708, USATel +1 919 660-8520Email tuan.vodinh@duke.eduAim: To develop an innovative 211At nanoplatform with high radiolabeling efficiency and low in vivo deastatination for future targeted alpha-particle therapy (TAT) to treat cancer.Methods: Star-shaped gold nanoparticles, gold nanostars (GNS), were used as the platform for 211At radiolabeling. Radiolabeling efficiency under different reaction conditions was tested. Uptake in the thyroid and stomach after systemic administration was used to evaluate the in vivo stability of 211At-labeled GNS. A subcutaneous U87MG human glioma xenograft murine model was used to preliminarily evaluate the therapeutic efficacy of 211At-labeled GNS after intratumoral administration.Results: The efficiency of labeling GNS with 211At was almost 100% using a simple and rapid synthesis process that was completed in only 1 min. In vitro stability test in serum showed that more than 99% of the 211At activity remained on the GNS after 24 h incubation at 37°C. In vivo biodistribution results showed low uptake in the thyroid (0.44– 0.64%ID) and stomach (0.21– 0.49%ID) between 0.5 and 21 h after intravenous injection, thus indicating excellent in vivo stability of 211At-labeled GNS. The preliminary therapeutic efficacy study demonstrated that 211At labeled GNS substantially reduced tumor growth (P < 0.001; two-way ANOVA) after intratumoral administration.Conclusion: The new 211At radiolabeling strategy based on GNS has the advantages of a simple process, high labeling efficiency, and minimal in vivo dissociation, making it an attractive potential platform for developing TAT agents that warrants further evaluation in future preclinical studies directed to evaluating prospects for clinical translation.Keywords: astatine-211, 211At, gold nanostars, GNS, cancer therapy, targeted alpha-particle therapy, TAT

Tatiana A Slastnikova,1,* Andrey A Rosenkranz,1,2,* Natalia B Morozova,3 Maria S Vorontsova,3 Vasiliy M Petriev,4,5 Tatiana N Lupanova,1 Alexey V Ulasov,1 Michael R Zalutsky,6 Raisa I Yakubovskaya,3 Alexander S Sobolev1,2 1Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Russian Academy of Sciences, 2Department of Biophysics, Biological Faculty, Lomonosov Moscow State University, 3Department of Anticancer Therapy Modifiers and Protectors, Moscow Hertsen Research Institute of Oncology, Russian Ministry of Health Care, Moscow, 4National Medical Research Radiological Center, Russian Ministry of Health Care, Obninsk, Moscow Region, 5Department of Nuclear Medicine, National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Moscow, Russia; 6Department of Radiology, Duke University Medical Center, Durham, NC, USA *These authors contributed equally to this work Purpose: Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting 111In for cancer therapy.Methods: Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with 111In in high specific activity. Cytotoxicity of the 111In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted 111In-labeled MNT in B16-F1 melanoma tumor-bearing mice.Results: The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of 111In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with 111In nontargeted control. In mice with B16-F1 tumors, prolonged retention of 111In by serial imaging and significant tumor growth delay (82% growth inhibition) were found.Conclusion: The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted 111In-NOTA–MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted 111In-NOTA–MNT suggests further applications of the MNT delivery strategy should be explored. Keywords: nuclear delivery, cancer, melanoma, radionuclide therapy, Auger electrons

Tatiana A Slastnikova1,2, Andrey A Rosenkranz1,2, Pavel V Gulak1, Raymond M Schiffelers3, Tatiana N Lupanova1,4, Yuri V Khramtsov1, Michael R Zalutsky5, Alexander S Sobolev1,21Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology, Moscow, Russia; 2Department of Biophysics, Biological Faculty, Moscow State University, Vorobyevy Gory, Moscow, Russia; 3Laboratory for Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, the Netherlands; 4Department of Bioengineering, Biological Faculty, Moscow State University, Vorobyevy Gory, Moscow, Russia; 5Department of Radiology, Duke University Medical Center, Durham, NC, USABackground: Modular nanotransporters (MNT) are recombinant multifunctional polypeptides created to exploit a cascade of cellular processes, initiated with membrane receptor recognition to deliver selective short-range and highly cytotoxic therapeutics to the cell nucleus. This research was designed for in vivo concept testing for this drug delivery platform using two modular nanotransporters, one targeted to the α-melanocyte-stimulating hormone (αMSH) receptor overexpressed on melanoma cells and the other to the epidermal growth factor (EGF) receptor overexpressed on several cancers, including glioblastoma, and head-and-neck and breast carcinoma cells.Methods: In vivo targeting of the modular nanotransporter was determined by immunofluorescence confocal laser scanning microscopy and by accumulation of 125I-labeled modular nanotransporters. The in vivo therapeutic effects of the modular nanotransporters were assessed by photodynamic therapy studies, given that the cytotoxicity of photosensitizers is critically dependent on their delivery to the cell nucleus.Results: Immunohistochemical analyses of tumor and neighboring normal tissues of mice injected with multifunctional nanotransporters demonstrated preferential uptake in tumor tissue, particularly in cell nuclei. With 125I-labeled MNT{αMSH}, optimal tumor:muscle and tumor:skin ratios of 8:1 and 9.8:1, respectively, were observed 3 hours after injection in B16-F1 melanoma-bearing mice. Treatment with bacteriochlorin p-MNT{αMSH} yielded 89%–98% tumor growth inhibition and a two-fold increase in survival for mice with B16-F1 and Cloudman S91 melanomas. Likewise, treatment of A431 human epidermoid carcinoma-bearing mice with chlorin e6- MNT{EGF} resulted in 94% tumor growth inhibition compared with free chlorin e6, with 75% of animals surviving at 3 months compared with 0% and 20% for untreated and free chlorin e6-treated groups, respectively.Conclusion: The multifunctional nanotransporter approach provides a new in vivo functional platform for drug development that could, in principle, be applicable to any combination of cell surface receptor and agent (photosensitizers, oligonucleotides, radionuclides) requiring nuclear delivery to achieve maximum effectiveness.Keywords: drug delivery, nanobiotechnology, nanomedicine, cancer therapy, photosensitizers, multifunctional nanotransporter

Purpose: To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR).Methods and Materials: The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines.Results: The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [211 At]astatide for all three cell lines.Conclusion: The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted alpha-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor. [ABSTRACT FROM AUTHOR]

Introduction: Single domain antibody fragments (sdAbs) are an appealing scaffold for radiopharmaceutical development due to their small size (~15 kDa), high solubility, high stability, and excellent tumor penetration. Previously, we developed NB7 sdAb, which has very high affinity for an epitope on PSMA that is different from those targeted by small molecule PSMA inhibitors. Herein, we evaluated NB7 after radioiodination using [*I]SGMIB (1,3,4-isomer) and iso-[*I]SGMIB (1,3,5-isomer), as well as their 211 At-labeled analogues., Methods: [*I]SGMIB, iso-[*I]SGMIB, [ 211 At]SAGMB, and iso-[ 211 At]SAGMB conjugates of NB7 sdAb were synthesized and their binding affinity, cell uptake and internalization were assessed in PSMA + PC3 PIP and PSMA - PC3 flu cells. Biodistribution studies were performed in mice bearing PSMA + PC3 PIP xenografts. First, a single-label experiment evaluated the tissue distribution of a NB7 bearing a His 6 -tag (NB7H6) and labeled with iso-[ 125 I]SGMIB. Three paired-label experiments then were performed to compare: a) NB7 labeled using [*I]SGMIB and iso-[*I]SGMIB, b) 131 I- vs 211 At-labeled NB7 conjugates and c) [ 125 I]SGMIB-NB7H6 to the small molecule PSMA inhibitor [ 131 I]YF2., Results: All NB7 radioconjugates bound specifically to PSMA with dissociation constants, K d , in the low nM range (1.4-6.4 nM). An initial biodistribution study demonstrated good tumor uptake for iso-[ 125 I]SGMIB-NB7H6 (7.2 ± 1.5 % ID/g at 1 h) and no deleterious effect of the His 6 -tag on renal activity levels, which declined to 3.1 ± 1.1 % ID/g by 4 h. Paired-label biodistribution found no distinction between the two SGMIB isomer NB7 conjugates with the [ 131 I]SGMIB-NB7-to-iso-[ 125 I]SGMIB-NB7 tumor uptake ratios not significantly different from unity: 1.06 ± 0.08 at 1 h, 1.04 ± 0.12 at 4 h, and 1.07 ± 0.09 at 24 h. Both isomer conjugates cleared rapidly from normal tissues and exhibited very low uptake in thyroid, lacrimal and salivary glands. Paired-label biodistribution of [ 131 I]SGMIB-NB7H6 and [ 211 At]SAGMB-NB7H6 demonstrated similar tumor uptake and kidney clearance for the two radioconjugates. However, levels of 211 At in thyroid, stomach, salivary and lacrimal glands were significantly higher (P < 0.05) that those for 131 I suggesting greater dehalogenation for [ 211 At]SAGMB-NB7H6. Finally, co-administration of [ 125 I]SGMIB-NB7H6 and [ 131 I]YF2 demonstrated good tumor uptake for both with considerably more rapid renal clearance for the NB7 radioconjugate., Conclusion: NB7 radioconjugates exhibited good accumulation in PSMA-positive xenografts with rapid clearance from kidney and other normal tissues. We conclude that NB7 is a potentially useful scaffold for developing PSMA-targeted theranostics with different characteristics than current small molecule and antibody-based approaches., Competing Interests: Declaration of competing interest LR and NP have filed a patent application that includes the NB7 single domain antibody fragment. The authors declare that they have no other known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Targeted α-particle therapy agents have shown promising responses in patients who have developed resistance to β - -particle emitting radionuclides, albeit off-target toxicity remains a concern. Astatine-211 emits only one α-particle per decay and may alleviate the toxicity from α-emitting daughter radionuclides. Previously, we developed the low-molecular-weight PSMA-targeted agent [ 211 At]L3-Lu that showed suitable therapeutic efficacy and was well tolerated in mice. Although [ 211 At]L3-Lu had good characteristics, we now have evaluated a closely related analogue, [ 211 At]YF2, to determine the better molecule for clinical translation., Methods: The tin precursors and unlabeled iodo standards for [ 211 At]YF2 and [ 211 At]L3-Lu each were synthesized and a new one-step labeling method was developed to produce [ 211 At]YF2 and [ 211 At]L3-Lu from the respective tin precursor. RCY and RCP were determined using RP-HPLC. Cell uptake, internalization and in vitro cell-killing (MTT) assays were performed on PSMA + PC-3 PIP cells in parallel experiments to compare [ 211 At]YF2 and [ 211 At]L3-Lu directly. A paired-label biodistribution study was performed in athymic mice with subcutaneous PSMA-positive PC-3 PIP xenografts as a head-to-head comparison of [ 131 I]YF2 and [ 125 I]L3-Lu. The tissue distribution of [ 211 At]YF2 and [ 211 At]L3-Lu were determined individually in the same animal model., Results: The syntheses of tin precursors and unlabeled iodo standards were accomplished in reasonable yields. A streamlined and scalable radiolabeling method (1 h total synthesis time) was developed for the radiosynthesis of both [ 211 At]YF2 and [ 211 At]L3-Lu with 86 ± 7 % (n = 10) and 87 ± 5 % (n = 7) RCY, respectively, and > 95 % RCP for both. The maximum activity of [ 211 At]YF2 produced to date was 666 MBq. An alternative method that did not involve HPLC purification was developed that provided similar RCY and RCP. Significantly higher cell uptake, internalization and cytotoxicity was seen for [ 211 At]YF2 compared with [ 211 At]L3-Lu. Significantly higher uptake and longer retention in tumor was seen for [ 131 I]YF2 than for co-administered [ 125 I]L3-Lu, while considerably higher renal uptake was seen for [ 131 I]YF2. The biodistribution of [ 211 At]YF2 was consistent with that of [ 131 I]YF2., Conclusion: [ 211 At]YF2 exhibited higher cellular uptake, internalization and cytotoxicity than [ 211 At]L3-Lu on PSMA-positive PC3 PIP cells. Likewise, higher uptake and longer retention in tumor was seen for [ 211 At]YF2. Experiments to evaluate the dosimetry and therapeutic efficacy of [ 211 At]YF2 are under way., Competing Interests: Declaration of competing interest Ganesan Vaidyanathan, Martin Pomper and Michael Zalutsky are co-inventors on a patent related to the labeled PSMA inhibitors described in this study and discussed in this publication. They are entitled to royalty distributions related to this technology, which was originally licensed by Precision Molecular, Inc. and now transferred to z-alpha, Inc. Martin Pomper and Michael Zalutsky are co-founders and paid consultants of z-alpha and are minority shareholders via stock options. No other potential competing interests related to this work are reported., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The present review aims to explore the potential targets/partners for future targeted radionuclide therapy (TRT) strategies, wherein cancer cells often are not killed effectively, despite receiving a high average tumor radiation dose. Here, we shall discuss the key factors in the cancer genome, especially those related to DNA damage response/repair and maintenance systems for escaping cell death in cancer cells. To overcome the current limitations of TRT effectiveness due to radiation/drug-tolerant cells and tumor heterogeneity, and to make TRT more effective, we propose that a promising strategy would be to target the DNA maintenance factors that are crucial for cancer survival. Considering their cancer-specific DNA damage response/repair ability and dysregulated transcription/epigenetic system, key factors such as PARP, ATM/ATR, amplified/overexpressed transcription factors, and DNA methyltransferases have the potential to be molecular targets for Auger electron therapy; moreover, their inhibition by non-radioactive molecules could be a partnering component for enhancing the therapeutic response of TRT.

Single-domain antibody fragments (sdAbs) are attractive for targeted α-particle therapy, particularly with 211 At, because of their rapid accumulation in tumor and clearance from normal tissues. Here, we evaluate the therapeutic potential of this strategy with 5F7 and VHH&#95;1028-2 sdAbs that bind with high affinity to domain IV of human epidermal growth factor receptor type 2 (HER2). Methods: The HER2-specific sdAbs and HER2-irrelevant VHH&#95;2001 were labeled using N -succinimidyl-3- 211 At-astato-5-guanidinomethyl benzoate ( iso - 211 At-SAGMB). The cytotoxicity of iso- 211 At-SAGMB-5F7 and iso- 211 At-SAGMB-VHH&#95;2001 were compared on HER2-expressing BT474 breast carcinoma cells. Three experiments in mice with subcutaneous BT474 xenografts were performed to evaluate the therapeutic effectiveness of single doses of iso- 211 At-SAGMB-5F7 (0.7-3.0 MBq), iso- 211 At-SAGMB-VHH&#95;1028 (1.0-3.0 MBq), and iso- 211 At-SAGMB-VHH&#95;1028 and iso- 211 At-SAGMB-VHH&#95;2001 (∼1.0 MBq). Results: Clonogenic survival of BT474 cells was reduced after exposure to iso- 211 At-SAGMB-5F7 (D 0  = 1.313 kBq/mL) whereas iso- 211 At-SAGMB-VHH&#95;2001 was ineffective. Dose-dependent tumor growth inhibition was observed with 211 At-labeled HER2-specific 5F7 and VHH&#95;1028 but not with HER2-irrelevant VHH&#95;2001. At the 3.0-MBq dose, complete tumor regression was seen in 3 of 4 mice treated with iso- 211 At-SAGMB-5F7 and 8 of 11 mice treated with iso- 211 At-SAGMB-VHH&#95;1028; prolongation in median survival was 495% and 414%, respectively. Conclusion: Combining rapidly internalizing, high-affinity HER2-targeted sdAbs with the iso- 211 At-SAGMB residualizing prosthetic agent is a promising strategy for targeted α-particle therapy of HER2-expressing cancers., (© 2023 by the Society of Nuclear Medicine and Molecular Imaging.)

Because of their rapid tumor accumulation and normal tissue clearance, single-domain antibody fragments (sdAbs) are an attractive vehicle for developing radiotherapeutics labeled with the α-emitter 211 At. Herein, we have evaluated iso -[ 211 At]AGMB-PODS, a prosthetic agent that combines a functionality for residualizing radiohalogens with a phenyloxadiazolyl methylsulfone (PODS) moiety for site-specific sdAb conjugation. Iso -[ 211 At]AGMB-PODS and its radioiodinated analogue were evaluated for thiol-selective conjugation to anti-HER2 5F7 sdAb bearing a C-terminus GGC tail. Both radiohalogenated PODS-5F7GGC conjugates were synthesized in good radiochemical yields and retained high binding affinity on HER2-positive BT474 breast carcinoma cells. Iso -[ 211 At]AGMB-PODS-5F7GGC was considerably more stable in vitro than its maleimide analogue in the presence of cysteine and human serum albumin (HSA) and exhibited excellent tumor uptake and high in vivo stability. Superior tumor-to-kidney activity ratios were seen for both radiohalogenated PODS-5F7GGC conjugates compared with [ 177 Lu]Lu-DOTA-PODS-5F7GGC. These results suggest that iso -[ 211 At]AGMB-PODS-5F7GGC warrants further evaluation for the treatment of HER2-expressing malignancies.

Locally advanced pancreatic tumours are highly resistant to conventional radiochemotherapy. Here we show that such resistance can be surmounted by an injectable depot of thermally responsive elastin-like polypeptide (ELP) conjugated with iodine-131 radionuclides ( 131 I-ELP) when combined with systemically delivered nanoparticle albumin-bound paclitaxel. This combination therapy induced complete tumour regressions in diverse subcutaneous and orthotopic mouse models of locoregional pancreatic tumours. 131 I-ELP brachytherapy was effective independently of the paclitaxel formulation and dose, but external beam radiotherapy (EBRT) only achieved tumour-growth inhibition when co-administered with nanoparticle paclitaxel. Histological analyses revealed that 131 I-ELP brachytherapy led to changes in the expression of intercellular collagen and junctional proteins within the tumour microenvironment. These changes, which differed from those of EBRT-treated tumours, correlated with the improved delivery and accumulation of paclitaxel nanoparticles within the tumour. Our findings support the further translational development of 131 I-ELP depots for the synergistic treatment of localized pancreatic cancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Single-domain antibody fragments (aka V H H, ~ 13 kDa) are promising delivery systems for brain tumor theranostics; however, achieving efficient delivery of V H H to intracranial lesions remains challenging due to the tumor-brain barrier. Here, we evaluate low-dose whole-brain irradiation as a strategy to increase the delivery of an anti- human epidermal growth factor receptor type 2 (HER2) V H H to breast cancer-derived intracranial tumors in mice., Methods: Mice with intracranial HER2-positive BT474BrM3 tumors received 10-Gy fractionated cranial irradiation and were evaluated by noninvasive imaging. Anti-HER2 V H H 5F7 was labeled with 18 F, administered intravenously to irradiated mice and controls, and PET/CT imaging was conducted periodically after irradiation. Tumor uptake of 18 F-labeled 5F7 in irradiated and control mice was compared by PET/CT image analysis and correlated with tumor volumes. In addition, longitudinal dynamic contrast-enhanced MRI (DCE-MRI) was conducted to visualize and quantify the potential effects of radiation on tumor perfusion and permeability., Results: Increased 18 F-labeled 5F7 intracranial tumor uptake was observed with PET in mice receiving cranial irradiation, with maximum tumor accumulation seen approximately 12 days post initial radiation treatment. No radiation-induced changes in HER2 expression were detected by Western blot, flow cytometry, or on tissue sections. DCE-MRI imaging demonstrated transiently increased tumor perfusion and permeability after irradiation, consistent with the higher tumor uptake of 18 F-labeled anti-HER2 5F7 in irradiated mice., Conclusion: Low-level brain irradiation induces dynamic changes in tumor vasculature that increase the intracranial tumor delivery of an anti-HER2 V H H, which could facilitate the use of radiolabeled V H H to detect, monitor, and treat HER2-expressing brain metastases., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Stapled peptides are promising scaffolds for inhibiting protein-protein interactions in cells, including between the intracellular oncoprotein MDM2 and p53. Herein, we have investigated the potential utility of a stapled peptide, VIP116, for developing radiolabeled agents targeting MDM2. VIP116 was radioiodinated using the prosthetic agent N-succinimidyl-3-[*I]iodobenzoate ([*I]SIB). The resulting labeled peptide [*I]SIB-VIP116 exhibited high uptake (165.3 ± 27.7%/mg protein) and specificity in SJSA-1 tumor cells. Tissue distribution studies of [*I]SIB-VIP116 revealed a peak tumor uptake of 2.19 ± 0.56 percent injected dose per gram (%ID/g) in SJSA-1 xenografts at 2 h post-injection, which was stable until 6 h. [*I]SIB-VIP116 exhibited high activity (8.33 ± 1.18%ID/g) in the blood pool but had high tumor-to-muscle ratios (12.0 ± 5.7), at 30 min. Metabolic stability studies in mice indicated that about 80% of the activity in plasma was intact [*I]SIB-VIP116 at 4 h. Our results confirm the cell permeability and specific binding of [*I]SIB-VIP116 to MDM2 and the suitability of the VIP116 scaffold for radiolabeled probe development., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Radiopharmaceutical therapy (RPT) is an attractive strategy for treatment of disseminated cancers including those overexpressing the HER2 receptor including breast, ovarian and gastroesophageal carcinomas. Single-domain antibody fragments (sdAbs) exemplified by the HER2-targeted VHH&#95;1028 evaluated herein are attractive for RPT because they rapidly accumulate in tumor and clear faster from normal tissues than intact antibodies. In this study, VHH&#95;1028 was labeled using the residualizing prosthetic agent N-succinimidyl 3-guanidinomethyl 5-[ 131 I]iodobenzoate (iso-[ 131 I]SGMIB) and its tissue distribution evaluated in the HER2-expressing SKOV-3 ovarian and BT474 breast carcinoma xenograft models. In head-to-head comparisons to [ 131 I]SGMIB-2Rs15d, a HER2-targeted radiopharmaceutical currently under clinical investigation, iso-[ 131 I]SGMIB-VHH&#95;1028 exhibited significantly higher tumor uptake and significantly lower kidney accumulation. The results demonstrated 2.9 and 6.3 times more favorable tumor-to-kidney radiation dose ratios in the SKOV-3 and BT474 xenograft models, respectively. Iso-[ 131 I]SGMIB-VHH&#95;1028 was prepared using a solid-phase extraction method for purification of the prosthetic agent intermediate Boc 2 -iso-[ 131 I]SGMIB that reproducibly scaled to therapeutic-level doses and obviated the need for its HPLC purification. Single-dose (SKOV-3) and multiple-dose (BT474) treatment regimens demonstrated that iso-[ 131 I]SGMIB-VHH&#95;1028 was well tolerated and provided significant tumor growth delay and survival prolongation. This study suggests that iso-[ 131 I]SGMIB-VHH&#95;1028 is a promising candidate for RPT of HER2-expressing cancers and further development is warranted., (© 2022. The Author(s).)

α-Particle emitters targeting the prostate-specific membrane antigen (PSMA) proved effective in treating patients with prostate cancer who were unresponsive to the corresponding β-particle therapy. 211 At is an α-emitter that may engender less toxicity than other α-emitting agents. We synthesized a new 211 At-labeled radiotracer targeting PSMA that resulted from the search for a pharmacokinetically optimized agent. Methods: A small series of 125 I-labeled compounds was synthesized from tin precursors to evaluate the effect of the location of the radiohalogen within the molecule and the presence of lutetium in the chelate on biodistribution. On that basis, 211 At- 3 -Lu was selected and evaluated in cell uptake and internalization studies, and biodistribution and PSMA-expressing (PSMA+) PC3 PIP tumor growth control were evaluated in experimental flank and metastatic (PC3-ML-Luc) models. A long-term (13-mo) toxicity study was performed for 211 At- 3 -Lu, including tissue chemistries and histopathology. Results: The radiochemical yield of 211 At- 3 -Lu was 17.8% ± 8.2%. Lead compound 211 At- 3 -Lu demonstrated total uptake within PSMA+ PC3 PIP cells of 13.4 ± 0.5% of the input dose after 4 h of incubation, with little uptake in control cells. In SCID mice, 211 At- 3 -Lu provided uptake that was 30.6 ± 4.8 percentage injected dose per gram (%ID/g) in PSMA+ PC3 PIP tumor at 1 h after injection, and this uptake decreased to 9.46 ± 0.96 %ID/g by 24 h. Tumor-to-salivary gland and tumor-to-kidney ratios were 129 ± 99 at 4 h and 130 ± 113 at 24 h, respectively. Deastatination was not significant (stomach, 0.34 ± 0.20 %ID/g at 4 h). Dose-dependent survival was demonstrated at higher doses (>1.48 MBq) in both flank and metastatic models. There was little off-target toxicity, as demonstrated by hematopoietic stability, unchanged tissue chemistries, weight gain rather than loss throughout treatment, and favorable histopathologic findings. Conclusion: Compound 211 At- 3 -Lu or close analogs may provide limited and acceptable toxicity while retaining efficacy in management of prostate cancer., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Single-domain antibody fragments (sdAbs) are promising vectors for immuno-PET; however, better methods for labeling sdAbs with 18 F are needed. Herein, we evaluate a site-specific strategy using an 18 F residualizing motif and the anti-epidermal growth factor receptor 2 (HER2) sdAb 5F7 bearing an engineered C-terminal GGC tail (5F7GGC). Methods: 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with 18 F by inverse electron demand Diels-Alder cycloaddition with a trans -cyclooctene attached to 6- 18 F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG 4 chain ( 18 F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, N -succinimidyl 3-(guanidinomethyl)-5- 125 I-iodobenzoate ( iso - 125 I-SGMIB). The 2 labeled sdAbs were compared in paired-label studies performed in the HER2-expressing BT474M1 breast carcinoma cell line and athymic mice bearing BT474M1 subcutaneous xenografts. Small-animal PET/CT imaging after administration of 18 F-5F7GGC in the above mouse model was also performed. Results: 18 F-5F7GGC was synthesized in an overall radiochemical yield of 8.9% ± 3.2% with retention of HER2 binding affinity and immunoreactivity. The total cell-associated and intracellular activity for 18 F-5F7GGC was similar to that for coincubated iso - 125 I-SGMIB-5F7. Likewise, the uptake of 18 F-5F7GGC in BT474M1 xenografts in mice was similar to that for iso - 125 I-SGMIB-5F7; however, 18 F-5F7GGC exhibited significantly more rapid clearance from the kidney. Small-animal PET/CT imaging confirmed high uptake and retention in the tumor with very little background activity at 3 h except in the bladder. Conclusion: This site-specific and residualizing 18 F-labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immuno-PET., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Aim: To develop an innovative 211 At nanoplatform with high radiolabeling efficiency and low in vivo deastatination for future targeted alpha-particle therapy (TAT) to treat cancer., Methods: Star-shaped gold nanoparticles, gold nanostars (GNS), were used as the platform for 211 At radiolabeling. Radiolabeling efficiency under different reaction conditions was tested. Uptake in the thyroid and stomach after systemic administration was used to evaluate the in vivo stability of 211 At-labeled GNS. A subcutaneous U87MG human glioma xenograft murine model was used to preliminarily evaluate the therapeutic efficacy of 211 At-labeled GNS after intratumoral administration., Results: The efficiency of labeling GNS with 211 At was almost 100% using a simple and rapid synthesis process that was completed in only 1 min. In vitro stability test in serum showed that more than 99% of the 211 At activity remained on the GNS after 24 h incubation at 37°C. In vivo biodistribution results showed low uptake in the thyroid (0.44-0.64%ID) and stomach (0.21-0.49%ID) between 0.5 and 21 h after intravenous injection, thus indicating excellent in vivo stability of 211 At-labeled GNS. The preliminary therapeutic efficacy study demonstrated that 211 At labeled GNS substantially reduced tumor growth (P < 0.001; two-way ANOVA) after intratumoral administration., Conclusion: The new 211 At radiolabeling strategy based on GNS has the advantages of a simple process, high labeling efficiency, and minimal in vivo dissociation, making it an attractive potential platform for developing TAT agents that warrants further evaluation in future preclinical studies directed to evaluating prospects for clinical translation., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Liu et al.)

RG7388 (Idasanutlin) is a potent inhibitor of oncoprotein murine double minute 2 (MDM2). Herein we investigated the feasibility of developing 18 F-labeled RG7388 as a radiotracer for imaging MDM2 expression in tumors with positron emission tomography (PET). Two fluorinated analogues of RG7388, 6 and 7 , were synthesized by attaching a fluoronicotinyl moiety to RG7388 via a polyethylene glycol (PEG 3 ) or a propyl linker. The inhibitory potency (IC 50 ) of 6 and 7 against MDM2 was determined by a fluorescence polarization (FP)-based assay. Next, compound 6 was labeled with 18 F using a trimethylammonium triflate precursor to obtain [ 18 F]FN-PEG 3 -RG7388 ([ 18 F] 6 ), and its properties were evaluated in MDM2 expressing wild-type p53 tumor cell lines (SJSA-1 and HepG2) in vitro and in tumor xenografts in vivo. The FP assays revealed an IC 50 against MDM2 of 119 nM and 160 nM for 6 and 7 , respectively. 18 F-labeling of 6 was achieved in 50.3 ± 7.5% radiochemical yield. [ 18 F] 6 exhibited a high uptake (∼70% of input dose) and specificity in SJSA-1 and HepG2 cell lines. Saturation binding assays revealed a binding affinity ( K d ) of 128 nM for [ 18 F] 6 on SJSA-1 cells. In mice, [ 18 F] 6 showed fast clearance from blood with a maximum tumor uptake of 3.80 ± 0.85% injected dose per gram (ID/g) in HepG2 xenografts at 30 min postinjection (p.i.) and 1.32 ± 0.32% ID/g in SJSA-1 xenografts at 1 h p.i. Specificity of [ 18 F] 6 uptake in tumors was demonstrated by pretreatment of mice with SJSA-xenografts with a blocking dose of RG7388 (35 mg/kg body weight, i.p.). In vivo stability studies in mice using HPLC showed ∼60% and ∼30% intact [ 18 F] 6 remaining in plasma at 30 min and 1 h p.i., respectively, with the remaining activity attributed to polar peaks. Our results suggest that RG7388 is a promising molecular scaffold for 18 F-labeled probe development for MDM2. Additional labeling strategies and functionalizing locations on RG7388 are under development to improve binding affinity and in vivo stability of the 18 F-labeled compound to make it more amenable for PET imaging of MDM2 in vivo.

The promising characteristics of the 7.2-h radiohalogen 211 At have long been recognized; including having chemical properties suitable for labeling targeting vectors ranging from small organic molecules to proteins, and the emission of only one α-particle per decay, providing greater control over off-target effects. Unfortunately, the impact of 211 At within the targeted α-particle therapy domain has been constrained by its limited availability. Paradoxically, the most commonly used production method - via the 209 Bi(α,2n) 211 At reaction - utilizes a widely available natural material (bismuth) as the target and straightforward cyclotron irradiation methodology. On the other hand, the most significant impediment to widespread 211 At availability is the need for an accelerator capable of generating ≥28 MeV α-particles with sufficient beam intensities to make clinically relevant levels of 211 At. In this review, current methodologies for the production and purification of 211 At - both by the direct production route noted above and via a 211 Rn generator system - will be discussed. The capabilities of cyclotrons that currently produce 211 At will be summarized and the characteristics of other accelerators that could be utilized for this purpose will be described. Finally, the logistics of networks, both academic and commercial, for facilitating 211 At distribution to locations remote from production sites will be addressed., Competing Interests: Declaration of competing interest M.R.Z. is a co-inventor on patents and patent applications for high-level (211)At labeling as well as (211)At-labeled compounds of potential utility for targeted radiotherapy. The authors declare no other known potential competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: Labeling single domain antibody fragments (sdAbs) with 18 F is an attractive strategy for immunoPET. Earlier, we developed a residualizing label, N-succinimidyl 3-((4-(4-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([ 18 F]RL-I), synthesized via a click reaction for labeling sdAbs with 18 F, that has attractive features but suffered from modest radiochemical yields and suboptimal hydrophobicity. Herein, we have evaluated the potential utility of an analogous agent, N-succinimidyl 3-(1-(2-(2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([ 18 F]SFETGMB; [ 18 F]RL-III) designed to address these limitations., Methods: [ 18 F]RL-III was synthesized by the click reaction between 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate and 1-azido-2-(2-(2-(2-[ 18 F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [ 18 F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[ 125 I]iodobenzoate (iso-[ 125 I]SGMIB). The 18 F-labeled sdAbs were evaluated in vitro using HER2-expressing breast and ovarian carcinoma cells (BT474/BT474M1 and SKOV-3) and in vivo in athymic mice bearing subcutaneous SKOV-3 or BT474 xenografts. PET imaging of athymic mice bearing either subcutaneous BT474 or intracranial BT474M1Br-Fluc xenografts after administration of [ 18 F]RL-III-5F7 also was performed., Results: Radiochemical yields for the synthesis of Boc 2 -[ 18 F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc 2 -[ 18 F]RL-I. The overall radiochemical yields for the synthesis of [ 18 F]RL-III-2Rs15d and [ 18 F]RL-III-5F7 from aqueous [ 18 F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [ 18 F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [ 18 F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [ 18 F]RL-III-2Rs15d. Although different xenograft models were used, [ 18 F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [ 18 F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [ 18 F]RL-III-5F7 in the BT474 subcutaneous model demonstrated clear delineation of the tumor (12.2 ± 5.1% ID/g) with minimal background activity except in kidneys. A tumor uptake (max) of 0.98%ID/g and a tumor-to-normal brain ratio of 9.8:1 were observed for [ 18 F]RL-III-5F7 in the intracranial model., Conclusions: Although higher radiochemical yields than that reported for [ 18 F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [ 18 F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern., Competing Interests: Declaration of competing interest Michael R. Zalutsky holds ownership interest and is a board member for Cereius, Inc. Ganesan Vaidyanathan is a consultant and shareholder for Cereius, Inc. There are no other potential conflicts of interest relevant to this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Murine double minute 2 (MDM2), a negative regulator of the p53 tumor suppressor protein, is overexpressed in several human cancers. Herein we investigate the feasibility of developing 18 F-labeled compounds based on the small molecule inhibitor SP-141 for imaging tumor MDM2 expression levels with positron emission tomography (PET). Three nonradioactive fluorinated SP-141 analogues, 1 - 3 , were synthesized, and their binding to the MDM2 protein was analyzed by surface plasmon resonance (SPR). One of these, a fluoroethoxy analogue, was labeled with fluorine-18 ( 18 F) using 18 F-fluorethyl bromide to provide [ 18 F] 1 and evaluated in vitro and in vivo. SPR analysis confirmed the binding of the fluorinated analogues to MDM2 at 1.25-20 µM concentrations. Cell uptake studies revealed high uptake (67.5-71.4%/mg protein) and specificity of [ 18 F] 1 in MCF7 and HepG2 cells. The uptake of [ 18 F] 1 in these cells could be modulated using 100 µM SP-141, potentially reflecting changes in MDM2 expression because of p53 activation by SP-141. [ 18 F] 1 exhibited stable uptake and retention in HepG2 tumor xenografts (~3 %ID/g) in vivo, but poor clearance from blood and other normal tissues, yielding low tumor-to-background ratios (<2) at 2 h post injection. Our results suggest that [ 18 F] 1 has suboptimal characteristics for in vivo evaluation as a PET tracer for MDM2, but warrant radiolabeling and assessment of the other fluorinated analogues synthesized in this work, 2 and 3 , and potentially other molecular scaffolds for developing MDM2 targeted radiotracers.

Introduction: The high potency and short tissue range of α-particles are attractive features for targeted radionuclide therapy, particularly for cancers with micro-metastases. In the current study, we describe the synthesis of a series of 211 At-labeled prostate-specific membrane antigen (PSMA) inhibitors and their preliminary evaluation as potential agents for metastatic prostate cancer treatment., Methods: Four novel Glu-urea based PSMA ligands containing a trialkyl stannyl group were synthesized and labeled with 211 At, and for comparative purposes, 131 I, via halodestannylation reactions with N-chlorosuccinimide as the oxidant. A PSMA inhibitory assay was performed to evaluate PSMA binding of the unlabeled, iodinated compounds. A series of paired-label biodistribution experiments were performed to compare each 211 At-labeled PSMA ligand to its 131 I-labeled counterpart in mice bearing subcutaneous PC3 PSMA+ PIP xenografts., Results: Radiochemical yields ranged from 32% to 65% for the 211 At-labeled PSMA inhibitors and were consistently lower than those obtained with the corresponding 131 I-labeled analogue. Good localization in PC3 PSMA+ PIP but not control xenografts was observed for all labeled molecules studied, which exhibited a variable degree of in vivo dehalogenation as reflected by thyroid and stomach activity levels. Normal tissue uptake and in vivo stability for several of the compounds was markedly improved compared with the previously evaluated compounds, [ 211 At]DCABzL and [*I]DCIBzL., Conclusions and Implications for Patient Care: Compared with the first generation compound [ 211 At]DCABzL, several of the novel 211 At-labeled PSMA ligands exhibited markedly improved stability in vivo and higher tumor-to-normal tissue ratios. [ 211 At]GV-620 has the most promising characteristics and warrants further evaluation as a targeted radiotherapeutic for prostate cancer., Competing Interests: Declaration of competing interest G.V., R.M., I.L., M.P. and M.R.Z. are co-inventors on filed patent applications that cover (211)At- and (131)I-labeled PSMA inhibitors including the molecules comprising this study. We declare no other competing interests related to this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: As a consequence of their small size, high stability and high affinity, single domain antibody fragments (sdAbs) are appealing targeting vectors for radiopharmaceutical development. With sdAbs binding to internalizing receptors like HER2, residualizing prosthetic agents can enhance tumor retention of radioiodine, which until now has been done with random labeling approaches. Herein we evaluate a site-specific strategy utilizing a radioiodinated, residualizing maleimido moiety and the anti-HER2 sdAb 5F7 bearing a GGC tail for conjugation., Methods: Maleimidoethyl 3-(guanidinomethyl)-5-iodobenzoate ([ 131 I]MEGMB) and its N-succinimidyl ester analogue, iso-[ 125 I]SGMIB, were labeled by halodestannylation and conjugated with 5F7GGC and 5F7, respectively. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label experiments directly compared iso-[ 125 I]SGMIB-5F7 and [ 131 I]MEGMIB-5F7GGC with regard to internalization/residualization and affinity on HER2-expressing SKOV-3 ovarian carcinoma cells as well as biodistribution and metabolite distribution in athymic mice with subcutaneous SKOV-3 xenografts., Results: [ 131 I]MEGMIB-5F7GGC had an immunoreactivity of 81.3% and K d  = 0.94 ± 0.27 nM. Internalization assays demonstrated high intracellular trapping for both conjugates, For example, at 1 h, intracellular retention was 50.30 ± 3.36% for [ 131 I]MEGMIB-5F7GGC and 55.95 ± 3.27% for iso-[ 125 I]SGMIB-5F7, while higher retention was seen for iso-[ 125 I]SGMIB-5F7 at later time points. Peak tumor uptake was similar for both conjugates (8.35 ± 2.66%ID/g and 8.43 ± 2.84%ID/g for iso-[ 125 I]SGMIB-5F7 and [ 131 I]MEGMIB-5F7GGC at 1 h, respectively); however, more rapid normal tissue clearance was seen for [ 131 I]MEGMIB-5F7GGC, with a 2-fold higher tumor-to-kidney ratio and a 3-fold higher tumor-to-liver ratio compared with co-injected iso-[ 125 I]SGMIB-5F7. Consisted with this, generation of labeled catabolites in the kidneys was higher for [ 131 I]MEGMIB-5F7GGC., Conclusion: [ 131 I]MEGMIB-5F7GGC offers similar tumor targeting as iso-[ 125 I]SGMIB-5F7 but with generally lower normal tissue uptake., Advances in Knowledge and Implication for Patient Care: The site specific nature of the [ 131 I]MEGMIB reagent may facilitate clinical translation, particularly for sdAb with compromised affinity after random labeling., Competing Interests: Declaration of competing interest G.V. and M.Z are consultants of Cereius, Inc. and hold ownership interest, including patents/patent applications in single domain antibody fragment radiolabeling methods., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Improvements in the diagnosis and treatment of systemic breast cancer have led to a prolongation in patient survival. Unfortunately, these advances are also associated with an increased incidence of brain metastases (BM), with the result that many patients succumb due to BM treatment failure. Intracranial delivery of many chemotherapeutic agents and other therapeutics is hindered by the presence of an impermeable blood-brain barrier (BBB) designed to protect the brain from harmful substances. The formation of BM compromises the integrity of the BBB, resulting in a highly heterogeneous blood-tumor barrier (BTB) with varying degrees of vascular permeability. Here, we discuss how blood vessels play an important role in the formation of brain micrometastases as well as in the transformation from poorly permeable BM to highly permeable BM. We then review the role of BTB vascular permeability in the diagnostics and the choice of treatment regimens for breast cancer brain metastases (BCBM) and discuss whether the vasculature of primary breast cancers can serve as a biomarker for BM. Specifically, we examine the association between the vascular permeability of BCBM and their accumulation of large molecules such as antibodies, which remains largely unexplored., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: In a previous clinical study, the authors evaluated the potential of antitenascin C monoclonal antibody (mAb) 81C6 labeled with 211 At via the prosthetic agent N -succinimidyl 3-[ 211 At]astatobenzoate (SAB) for the treatment of primary brain tumors. Although encouraging results were obtained, labeling chemistry failed while attempting to escalate the dose to 370 MBq. The goal of the current study was to develop a revised procedure less susceptible to radiolysis-mediated effects on 211 At labeling that would be suitable for use at higher activity levels of this α-emitter. Materials and Methods: Addition of N -chlorosuccinimide to the methanol used to remove the 211 At from the cryotrap after bismuth target distillation was done to thwart radiolytic decomposition of reactive 211 At and the tin precursor. A series of 11 reactions were performed to produce SAB at initial 211 At activity levels of 0.31-2.74 GBq from 50 μg of N -succinimidyl 3-trimethylstannylbenzoate (Me-STB), which was then reacted with murine 81C6 mAb without purification of the SAB intermediate. Radiochemical purity, immunoreactive fraction, sterility, and apyrogenicity of the 211 At-labeled 81C6 preparations were evaluated. Results: Murine 81C6 mAb was successfully labeled with 211 At using these revised procedures with improved radiochemical yields and decreased overall synthesis time compared with the original clinical labeling procedure. Conclusions: With 2.74 GBq of 211 At, it was possible to produce 1.0 GBq of 211 At-labeled 81C6 with an immunoreactive fraction of 92%. These revised procedures permit production of 211 At-labeled mAbs suitable for use at clinically relevant activity levels.

Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with 18 F (t ½  = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for 18 F-labeling of sdAbs via trans-cyclooctene (TCO)-tetrazine (Tz) inverse electron demand Diel's Alder cycloaddition reaction (IEDDAR) that incorporated a renal brush border enzyme (RBBE)-cleavable linker. Although >15 fold reduction in kidney activity levels was achieved, tumor uptake was compromised. Here we investigate whether replacing the [ 18 F]AlF-NOTA moiety with [ 18 F]fluoronicotinyl would rectify this problem. Anti-HER2 sdAb 5F7 was first derivatized with a TCO-containing agent that included the RBBE-cleavable linker GlyLys (GK) and a PEG chain, and then subjected to IEDDAR with 6-[ 18 F]fluoronicotinyl-PEG 4 -methyltetrazine to provide [ 18 F]FN-PEG 4 -Tz-TCO-GK-PEG 4 -5F7 ([ 18 F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[ 125 I]iodobenzoate (iso-[ 125 I]SGMIB) also were synthesized. Radiochemical purity, affinity (K D ) and immunoreactive fraction of [ 18 F]FN-GK-5F7 were 99%, 5.4 ± 0.7 nM and 72.5 ± 4.3%, respectively. Tumor uptake of [ 18 F]FN-GK-5F7 in athymic mice bearing subcutaneous SKOV3 xenografts (3.7 ± 1.2% ID/g and 3.4 ± 1.0% ID/g at 1 h and 3 h, respectively) was 2- to 3-fold lower than for co-injected iso-[ 125 I]SGMIB-5F7 (6.9 ± 1.9 %ID/g and 8.7 ± 3.0 %ID/g). However, due to its 6-fold lower kidney activity levels, tumor-to-kidney ratios for [ 18 F]FN-GK-5F7 were 3-4 times higher than those for co-injected iso-[ 125 I]SGMIB-5F7 as well as those observed for the 18 F conjugate lacking the RBBE-cleavable linker. Micro-PET/CT imaging of [ 18 F]FN-GK-5F7 in mice with SKOV-3 subcutaneous xenografts clearly delineated tumor as early as 1 h with minimal activity in the kidneys; however, there was considerable activity in gallbladder and intestines. Although the tumor uptake of [ 18 F]FN-GK-5F7 was unexpectedly disappointing, incorporating an alternative RBBE-cleavable linker into this labeling strategy may ameliorate this problem., Competing Interests: Declaration of Competing Interest Michael R. Zalutsky is the founder of Cereius, Inc., a company that develops radiolabeling methodologies, including those described herein, for biomolecules including sdAbs. Ganesan Vaidyanathan is a consultant and shareholder of Cereius, Inc., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Poly(ADP-ribose)polymerase-1 (PARP1) is a DNA repair enzyme highly expressed in the nuclei of mammalian cells, with a structure and function that have attracted interest since its discovery. PARP inhibitors, moreover, can be used to induce synthetic lethality in cells where the homologous recombination (HR) pathway is deficient. Several small molecule PARP inhibitors have been approved by the FDA for multiple cancers bearing this deficiency These PARP inhibitors also act as radiosensitizing agents by delaying single strand break (SSB) repair and causing subsequent double strand break (DSB) generation, a concept that has been leveraged in various preclinical models of combination therapy with PARP inhibitors and ionizing radiation. Researchers have determined the efficacy of various PARP inhibitors at sub-cytotoxic concentrations in radiosensitizing multiple human cancer cell lines to ionizing radiation. Furthermore, several groups have begun evaluating combination therapy strategies in mouse models of cancer, and a fluorescent imaging agent that allows for subcellular imaging in real time has been developed from a PARP inhibitor scaffold. Other PARP inhibitor scaffolds have been radiolabeled to create PET imaging agents, some of which have also entered clinical trials. Most recently, these highly targeted small molecules have been radiolabeled with therapeutic isotopes to create radiotherapeutics and radiotheranostics in cancers whose primary interventions are surgical resection and whole-body radiotherapy. In this review we discuss the utilization of these small molecules in combination therapies and in scaffolds for imaging agents, radiotherapeutics, and radiotheranostics. Development of these radiolabeled PARP inhibitors has presented promising results for new interventions in the fight against some of the most intractable cancers., (Copyright © 2020 Jannetti, Zeglis, Zalutsky and Reiner.)

The presence of Auger electrons (AE) among the decay products of a number of radionuclides makes these radionuclides an attractive means for treating cancer because these short-range electrons can cause significant damage in the immediate vicinity of the decomposition site. Moreover, the extreme locality of the effect provides a potential for selective eradication of cancer cells with minimal damage to adjacent normal cells provided that the delivery of the AE emitter to the most vulnerable parts of the cell can be achieved. Few cellular compartments have been regarded as the desired target site for AE emitters, with the cell nucleus generally recognized as the preferred site for AE decay due to the extreme sensitivity of nuclear DNA to direct damage by radiation of high linear energy transfer. Thus, the advantages of AE emitters for cancer therapy are most likely to be realized by their selective delivery into the nucleus of the malignant cells. To achieve this goal, delivery systems must combine a challenging complex of properties that not only provide cancer cell preferential recognition but also cell entry followed by transport into the cell nucleus. A promising strategy for achieving this is the recruitment of natural cell transport processes of macromolecules, involved in each of the aforementioned steps. To date, a number of constructs exploiting intracellular transport systems have been proposed for AE emitter delivery to the nucleus of a targeted cell. An example of such a multifunctional vehicle that provides smart step-by-step delivery is the so-called modular nanotransporter, which accomplishes selective recognition, binding, internalization, and endosomal escape followed by nuclear import of the delivered radionuclide. The current review will focus on delivery systems utilizing various intracellular transport pathways and their combinations in order to provide efficient targeting of AE to the cancer cell nucleus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Trastuzumab is an antibody used for the treatment of human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancers. Since trastuzumab is an internalizing antibody, two factors could play an important role in achieving high uptake and prolonged retention of radioactivity in HER2-positive tumors after radioiodination-residualizing capacity after receptor-mediated internalization and susceptibility to dehalogenation. To evaluate the contribution of these two factors, trastuzumab was radiolabeled using the residualizing reagent N -succinimidyl 4-guanidinomethyl-3-[*I]iodobenzoate ([*I]SGMIB) and the nonresidualizing reagent N -succinimidyl-3-[*I]iodobenzoate ([*I]SIB), both of which are highly dehalogenation-resistant. Paired-label uptake and intracellular retention of [ 125 I]SGMIB-trastuzumab and [ 131 I]SIB-trastuzumab was compared on HER2-expressing BT474 human breast carcinoma cells. Tumor uptake and normal tissue distribution characteristics for the two labeled conjugates were assessed in mice bearing BT474M1 xenografts. The internalization and intracellular retention of initially-bound radioactivity in BT474 cells was similar for the two labeled conjugates up to 4 h, but were significantly higher for [ 125 I]SGMIB-trastuzumab at 6 and 24 h. Similarly, [*I]SGMIB labeling resulted in significantly higher uptake and retention of radioactivity in BT474M1 xenografts at all studied time points. Moreover, tumor-to-tissue ratios for [ 125 I]SGMIB-trastuzumab were consistently higher than those for [ 131 I]SIB-trastuzumab starting at 12 h postinjection. Thus, optimal targeting of HER2-positive breast cancers with a radioiodinated trastuzumab conjugate requires an acylation agent that imparts residualizing capacity in addition to high stability towards dehalogenation in vivo.

Despite decades of efforts, non-invasive sensitive detection of small malignant brain tumors still remains challenging. Here we report a dual-modality 124 I-labeled gold nanostar ( 124 I-GNS) probe for sensitive brain tumor imaging with positron emission tomography (PET) and subcellular tracking with two-photon photoluminescence (TPL) and electron microscopy (EM). Experiment results showed that the developed nanoprobe has potential to reach sub-millimeter intracranial brain tumor detection using PET scan, which is superior to any currently available non-invasive imaging modality. Microscopic examination using TPL and EM further confirmed that GNS nanoparticles permeated the brain tumor leaky vasculature and accumulated inside brain tumor cells following systemic administration. Selective brain tumor targeting by enhanced permeability and retention effect and ultrasensitive imaging render 124 I-GNS nanoprobe promise for future brain tumor-related preclinical and translational applications.

Background: Radium-223 is a targeted alpha-particle therapy that improves survival in men with metastatic castration resistant prostate cancer (mCRPC), particularly in men with elevated serum levels of bone alkaline phosphatase (B-ALP). We hypothesized that osteomimicry, a form of epithelial plasticity leading to an osteoblastic phenotype, may contribute to intralesional deposition of radium-223 and subsequent irradiation of the tumor microenvironment., Methods: We conducted a pharmacodynamic study (NCT02204943) of radium-223 in men with bone mCRPC. Prior to and three and six months after radium-223 treatment initiation, we collected CTCs and metastatic biopsies for phenotypic characterization and CTC genomic analysis. The primary objective was to describe the impact of radium-223 on the prevalence of CTC B-ALP over time. We measured radium-223 decay products in tumor and surrounding normal bone during treatment. We validated genomic findings in a separate independent study of men with bone metastatic mCRPC (n = 45) and publicly accessible data of metastatic CRPC tissues., Results: We enrolled 20 men with symptomatic bone predominant mCRPC and treated with radium-223. We observed greater radium-223 radioactivity levels in metastatic bone tumor containing biopsies compared with adjacent normal bone. We found evidence of persistent Cellsearch CTCs and B-ALP (+) CTCs in the majority of men over time during radium-223 therapy despite serum B-ALP normalization. We identified genomic gains in osteoblast mimicry genes including gains of ALPL, osteopontin, SPARC, OB-cadherin and loss of RUNX2, and validated genomic alterations or increased expression at the DNA and RNA level in an independent cohort of 45 men with bone-metastatic CRPC and in 150 metastatic biopsies from men with mCRPC., Conclusions: Osteomimicry may contribute in part to the uptake of radium-223 within bone metastases and may thereby enhance the therapeutic benefit of this bone targeting radiotherapy., Competing Interests: Drs. Armstrong and George receive consulting support and research support to Duke University from Bayer and serve as speakers for Bayer. Competing interest statement: Drs. George and Armstrong receive consulting and speaking income from Bayer as well as research funding (to Duke University) from Bayer. No other conflicts of interest to report. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.this does not alter our adherence to PLOS ONE policies on sharing data and materials.

ImmunoPET agents are being investigated to assess the status of epidermal growth factor receptor 2 (HER2) in breast cancer patients with the goal of selecting those likely to benefit from HER2-targeted therapies and monitoring their progress after these treatments. We have been exploring the use of single domain antibody fragments (sdAbs) labeled with 18 F using residualizing prosthetic agents for this purpose. In this study, we have labeled two sdAbs that bind to different domains on the HER2 receptor, 2Rs15d and 5F7, using 2,3,5,6-tetrafluorophenyl 6-[ 18 F]fluoronicotinate ([ 18 F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [ 18 F]TFPFN-2Rs15d and [ 18 F]TFPFN-5F7 was 5.7 ± 3.6 and 4.0 ± 2.0%, respectively. The radiochemical purity of labeled sdAbs was >95%, immunoreactive fractions were about 60%, and affinity was in the low nanomolar range. Intracellularly trapped activity from [ 18 F]TFPFN-2Rs15d and [ 18 F]TFPFN-5F7 in HER2-expressing SKOV-3 ovarian and BT474M1 breast carcinoma cells were similar to the sdAbs labeled using the previously validated radioiodination residualizing prosthetic agents N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[ 125 I]iodobenzoate ( iso-[ 125 I]SGMIB). Intracellular activity was about 2-fold higher for radiolabeled 5F7 compared with 2Rs15d for both 18 F and 125 I. While tumor uptake of both [ 18 F]TFPFN-2Rs15d and [ 18 F]TFPFN-5F7 was comparable to those for the coadministered 125 I-labeled sdAb, renal uptake of the 18 F-labeled sdAbs was substantially lower. In microPET images, the tumor was clearly delineated in SKOV-3 and BT474 xenograft-bearing athymic mice with low levels of background activity in normal tissues, except the bladder. These results indicate that the [ 18 F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.

Single domain antibody fragments (sdAbs) labeled with 18 F have shown promise for assessing the status of oncological targets such as the human epidermal growth factor receptor 2 (HER2) by positron emission tomography (PET). Earlier, we evaluated two residualizing prosthetic agents for 18 F-labeling of anti-HER2 sdAbs; however, these methods resulted in poor labeling yields and high uptake of 18 F activity in the kidneys. To potentially mitigate these limitations, we have now developed an 18 F labeling method that utilizes the trans-cyclooctene (TCO)-tetrazine (Tz)-based inverse-electron demand Diels-Alder reaction (IEDDAR) in tandem with a renal brush border enzyme-cleavable glycine-lysine (GK) linker in the prosthetic moiety. The HER2-targeted sdAb 2Rs15d was derivatized with TCO-GK-PEG 4 -NHS or TCO-PEG 4 -NHS, which lacks the cleavable linker. As an additional control, the non HER2-specific sdAb R3B23 was derivatized with TCO-GK-PEG 4 -NHS. The resultant sdAb conjugates were labeled with 18 F by IEDDAR using [ 18 F]AlF-NOTA-PEG 4 -methyltetrazine. As a positive control, the 2Rs15d sdAb was radioiodinated using the well-characterized residualizing prosthetic agent, N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB). Synthesis of [ 18 F]AlF-NOTA-Tz-TCO-GK-2Rs15d was achieved with an overall radiochemical yield (RCY) of 17.8 ± 1.5% ( n = 5) in 90 min, a significant improvement over prior methods (3-4% in 2-3 h). In vitro assays indicated that [ 18 F]AlF-NOTA-Tz-TCO-GK-2Rs15d bound with high affinity and immunoreactivity to HER2. In normal mice, when normalized to coinjected [ 125 I]SGMIB-2Rs15d, the kidney uptake of [ 18 F]AlF-NOTA-Tz-TCO-GK-2Rs15d was 15- and 28-fold lower ( P < 0.001) than that seen for the noncleavable control ([ 18 F]AlF-NOTA-Tz-TCO-2Rs15d) at 1 and 3 h, respectively. Uptake of [ 18 F]AlF-NOTA-Tz-TCO-GK-2Rs15d in HER2-expressing SKOV-3 ovarian carcinoma xenografts implanted in athymic mice was about 80% of that seen for coinjected [ 125 I]SGMIB-2Rs15d. On the other hand, kidney uptake was 5-6-fold lower, and as a result, tumor-to-kidney ratios were 4-fold higher for [ 18 F]AlF-NOTA-Tz-TCO-GK-2Rs15d than those for [ 125 I]SGMIB-2Rs15d. SKOV-3 xenografts were clearly delineated even at 1 h after administration of [ 18 F]AlF-NOTA-Tz-TCO-GK-2Rs15d by Micro-PET/CT imaging with even higher contrast observed thereafter. In conclusion, this strategy warrants further evaluation for labeling small proteins such as sdAbs because it offers the benefits of good radiochemical yields and enhanced tumor-to-normal tissue ratios, particularly in the kidney.

Introduction: Radiolabeled, low-molecular-weight prostate-specific membrane antigen (PSMA) inhibitors based on the Glu-ureido pharmacophore show promise for the detection and treatment of castration-resistant prostate cancer; however, high renal retention of activity, related in part to overexpression of PSMA in kidneys can be problematic. The goal of the current study was to investigate the use of brush border enzyme-cleavable linkers as a strategy for reducing kidney activity levels from radiolabeled PSMA inhibitors., Methods: PSMA-769 (6), a derivative of the prototypical PSMA inhibitor (((S)‑1‑carboxy‑5‑(4‑iodobenzamido)pentyl)carbamoyl)glutamate (12) modified to contain a Gly-Tyr linker, and its protected tin precursor (11) were synthesized starting from the basic pharmacophore molecule Lys-urea-Glu. An analogue of 6 containing d‑tyrosine in lieu of l‑tyrosine (PSMA-769-d-tyrosine) and the corresponding tin precursor (d-11) also were synthesized. Both radioiodinated and 211 At-labeled 6 were synthesized by radiohalogenation of 11 and deprotection in situ. Similarly, radioiodinated d-6 was synthesized from d-11. Paired label biodistribution of [ 125 I]12 and [ 131 I]6 was performed in normal mice and in SCID mice bearing both PC3 PIP (PSMA+) and PC3 flu (PSMA-) subcutaneous prostate carcinoma xenografts. The biodistribution of [ 131 I]6 and [ 211 At]6 was also evaluated in this tumor model. Biodistribution of the two radioiodinated diastereomers of 6 was evaluated in normal mice and urine samples were analyzed for the presence of 4‑iodohippuric acid., Results: Compounds [ 131 I]6 and [ 211 At]6 were synthesized from 11 in overall radiochemical yields of 32.5 ± 0.1% (n = 4) and 22% (n = 1), respectively; radiochemical purity was >95%. In normal mice, renal uptake of [ 131 I]6 was 1.4-, 2.8- and 161-fold lower than that seen for co-injected [ 125 I]12 at 1 h, 4 h and 21 h, respectively. In tumor-bearing mice, kidney uptake of [ 131 I]6 was similar to that for [ 125 I]12 (P > 0.05) at 1 h and 4 h but was 6- to 7-fold lower at 21 h; however, [ 131 I]6 uptake in PC3 PIP tumors was also lower than that seen for [ 125 I]12 at 21 h (12.6 ± 3.4%ID/g vs. 36.8 ± 12.4%ID/g). Uptake of [ 211 At]PSMA-769 in PC3 PIP tumors was slightly higher than that seen for [ 131 I]PSMA-769 at 4 h (9.6 ± 1.6%ID/g versus 7.8 ± 1.6%ID/g; P = 0.002); its uptake in a number of normal tissues also was higher. In normal mice, kidney uptake of [ 125 I]PSMA-769 at 4 h was about 73% of that seen for [ 131 I]PSMA-769-d-tyrosine. Activity in the urine of mice receiving [ 125 I]PSMA-769 contained mainly 4‑[ 125 I]iodohippuric acid while unmetabolized intact molecule was present in the case of [ 125 I]PSMA-769-d-tyrosine., Conclusion: Use of this brush border enzyme-cleavable linker reduced kidney uptake and resulted in improved tumor:kidney uptake ratios. Although further structural improvements are needed, this linker approach might be useful as a component in strategies for reducing renal uptake of radiolabeled PSMA inhibitors., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The residualizing prosthetic agent N ε -(3-[ * I]iodobenzoyl)-Lys⁵- N α -maleimido-Gly¹-d-GEEEK ([ * I]IB-Mal-d-GEEEK) showed promise for the radioiodination of monoclonal antibodies (mAbs) that bind to internalizing molecular targets. Although enhanced tumor uptake was achieved in these studies, elevated kidney accumulation also was observed, particularly with low-molecular-weight, single-domain antibody fragments (sdAbs). Here, we developed an analogous agent (IB-Mal-d-GDDDK), in which glutamate residues (E) were replaced with aspartates (D) to determine whether this modification could decrease renal uptake. [ 125 I]IB-Mal-d-GDDDK and [ 131 I]IB-Mal-d-GEEEK were synthesized with similar radiochemical yields (60⁻80%) and coupled to the anti-HER2 sdAb 5F7 at 50⁻60% efficiency. Paired-label internalization assays in vitro indicated similar levels of intracellular activity residualization in HER2-expressing BT474M1 cells for [ 125 I]IB-Mal-d-GDDDK-5F7 and [ 131 I]IB-Mal-d-GEEEK-5F7. A paired-label biodistribution comparison of the two labeled conjugates was performed in mice with HER2-expressing SKOV-3 xenografts, and the results of this study indicated that renal uptake at 1 h was 127.5 ± 18.7% ID/g and 271.4 ± 66.6% ID/g for [ 125 I]IB-Mal-d-GDDDK-5F7 and [ 131 I]IB-Mal-d-GEEEK-5F7, respectively. The tumor uptake of the two radioconjugates was not significantly different. These results demonstrate that substitution of E with D in the IB-Mal-d-GEEEK construct reduced kidney accumulation of the sdAb. However, renal activity levels need to be reduced further if d-amino acid derived prosthetic agents are to be of practical value for labeling low molecular weight biomolecules such as sdAbs.

Mutations in the isocitrate dehydrogenase gene 1 (IDH1) are common in gliomas. Studies suggest that IDH1 mutations are early events in glioma formation and are important drivers of malignant progression. Herein, we report the synthesis and evaluation of a 18 F-labeled triazinediamine analogue, [ 18 F] 1 , as a candidate radiotracer for noninvasive imaging of IDH1 mutations in gliomas by positron emission tomography (PET). In vitro studies revealed good binding inhibition potency and binding affinity for [ 18 F] 1 in IDH1 mutant glioma cell lines, with a half-maximal inhibitory concentration value (IC 50 ) of 54 nM and an equilibrium dissociation constant ( K d ) of 40 nM. In vivo studies using mutant IDH1 glioma xenografts showed good tumor uptake of [ 18 F] 1 and specific inhibition by the unlabeled 1 , but also elevated radioactivity uptake in the bone, suggesting significant defluorination. The results support further optimization of the triazinediamine scaffold to develop a more stable and potent 18 F-labeled analogue for PET imaging of IDH1 mutations in gliomas., Competing Interests: The authors declare no competing financial interest.

In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with 18 F via conjugation of a residualizing prosthetic agent that was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In order to potentially increase overall efficiency and decrease the time required for labeling, we now investigate the use of a strain-promoted azide-alkyne cycloaddition (SPAAC) between the 2Rs15d sdAb, which had been pre-derivatized with an azide-containing residualizing moiety, and an 18 F-labeled aza-dibenzocyclooctyne derivative. The HER2-targeted sdAb 2Rs15d and a nonspecific sdAb R3B23 were pre-conjugated with a moiety containing both azide- and guanidine functionalities. The thus derivatized sdAbs were radiolabeled with 18 F using an 18 F-labeled aza-dibenzocyclooctyne derivative ([ 18 F]F-ADIBO) via SPAAC, generating the desired conjugate ([ 18 F]RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB), the prototypical residualizing agent for radioiodination. Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake of [ 18 F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [ 18 F]RL-II-2Rs15d and [ 125 I]SGMIB-2Rs15d, and microPET/CT imaging of [ 18 F]RL-II-2Rs15d and the [ 18 F]RL-II-R3B23 control sdAb were performed in nude mice bearing HER2-expressing SKOV-3 xenografts. A radiochemical yield of 23.9 ± 6.9% (n = 8) was achieved for the SPAAC reaction between [ 18 F]F-ADIBO and azide-modified 2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (K d ) and IRF for the binding of [ 18 F]RL-II-2Rs15d to HER2 were 5.6 ± 1.3 nM and 73.1 ± 22.5% (n = 3), respectively. The specific uptake of [ 18 F]RL-II-2Rs15d by HER2-expressing BT474M1 breast carcinoma cells in vitro was 14-17% of the input dose at 1, 2, and 4 h, slightly higher than seen for co-incubated [ 125 I]SGMIB-2Rs15d. The uptake of [ 18 F]RL-II-2Rs15d in SKOV-3 xenografts at 1 h and 2 h p.i. were 5.54 ± 0.77% ID/g and 6.42 ± 1.70% ID/g, respectively, slightly higher than those for co-administered [ 125 I]SGMIB-2Rs15d (4.80 ± 0.78% ID/g and 4.78 ± 1.39% ID/g). MicroPET/CT imaging with [ 18 F]RL-II-2Rs15d at 1-3 h p.i. clearly delineated SKOV-3 tumors while no significant accumulation of activity in tumor was seen for [ 18 F]RL-II-R3B23. With the exception of kidneys, normal tissue levels for [ 18 F]RL-II-2Rs15d were low and cleared rapidly. To our knowledge, this is the first time SPAAC method has been used to label an sdAb with 18 F, especially with residualizing functionality., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are commonly found in gliomas. AGI-5198, a potent and selective inhibitor of the mutant IDH1 enzyme, was radiolabeled with radioiodine and fluorine-18. These radiotracers were evaluated as potential probes for imaging mutant IDH1 expression in tumors with positron emission tomography (PET). Radioiodination of AGI-5198 was achieved using a tin precursor in 79 ± 6% yield (n = 9), and 18 F-labeling was accomplished by the Ugi reaction in a decay-corrected radiochemical yield of 2.6 ± 1.6% (n = 5). The inhibitory potency of the analogous nonradioactive compounds against mutant IDH1 (IDH1-R132H) was determined in enzymatic assays. Cell uptake studies using radiolabeled AGI-5198 analogues revealed somewhat higher uptake in IDH1-mutated cells than that in wild-type IDH1 cells. The radiolabeled compounds displayed favorable tissue distribution characteristics in vivo, and good initial uptake in IDH1-mutated tumor xenografts; however, tumor uptake decreased with time. Radioiodinated AGI-5198 exhibited higher tumor-to-background ratios compared with 18 F-labeled AGI-5198; unfortunately, similar results were observed in wild-type IDH1 tumor xenografts as well, indicating lack of selectivity for mutant IDH1 for this tracer. These results suggest that AGI-5198 analogues are not a promising platform for radiotracer development. Nonetheless, insights gained from this study may help in design and optimization of novel chemical scaffolds for developing radiotracers for imaging the mutant IDH1 enzyme., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Derived from heavy chain only camelid antibodies, ~15-kDa single-domain antibody fragments (sdAbs) are an attractive platform for developing molecularly specific imaging probes and targeted radiotherapeutics. The rapid tumor accumulation and normal tissue clearance of sdAbs might be ideal for use with 211 At, a 7.2-h half-life α-emitter, if appropriate labeling chemistry can be devised to trap 211 At in cancer cells after sdAb binding. This study evaluated two reagents, [ 211 At]SAGMB and iso-[ 211 At]SAGMB, for this purpose., Methods: [ 211 At]SAGMB and iso-[ 211 At]SAGMB, and their radioiodinated analogues [ 131 I]SGMIB and iso-[ 131 I]SGMIB, were synthesized by halodestannylation and reacted with the anti-HER2 sdAb 5F7. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label internalization assays on HER2-expressing BT474M1 breast carcinoma cells directly compared [ 131 I]SGMIB-5F7/[ 211 At]SAGMB-5F7 and iso-[ 131 I]SGMIB-5F7/iso-[ 211 At]SAGMB-5F7 tandems. The biodistribution of the two tandems was evaluated in SCID mice with subcutaneous BT474M1 xenografts., Results: Radiochemical yields for Boc 2 -iso-[ 211 At]SAGMB and Boc 2 -[ 211 At]SAGMB synthesis, and efficiencies for coupling of iso-[ 211 At]SAGMB and [ 211 At]SAGMB to 5F7 were similar, with radiochemical purities of [ 211 At]SAGMB-5F7 and iso-[ 211 At]SAGMB-5F7 >98%. iso-[ 211 At]SAGMB-5F7 and [ 211 At]SAGMB-5F7 had immunoreactive fractions >80% and HER2 binding affinities of less than 5 nM. Internalization assays demonstrated high intracellular trapping of radioactivity, with little difference observed between corresponding 211 At- and 131 I-labeled 5F7 conjugates. Higher BT474M1 intracellular retention was observed from 1-6 h for the iso-conjugates (iso-[ 211 At]SAGMB-5F7, 74.3 ± 2.8%, vs. [ 211 At]SAGMB-5F7, 63.7 ± 0.4% at 2 h) with the opposite behavior observed at 24 h. Peak tumor uptake for iso-[ 211 At]SAGMB-5F7 was 23.4 ± 2.2% ID/g at 4 h, slightly lower than its radioiodinated counterpart, but significantly higher than observed with [ 211 At]SAGMB-5F7. Except in kidneys and lungs, tumor-to-normal organ ratios for iso-[ 211 At]SAGMB-5F7 were greater than 10:1 by 2 h, and significantly higher than those for [ 211 At]SAGMB-5F7., Conclusion: These 211 At-labeled sdAb conjugates, particularly iso-[ 211 At]SAGMB-5F7, warrant further evaluation for targeted α-particle radiotherapy of HER2-expressing cancers., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Our previous studies with F-18-labeled anti-HER2 single-domain antibodies (sdAbs) utilized 5F7, which binds to the same epitope on HER2 as trastuzumab, complicating its use for positron emission tomography (PET) imaging of patients undergoing trastuzumab therapy. On the other hand, sdAb 2Rs15d binds to a different epitope on HER2 and thus might be a preferable vector for imaging in these patients. The aim of this study was to evaluate the tumor targeting of F-18 -labeled 2Rs15d in HER2-expressing breast carcinoma cells and xenografts., Procedures: sdAb 2Rs15d was labeled with the residualizing labels N-succinimidyl 3-((4-(4-[ 18 F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([ 18 F]RL-I) and N-succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB), and the purity and HER2-specific binding affinity and immunoreactivity were assessed after labeling. The biodistribution of I-125- and F-18-labeled 2Rs15d was determined in SCID mice bearing subcutaneous BT474M1 xenografts. MicroPET/x-ray computed tomograph (CT) imaging of [ 18 F]RL-I-2Rs15d was performed in this model and compared to that of nonspecific sdAb [ 18 F]RL-I-R3B23. MicroPET/CT imaging was also done in an intracranial HER2-positive breast cancer brain metastasis model after administration of 2Rs15d-, 5F7-, and R3B23-[ 18 F]RL-I conjugates., Results: [ 18 F]RL-I was conjugated to 2Rs15d in 40.8 ± 9.1 % yield and with a radiochemical purity of 97-100 %. Its immunoreactive fraction (IRF) and affinity for HER2-specific binding were 79.2 ± 5.4 % and 7.1 ± 0.4 nM, respectively. [ 125 I]SGMIB was conjugated to 2Rs15d in 58.4 ± 8.2 % yield and with a radiochemical purity of 95-99 %; its IRF and affinity for HER2-specific binding were 79.0 ± 12.9 % and 4.5 ± 0.8 nM, respectively. Internalized radioactivity in BT474M1 cells in vitro for [ 18 F]RL-I-2Rs15d was 43.7 ± 3.6, 36.5 ± 2.6, and 21.7 ± 1.2 % of initially bound radioactivity at 1, 2, and 4 h, respectively, and was similar to that seen for [ 125 I]SGMIB-2Rs15d. Uptake of [ 18 F]RL-I-2Rs15d in subcutaneous xenografts was 16-20 %ID/g over 1-3 h. Subcutaneous tumor could be clearly delineated by microPET/CT imaging with [ 18 F]RL-I-2Rs15d but not with [ 18 F]RL-I-R3B23. Intracranial breast cancer brain metastases could be visualized after intravenous administration of both [ 18 F]RL-I-2Rs15d and [ 18 F]RL-I-5F7., Conclusions: Although radiolabeled 2Rs15d conjugates exhibited lower tumor cell retention both in vitro and in vivo than that observed previously for 5F7, given that it binds to a different epitope on HER2 from those targeted by the clinically utilized HER2-targeted therapeutic antibodies trastuzumab and pertuzumab, F-18-labeled 2Rs15d has potential for assessing HER2 status by PET imaging after trastuzumab and/or pertuzumab therapy.

Purpose: Camelid single-domain antibody-fragments (sdAb) have beneficial pharmacokinetic properties, and those targeted to HER2 can be used for imaging of HER2-overexpressing cancer. Labeled with a therapeutic radionuclide, they may be used for HER2-targeted therapy. Here, we describe the generation of a 131 I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer. Experimental Design: Anti-HER2 sdAb 2Rs15d was labeled with 131 I using [ 131 I]SGMIB and evaluated in vitro Biodistribution was evaluated in two HER2 + murine xenograft models by micro-SPECT/CT imaging and at necropsy, and under challenge with trastuzumab and pertuzumab. The therapeutic potential of [ 131 I]SGMIB-2Rs15d was investigated in two HER2 + tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [ 127 I]SGMIB-sdAb at 1.4 mg/kg. The structure of the 2Rs15d-HER2 complex was determined by X-ray crystallography. Results: [ 131 I]SGMIB-2Rs15d bound specifically to HER2 + cells ( K d = 4.74 ± 0.39 nmol/L). High and specific tumor uptake was observed in both BT474/M1 and SKOV-3 tumor xenografted mice and surpassed kidney levels by 3 hours. Extremely low uptake values were observed in other normal tissues at all time points. The crystal structure revealed that 2Rs15d recognizes HER2 Domain 1, consistent with the lack of competition with trastuzumab and pertuzumab observed in vivo [ 131 I]SGMIB-2Rs15d alone, or in combination with trastuzumab, extended median survival significantly. No toxicity was observed after injecting [ 127 I]SGMIB-2Rs15d. Conclusions: These findings demonstrate the theranostic potential of [ 131 I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [ 131 I]SGMIB-2Rs15d and could thereby impact therapy outcome on HER2 + breast cancer patients. Clin Cancer Res; 23(21); 6616-28. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Radionuclides conjugated to molecules that bind specifically to cancer cells are of great interest as a means to increase the specificity of radiotherapy. Currently, the methods to disseminate these targeted radiotherapeutics have been either systemic delivery or by bolus injection into the tumor or tumor resection cavity. Herein we model a potentially more efficient method of delivery, namely pressure-driven fluid flow, called convection-enhanced delivery (CED), where a device infuses the molecules in solution (or suspension) directly into the tissue of interest. In particular, we focus on the setting of primary brain cancer after debulking surgery, where the tissue margins surrounding the surgical resection cavity are infiltrated with tumor cells and the most frequent sites of tumor recurrence. We develop the combination of fluid flow, chemical kinetics, and radiation dose models needed to examine such protocols. We focus on Auger electron-emitting radionuclides (e.g. 67 Ga, 77 Br, 111 In, 125 I, 123 I, 193m Pt, 195m Pt) whose short range makes them ideal for targeted therapy in this setting of small foci of tumor spread within normal tissue. By solving these model equations, we confirm that a CED protocol is promising in allowing sufficient absorbed dose to destroy cancer cells with minimal absorbed dose to normal cells at clinically feasible activity levels. We also show that Auger emitters are ideal for this purpose while the longer range alpha particle emitters fail to meet criteria for effective therapy (as neither would energetic beta particle emitters). The model is used with simplified assumptions on the geometry and homogeneity of brain tissue to allow semi-analytic solutions to be displayed, and with the purpose of a first examination of this new delivery protocol proposed for radionuclide therapy. However, we emphasize that it is immediately extensible to personalized therapy treatment planning as we have previously shown for conventional CED, at the price of requiring a fully numerical computerized approach.

Introduction: Alpha particles are radiation of high energy and short range, properties that can lead to radiolysis-mediated complications in labeling chemistry at the high radioactivity levels required for clinical application. In previous papers in this series, we have shown that radiation dose has a profound effect on the astatine species that are present in the labeling reaction and their suitability for the synthesis of N-succinimidyl 3-[ 211 At]astatobenzoate. The purpose of this study was to evaluate the effects of adding N-chlorosuccinimide (NCS) to the methanol solution used for initial isolation of 211 At after distillation, a process referred to as 211 At stabilization, on 211 At chemistry after exposure to high radiation doses., Methods: High performance liquid chromatography was used to evaluate the distribution of 211 At species present in methanol in the 500-65,000Gy radiation dose range and the synthesis of SAB from N-succinimidyl 3-(tri-n-butylstannyl)benzoate in the 500-120,000Gy radiation dose range using different 211 At timeactivity combinations under conditions with/without 211 At stabilization., Results: In the absence of NCS stabilization, a reduced form of astatine, At(2), increased with increasing radiation dose, accounting for about half the total activity by about 15,000Gy, while with stabilization, At(2) accounted for <10% of 211 At activity even at doses >60,000Gy. SAB yields without stabilization rapidly declined with increasing dose, falling to ~20% at about 5000Gy while with stabilization, yields >80% were obtained with 211 At solutions stored for more than 23h and receiving radiation doses >100,000Gy., Conclusions: Adding NCS to the methanol solution used for initial isolation of 211 At is a promising strategy for countering the deleterious effects of radiolysis on 211 At chemistry., Advances in Knowledge and Implications for Patient Care: This strategy could facilitate the ability to perform 211 At labeling at sites remote from its production and at the high activity levels required for clinical applications., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting 111 In for cancer therapy., Methods: Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with 111 In in high specific activity. Cytotoxicity of the 111 In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted 111 In-labeled MNT in B16-F1 melanoma tumor-bearing mice., Results: The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of 111 In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with 111 In nontargeted control. In mice with B16-F1 tumors, prolonged retention of 111 In by serial imaging and significant tumor growth delay (82% growth inhibition) were found., Conclusion: The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted 111 In-NOTA-MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted 111 In-NOTA-MNT suggests further applications of the MNT delivery strategy should be explored., Competing Interests: The authors report no conflicts of interest in this work.

Background: Amino acid transporters, such as LAT1, are overexpressed in aggressive prostate and breast carcinomas, directly influencing pathways of growth and proliferation., Objective: The purpose of this study was to synthesize and characterize a novel 18F labeled leucine analog, 5-[18F]fluoroleucine, as a potential imaging agent for aggressive tumors which may not be amenable to imaging by FDG PET., Methods: 5-fluoroleucine was synthesized and characterized, and its 18F-labeled analog was synthesized from a mesylate precursor. First, breast cancer cell line assays were performed to evaluate uptake of 3H- or 14C-labeled L-leucine and other essential amino acids. Both L-leucine and 5- [18F]fluoroleucine were tested for uptake and accumulation over time, and for uptake via LAT1. Biodistribution studies were performed to estimate radiation dosimetry for human studies. Small animal PET / CT studies of a breast cancer were performed to evaluate in vivo 5-[18F]fluoroleucine tumor uptake., Results: Breast cancer cell lines showed increasing high net accumulation of L-[14C]leucine. Both L-leucine and 5-[18F]fluoroleucine showed increasing uptake over time in in vitro tumor cell assays, and uptake was also shown to occur via LAT1. The biodistribution study of 5-[18F]fluoroleucine showed rapid renal excretion, no significant in vivo metabolism, and acceptable dosimetry for use in humans. In vivo small animal PET / CT imaging of a breast cancer xenograft showed uptake of 5- [18F]fluoroleucine in the tumor, which progressively increased over time., Conclusion: 5-[18F]fluoroleucine is a leucine analog which may be useful in identifying tumors with high or upregulated expression of amino acid transporters, providing additional information that may not be provided by FDG PET., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Radiohalogenated agents are often the first line of pursuit in the development of new radiopharmaceuticals-whether antibodies, peptides, or small molecules-because of their ease of synthesis, lack of substantial steric perturbation of the original affinity agent (in some cases, providing enhanced affinity), and capacity to be transformed into therapeutics (in some cases, with a mere switch of an isotope). They often provide proof of a principle before optimization for pharmacokinetics or generation of radiometallated agents, when the latter are necessary. In particular, 18 F has been well integrated into normal clinical work flow in the form of 18 F-FDG for oncologic imaging, with reliable daily production and distribution to sites for immediate use, without the need for on-site preparation. Here we discuss radiohalogenated versions of imaging and therapeutic agents targeting the prostate-specific membrane antigen (PSMA); these were among the first such agents to be synthesized and used clinically. PSMA is highly expressed on prostate cancer epithelial cells and is currently being extensively investigated around the world as a target for imaging and therapy of prostate cancer. Additionally, the presence of PSMA on nonprostate tumor neovasculature has opened the possibility of PSMA-targeted molecules as generalizable cancer imaging and therapy agents. We focus on 18 F-labeled agents for PET, as they begin to redefine-along with the corresponding 68 Ga-labeled agents discussed elsewhere in this supplement to The Journal of Nuclear Medicine-the management of prostate cancer across a variety of clinical contexts., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)