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Supplementary Table S5 shows the ex vivo biodistribution of 131I-2Rs15d after 1h in mice with BT474/M1 xenografts that were pre-treated 72h earlier with a 100-fold molar excess of trastuzumab, pertuzumab or a combination of both.

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 131I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer.Experimental Design: Anti-HER2 sdAb 2Rs15d was labeled with 131I using [131I]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 [131I]SGMIB-2Rs15d was investigated in two HER2+ tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [127I]SGMIB-sdAb at 1.4 mg/kg. The structure of the 2Rs15d–HER2 complex was determined by X-ray crystallography.Results: [131I]SGMIB-2Rs15d bound specifically to HER2+ cells (Kd = 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. [131I]SGMIB-2Rs15d alone, or in combination with trastuzumab, extended median survival significantly. No toxicity was observed after injecting [127I]SGMIB-2Rs15d.Conclusions: These findings demonstrate the theranostic potential of [131I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [131I]SGMIB-2Rs15d and could thereby impact therapy outcome on HER2+ breast cancer patients. Clin Cancer Res; 23(21); 6616–28. ©2017 AACR.

A detailed description of the materials and methods used for the HER2-2Rs15d structure determination, the radioiodination of sdAbs, and all flow cytometry experiments.

Supplementary figure S1 details the HER2-epitope recognized by 2Rs15d compared to those reported for other described HER2-binders and a view of HER2-ÂÃ,â­2Rs15d interactions.

Because of their rapid tumor accumulation and normal tissue clearance, ~15-kDa single domain antibody fragments (sdAbs, also known as nanobodies) are an attractive vehicle for developing targeted radiotherapeutics labeled with the 7.2-h α-particle emitting radiohalogen (211)At. We have developed a site-specific (211)At-labeled prosthetic agent — iso-[(211)At]AGMB-PODS — that combines a functionality for residualizing radioactivity in tumor cells with a phenyloxadiazolyl methylsulfone (PODS) moiety for linking it to an sdAb. This reagent was evaluated for thiol-selective conjugation to anti-HER2 5F7 sdAb modified to have a C-terminus GGC tail for this purpose. The iso-[(211)At]AGMB-PODS-5F7GGC conjugate and its (131)I-labeled analogue were synthesized in good radiochemical yields with retention of high binding affinity and immunoreactivity. The PODS radioconjugates exhibited high specific binding, internalization and intracellular trapping of radioactivity on HER2-positive BT474 breast carcinoma cells in vitro. Iso-[(211)At]AGMB-PODS-5F7GGC displayed considerably higher in vitro stability in the presence of cysteine and HSA compared with the maleimide analogue, [(211)At]MEAGMB-5F7GGC, excellent tumor uptake and high in vivo stability. Superior tumor-to-kidney activity ratios were seen for iso-[(211)At]AGMB-PODS-5F7GGC and its radioiodinated analogue compared with [(177)Lu]Lu-DOTA-PODS-5F7GGC. These results suggest that iso-[(211)At]AGMB-PODS-5F7GGC warrants further evaluation as a targeted α-particle emitting agent for the treatment of HER2-expressing malignancies.

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

Introduction Labeling single domain antibody fragments (sdAbs) with 18F 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 ([18F]RL-I), synthesized via a click reaction for labeling sdAbs with 18F, 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-[18F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([18F]SFETGMB; [18F]RL-III) designed to address these limitations. Methods [18F]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-[18F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [18F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB). The 18F-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 [18F]RL-III-5F7 also was performed. Results Radiochemical yields for the synthesis of Boc2-[18F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc2-[18F]RL-I. The overall radiochemical yields for the synthesis of [18F]RL-III-2Rs15d and [18F]RL-III-5F7 from aqueous [18F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [18F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [18F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [18F]RL-III-2Rs15d. Although different xenograft models were used, [18F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [18F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [18F]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 [18F]RL-III-5F7 in the intracranial model. Conclusions Although higher radiochemical yields than that reported for [18F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [18F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern.

Background Single-domain antibody fragments (aka VHH, ~ 13 kDa) are promising delivery systems for brain tumor theranostics; however, achieving efficient delivery of VHH 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) VHH 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 VHH 5F7 was labeled with 18F, administered intravenously to irradiated mice and controls, and PET/CT imaging was conducted periodically after irradiation. Tumor uptake of 18F-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 18F-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 18F-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 VHH, which could facilitate the use of radiolabeled VHH to detect, monitor, and treat HER2-expressing brain metastases.

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. Astatine-211 is an α-emitter that may engender less toxicity than other α-emitting agents. We synthesized a new 211At-labeled radiotracer targeting PSMA that resulted from the search for a pharmacokinetically optimized agent. Methods: A small series of 125I-labeled compounds were synthesized from their tin precursors to evaluate the effect of location of radiohalogen within the molecule and the presence of lutetium in the chelate on biodistribution. On that basis, 211At-VK-02-90-Lu was selected and evaluated in cell uptake and internalization studies, biodistribution and PSMA+ PC3 PIP tumor growth control in experimental flank and metastatic (PC3-ML-Luc) models. A long-term (13-month) toxicity study was performed for 211At-VK-02-90-Lu, including tissue chemistries and histopathology. Results: The radiochemical yield of 211At-VK-02-90-Lu was 17.8 ± 8.2%. Lead compound 211At-VK-02-90-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, 211At-VK-02-90-Lu provided 30.6 ± 4.8 percentage of injected dose per gram (%ID/g) of uptake in PSMA+ PC3 PIP tumor at 1 h post-injection that 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. De-astatination 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 histopathology. Conclusion: Compound 211At-VK-02-90-Lu or close analogs may provide limited and acceptable toxicity while retaining efficacy in management of prostate cancer.

1624 Objectives: Positive allosteric modulators of muscarinic acetylcholine receptor subtype 4 (M4) have been identified as promising activators for the treatment of neurological disorders and neurodegenerative diseases, including schizophrenia, Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). We have identified 1-(6,7-dimethyl-4-(methylamino) -1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-yl)-2-(trans-2-(6-fluoropyridin-3-yl)cyclopropyl)ethan-1-one (7) as a lead molecule for M4-specific PET ligand attributed to its high potency (IC50 = 0.8 nM),[1] feasibility for 18F-labeling on 2-substituted pyridine scaffold and favorable in vitro brain permeability prediction (MPO score of 5.83 and logBB of -0.16). The goal of the project was to synthesize the standard and precursor molecules and perform radiolabeling of compound 7 with fluorine-18 as well as preliminary evaluation of [18F]7 in rodents. Methods: The leading compound 7 was synthesized as summarized in Scheme 1A.The cyclization of compound 1 with Et2Zn and CH2I2 led to intermediate 2 in 80% yield. Through Suzuki coupling reaction of compound 2 and 5-bromo-2-fluoropyridine 3, compound 4 was obtained in 25% yield. By deprotection and oxidation with RuCl3 and NaIO4, compound 4 wasconverted to carboxylic acid 5 in 53% yield. Through condensation reactions between acid 5 and amine 6, the desired product 7 was obtained in 86% yield. The synthesis of the labeling precursor for compound 7 was showed in Scheme 1B.Through Suzuki coupling reaction of compound 2 and 5-bromo-2-nitropyridine 8, compound 9 was obtained in 73% yield. By deprotection and oxidation with RuCl3 and NaIO4, compound 9 wasconverted to carboxylic acid 10 in 95% yield. After condensation reaction of acid 10 and amine 6, the precursor 11 was obtained in 63% yield. 18F-labeled radiotracer [18F]7 was obtained by reacting with nitro precursor 11 with [18F]fluoride using potassium carbonate as eluting base (Scheme 1C). Then the binding specificity in vitro of [18F]7 was evaluated by autoradiography studies on Sprague-Dawley rat brain. Subsequently, dynamic PET acquisitions (0-60 min) were conducted with [18F]7 in Sprague-Dawley rats. Results: The synthesis of candidate compound 7 was obtained from commercially available compound 1 in four steps with overall yield of 9%. The radiochemical yield of [18F]7 was 2% (non-decay corrected) with greater than 99% radiochemical purity, and the molar activity was higher than 37 GBq/μmol (1.0 Ci/μmol). The results of autoradiography studies are shown in Figure 2. At the baseline, the distribution of radioactivity was mainly concentrated in the striatum, cerebral cortex, thalamus and hippocampus, which was consistent with the expression pattern of M4 in the brain. In the blocking study, pretreatment with unlabeled 7 resulted in visible reduction of radioactivity in striatum and cerebral cortex which are reported as the M4 rich brain regions. PET images of [18F]7 aredisplayed inFigure 1B. [18F]7 showed limited brain uptake (ca. 0.55 SUV peak uptake) with marginal difference in the cerebral cortex, cerebellum, striatum, hippocampus and thalamus. Conclusions: The 18F-labeling ligand [18F]7 (also named [18F]M4R-1911)was obtained in a reasonable radiochemical yield, high radiochemical purity and high molar activity. While in vitro autoradiography studies showed that [18F]7 hadlow-to-moderate in vitro binding specificity, PET studies confirmed that [18F]7 was not a suitable tracer for in vivo imaging of M4 due to limited brain permeability. Further in-depth medicinal chemistry studies are necessary to design new M4 ligands with improved brain permeability and high specific binding for PET imaging studies. References: [1] WO2018234953.

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.

Background: In a previous clinical study, the authors evaluated the potential of antitenascin C monoclonal antibody (mAb) 81C6 labeled with 211At via the prosthetic agent N-succinimidyl 3-[211At]as...

BackgroundSingle-domain antibody fragments (aka VHH, ∼13 kDa) are promising delivery systems for brain tumor theranostics; however, achieving efficient delivery of VHH 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-HER2 VHH to breast cancer-derived intracranial tumors in mice.MethodsMice with intracranial HER2-positive BT474BrM3 tumors received 10-Gy fractionated cranial irradiation and evaluated using non-invasive imaging methods. The anti-HER2 VHH 5F7 was labeled with 18F, administered intravenously to irradiated mice and controls, and PET/CT imaging was conducted at various intervals after irradiation. Tumor uptake of 18F-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.ResultsIncreased 18F-labeled 5F7 intracranial tumor uptake was observed with PET in mice that received 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 18F-labeled anti-HER2 5F7 in irradiated mice.ConclusionLow-level brain irradiation induces dynamic changes in tumor vasculature that increase the intracranial tumor delivery of an anti-HER2 VHH, which could facilitate the use of labeled sdAb to detect, monitor, and treat HER2-expressing brain metastases.Key pointsLow-level radiation enhances uptake of HER2-specific VHH in intracranial tumors.XRT + radiolabeled VHH shows promise as a treatment strategy for breast cancer brain metastases.Importance of the StudyImproving the detection and treatment of brain metastases (BM) that overexpress human epidermal growth factor receptor type 2 (HER2) is an urgent medical need. Drug delivery to BM is confounded by their tumor vasculature, which is more restrictive than in GBM. Single domain antibody fragments, about one-tenth the size of antibodies, could be promising theranostic vectors for BM provided sufficient BM uptake could be achieved. In this study, we utilized longitudinal PET imaging to demonstrate that low-dose whole-brain irradiation (WBRT) significantly increased 18F-labeled HER2-specific 5F7 VHH uptake in intracranial HER2-positive tumors in mice. Combining low dose WBRT with 5F7 VHH labeled with α-or β-particle emitting radionuclides could provide an effective and specific targeted radiotherapeutic strategy for treating patients with HER2-expressing BM.

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.

RG7388 (Idasanutlin) has been investigated as a small molecule 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 post-injection (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 pre-treatment 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 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.

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 protected Boc(2)-[(18)F]RL-III (21.5 ± 3.4%) were significantly higher than reported for protected 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.

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 211At-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 211At, and for comparative purposes, 131I, 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 211At-labeled PSMA ligand to its 131I-labeled counterpart in mice bearing subcutaneous PC3 PSMA+ PIP xenografts. Results Radiochemical yields ranged from 32% to 65% for the 211At-labeled PSMA inhibitors and were consistently lower than those obtained with the corresponding 131I-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, [211At]DCABzL and [*I]DCIBzL. Conclusions and implications for patient care Compared with the first generation compound [211At]DCABzL, several of the novel 211At-labeled PSMA ligands exhibited markedly improved stability in vivo and higher tumor-to-normal tissue ratios. [211At]GV-620 has the most promising characteristics and warrants further evaluation as a targeted radiotherapeutic for prostate cancer.

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.

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.

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.

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 trimethyl 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.

Single domain antibody fragments (sdAbs) exhibit a rapid tumor uptake and fast blood clearance amenable for labeling with 18F (t½ = 110 min) but suffer from high kidney accumulation. Previously, we developed a method for 18F-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-PEG4-methyltetrazine to provide [ 18 F]FN-PEG4-Tz-TCO-GK-PEG4-5F7 ([ 18 F]FN-GK-5F7). For comparisons, a control lacking GK linker and 5F7 labeled using residualizing N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB) also were synthesized. Radiochemical purity, affinity (KD) 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-[125I]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-[125I]SGMIB-5F7 as well as those observed for the 18F 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.

INTRODUCTION: As a consequence of their small size, high stability and high affinity, single domain antibody fragments (sdAb) 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.

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.

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 co-injected [(125)I]SGMIB-2Rs15d, the kidney uptake of [(18)F]AlF-NOTA-Tz-TCO-GK-2Rs15d was 15- and 28-fold lower (P

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 18F-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 18F-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.

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 131I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer.Experimental Design: Anti-HER2 sdAb 2Rs15d was labeled with 131I using [131I]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 [131I]SGMIB-2Rs15d was investigated in two HER2+ tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [127I]SGMIB-sdAb at 1.4 mg/kg. The structure of the 2Rs15d–HER2 complex was determined by X-ray crystallography.Results: [131I]SGMIB-2Rs15d bound specifically to HER2+ cells (Kd = 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. [131I]SGMIB-2Rs15d alone, or in combination with trastuzumab, extended median survival significantly. No toxicity was observed after injecting [127I]SGMIB-2Rs15d.Conclusions: These findings demonstrate the theranostic potential of [131I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [131I]SGMIB-2Rs15d and could thereby impact therapy outcome on HER2+ breast cancer patients. Clin Cancer Res; 23(21); 6616–28. ©2017 AACR.

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 (sup131/supI-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.sup131/supI-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 thatsup131/supI-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 ofsup131/supI-ELP depots for the synergistic treatment of localized pancreatic cancer.

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&mdash, 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 [125I]SGMIB-trastuzumab and [131I]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 [125I]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 [125I]SGMIB-trastuzumab were consistently higher than those for [131I]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.

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.

Introduction Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). Methods A small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC 50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4 , were labeled with radioiodine ( 125 I) and/or 18 F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [ 125 I] 1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. Results Enzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC 50 values of 1.7 μM and 2.3 μM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [ 125 I] 1 (n = 4) and 67.5 ± 6.6% for [ 18 F] 4 (n = 3). [ 125 I] 1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [ 125 I] 1 in tumor cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled butyl-phenyl sulfonamide inhibitors. Conclusions These data demonstrate the feasibility of developing radiolabeled probes for the mutant IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with improved characteristics as probes for imaging mutant IDH1 expression in tumors.

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 18F 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-[ 18F]fluoronicotinate ([ 18F]TFPFN) and evaluated their HER2 targeting properties in vitro and in vivo. The overall decay-corrected radiochemical yield for the synthesis of [ 18F]TFPFN-2Rs15d and [ 18F]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 [ 18F]TFPFN-2Rs15d and [ 18F]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-[ 125I]iodobenzoate ([ 125I]SGMIB) and N-succinimidyl 3-guanidinomethyl-5-[ 125I]iodobenzoate (iso-[ 125I]SGMIB). Intracellular activity was about 2-fold higher for radiolabeled 5F7 compared with 2Rs15d for both 18F and 125I. While tumor uptake of both [ 18F]TFPFN-2Rs15d and [ 18F]TFPFN-5F7 was comparable to those for the coadministered 125I-labeled sdAb, renal uptake of the 18F-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 [ 18F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.

Radioisotopes of iodine have been incorporated into a wide variety of radiopharmaceuticals ranging from small, low molecular weight compounds to large molecules like antibodies. Because of the routine availability of radioisotopes of iodine with different nuclear decay properties, radioiodination is an attractive strategy because the same chemistry can be utilized for both radionuclide imaging and targeted radiotherapy. Over the years, various methods have been developed for the synthesis of radioiodinated compounds. This chapter gives an overview of these methods as well as their potential advantages and disadvantages. Some useful tips and tactics for the radioiodination chemistry are provided. Important milestones in radioiodination chemistry are summarized and some thoughts about the future of radioiodination as a radiopharmaceutical chemistry strategy are provided.

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. 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 co-administered (125)I-labeled sdAb, renal uptake of the (18)F-labeled sdAbs was substantially lower. In microPET images, tumor was clearly delineated in SKOV-3 and BT474 xenograft-bearing athymic mice with low levels of background activity in normal tissues except bladder. These results indicate that the [(18)F]TFPFN prosthetic group could be a valuable reagent for developing sdAb-based immunoPET imaging agents.

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 18F-labeled triazinediamine analogue, [18F]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 [18F]1 in IDH1 mutant glioma cell lines, with a half-maximal inhibitory concentration value (IC50) of 54 nM and an equilibrium dissociation constant (Kd) of 40 nM. In vivo studies using mutant IDH1 glioma xenografts showed good tumor uptake of [18F]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 18F-labeled analogue for PET imaging of IDH1 mutations in gliomas.

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 micro-PET/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 ± 1.2 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). Micro-PET/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.

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel immunotoxin that reacts with wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFRvIII proteins overexpressed in glioblastomas. This study assessed the toxicity of intracerebral administration of D2C7-IT to support an initial Food and Drug Administration Investigational New Drug application. After the optimization of the formulation and administration, two cohorts (an acute and chronic cohort necropsied on study days 5 and 34) of Sprague-Dawley (SD) rats (four groups of 5 males and 5 females) were infused with the D2C7-IT formulation at total doses of 0, 0.05, 0.1, 0.4 µg (the acute cohort) and 0, 0.05, 0.1, 0.35 µg (the chronic cohort) for approximately 72 hours by intracerebral convection-enhanced delivery using osmotic pumps. Mortality was observed in the 0.40 µg (5/10 rats) and 0.35 µg (4/10 rats) high-dose groups of each cohort. Body weight loss and abnormal behavior were only revealed in the rats treated with high doses of D2C7-IT. No dose-related effects were observed in clinical laboratory tests in either cohort. A gross pathologic examination of systemic tissues from the high-dose and control groups in both cohorts exhibited no dose-related or drug-related pathologic findings. Brain histopathology revealed the frequent occurrence of dose-related encephalomalacia, edema, and demyelination in the high-dose groups of both cohorts. In this study, the maximum tolerated dose of D2C7-IT was determined to be between 0.10 and 0.35 µg, and the no observed adverse effect level was 0.05 µg in SD rats. Both parameters were utilized to design the Phase I/II D2C7-IT clinical trial.

INTRODUCTION: Radioiodinated meta-iodobenzylguanidine (MIBG), a norepinephrine transporter (NET) substrate, has been extensively used as an imaging agent to study the pathophysiology of the heart and for the diagnosis and treatment of neuroendocrine tumors. The goal of this study was to develop an (18)F-labeled analogue of MIBG that like MIBG itself could be synthesized in a single radiochemical step. Towards this end, we designed 4-fluoropropoxy-3-iodobenzylguanidine (FPOIBG). METHODS: Standards of FPOIBG and 4-fluoropropoxy-3-bromobenzylguanidine (FPOBBG) as well as their tosylate precursors for labeling with (18)F, and a tin precursor for the preparation of radioiodinated FPOIBG were synthesized. Radiolabeled derivatives were synthesized by nucleophilic substitution and electrophilic iododestannylation from the corresponding precursors. Labeled compounds were evaluated for NET transporter recognition in in vitro assays using three NET-expressing cell lines and in biodistribution experiments in normal mice, with all studies performed in a paired-label format. Competitive inhibition of [(125)I]MIBG uptake by unlabeled benzylguanidine compounds was performed in UVW-NAT cell line to determine IC50 values. RESULTS: [(18)F]FPOIBG was synthesized from the corresponding tosylate precursor in 5.2 ± 0.5% (n = 6) overall radiochemical yields starting with aqueous fluoride in about 105 min. In a paired-label in vitro assay, the uptake of [(18)F]FPOIBG at 2h was 10.2 ± 1.5%, 39.6 ± 13.4%, and 13.3 ± 2.5%, in NET-expressing SK-N-SH, UVW-NAT, and SK-N-BE(2c) cells, respectively, while these values for [(125)I]MIBG were 57.3 ± 8.1%, 82.7 ± 8.9%, and 66.3 ± 3.6%. The specificity of uptake of both tracers was demonstrated by blocking with desipramine. The (125)I-labeled congener of FPOIBG gave similar results. On the other hand, [(18)F]FPOBBG, a compound recently reported in the literature, demonstrated much higher uptake, albeit less than that of co-incubated [(125)I]MIBG. IC50 values for FPOIBG were higher than those obtained for MIBG and FPOBBG. Unlike the case with [(18)F]FPOBBG, the heart uptake [(18)F]FPOIBG in normal mice was significantly lower than that of MIBG. CONCLUSION: Although [(18)F]FPOIBG does not appear to warrant further consideration as an (18)F-labeled MIBG analogue, analogues wherein the iodine in it is replaced with a chlorine, fluorine or hydrogen might be worth pursuing. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: An (18)F-labeled analogue of the well-known radiopharmaceutical MIBG could have significant impact, potentially improving imaging of NET related disease in cardiology and in the imaging of neuroendocrine tumors. Although (18)F-labeled analogues of MIBG have been reported including LMI1195, we undertook this work hypothesizing that based on its greater structural similarity to MIBG, FPOIBG might be a better analogue than LMI1195.

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. A radioiodinated analogue of 11 containing d-tyrosine in lieu of l-tyrosine (PSMA-769-d-tyrosine) also was synthesized from its corresponding tin precursor (d-11). 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.