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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.

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.

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.

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.

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.

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.

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.

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 systemically administered 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 (EPR) effect and ultrasensitive imaging render (124)I-GNS nanoprobe promise for future brain tumor-related preclinical and translational applications.

Hypoxia is a significant therapeutic problem for solid tumors because hypoxic cells are treatment-resistant and more aggressive. Hypoxia-activated prodrugs such as SN30000 use a mechanism of activation in hypoxic cells similar to that of 2-nitroimidazole hypoxia PET tracers. Therefore, we have evaluated the usefulness of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-(18)F-pentafluoropropyl)-acetamide ((18)F-EF5) PET to monitor and predict tumor response to SN30000 plus radiation treatment (RT).Human non-small cell lung cancer xenografts (H460) in athymic rats were imaged with (18)F-EF5 PET before and after treatment with SN30000 (90 mg/kg), with or without 15-Gy RT. The feasibility of imaging early changes in hypoxia in response to SN30000 was examined 24 h after treatment, followed by ex vivo γ-counting and immunohistochemical examination to study drug-induced apoptosis. Subsequently, the therapeutic effects of SN30000 with or without RT were evaluated in tumor growth delay studies and compared with early treatment-induced changes observed by (18)F-EF5 PET. Changes in tumor hemoglobin oxygen saturation as a function of time after treatment measured by optical spectroscopy were compared with PET data.The uptake of (18)F-EF5 was significantly lower in SN30000-treated tumors than in saline controls 24 h after treatment (mean standardized uptake value, 0.44 ± 0.08 vs. 0.56 ± 0.08 for control group; P0.05). Apoptosis was significantly higher in SN30000-treated tumors than in controls. Early treatment-induced changes in (18)F-EF5 uptake were indicative of tumor response in growth delay studies at the group level. SN30000 plus RT significantly decreased (18)F-EF5 uptake relative to baseline and resulted in complete tumor remission in 5 of 7 animals. SN30000 alone decreased (18)F-EF5 uptake, generally in tumors with high initial standardized uptake values, and showed a minor tumor growth delay effect. The changes induced by SN30000 with or without RT in (18)F-EF5 uptake correlated with baseline hypoxia levels. RT caused significant increases in tumor oxygen concentration and hemoglobin oxygen saturation.A hypoxia PET imaging agent can measure changes in tumor hypoxic fraction in response to SN30000. These results suggest the utility of (18)F-EF5 PET for monitoring early response to tumor treatment with SN30000 plus RT in the clinical development of this novel hypoxia-activated prodrug.

Introduction With a molecular weight an order of magnitude lower than antibodies but possessing comparable affinities, Nanobodies (Nbs) are attractive as targeting agents for cancer diagnosis and therapy. An anti-HER2 Nb could be utilized to determine HER2 status in breast cancer patients prior to trastuzumab treatment. This provided motivation for the generation of HER2-specific 5F7GGC Nb, its radioiodination and evaluation for targeting HER2 expressing tumors. Methods 5F7GGC Nb was radioiodinated with 125 I using Iodogen and with 131 I using the residualizing agent N ɛ -(3-[ 131 I]iodobenzoyl)-Lys 5 -N α -maleimido-Gly 1 -GEEEK ([ 131 I]IB-Mal-d-GEEEK) used previously successfully with intact antibodies. Paired-label internalization assays using BT474M1 cells and tissue distribution experiments in athymic mice bearing BT474M1 xenografts were performed to compare the two labeled Nb preparations. Results The radiochemical yields for Iodogen and [ 131 I]IB-Mal-d-GEEEK labeling were 83.6±5.0% (n=10) and 59.6±9.4% (n=15), respectively. The immunoreactivity of labeled proteins was preserved as confirmed by in vitro and in vivo binding to tumor cells. Biodistribution studies showed that Nb radiolabeled using [ 131 I]IB-Mal-d-GEEEK, compared with the directly labeled Nb, had a higher tumor uptake (4.65±0.61% ID/g vs. 2.92±0.24% ID/g at 8h), faster blood clearance, lower accumulation in non-target organs except kidneys, and as a result, higher concomitant tumor-to-blood and tumor-to-tissue ratios. Conclusions Taken together, these results demonstrate that 5F7GGC anti-HER2 Nb labeled with residualizing [ 131 I]IB-Mal-d-GEEEK had better tumor targeting properties compared to the directly labeled Nb suggesting the potential utility of this Nb conjugate for SPECT ( 129 I) and PET imaging ( 124 I) of patients with HER2-expressing tumors.

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.

Intratumoral radiation therapy – ‘brachytherapy’ – is a highly effective treatment for solid tumors, particularly prostate cancer. Current titanium seed implants, however, are permanent and are limited in clinical application to indolent malignancies of low- to intermediate-risk. Attempts to develop polymeric alternatives, however, have been plagued by poor retention and off-target toxicity due to degradation. Herein, we report on a new approach whereby thermally sensitive micelles composed of an elastin-like polypeptide (ELP) are labeled with the radionuclide 131I to form an in situ hydrogel that is stabilized by two independent mechanisms: first, body heat triggers the radioactive ELP micelles to rapidly phase transition into an insoluble, viscous coacervate in under 2 minutes; second, the high energy β-emissions of 131I further stabilize the depot by introducing crosslinks within the ELP depot over 24 hours. These injectable brachytherapy hydrogels were used to treat two aggressive orthotopic tumor models in athymic nude mice: a human PC-3M-luc-C6 prostate tumor and a human BxPc3-luc2 pancreatic tumor model. The ELP depots retained greater than 52% and 70% of their radioactivity through 60 days in the prostate and pancreatic tumors with no appreciable radioactive accumulation (≤ 0.1% ID) in off-target tissues after 72 hours. The 131I-ELP depots achieved >95% tumor regression in the prostate tumors (n=8); with a median survival of more than 60 days compared to 12 days for control mice. For the pancreatic tumors, ELP brachytherapy (n=6) induced significant growth inhibition (p = 0.001, ANOVA) and enhanced median survival to 27 days over controls.

Pancreatic tumors remain one of the deadliest manifestations of cancer in clinical oncology. Even when tumors are diagnosed early, remain loco-regionalized, and receive cutting-edge cancer treatment, the 5-year survival rate remains less than 27%. This high mortality rate arises from the significant biologic barriers that inhibit drug delivery effectiveness in pancreatic tumors, as well as their underlying genetic mutations that confer resistance to traditional radiation and chemotherapeutic standards of care. In this work, we hypothesized that a new form of radiation brachytherapy might prove highly synergistic for overcoming the resistance native to pancreatic tumors. Utilizing the thermally stabilized biopolymer ELP, 131iodine radionuclides can be delivered intratumorally to provide continuous, focalized, high-dose β-radiation to treat pancreatic cancer from the inside out. Dose-escalation studies revealed that the two important variables for improving outcomes were the 131I-ELP radioactivity dose and the duration of concurrent paclitaxel therapy. 100% tumor regression was achieved across multiple genotypic models of pancreatic cancer models: MIA PaCa-2, AsPc-1, and BxPc3 (both subcutaneous and orthotopic). The potent synergistic results of 131I-ELP brachytherapy were replicated using both a novel micelle formulation of paclitaxel and the clinical standard Abraxane. Multigroup analysis revealed true molecular synergy as assessed by the Bliss Independence framework (p < 0.01). External beam irradiation, when similarly employed as comparison, merely induced growth inhibition when combined with paclitaxel utilizing a 5Gy hypofractionation regimen. Immunohistochemical analysis of treated tumors revealed differential modulation of biomolecular components that regulate the effectiveness of drug delivery and radiotoxicity. Levels of PECAM, VE-cadherin, claudin-4, HIF-1α, and stromal collagen were all specifically investigated, while macroscopic analysis revealed significantly enhanced accumulation and retention of fluorescently tagged paclitaxel when tumors were treated with biopolymer brachytherapy. Standard x-ray therapy indicated little to no improvement. No signs of any toxic side effects were observed in serologic enzyme analysis and multiorgan biodistribution studies. Citation Format: Jeffrey L. Schaal, Jayanta Bhattacharyya, Xinghai Li, Samagya Banskota, Jeremy Brownstein, David Kirsch, Michael Zalutsky, Ashutosh Chilkoti. Biopolymer β-brachytherapy delivered concomitantly with systemic paclitaxel outperforms traditional x-ray radiation to induce complete regression in multiple pancreatic tumor xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5831.

Medulloblastoma, the most common pediatric brain tumor, is difficult to treat because conventional therapeutic approaches result in significant toxicity to normal central nervous system tissues, compromising quality of life. Given the fact that medulloblastomas express the somatostatin subtype 2 receptor, [177Lu-DOTA0,Tyr3]octreotate ([177Lu]DOTA-TATE) could be a potentially useful targeted radiotherapeutic for the treatment of this malignancy. The current study was undertaken to evaluate this possibility in preclinical models of D341 MED human medulloblastoma by comparing the properties of [177Lu]DOTA-TATE to those of glucose-[125I-Tyr3]-octreotate ([125I]Gluc-TOCA), a radiopeptide previously shown to target this cell line. In vitro assays indicated that both labeled peptides exhibited similar cell-associated and internalized radioactivity after a 30-min incubation at 37° C; however, at the end of the 4 h incubation period, the internalized radioactivity for [177Lu]DOTA-TATE (6.22 ± 0.75%) was nearly twice that for [125I]Gluc-TOCA (3.16 ± 0.27%), with similar differences seen in total cell-associated radioactivity levels. Consistent with the results from the internalization assays, results from paired-label tissue distribution studies in athymic mice with subcutaneous D341 MED medulloblastoma xenografts showed a similar degree of tumor accumulation for [177Lu]DOTA-TATE and [125I]Gluc-TOCA at early time points but by 24 h, a more than 5-fold advantage was observed for the 177Lu-labeled peptide. Tumor-to-normal tissue ratios generally were more favorable for [177Lu]DOTA-TATE at all time points, due in part to its lower accumulation in normal tissues except kidneys. Taken together, these results suggest that [177Lu]DOTA-TATE warrants further investigation as a targeted radiotherapeutic for medulloblastoma treatment.

Adults with primary malignant glioma have an unacceptably poor outcome. Although temozolomide plus radiotherapy improves survival of newly diagnosed glioblastoma multiforme (GBM) patients,1 most patients develop tumor progression within 1 – 2 years. Outcome following recurrence is poor.2 Most tumors recur at or adjacent to the site of origin, indicating that failure to eradicate local tumor growth is a major factor contributing to poor outcome.3 For this reason, we have focused on augmenting local control to improve overall outcome by administering tumor-associated radiolabeled monoclonal antibodies (mAbs) directly into spontaneous tumor cysts, surgically created resection cavities (SCRCs), the intrathecal space, and solid tumors.4–8 Tenascin, an extracellular matrix hexabrachion glycoprotein, is expressed ubiquitously in several cancers, including high-grade gliomas, but not in normal brain.9,10 mAb 81C6, a murine isotype 2b immunoglobulin G (IgG2b) that binds to an alternatively spliced region of tenascin,9–12 reacts specifically with tenascin-expressing tumors.13 When labeled with 131I, 81C6 delays tumor growth and prolongs survival in flank and intracranial human xenograft models.14,15 An initial human experience demonstrated the specificity of 131I-labeled murine 81C6 (131I-81C6) mAb compared to 125I-labeled nonspecific IgG2b mAb but also revealed limited intratumor penetration following intravenous or intraarterial administration.16 Hence, subsequent studies incorporated administration into an SCRC, tumor cyst, or intrathecal space. Prior phase I studies established the maximum tolerated dose (MTD) of 131I-81C6 injected into the SCRC of patients with newly diagnosed and recurrent malignant brain tumors to be 120 mCi and 100 mCi, respectively.7,8 Phase II studies demonstrated that patients treated with 131I-81C6 had favorable overall survival compared to established historical controls.4,5 Based on constraints of our U.S. Food and Drug Administration (FDA)-approved phase I and II studies, patients received a “fixed” dose of 131I-81C6 in which the administered level of radioactivity was the same among groups of patients. As a result of this design, 131I dosages were not adjusted to compensate for patient-specific variables such as SCRC volume and SCRC residence time. Dosimetry of patients treated on these studies revealed a wide range of radiation absorbed doses at the SCRC margin. Moreover, outcome correlated with delivered radiation dose to the SCRC. Specifically, patients who received less than 44 Gy were more likely to develop recurrent tumor, whereas those who received significantly more than 44 Gy were more likely to develop radionecrosis. Therefore, a 44-Gy boost to the SCRC margin was considered optimal.17 We now report a pilot study using a novel, patient-specific dosing strategy of 131I-81C6 designed to achieve a 44-Gy boost to the 2-cm SCRC margin in adults with newly diagnosed malignant gliomas.

Alpha-particle emitters have a high linear energy transfer and short range, offering the potential for treating micrometastases while sparing normal tissues. We developed a urea-based, 211At-labeled small molecule targeting prostate-specific membrane antigen (PSMA) for the treatment of micrometastases due to prostate cancer (PC). Methods: PSMA-targeted (2S)-2-(3-(1-carboxy-5-(4-211At-astatobenzamido)pentyl)ureido)-pentanedioic acid (211At-6) was synthesized. Cellular uptake and clonogenic survival were tested in PSMA-positive (PSMA+) PC3 PIP and PSMA-negative (PSMA−) PC3 flu human PC cells after 211At-6 treatment. The antitumor efficacy of 211At-6 was evaluated in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts at a 740-kBq dose and in mice bearing PSMA+, luciferase-expressing PC3-ML micrometastases. Biodistribution was determined in mice bearing PSMA+ PC3 PIP and PSMA– PC3 flu flank xenografts. Suborgan distribution was evaluated using α-camera images, and microscale dosimetry was modeled. Long-term toxicity was assessed in mice for 12 mo. Results: 211At-6 treatment resulted in PSMA-specific cellular uptake and decreased clonogenic survival in PSMA+ PC3 PIP cells and caused significant tumor growth delay in PSMA+ PC3 PIP flank tumors. Significantly improved survival was achieved in the newly developed PSMA+ micrometastatic PC model. Biodistribution showed uptake of 211At-6 in PSMA+ PC3 PIP tumors and in kidneys. Microscale kidney dosimetry based on α-camera images and a nephron model revealed hot spots in the proximal renal tubules. Long-term toxicity studies confirmed that the dose-limiting toxicity was late radiation nephropathy. Conclusion: PSMA-targeted 211At-6 α-particle radiotherapy yielded significantly improved survival in mice bearing PC micrometastases after systemic administration. 211At-6 also showed uptake in renal proximal tubules resulting in late nephrotoxicity, highlighting the importance of long-term toxicity studies and microscale dosimetry.

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.

alpha-Particle-emitting radionuclides, such as (211)At, with a 7.2-h half-life, may be optimally suited for the molecularly targeted radiotherapy of strategically sensitive tumor sites, such as those in the central nervous system. Because of the much shorter range and more potent cytotoxicity of alpha-particles than of beta-particles, (211)At-labeled agents may be ideal for the eradication of tumor cells remaining after surgical debulking of malignant brain tumors. The main goal of this study was to investigate the feasibility and safety of this approach in patients with recurrent malignant brain tumors.Chimeric antitenascin monoclonal antibody 81C6 (ch81C6) (10 mg) was labeled with 71-347 MBq of (211)At by use of N-succinimidyl 3-[(211)At]astatobenzoate. Eighteen patients were treated with (211)At-labeled ch81C6 ((211)At-ch81C6) administered into a surgically created resection cavity (SCRC) and then with salvage chemotherapy. Serial gamma-camera imaging and blood sampling over 24 h were performed.A total of 96.7% +/- 3.6% (mean +/- SD) of (211)At decays occurred in the SCRC, and the mean blood-pool percentage injected dose wasor = 0.3. No patient experienced dose-limiting toxicity, and the maximum tolerated dose was not identified. Six patients experienced grade 2 neurotoxicity within 6 wk of (211)At-ch81C6 administration; this neurotoxicity resolved fully in all but 1 patient. No toxicities of grade 3 or higher were attributable to the treatment. No patient required repeat surgery for radionecrosis. The median survival times for all patients, those with glioblastoma multiforme, and those with anaplastic astrocytoma or oligodendroglioma were 54, 52, and 116 wk, respectively.This study provides proof of concept for regional targeted radiotherapy with (211)At-labeled molecules in oncology. Specifically, the regional administration of (211)At-ch81C6 is feasible, safe, and associated with a promising antitumor benefit in patients with malignant central nervous system tumors.

ELPs are genetically engineered, thermally responsive polypeptides that preferentially accumulate in solid tumors subjected to focused, mild hyperthermia. In this paper, we report the biodegradation, pharmacokinetics, tumor localization, and tumor spatial distribution of (14)C-labeled ELPs that were radiolabeled during their biosynthesis in Escheriehia coli. The in vitro degradation rate of a thermally responsive (14)C-labeled ELP1 ([(14)C] ELP1) with a molecular weight of 59.4 kDa, upon exposure to murine serum, was 2.49 wt.%/day. The apparent in vivo degradation rate of ELP1 after intravenous injection of nude mice was 2.46 wt.%/day and its terminal half-life was 8.7 h. The tumor accumulation and spatial distribution of intravenously administered ELP1 and a control ELP that was designed to remain soluble in heated tumors (ELP2) were examined in both heated (41.5 degrees C) and unheated tumors. ELP1 accumulated at a significantly higher concentration in heated tumors than ELP1 in unheated tumors and ELP2 in heated tumors. Quantitative autoradiography of tumor sections provided similar tumor accumulation results as the whole tumor analysis but, in addition, showed that ELP1 had a more homogeneous distribution in heated tumors and a greater concentration in the tumor center than either control treatment.

Intra-articular drug delivery is the preferred standard for targeting pharmacologic treatment directly to joints to reduce undesirable side effects associated with systemic drug delivery. In this study, a biologically based drug delivery vehicle was designed for intra-articular drug delivery using elastin-like polypeptides (ELPs), a biopolymer composed of repeating pentapeptides that undergo a phase transition to form aggregates above their transition temperature. The ELP drug delivery vehicle was designed to aggregate upon intra-articular injection at 37 degrees C, and form a drug 'depot' that could slowly disaggregate and be cleared from the joint space over time. We evaluated the in vivo biodistribution and joint half-life of radiolabeled ELPs, with and without the ability to aggregate, at physiological temperatures encountered after intra-articular injection in a rat knee. Biodistribution studies revealed that the aggregating ELP had a 25-fold longer half-life in the injected joint than a similar molecular weight protein that remained soluble and did not aggregate. These results suggest that the intra-articular joint delivery of ELP-based fusion proteins may be a viable strategy for the prolonged release of disease-modifying protein drugs for osteoarthritis and other arthritides.

Purpose To assess the efficacy and toxicity of intraresection cavity iodine-131–labeled murine antitenascin monoclonal antibody 81C6 (131I-m81C6) among recurrent malignant brain tumor patients. Patients and Methods In this phase II trial, 100 mCi of 131I-m81C6 was injected directly into the surgically created resection cavity (SCRC) of 43 patients with recurrent malignant glioma (glioblastoma multiforme [GBM], n = 33; anaplastic astrocytoma [AA], n = 6; anaplastic oligodendroglioma [AO], n = 2; gliosarcoma [GS], n = 1; and metastatic adenocarcinoma, n = 1) followed by chemotherapy. Results With a median follow-up of 172 weeks, 63% and 59% of patients with GBM/GS and AA/AO tumors were alive at 1 year. Median overall survival for patients with GBM/GS and AA/AO tumors was 64 and 99 weeks, respectively. Ten patients (23%) developed acute hematologic toxicity. Five patients (12%) developed acute reversible neurotoxicity. One patient (2%) developed irreversible neurotoxicity. No patients required reoperation for radionecrosis. Conclusion In this single-institution phase II study, administration of 100 mCi of 131I-m81C6 to recurrent malignant glioma patients followed by chemotherapy is associated with a median survival that is greater than that of historical controls treated with surgery plus iodine-125 brachytherapy. Furthermore, toxicity was acceptable. Administration of a fixed millicurie dose resulted in a wide range of absorbed radiation doses to the SCRC. We are now conducting a phase II trial, approved by the US Food and Drug Administration, using patient-specific 131I-m81C6 dosing, to deliver 44 Gy to the SCRC followed by standardized chemotherapy. A phase III multicenter trial with patient-specific dosing is planned.

Radioiodinated meta-iodobenzylguanidine (MIBG) is used in the diagnosis and therapy of various neuroendocrine tumors. As a part of our efforts to develop an MIBG analogue with improved characteristics for these applications, a synthesis of 3-[131I]iodo-4-methylbenzylguanidine ([131I]MeIBG) was developed. Unlabeled MeIBG and the tin precursor, N, N'-(bis-tert-butyloxycarbonyl)-N-(4-methyl-3-trimethylstannylbenzyl) guanidine were synthesized in two steps from 3-iodo-4-methylbenzylalcohol. Radioiodinated MeIBG was synthesized at a no-carrier-added level by the iododestannylation of the tin precursor in about 85% radiochemical yield. The accumulation of [131I]MeIBG (38.9+/-3.0% of input counts) by human neuroblastoma SK-N-SH cells in vitro was 87% that of [125I]MIBG (44.5+/-3.0%) and a number of Uptake-1 inhibiting conditions reduced the uptake of both tracers in this cell line to a similar degree suggesting that introduction of a methyl substituent at the 4-position of MIBG did not adversely affect its biological characteristics.

Improving drug and macromolecular delivery of anti-cancer agents to tumours results in greater efficacy without increased toxicity. The current study was undertaken to assess the effects of the timing of injection of tumour specific and non-specific monoclonal antibodies (mAbs) relative to a hyperthermia treatment on tumour and normal tissue uptake. Using a local hyperthermia protocol of 45 min at 43 degrees C, uptake in tumour and normal tissues was measured at 1, 4, 12, 24, 48 and 72 h after injection. An anti-tenascin chimeric mAb, ch81C6, served as the specific mAb in a D-54 MG glioma xenograft mouse model. The chimeric mAb chTPS3.2 served as the control. A five-to-eight-fold increase in uptake of the tumour-targeted mAb was achieved in the heated tumours when compared with the non-heated tumours at 1 h. Differences in absolute tumour uptake of the specific mAb between the mice injected prior to hyperthermia and mice injected post-hyperthermia were seen only at 1 and 12 h. The median uptakes in the tumours of mice injected pre-heat were 25%ID/g at 1 h and 43.5%ID/g at 12 h, while in the animals injected post-hyperthermia the median uptakes were 45.5%ID/g and 80.2%ID/g, respectively. Blood levels of both the specific and non-specific mAbs were consistently higher over the initial 12 h period in the mice injected post-hyperthermia. Normal tissue uptake was also increased at most time points in the mice injected post-hyperthermia. The clinical importance of the differences in specific mAb uptake in tumour detected statistically at 1 and 12 h is questionable, given the highly variable nature of mAb uptake in vivo. Tumour targeting mAbs administered in combination with heat may be injected either prior to or immediately following hyperthermia treatment, with the expectation that levels of uptake in tumour will be relatively equivalent. Absolute normal tissue levels will be higher in patients receiving the mAb post-hyperthermia.

Radioiodinated meta -iodobenzylguanidine (MIBG) is used in the diagnosis and therapy of various neuroendocrine tumors. To investigate whether an additional guanidine function in the structure of MIBG will yield analogues that may potentially enhance tumor-to-target ratios, two derivatives—one with a guanidine moiety and another with a guanidinomethyl group at the 4-position of MIBG—were prepared. In the absence of any uptake-1 inhibiting conditions, the uptake of 4-guanidinomethyl-3-[ 131 I]iodobenzylguanidine ([ 131 I]GMIBG) by SK-N-SH cells in vitro was 1.7±0.1% of input counts, compared to a value of 40.3±1.4% for [ 125 I[MIBG suggesting that guanidinomethyl group at the 4-position negated the biological properties of MIBG. On the other hand, 4-guanidino-3-[ 131 I]iodobenzylguanidine ([ 131 I]GIBG) had an uptake (5.6±0.3%) that was 12–13% that of [ 125 I]MIBG (46.1±2.7%), and the ratio of uptake by control over DMI-treated (nonspecific) cultures was higher for [ 131 I]GIBG (20.9±0.3) than [ 125 I]MIBG itself (15.0±2.7). The exocytosis of [ 131 I]GIBG and [ 125 I]MIBG from SK-N-SH cells was similar. The uptake of [ 131 I]GIBG in the mouse target tissues, heart and adrenals, as well as in a number of other tissues was about half that of [ 125 I]MIBG. These results suggest that substitution of guanidine functions, especially a guanidinomethyl group, in MIBG structure may not be advantageous.

The utility of external beam radiotherapy for the treatment of malignant brain tumours is compromised by the need to avoid excessive radiation damage to normal CNS tissues. This review describes the current status of targeted radiotherapy, an alternative strategy for brain tumour treatment that offers the exciting prospect of increasing the specificity of tumour cell irradiation.

Meta-( 131 I)iodobenzylguanidine (( 131 I)MIBG), used for neuroblastoma treatment, binds to the noradrenaline transporter (NAT). After NAT gene transfection, other tumour types might be treatable with ( 131 I)MIBG. At 14 MBq, carrier-added ( 131 I)MIBG (1110 MBq/mg) delayed tumour regrowth in NAT gene-transfected xenografts by 39-days, while no-carrier-added ( 131 I)MIBG (>104 GBq/mg) cured 100% of tumours.

160 Background: Radium-223 is a targeted alpha therapy that improves survival in men with mCRPC. The biologic basis for radium-223 efficacy is not completely understood. We hypothesized that PC osteomimicry, a form of epithelial plasticity leading to an osteoblastic phenotype, may contribute to the intralesional deposition of radium-223 and subsequent irradiation of the tumor microenvironment. Methods: We conducted a pharmacodynamic study of radium-223 in men with bone predominant mCRPC to investigate genomic and phenotypic alterations in circulating tumor cells (CTCs), ctDNA, and metastases. Prior to radium and 3 and 6 months after radium, liquid and metastatic biopsies were collected, including CTCs for phenotypic characterization and CTC/ctDNA genomic analysis. The primary objective was to describe the prevalence of CTC bone alkaline phosphatase (BAP) over time. We measured radium-223 decay products in tumor and surrounding normal bone during treatment. Results: We enrolled 20 men with heavily pre-treated symptomatic bone predominant mCRPC and treated with radium-223 over a median of 6 doses. 55% had elevated serum BAP. PFS was 5.5 mo; OS was 13.3 mo; 10% had unfavorable (≥5) to favorable ( < 5) Cellsearch CTC conversion; 5% had ≥30% PSA decline. We found evidence of persistent CTC BAP expression in both EpCAM+ and EpCAM- CTCs in the majority of men over time during radium treatment. We identified genomic gain of key osteomimicry regions in CTC DNA, including loci for BAP, osteopontin, and OB-cadherin. Radium-223 uptake was observed in tumor to a greater degree than surrounding normal bone. CTC DNA and matched ctDNA and CTC cultures suggested persistence of CTCs with aggressive genomic alterations such as AR, FOXA1, and MYC gain and PTEN and RB1 loss. We established multiple CTC cultures and one CTC PDX; cells exhibited evidence of epithelial plasticity with BAP expression. Conclusions: Osteomimicry may contribute to the uptake of Radium-223 within bone metastases and may thereby enhance the therapeutic benefit of radium-223. We found genomic and phenotypic evidence of osteomimicry in CTCs and CTC cultures from men with mCRPC. Clinical trial information: NCT02204943.

p-[18F]Fluorobenzylguanidine ([18F]PFBG) is a norepinephrine analog that has been developed as a positron emission tomography (PET) imaging radiopharmaceutical. Myocardial sympathetic innervation, neuroendocrine structures, and tumors can be noninvasively imaged with [18F]PFBG. In this study, the uptake characteristics of [18F]PFBG were investigated in 2 dogs with a spontaneous pheochromocytoma. The extent of the pheochromocytoma was well documented in both dogs on the PET study. The standardized uptake values within the pheochromocytomas were greater than 25 by 10 min, and were 37 and 50 by 45 min in each dog. A third dog that was suspected to have an adrenal mass was also studied. In this dog, the [18F]PFBG study was normal. Surgical exploration and adrenal biopsy confirmed the [15F]PFBG imaging findings in both dogs. In each dog, there was rapid blood-pool clearance (within 10 min after intravenous administration of the [18F]PFBG), with high uptake specific within the myocardium and adrenal medulla. The results indicate that [18F]PFBG may be useful for imaging canine pheochromocytomas and aid in differentiating adrenal masses.

Monoclonal antibodies (mAbs) such as the tumor-specific anti-epidermal growth factor receptor variant III (EGFRvIII) that are internalized and degraded after cell binding necessitate the use of radioiodination methods that minimize the loss of radioactivity from the tumor cell after intracellular processing. The purpose of the current study was to determine the suitability of N -succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) for labeling this internalizing mAb. A series of paired-label biodistribution experiments were performed in athymic mice bearing subcutaneous, EGFRvIII-expressing, D-256 human glioma and U87ΔEGFR xenografts. The tissue distribution of radioiodine activity following injection of anti-EGFRvIII mAb L8A4 labeled using N -succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) were compared to those for mAb labeled using Iodogen, N -succinimidyl 3-iodo-5-pyridinecarboxylate (SIPC) as well as the Boc-protected precursor of SGMIB. Tumor uptake of radioiodine activity for mAb labeled via SGMIB was significantly higher than co-administered L8A4 radioiodinated by other methods. For example, 3 days after injection, D-256 tumor uptake of L8A4 labeled via SGMIB was 20.4 ± 4.6% ID/g compared with 11.7 ± 5.5% ID/g when the SIPC method was used. Thyroid uptake for L8A4 (SGMIB) was up to 36 times lower than L8A4 (Iodogen) and less than 0.35% in all experiments, indicating a low degree of deiodination in vivo . These results suggest that SGMIB may be a useful reagent for the radioiodination of this internalizing anti-EGFRvIII mAb.

Previously published reports indicate that serum copper levels are elevated in patients with prostate cancer and that increased copper uptake can be used as a means to image prostate tumors. It is unclear, however, to what extent copper is required for prostate cancer cell function as we observed only modest effects of chelation strategies on the growth of these cells in vitro. With the goal of exploiting prostate cancer cell proclivity for copper uptake, we developed a “conditional lethal” screen to identify compounds whose cytotoxic actions were manifested in a copper-dependent manner. Emerging from this screen was a series of dithiocarbamates, which, when complexed with copper, induced reactive oxygen species–dependent apoptosis of malignant, but not normal, prostate cells. One of the dithiocarbamates identified, disulfiram (DSF), is an FDA-approved drug that has previously yielded disappointing results in clinical trials in patients with recurrent prostate cancer. Similarly, in our studies, DSF alone had a minimal effect on the growth of prostate cancer tumors when propagated as xenografts. However, when DSF was coadministered with copper, a very dramatic inhibition of tumor growth in models of hormone-sensitive and of castrate-resistant disease was observed. Furthermore, we determined that prostate cancer cells express high levels of CTR1, the primary copper transporter, and additional chaperones that are required to maintain intracellular copper homeostasis. The expression levels of most of these proteins are increased further upon treatment of androgen receptor (AR)–positive prostate cancer cell lines with androgens. Not surprisingly, robust CTR1-dependent uptake of copper into prostate cancer cells was observed, an activity that was accentuated by activation of AR. Given these data linking AR to intracellular copper uptake, we believe that dithiocarbamate/copper complexes are likely to be effective for the treatment of patients with prostate cancer whose disease is resistant to classical androgen ablation therapies. Cancer Res; 74(20); 5819–31. ©2014 AACR.

Even with novel chemotherapeutic agents and external beam radiation therapy, the prognosis of neoplastic meningitis secondary to malignant melanoma is still dismal. The authors report a case study of a 46-year-old white female who presented with progressive hearing loss, severe headaches, nausea, vomiting, and a rapid decline in neurologic status. She was referred to Duke University Medical Center after conventional chemotherapy for malignant melanoma failed. She was enrolled in a Phase I trial of 131I-labeled monoclonal antibody Mel-14 F(ab′)2 fragment administered intrathecally. Within a year after her treatment, she recovered, having a normal neurologic exam except for residual bilateral hearing loss. The authors discuss dosimetry, preclinical, and clinical studies conducted with Mel-14 F(ab′)2 and introduce a potentially promising therapy option in the treatment of neoplastic meningitis in patients with malignant melanoma. Currently, the patient remains neurologically normal except for a mild bilateral hearing loss more than 4 years after treatment and has no radiographic evidence of neoplastic meningitis. Cancer 2001;91:1809–13. © 2001 American Cancer Society.

PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine-131 (131I)–labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment. PATIENTS AND METHODS: In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of 131I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment. RESULTS: We treated 42 patients with 131I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively. CONCLUSION: The MTD for administration of 131I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.

Monoclonal antibody (MAb) internalization can have a major effect on tumor retention of radiolabel. Two anti-HER-2/neu MAbs (TA1 and 520C9) were radioiodinated using the iodogen, N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC), and tyramine-cellobiose (TCB) methods. Paired-label studies compared internalization and cellular processing of the labeled MAbs by SKOv3 9002-18 ovarian cancer cells in vitro. Intracellular radioiodine activity for 520C9 was up to 2.6 and 3.0 times higher for SIPC and TCB labeling, respectively, compared with iodogen. Likewise, intracellular activity for TA1 was up to 2.3 and 2.9 times higher with the SIPC and TCB methods compared with iodogen labeling. Unfortunately, similar advantages in tumor accumulation were not achieved in athymic mice bearing SKOv3 9008-18 ovarian cancer xenografts.

Nanobodies are approximately 15-kDa proteins based on the smallest functional fragments of naturally occurring heavy chain-only antibodies and represent an attractive platform for the development of molecularly targeted agents for cancer diagnosis and therapy. Because the human epidermal growth factor receptor type 2 (HER2) is overexpressed in breast and ovarian carcinoma, as well as in other malignancies, HER2-specific Nanobodies may be valuable radiodiagnostics and therapeutics for these diseases. The aim of the present study was to evaluate the tumor-targeting potential of anti-HER2 5F7GGC Nanobody after radioiodination with the residualizing agent N-succinimidyl 4-guanidinomethyl 3-(125/131)I-iodobenzoate (*I-SGMIB). METHODS: The 5F7GGC Nanobody was radiolabeled using *I-SGMIB and, for comparison, with N(?)-(3-*I-iodobenzoyl)-Lys(5)-N(?)-maleimido-Gly(1)-GEEEK (*I-IB-Mal-d-GEEEK), another residualizing agent, and by direct radioiodination using IODO-GEN ((125)I-Nanobody). The 3 labeled Nanobodies wereevaluated in affinity measurements, and paired-label internalization assays were performed on HER2-expressing BT474M1 breast carcinoma cells and in paired-label tissue distribution measurements in mice bearing subcutaneous BT474M1 xenografts. RESULTS: *I-SGMIB-Nanobody was produced in 50.4% ± 3.6% radiochemical yield and exhibited a dissociation constant of 1.5 ± 0.5 nM. Internalization assays demonstrated that intracellular retention of radioactivity was up to 1.5-fold higher for *I-SGMIB-Nanobody than for coincubated (125)I-Nanobody or *I-IB-Mal-d-GEEEK-Nanobody. Peak tumor uptake for *I-SGMIB-Nanobody was 24.50% ± 9.89% injected dose/g at 2 h, 2- to 4-fold higher than observed with other labeling methods, and was reduced by 90% with trastuzumab blocking, confirming the HER2 specificity of localization. Moreover, normal-organ clearance was fastest for *I-SGMIB-Nanobody, such that tumor-to-normal-organ ratios greater than 50:1 were reached by 24 h in all tissues except lungs and kidneys, for which the values were 10.4 ± 4.5 and 5.2 ± 1.5, respectively. CONCLUSION: Labeling anti-HER2 Nanobody 5F7GGC with *I-SGMIB yields a promising new conjugate for targeting HER2-expressing malignancies. Further research is needed to determine the potential utility of *I-SGMIB-5F7GGC labeled with (124)I, (123)I, and (131)I for PET and SPECT imaging and for targeted radiotherapy, respectively.

A paired-label study was performed in athymic mice bearing subcutaneous D-54 MG human glioma xenografts to compare the localization of human/mouse anti-tenascin chimeric antibody 81C6 labeled by reaction with N-succinimidyl 3-[211At]astatobenzoate and N-succinimidyl 3-[131I]iodobenzoate. Over the 48-h observation period, the distribution of 211At- and 131I-labeled antibody were quite similar in tumor and normal tissues except stomach. These data were used to calculate human radiation doses for both intravenously and intrathecal administered 211At-labeled chimeric 81C6 using a quality factor of 5 for alpha-emissions.

An analogue of meta-iodobenzylguanidine (MIBG) in which an aromatic hydrogen was replaced with fluorine has been found to possess many properties similar to those of the parent compound. Moreover, 4-fluoro-3-iodobenzylguanidine (FIBG) was retained in vitro by human neuroblastoma cells to a much greater extent than MIBG itself. Since alpha-emitters such as 211At could be valuable for the treatment of micrometastatic disease, an FIBG analogue in which the iodine atom is replaced by 211At would be of interest. In this study, we have evaluated the in vitro and in vivo properties of 3-[211At]astato-4-fluorobenzylguanidine ([211At]AFBG). The specific binding of [211At]AFBG to SK-N-SH human neuroblastoma cells remained fairly constant over 2- to 3-log activity range and was similar to that of [131I]MIBG. The uptake of [211At]AFBG by this cell line was reduced by desipramine, ouabain, 4 degrees C incubation, noradrenaline, unlabelled MIBG and FIBG, suggesting that its uptake is specifically mediated through an active uptake-1 mechanism. Over the 16 h period studied, the amount of [211At]AFBG retained was similar to that of [131I]FIBG, whereas the per cent of retained meta-[211At]astatobenzylguanidine ([211At]MABG) was considerably less than that of [131I]FIBG (53% vs 75%; P < 0.05). The IC50 values for the inhibition of uptake of [131I]MIBG, [211At]MABG, [125I]FIBG and [211At]AFBG by unlabelled MIBG were 209, 300, 407 and 661 nM respectively, suggesting that the affinities of these tracers for the noradrenaline transporter in SK-N-SH cells increase in that order. Compared with [211At]MABG, higher uptake of [211At]AFBG was seen in vivo in normal mouse target tissues such as heart and, to a certain extent, in adrenals. That the uptake of [211At]AFBG in these tissues was related to the uptake-1 mechanism was demonstrated by its reduction when mice were pretreated with desipramine. However, the stability of [211At]AFBG towards in vivo dehalogenation was less than that of [211At]MABG, as evidenced by the higher uptake of 211At in thyroid, spleen, lungs and stomach.

Solid tumours have an elevated interstitial fluid pressure (IFP) due to the lack of normal lymphatics, increased permeability of tumour vasculature and an expanding cell population within a potentially limited space. This elevated IFP has been proposed to be an important barrier to the delivery of drugs and marcromolecules. We have demonstrated that local hyperthermia (4 h, 41.8 degrees C) is capable of significantly enhancing the uptake of radiolabelled monoclonal antibodies (mAbs) in D-54 MG glioma xenografts grown subcutaneously in athymic mice. To determine if this increased uptake was attributable to alterations in the tumour IFP, pressure measurements using the wick-in-needle technique were made in tumours after hyperthermia treatment. These pressure measurements were taken at various time points from 4 to 90 h following the initiation of the hyperthermia and compared with pressures taken concurrently in untreated tumours. In addition, pressures were measured following a 2 h, 41.8 degrees C hyperthermia treatment, a protocol which does not result in elevated uptake of radiolabeled mAbs. No significant differences were seen at any time point in IFP measured in the tumours receiving either hyperthermia treatment when compared with untreated tumours. Thus, we conclude that the mechanism by which this hyperthermia regimen enhances mAb uptake in this human glioma xenograft model is not due to alternations in tumour IFP.

SummaryInhibitors of heat-shock protein 90 (Hsp90) have demonstrated an unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained, but could be influenced by ectopically expressed Hsp90 in tumors. In this work, we synthesized Hsp90 inhibitors that can carry optical or radioiodinated probes via a polyethyleneglycol tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein occurs at the plasma membrane. In mice, we observed exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors that target ectopic Hsp90 can be used to detect breast cancer malignancies through noninvasive imaging.

Radionuclides such as 211At and 212Bi which decay by the emission of alpha-particles are attractive for certain applications of targeted radiotherapy. The tissue penetration of 212Bi and 211At alpha-particles is equivalent to only a few cell diameters, offering the possibility of combining cell-specific targeting with radiation of similar range. Unlike the beta-particles emitted by radionuclides such as 131I and 90Y, alpha-particles are radiation of high linear energy transfer and thus greater biological effectiveness. Several approaches have been explored for targeted radiotherapy with 212Bi- and 211At-labelled substances including colloids, monoclonal antibodies, metabolic precursors, receptor-avid ligands and other lower molecular weight molecules. An additional agent which exemplifies the promise of alpha-emitting radiopharmaceuticals is meta-[211At]astatobenzylguanidine. The toxicity of this compound under single-cell conditions, determined both by [3H]thymidine incorporation and by limiting dilution clonogenic assays, for human neuroblastoma cells is of the order of 1000 times higher than that of meta-[131I] iodobenzylguanidine. For meta-[211At] astatobenzylguanidine, the Do value was equivalent to only 6-7 211At atoms bound per cell. These results suggest that meta-[211At] astatobenzylguanidine might be valuable for the targeted radiotherapy of micrometastatic neuroblastomas.

A paired-label biodistribution was performed in athymic mice bearing SK-N-SH human neuroblastoma xenografts to compare the tissue uptake of meta-[211At]astatobenzylguanidine ([211At]MABG) and [131I]MIBG. Significantly higher (p < 0.05) uptake of [211At]MABG was seen in tumor (3.8 +/- 0.8% ID/g vs. 3.1 +/- 0.7% ID/g at 8 h) compared to [131I]MIBG. Desipramine reduced tumor uptake of [211At] MABG by 43%, suggesting that its accumulation was related to the specific uptake-1 mechanism. Higher uptake of [211At]MABG was also seen in normal tissue targets such as heart (6.0 +/- 0.9% ID/g vs. 4.5 +/- 0.8% ID/g at 8 h; p < 0.05). Pretreatment of mice with unlabeled MIBG increased tumor uptake of [211At]MABG by 1.5-fold while reducing uptake in heart and several other normal tissues. The vesicular uptake inhibitor tetrabenazine reduced heart uptake by 30% without reducing the tumor uptake. These results suggest such strategies might be useful for improving [211At]MABG tumor-to-normal tissue ratios.

Hyperthermia is a therapeutic modality under investigation for its ability to increase absolute levels of tumor uptake of radiolabeled monoclonal antibodies (MAbs). We have investigated whether hyperthermia may affect the binding parameters of MAbs. The effects of clinically relevant levels of hyperthermia on the kinetic binding parameters were investigated for 81C6, an antibody undergoing Phase I/II clinical trials for the treatment of brain tumors and neoplastic meningitis. No obvious effects of temperature in either the association or dissociation rate constants, nor in the equilibrium constants, were apparent between 37 degrees and 45 degrees C. The improved binding stability of the bivalent form of the MAb was apparent when compared with its monovalent Fab fragment.

When labeled using the Iodogen method, a chimeric antibody composed of the human IgG 2 constant region and the variable regions of murine anti-tenascin 81C6 exhibited superior uptake in human glioma xenografts compared with its murine parent. In the current study, three paired-label experiments were performed in athymic mice with subcutaneous D-54 MG human glioma xenografts to evaluate further the properties of radioiodinated chimeric 81C6. These studies demonstrated that (a) the enhanced tumor uptake of chimeric 81C6 is specific; (b) when labeling was performed using N -succinimidyl 3-iodobenzoate, chimeric 81C6 again showed preferential accumulation in tumor compared with murine 81C6; and (c) the tumor uptake advantage observed previously with murine 81C6 for N -succinimidyl 3-iodobenzoate compared with lodogen labeling did not occur with chimeric 81C6.

The present study undertook to establish the dose (LD) of systematically administered (via tail vein) sodium [211At]astatide that would kill 10% (LD10) of exposed animals in two mouse models and to evaluate the resulting histologic lesions.Three dose escalation experiments were carried out using groups of 10 3- to 4-week-old, 20 +/- 2 g B6C3F1 mice, and one dose escalation experiment was carried out with groups of 10 4- to 6-week-old, 22 +/- 2 g BALB/c (nu/nu) mice. All animals were weighed daily and checked twice daily for general health; autopsies were performed within 12 h of death.The LD10 (95% confidence interval) level of free [211At]astatide at 360 days was 15.1 microCi (5.2-19.1 microCi) in B6C3F1 mice and was associated with a 37.8% weight difference from saline controls (p0.001). In the BALB/c (nu/nu) mice, the LD10 at 360 days was 7.7 microCi (0-14.2 microCi), while a dose of 10 microCi (0.42 microCi g(-1)) was associated with a 9.44% weight difference vs. saline controls (p0.05). Exclusive of the well-known effects on thyroid, [211At]astatide activity levels were associated with severe bone marrow depression, testicular atrophy, focal alopecia, and nuclear atypia of the epidermoid mucosa of the fore-stomach in the B6C3F1 mice; at activity levels approximating LD10 at 360 days, mild changes in the heart, liver, stomach, and spleen were observed. For BALB/c (nu/nu) mice, administration of 10 microCi was associated at autopsy with mild histologic lesions in the heart, stomach, liver, and spleen.These studies provide a basis for the design of further investigations of [211At]-labeled compounds as therapeutic agents.

The kinetics of para-[18F]fluorobenzylguanidine ([18F]PFBG) were investigated in a canine coronary artery occlusion model. Five dogs were imaged by positron emission tomography (PET) before and after complete surgical ligation of the left anterior descending coronary artery. PET studies included a 10-minute dynamic [13N]NH3 perfusion scan, followed 1 hour later by 3-hour dynamic [18F]PFBG scanning. [18F]PFBG and [13N]NH3 images demonstrated homogeneous myocardial uptake/perfusion before infarction. One hundred eighty minutes after [18F]PFBG administration, myocardial accumulation was decreased by 60% (day, 2, 0.0065% ±0.0015% injected dose/ml) and 58% (day 16, 0.0069%±0.003% injected dose/ml) compared with a similar myocardial region of interest from the preinfarction (0.016%±0.005% injected dose/ml) study. Myocardial accumulation of [13N]NH3 at 9 minutes showed a 52% (day 2) and 7% (day 16) decrease compared with the preinfarction study. The accumulation of [18F]PFBG in the infarction was decreased significantly at 120 and 180 minutes on all postinfarction studies (p=0.01). In three dogs a significant decrease in the myocardial norepinephrine concentration was documented in the area of infarction (237±94 ng/gm) versus the noninfarcted (1018±48 ng/gm) myocardium (p=0.001). A decreased accumulation of [18F]PFBG was observed in the area of myocardial infarct in this canine model. The magnitude of the decrease in [18F]PFBG was larger than that seen with [13N]NH3 on day 16 after infarction suggesting reperfusion and persistent sympathetic neuronal dysfunction.

Radionuclides which decay by the emission of alpha-particles are attractive for certain radioimmunotherapeutic applications. These include the treatment of lymphomas, compartmentally spread malignancies such as ovarian cancer and neoplastic meningitis, and micrometastatic disease. Two alpha-emitting radionuclides of interest for this purpose are 212Bi (60.6 min half life) and 211At (7.2 hr half life). Compared with the beta-emitters commonly used for radiotherapy, the alpha-particles of 212Bi and 211At are of higher energy, much shorter range (less than 100 microm), and considerably higher linear energy transfer. Preliminary results obtained in a variety of in vitro systems and in vivo models have documented the exquisite toxicity of alpha-particles and have established a basis for initiating radiotherapy trials in humans with monoclonal antibodies labeled with alpha-emitting radionuclides.

Two peptides of potential utility for targeting melanoma cells, α-melanocyte-stimulating hormone (α-MSH) and its more potent analogue [Nle4,d-Phe7]-α-MSH, were radioiodinated in 45−65% yield using N-succinimidyl 3-[125I]iodobenzoate (SIB). To determine whether this labeling method resulted in improved in vitro and in vivo characteristics, these peptides also were labeled with 131I by direct iodination with the iodogen method. For α-MSH, the rapid tissue clearance of both radionuclides in mice was consistent with rapid degradation of the peptide; however, significantly lower levels of 125I were observed in thyroid and stomach, reflecting a greater inertness to deiodination. More extensive comparisons were performed with [Nle4,d-Phe7]-α-MSH. The in vitro binding of [Nle4,d-Phe7,Lys11−(125I)IBA]-α-MSH (prepared using SIB) to the murine B-16 melanoma cell line, 34.1 ± 4.7%, was more than twice as high as that for [Tyr2(131I),Nle4,d-Phe7]-α-MSH (15.0 ± 0.1%), and its KD was more than 10-fold lower than that fo...

Malignant brain tumors are one of the most lethal cancer forms causing over 14,000 deaths each year. In spite of extensive research efforts over many decades, the median overall survival (OS) still remains at just 15 months for the common malignant giloma, glioblastoma multiforme (GBM). We have developed a multifunctional gold nanostar (GNS) probe that shows potential to improve cancer management by providing personalized cancer theranostics, a combination of diagnostic and therapeutic functions. The GNS nanoprobe has a tunable plasmon absorption peak in the near infrared (NIR) “tissue optical window” for deep-tissue penetration and tip-enhanced surface plasmon resonance, which makes the GNS as a superior nanoprobe for cancer detection using surface-enhanced Raman spectroscopy (SERS), two-photon photoluminescence (TPL) imaging and photothermal therapy with NIR light. We have also functionalized the GNS with iodine-124 radioisotopes for sensitive 3D positron emission tomography (PET) and achieved a 10-picomolar limit of detection (LOD). The GNS nanoprobe accumulates selectively in brain tumor due to the enhanced permeability and retention (EPR) effect, which is caused by defective endothelial tight junctions in blood vessels surrounding the tumor. On the contrary, the GNS nanoprobe has minimum uptake in normal brain tissue due to the intact blood-brain-barrier (BBB). The GNS uptake ratio of tumor-to-normal (T/N) brain tissue is measured to be up to 30:1 by using inductively coupled plasma mass spectrometry (ICP-MS). By combining the high detection sensitivity of PET imaging and specific accumulation of GNS in brain tumor, we have demonstrated the feasibility of 1-mm brain tumor detection with the I-124 labeled GNS nano radiotracer. The obtained T/N ratio is up to 10:1 with our GNS nanoprobe labeled with I-124, which is much better than that (typically less than 1.5:1) for traditional F-18 fluorodeoxyglucose (FDG) PET contrast agent due to high glucose uptake in normal brain tissue. Furthermore, we have utilized GNS's high TPL intensity to delineate brain tumor margin with excellent sensitivity and high spatial resolution (μm level). In addition to brain tumor imaging, we have successfully demonstrated photothermal ablation with a NIR laser taking advantage of GNS's superior photothermal conversion efficiency. We have also performed a 6-month toxicity study for the GNS nanoprobe and experiment results, including body weight monitoring, blood chemistry test, and H&E histopathology examination, show no deleterious effect after infusion of GNS nanoprobe up to 100 mg/kg dose. In summary, the GNS nanoprobe, with limited toxicity and multifunctional capabilities, exhibits great promise to fulfill the unmet needs in brain tumor management in future translational medicine investigations. Citation Format: Yang Liu, Austin Carpenter, Hsiangkuo Yuan, Zhengyuan Zhou, Michael Zalutsky, Ganesan Vaidyanathan, Hai Yan, Tuan Vo-Dinh. Gold nanostar as theranostic probe for brain tumor sensitive PET-optical imaging and image-guided specific photothermal therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4213.

In vitro and in vivo neuroblastoma models were used to determine whether improvements in tumour targeting in vivo and therapeutic efficacy in vitro could result from the use of no-carrier-added (n.c.a.) [131I]MIBG. Results were compared with use of the conventional therapy MIBG preparation (ex. [131I]MIBG) of lower specific activity which is produced by iodide exchange reaction. The efficacy of n.c.a. [131I]MIBG was compared with that of [131I]MIBG over a range of specific activities by the assessment of neuroblastoma spheroid growth delay. Whereas n.c.a. [131I]MIBG at a radioactivity concentration of 2 MBq/ml prevented the regrowth of 84% of spheroids, toxicity was significantly reduced by the addition of non-radiolabelled MIBG to the incubation medium. The time-dependent biodistribution of n.c.a. [131I]MIBG in nude mice bearing human neuroblastoma xenografts was compared with that of the conventional therapy radiopharmaceutical. The n.c.a. agent gave improved tumour uptake but also significantly greater accumulation in normal tissues known to accumulate MIBG such as heart, adrenal and skin. However, uptake and retention in the blood was unaltered. For all tissues examined, the 3-day calculations were undertaken to predict organ to tumour dose ratios which would result in human neuroblastoma patients with each of the [131I]MIBG preparations. These results suggest that significant therapeutic gain may be achieved by the use of n.c.a. [131I]MIBG as a treatment agent in neuroblastoma. neuroblastoma.