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A set of 211 At-astatoarenes were synthesized from corresponding trimethylgermyl arenes with an average radiochemical conversion (RCC) of ca. 50 % for electron-rich and approx. 70 % in case of electron-deficient arenes. Both electron rich and electron poor substrates were successfully radiolabeled at room temperature (RT) using relatively low precursor amounts (0.15 μmol/0.02 mL solvent (7.5 mM)). Ready access to ortho-, para- and meta- astatinated arenes was achievable. Optimized reaction conditions were successfully applied to label a poly (ADP-ribose) polymerase (PARP) inhibitor with a RCC of approx. 50 %. We believe that trimethylgermyl derivatives are a viable addition to the astatination precursor toolbox and facilitate astatination of arenes. The developed labeling method should easily be applicable for productions under good manufacturing practice (GMP)., (© 2024 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

Background: A significant challenge in cancer therapy lies in eradicating hidden disseminated tumor cells. Within Nuclear Medicine, Targeted Alpha Therapy is a promising approach for cancer treatment tackling disseminated cancer. As tumor size decreases, alpha-particles gain prominence due to their high Linear Energy Transfer (LET) and short path length. Among alpha-particle emitters, 211 At stands out with its 7.2 hour half-life and 100% alpha emission decay. However, optimizing the pharmacokinetics of radiopharmaceuticals with short lived radionuclides such as 211 At is pivotal, and in this regard, pretargeting is a valuable tool. This method involves priming the tumor with a modified monoclonal antibody capable of binding both the tumor antigen and the radiolabeled carrier, termed the "effector molecule. This smaller, faster-clearing molecule improves efficacy. Utilizing the Diels Alder click reaction between Tetrazine (Tz) and Trans-cyclooctene (TCO), the Tz-substituted effector molecule combines seamlessly with the TCO-modified antibody. This study aims to evaluate the in vivo biodistribution of two Poly-L-Lysine-based effector molecule sizes (10 and 21 kDa), labelled with 211 At, and the in vitro binding of the most favorable polymer size, in order to optimize the pretargeted radioimmunotherapy with 211 At., Results: In vivo results favor the smaller polymer's biodistribution pattern over the larger one, which accumulates in organs like the liver and spleen. This is especially evident when comparing the biodistribution of the smaller polymer to a directly labelled monoclonal antibody. The smaller variant also shows rapid and efficient binding to SKOV-3 cells preloaded with TCO-modified Trastuzumab in vitro, emphasizing its potential. Both polymer sizes showed equal or better in vivo stability of the astatine-carbon bond compared to a monoclonal antibody labelled with the same prosthetic group., Conclusions: Overall, the small Poly-L-Lysine-based effector molecule (10 kDa) holds the most promise for future research, exhibiting significantly lower uptake in the kidneys and spleen compared to the larger effector (21 kDa) while maintaining an in vivo stability of the astatine-carbon bond comparable to or better than intact antibodies. A proof of concept in vitro cell study demonstrates rapid reaction between the small astatinated effector and a TCO-labelled antibody, indicating the potential of this novel Poly-L-Lysine-based pretargeting system for further investigation in an in vivo tumor model., (© 2024. The Author(s).)

Targeted alpha therapy of disseminated cancer is an emerging technique where astatine-211 is one of the most promising candidate nuclides. Astatine-211 can be produced in medium energy cyclotrons by alpha particle bombardment of natural bismuth. The produced astatine is then commonly recovered from the irradiated solid target material through dry distillation. The dry distillation process often includes elution and solvation of condensed astatine with chloroform, forming Chloroform Eluate. In this work the handling and safe use of the high activity concentration Chloroform Eluate has been investigated. Correctly performed, evaporation of Chloroform Eluate results in a dry residue with complete recovery of the astatine. The dry residue can then serve as a versatile starting material, using appropriate oxidizing or reducing conditions, for subsequent downstream chemistry. However, it has been found that when evaporating the Chloroform Eluate, astatine can be volatilized if continuing the process beyond the point of dryness. This behavior is more pronounced when the Chloroform Eluate has received a higher absorbed dose. Upon water phase contact of the Chloroform Eluate, a major part of the astatine activity becomes water soluble, leaving the organic phase. A behavior which is also dependent on dose to the solvent., (© 2024. The Author(s).)

The application of prostate-specific membrane antigen (PSMA)-targeted α-therapy is a promising alternative to β - -particle-based treatments. 211 At is among the potential α-emitters that are favorable for this concept. Herein, 211 At-based PSMA radiopharmaceuticals were designed, developed, and evaluated. Methods: To identify a 211 At-labeled lead, a surrogate strategy was applied. Because astatine does not exist as a stable nuclide, it is commonly replaced with iodine to mimic the pharmacokinetic behavior of the corresponding 211 At-labeled compounds. To facilitate the process of structural design, iodine-based candidates were radiolabeled with the PET radionuclide 68 Ga to study their preliminary in vitro and in vivo properties before the desired 211 At-labeled lead compound was formed. The most promising candidate from this evaluation was chosen to be 211 At-labeled and tested in biodistribution studies. Results: All 68 Ga-labeled surrogates displayed affinities in the nanomolar range and specific internalization in PSMA-positive LNCaP cells. PET imaging of these compounds identified [ 68 Ga]PSGa- 3 as the lead compound. Subsequently, [ 211 At]PSAt- 3 -Ga was synthesized in a radiochemical yield of 35% and showed tumor uptake of 19 ± 8 percentage injected dose per gram of tissue (%ID/g) at 1 h after injection and 7.6 ± 2.9 %ID/g after 24 h. Uptake in off-target tissues such as the thyroid (2.0 ± 1.1 %ID/g), spleen (3.0 ± 0.6 %ID/g), or stomach (2.0 ± 0.4 %ID/g) was low, indicating low in vivo deastatination of [ 211 At]PSAt- 3 -Ga. Conclusion: The reported findings support the use of iodine-based and 68 Ga-labeled variants as a convenient strategy for developing astatinated compounds and confirm [ 211 At]PSAt- 3 as a promising radiopharmaceutical for targeted α-therapy., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Objective: Hypokalemia is associated with increased risk of arrhythmias and it is recommended to monitor plasma potassium (p-K) regularly in at-risk patients with cardiovascular diseases. It is poorly understood if administration of potassium supplements and mineralocorticoid receptor antagonists (MRA) aimed at increasing p-K also increases intracellular potassium., Methods: Adults aged≥18 years with an implantable cardioverter defibrillator (ICD) were randomized (1:1) to a control group or to an intervention that included guidance on potassium rich diets, potassium supplements, and MRA to increase p-K to target levels of 4.5-5.0 mmol/l for six months. Total-body-potassium (TBK) was measured by a Whole-Body-Counter along with p-K at baseline, after six weeks, and after six months., Results: Fourteen patients (mean age: 59 years (standard deviation 14), 79% men) were included. Mean p-K was 3.8 mmol/l (0.2), and mean TBK was 1.50 g/kg (0.20) at baseline. After six-weeks, p-K had increased by 0.47 mmol/l (95%CI:0.14;0.81), p = 0.008 in the intervention group compared to controls, whereas no significant difference was found in TBK (44 mg/kg (-20;108), p = 0.17). After six-months, no significant difference was found in p-K as compared to baseline (0.16 mmol/l (-0.18;0.51), p = 0.36), but a significant increase in TBK of 82 mg/kg (16;148), p = 0.017 was found in the intervention group compared to controls., Conclusions: Increased potassium intake and MRAs increased TBK gradually and a significant increase was seen after six months. The differentially regulated p-K and TBK challenges current knowledge on potassium homeostasis and the time required before the full potential of p-K increasing treatment can be anticipated., Trial Registration: www.clinicaltrials.gov (NCT03833089)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Winsløw et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To enhance targeting efficacy in the radioimmunotherapy of disseminated cancer, several pretargeting strategies have been developed. In pretargeted radioimmunotherapy, the tumor is pretargeted with a modified monoclonal antibody that has an affinity for both tumor antigens and radiolabeled carriers. In this work, we aimed to synthesize and evaluate poly-L-lysine-based effector molecules for pretargeting applications based on the tetrazine and trans-cyclooctene reaction using 211 At for targeted alpha therapy and 125 I as a surrogate for the imaging radionuclides 123, 124 I. Poly-L-lysine in two sizes was functionalized with a prosthetic group, for the attachment of both radiohalogens, and tetrazine, to allow binding to the trans-cyclooctene-modified pretargeting agent, maintaining the structural integrity of the polymer. Radiolabeling resulted in a radiochemical yield of over 80% for astatinated poly-L-lysines and a range of 66-91% for iodinated poly-L-lysines. High specific astatine activity was achieved without affecting the stability of the radiopharmaceutical or the binding between tetrazine and transcyclooctene. Two sizes of poly-L-lysine were evaluated, which displayed similar blood clearance profiles in a pilot in vivo study. This work is a first step toward creating a pretargeting system optimized for targeted alpha therapy with 211 At.

Astatine-211 ( 211 At) is one of the most promising α-emitters for targeted alpha therapy, especially of cancer metastases. However, the lack of a stable isotope, frequent in vivo deastatination, and limited radiochemical knowledge makes it challenging to apply. Here, we report a new strategy for radiolabeling the lipophilic core of polymeric micelles (PMs) with covalently bound 211 At. The PMs were radiolabeled via either an indirect synthon-based method or directly on the amphipathic block copolymer. The radiochemistry was optimized with iodine-125 ( 125 I) and then adapted for 211 At, enabling the use of both elements as a potential theranostic pair. PMs that were core-radiolabeled with both 125 I or 211 At were prepared and characterized, based on a PEG(5k)-PLGA(10k) co-polymer. The stability of the radiolabeled PMs was evaluated in mouse serum for 21 h, showing radiochemical stability above 85%. After in vivo evaluation of the 211 At- labeled PMs, 4-5 % ID/g of the 211 At could still be detected in the blood, showing a promising in vivo stability of the PMs. Further, 211 At-labeled PMs accumulated in the spleen (20-30 %ID/g) and the liver (2.5- 5.5 %ID/g), along with some detection of 211 At in the thyroid (3.5-9 %ID/g). This led to the hypothesis that deastatination takes place in the liver, whereas good stability of the 211 At core-radiolabel was observed in the blood., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Introduction: Antibodies labeled with alpha-emitter astatine-211 have previously shown effective in intraperitoneal (i.p.) treatments of ovarian cancer. In the present work we explore the use of investigational farletuzumab, aimed at the folate receptor alpha. The aim was to evaluate the biodistribution and therapeutic effect of 211 At-farletuzumab in in-vitro and in-vivo experiments and, using models for radiation dosimetry, to translate the findings to expected clinical result. The activity concentration used for therapy in mice (170 kBq/mL) was chosen to be in agreement with an activity concentration that is anticipated to be clinically relevant in patients (200 MBq/L)., Methods: For biodistribution, using intravenous injections and mice carrying subcutaneous (s.c.) tumors, the animals were administered either 211 At-farletuzumab (n = 16); or with a combination of 125 I-farletuzumab and 211 At-MX35 (n = 12). At 1, 3, 10 and 22 h, mice were euthanized and s.c.-tumors and organs weighted and measured for radioactivity. To evaluate therapeutic efficacy, mice were inoculated i.p. with 2 × 10 6 NIH:OVCAR-3 cells. Twelve days later, the treatments were initiated by i.p.-administration. Specific treatment was given by 211 At-labeled farletuzumab (group A; n = 22, 170 kBq/mL) which is specific for OVCAR-3 cells. Control treatments were given by either 211 At-labeled rituximab which is unspecific for OVCAR-3 (group B; n = 22, 170 kBq/mL), non-radiolabeled farletuzumab (group C; n = 11) or PBS only (group D; n = 8)., Results: The biodistribution of 211 At-farletuzumab was similar to that with 125 I as radiolabel, and also to that of 211 At-labeled MX35 antibody. The tumor-free fraction (TFF) of the three control groups were all low (PBS 12%, unlabeled specific farletuzumab 9% and unspecific 211 At-rituximab 14%). TFF following treatment with 211 At-farletuzumab was 91%., Conclusion: The current investigation of intraperitoneal therapy with 211 At-farletuzumab, delivered at clinically relevant 211 At-mAb radioactivity concentrations and specific activities, showed a 6 to 10-fold increase (treated versus controls) in antitumor efficacy. This observation warrants further clinical testing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stig Palm, Tom Bäck, Lars Jacobsson, Sture Lindegren and Per Albertsson declare ownership in the Swedish commercial company Aprit Biotech AB. The data in this manuscript was derived prior to the declared ownership., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the consensus around the clinical potential of the α-emitting radionuclide astatine-211 ( 211 At), there are only a limited number of research facilities that work with this nuclide. There are three main reasons for this: (1) Scarce availability of the nuclide. Despite a relatively large number of globally existing cyclotrons capable of producing 211 At, few cyclotron facilities produce the nuclide on a regular basis. (2) Lack of a chemical infrastructure, that is, isolation of 211 At from irradiated targets and the subsequent synthesis of an astatinated product. At present, the research groups that work with 211 At depend on custom systems for recovering 211 At from the irradiated targets. Setting up and implementing such custom units require long lead times to provide a proper working system. (3) The chemistry of 211 At. Compared with radiometals there are no well-established and generally accepted synthesis methods for forming sufficiently stable bonds between 211 At and the tumor-specific vector to allow for systemic applications. Herein we present an overview of the infrastructure of producing 211 At radiopharmaceuticals, from target to radiolabeled product including chemical strategies to overcome hurdles for advancement into clinical trials with 211 At.

Purpose: Targeted alpha therapy (TAT) is a promising treatment for micrometastatic and minimal residual cancer. We evaluated systemic α-radioimmunotherapy (α-RIT) of metastatic castration-resistant prostate cancer (mCRPC) using the α-particle emitter 211 At-labeled to the anti-PSCA A11 minibody. A11 is specific for prostate stem cell antigen (PSCA), a cell surface glycoprotein which is overexpressed in more than 90% of both localized prostate cancer and bone metastases., Methods: PC3-PSCA cells were implanted subcutaneously (s.c.) and intratibially (i.t) in nude mice. Efficacy of α-RIT (two fractions-14-day interval) was studied on s.c. macrotumors (0, 1.5 and 1.9 MBq) and on i.t. microtumors (~100-200 μm; 0, 0.8 or 1.5 MBq) by tumor-volume measurements. The injected activities for therapies were estimated from separate biodistribution and myelotoxicity studies., Results: Tumor targeting of 211 At-A11 was efficient and the effect on s.c. macrotumors was strong and dose-dependent. At 6 weeks, the mean tumor volumes for the treated groups, compared with controls, were reduced by approximately 85%. The separate myelotoxicity study following one single fraction showed reduced white blood cells (WBC) for all treated groups on day 6 after treatment. For the 0.8 and 1.5 MBq, the WBC reductions were transient and followed by recovery at day 13. For 2.4 MBq, a clear toxicity was observed and the mice were sacrificed on day 7. In the long-term follow-up of the 0.8 and 1.5 MBq-groups, blood counts on day 252 were normal and no signs of radiotoxicity observed. Efficacy on i.t. microtumors was evaluated in two experiments. In experiment 1, the tumor-free fraction (TFF) was 95% for both treated groups and significantly different (p < 0.05) from the controls at a TFF of 66%). In experiment 2, the difference in TFF was smaller, 32% for the treated group versus 20% for the controls. However, the difference in microtumor volume in experiment 2 was highly significant, 0.010 ± 0.003 mm 3 versus 3.79 ± 1.24 mm 3 (treated versus controls, respectively), i.e., a 99.7% reduction (p < 0.001). The different outcome in experiment 1 and 2 is most likely due to differences in microtumor sizes at therapy, or higher tumor-take in experiment 2 (where more cells were implanted)., Conclusion: Evaluating fractionated α-RIT with 211 At-labeled anti-PSCA A11 minibody, we found clear growth inhibition on both macrotumors and intratibial microtumors. For mice treated with multiple fractions, we also observed radiotoxicity manifested by progressive loss in body weight at 30 to 90 days after treatment. Our findings are conceptually promising for a systemic TAT of mCRPC and warrant further investigations of 211 At-labeled PSCA-directed vectors. Such studies should include methods to improve the therapeutic window, e.g., by implementing a pretargeted regimen of α-RIT or by altering the size of the targeting vector.

The use of nanobodies (Nbs) as vehicles in targeted alpha therapy (TAT) has gained great interest because of their excellent properties. They combine high in vivo affinity and specificity of binding with fast kinetics. This research investigates a novel targeted therapy that combines the α-particle emitter astatine-211 ( 211 At) and the anti-HER2 Nb 2Rs15d to selectively target HER2+ cancer cells. Two distinctive radiochemical methodologies are investigated using three different coupling reagents. The first method uses the coupling reagents, N -succinimidyl 4-(1,2-bis- tert -butoxycarbonyl)guanidinomethyl-3-(trimethylstannyl)benzoate (Boc 2 -SGMTB) and N -succinimidyl-3-(trimethylstannyl)benzoate ( m -MeATE), which are both directed to amino groups on the Nb, resulting in random conjugation. The second method aims at obtaining a homogeneous tracer population, via a site-specific conjugation of the N -[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide (MSB) reagent onto the carboxyl-terminal cysteine of the Nb. The resulting radioconjugates are evaluated in vitro and in vivo. 2Rs15d is labeled with 211 At using Boc 2 -SGMTB, m -MeATE, and MSB. After astatination and purification, the binding specificity of the radioconjugates is validated on HER2+ cells, followed by an in vivo biodistribution assessment in SKOV-3 xenografted mice. α-camera imaging is performed to determine uptake and activity distribution in kidneys/tumors. 2Rs15d astatination resulted in a high radiochemical purity >95% for all radioconjugates. The biodistribution studies of all radioconjugates revealed comparable tumor uptake (higher than 8% ID/g at 1 h). [ 211 At]SAGMB-2Rs15d showed minor uptake in normal tissues. Only in the kidneys, a higher uptake was measured after 1 h, but decreased rapidly after 3 h. Astatinated Nbs consisting of m -MeATE or MSB reagents revealed elevated uptake in lungs and stomach, indicating the presence of released 211 At. α-Camera imaging of tumors revealed a homogeneous activity distribution. The radioactivity in the kidneys was initially concentrated in the renal cortex, while after 3 h most radioactivity was measured in the medulla, confirming the fast washout into urine. Changing the reagents for Nb astatination resulted in different in vivo biodistribution profiles, while keeping the targeting moiety identical. Boc 2 -SGMTB is the preferred reagent for Nb astatination because of its high tumor uptake, its low background signals, and its fast renal excretion. We envision [ 211 At]SAGMB-2Rs15d to be a promising therapeutic agent for TAT and aim toward efficacy evaluation.

Eliminating microscopic residual disease with α-particle radiation is theoretically appealing. After extensive preclinical work with α-particle-emitting 211 At, we performed a phase I trial with intraperitoneal α-particle therapy in epithelial ovarian cancer using 211 At conjugated to MX35, the antigen-binding fragments-F(ab') 2 -of a mouse monoclonal antibody. We now present clinical outcome data and toxicity in a long-term follow-up with individual absorbed dose estimations. Methods: Twelve patients with relapsed epithelial ovarian cancer, achieving a second complete or nearly complete response with chemotherapy, received intraperitoneal treatment with escalating (20-215 MBq/L) activity concentrations of 211 At-MX35 F(ab') 2. Results: The activity concentration was escalated to 215 MBq/L without any dose-limiting toxicities. Most toxicities were low-grade and likely related to the treatment procedure, not clearly linked to the α-particle irradiation, with no observed hematologic toxicity. One grade 3 fatigue and 1 grade 4 intestinal perforation during catheter implantation were observed. Four patients had a survival of more than 6 y, one of whom did not relapse. At progression, chemotherapy was given without signs of reduced tolerability. Overall median survival was 35 mo, with a 1-, 2-, 5-, and 10-y survival of 100%, 83%, 50%, and 25%, respectively. Calculations of the absorbed doses showed that a lower specific activity is associated with a lower single-cell dose, whereas a high specific activity may result in a lower central dose in microtumors. Individual differences in absorbed dose to possible microtumors were due to variations in administered activity and the specific activity. Conclusion : No apparent signs of radiation-induced toxicity or decreased tolerance to relapse therapy were observed. The dosimetric calculations show that further optimization is advisable to increase the efficacy and reduce possible long-term toxicity., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

The goal of this study was to investigate whether targeted α-therapy can be used to successfully treat macrotumors, in addition to its established role for treating micrometastatic and minimal disease. We used an intravenous fractionated regimen of α-radioimmunotherapy in a subcutaneous tumor model in mice. We aimed to evaluate the absorbed dose levels required for tumor eradication and growth monitoring, as well as to evaluate long-term survival after treatment. Methods: Mice bearing subcutaneous tumors (50 mm 3 , NIH:OVCAR-3) were injected repeatedly (1-3 intravenous injections 7-10 d apart, allowing bone marrow recovery) with 211 At-MX35-F(ab') 2 at different activities (close to acute myelotoxicity). Mean absorbed doses to tumors and organs were estimated from biodistribution data and summed for the fractions. Tumor growth was monitored for 100 d and survival for 1 y after treatment. Toxicity analysis included body weight, white blood cell count, and hematocrit. Results: Effects on tumor growth after fractionated α-radioimmunotherapy with 211 At-MX35-F(ab') 2 was strong and dose-dependent. Complete remission (tumor-free fraction, 100%) was found for tumor doses of 12.4 and 16.4 Gy. The administered activities were high, and long-term toxicity effects (≤60 wk) were clear. Above 1 MBq, the median survival decreased linearly with injected activity, from 44 to 11 wk. Toxicity was also seen by reduced body weight. White blood cell count analysis after α-radioimmunotherapy indicated bone marrow recovery for the low-activity groups, whereas for high-activity groups the reduction was close to acute myelotoxicity. A decrease in hematocrit was seen at a late interval (34-59 wk after therapy). The main external indication of poor health was dehydration. Conclusion: Having observed complete eradication of solid tumor xenografts, we conclude that targeted α-therapy regimens may stretch beyond the realm of micrometastatic disease and be eradicative also for macrotumors. Our observations indicate that at least 10 Gy are required. This agrees well with the calculated tumor control probability. Considering a relative biological effectiveness of 5, this dose level seems reasonable. However, complete remission was achieved first at activity levels close to lethal and was accompanied by biologic effects that reduced long-term survival., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Effective treatment of metastasis is a great challenge in the treatment of different types of cancers. Targeted alpha therapy utilizes the short tissue range (50-100 μm) of α particles, making the method suitable for treatment of disseminated occult cancers in the form of microtumors or even single cancer cells. A promising radioactive nuclide for this type of therapy is astatine-211. Astatine-211 attached to tumor-specific antibodies as carrier molecules is a system currently under investigation for use in targeted alpha therapy. In the common radiolabeling procedure, astatine is coupled to the antibody arbitrarily on lysine residues. By instead coupling astatine to disulfide bridges in the antibody structure, the immunoreactivity of the antibody conjugates could possibly be increased. Here, the disulfide-based conjugation was performed using a new coupling reagent, maleimidoethyl 3-(trimethylstannyl)benzamide (MSB), and evaluated for chemical stability in vitro. The immunoconjugates were subsequently astatinated, resulting in both high radiochemical yield and high specific activity. The MSB-conjugate was shown to be stable with a long shelf life prior to the astatination. In a comparison of the in vivo distribution of the new immunoconjugate with other tin-based immunoconjugates in tumor-bearing mice, the MSB conjugation method was found to be a viable option for successful astatine labeling of different monoclonal antibodies.

In this work a new coupling reagent, N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide, for radiohalogenation has been synthesized and characterized. The reagent is intended to either be attached to reduced disulfide bridges of proteins (making the halogenation site-specific) or to free terminal cysteine groups on peptides. The new reagent was also shown to be easily halogenated with inactive bromine and iodine as well as (125)I and (211)At, indicating potential use within targeted radiotherapy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Astatine-211 is possibly the most promising radionuclide for targeted α-particle therapy when it comes to the treatment of occult disseminated cancer. Preclinical research has proven effective, and patient studies have been initiated based on these results. However, a lack of production capacity and the complex radiochemistry of (211)At are major obstacles for research and prospective clinical applications. In the present study, astatination of immunoconjugates, already prepared well in advance before radiolabeling, was performed to investigate the possibility of formulating a kit-like reagent for the production of (211)At radiopharmaceuticals. The shelf-life of ɛ-lysyl-3-(trimethylstannyl)benzamide immunoconjugates was evaluated, that is, the effect of different storage times on the quality of the immunoconjugates. The quality being referred to is the capacity to maintain a good radiochemical yield and good cell-binding property after labeling with (211)At. The stability of the conjugates was found to be pH dependent with high stability at pH≥7 and less stability at pH≤5.5. The immunoconjugates (based on trastuzumab) could be kept for more than 3 months in a phosphate buffered saline solution (pH 7.4) at 4°C before labeling, without compromising the quality of the labeled product. The conjugates are also unaffected by storage at -20°C. Conjugates with a good shelf-life compatible with distant shipping as well as improved radiochemistry are important steps to facilitate further clinical progress with (211)At.

Unlabelled: Targeted α-therapy (TAT) appears to be an ideal therapeutic technique for eliminating malignant circulating, minimal residual, or micrometastatic cells. These types of malignancies are typically infraclinical, complicating the evaluation of potential treatments. This study presents a method of ex vivo activity quantification with an α-camera device, allowing measurement of the activity taken up by tumor cells in biologic structures a few tens of microns., Methods: We examined micrometastases from a murine model of ovarian carcinoma after injection of a radioimmunoconjugate labeled with (211)At for TAT. At different time points, biologic samples were excised and cryosectioned. The activity level and the number of tumor cells were determined by combined information from 2 adjacent sections: one exposed to the α-camera and the other stained with hematoxylin and eosin. The time-activity curves for tumor cell clusters, comprising fewer than 10 cells, were derived for 2 different injected activities (6 and 1 MBq)., Results: High uptake and good retention of the radioimmunoconjugate were observed at the surface of tumor cells. Dosimetric calculations based on the measured time-integrated activity indicated that for an injected activity of 1 MBq, isolated tumor cells received at least 12 Gy. In larger micrometastases (≤ 100 μm in diameter), the activity uptake per cell was lower, possibly because of hindered penetration of radiolabeled antibodies; however, the mean absorbed dose delivered to tumor cells was above 30 Gy, due to cross-fire irradiation., Conclusion: Using the α-camera, we developed a method of ex vivo activity quantification at the cellular scale, which was further applied to characterize the behavior of a radiolabeled antibody administered in vivo against ovarian carcinoma. This study demonstrated a reliable measurement of activity. This method of activity quantification, based on experimentally measured data, is expected to improve the relevance of small-scale dosimetry studies and thus to accelerate the optimization of TAT.

Background: Most carcinomas are prone to metastasize despite successful treatment of the primary tumor. One way to address this clinical challenge may be targeted therapy with α-emitting radionuclides such as astatine-211 (211At). Radioimmunotherapy utilizing α-particle emitting radionuclides is considered especially suitable for the treatment of small cell clusters and single cells, although lesions of different sizes may also be present in the patient. The aim of this study was primarily to evaluate the toxicity and secondarily in vivo efficacy of a 211At-labeled monoclonal antibody (mAb) directed against colon carcinoma with tumor diameters of approximately 10 mm., Methods: Eighteen rats with subperitoneal syngeneic colon carcinoma were allocated to three groups of six animals together with three healthy rats in each group. The groups were injected intravenously with either 150 μg of unlabeled mAbs (controls) or 2.5 or 5 MBq 211At-mAbs directed towards the Lewis Y antigen expressed on the cell membrane of several carcinomas. Tumor volume, body weight, and blood cell counts were monitored for 100 days after treatment., Results: Local tumors were non-palpable in five out of six rats after treatment with both activities of 211At-mAbs, compared to one out of six in the control group. At the study end, half of the animals in each group given 211At-BR96 and one animal in the control group were free from disease. Radioimmunotherapy resulted in dose-dependent, transient weight loss and myelotoxicity. Survival was significantly better in the groups receiving targeted alpha therapy than in those receiving unlabeled mAbs., Conclusions: This study demonstrates the possibility of treating small, solid colon carcinoma tumors with α-emitting radionuclides such as 211At bound to mAbs, with tolerable toxicity.

Purpose: The aim of this study was to identify gene expression profiles distinguishing alpha-particle (211)At and (60)Co irradiation., Materials and Methods: Gene expression microarray profiling was performed using total RNA from confluent human fibroblasts 5 hours after exposure to (211)At labeled trastuzumab monoclonal antibody (0.25, 0.5, and 1 Gy) and (60)Co (1, 2, and 3 Gy)., Results: We report gene expression profiles that distinguish the effect different radiation qualities and absorbed doses have on cellular functions in human fibroblasts. In addition, we identified commonly expressed transcripts between (211)At and (60)Co irradiation. A greater number of transcripts were modulated by (211)At than (60)Co irradiation. In addition, down-regulation was more prevalent than up-regulation following (211)At irradiation. Several biological processes were enriched for both irradiation qualities such as transcription, cell cycle regulation, and cell cycle arrest, whereas mitosis, spindle assembly checkpoint, and apoptotic chromosome condensation were uniquely enriched for alpha particle irradiation., Conclusions: LET-dependent transcriptional modulations were observed in human fibroblasts 5 hours after irradiation exposure. These findings suggest that in comparison with (60)Co, (211)At has the clearest influence on both tumor protein p53-activated and repressed genes, which impose a greater overall burden to the cell following alpha particle irradiation.

Introduction: The purpose of this study was to compare the therapeutic efficacy and biodistribution of the monoclonal antibody MX35 labeled with either (213)Bi or (211)At, both α-emitters, in an ovarian cancer model., Methods: One hundred female nude BALB/c (nu/nu) mice were inoculated intraperitoneally with human ovarian cancer cells (OVCAR-3). Two weeks later, 40 of these mice were injected intraperitoneally with ~2.7 MBq of (213)Bi-MX35 (n=20) or ~0.44 MBq of (211)At-MX35 (n=20). Four weeks after inoculation, 40 new OVCAR-3-inoculated mice were injected with the same activities of (213)Bi-MX35 (n=20) or (211)At-MX35 (n=20). Presence of tumors and ascites was investigated 8 weeks after therapy. Biodistributions of intraperitoneally injected (213)Bi-MX35 and (211)At-MX35 were studied in tumor-free nude BALB/c (nu/nu) mice (n=16)., Results: The animals injected with (213)Bi-MX35 or (211)At-MX35 2 weeks after cell inoculation had tumor-free fractions (TFFs) of 0.60 and 0.90, respectively. The untreated reference group had a TFF of 0.20. The groups treated with (213)Bi-MX35 or (211)At-MX35 4 weeks after inoculation both had TFFs of 0.25, and the reference animals all exhibited evidence of disease. The biodistributions of (213)Bi-MX35 and (211)At-MX35 were very similar to each other and displayed no alarming activity levels in the investigated organs., Conclusions: Micrometastatic growth of an ovarian cancer cell line was reduced in nude mice after treatment with (213)Bi-MX35or (211)At-MX35. Treatment with (211)At-MX35 provided a non-significantly better result for the chosen activity levels. The radiolabeled MX35 did not accumulate to a high extent in the investigated organs. No considerable signs of toxicity were observed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Avidin-coupled monoclonal antibody MX35 (avidin-MX35) and astatine-211-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PL(suc)) were administered in mice to assess potential efficacy as an intraperitoneal (i.p.) therapy for microscopic tumors. We aimed to establish a timeline for pretargeted radioimmunotherapy using these substances, and estimate the maximum tolerable activity., Methods: (125)I-avidin-MX35 and (211)At-B-PL(suc) were administered i.p. in nude mice. Tissue distributions were studied at various time points and mean absorbed doses were estimated from organ uptake of (211)At-B-PL(suc). Studies of myelotoxicity were performed after administration of different activities of (211)At-B-PL(suc)., Results: We observed low blood content of both (125)I-avidin-MX35 and (211)At-B-PL(suc), indicating fast clearance. After sodium perchlorate blocking, the highest (211)At uptake was found in kidneys. Red bone marrow (RBM) accumulated some (211)At activity. Mean absorbed doses of special interest were 2.3 Gy/MBq for kidneys, 0.4 Gy/MBq for blood, and 0.9 Gy/MBq for RBM. An absorbed dose of 0.9 Gy to the RBM was found to be safe. These values suggested that RBM would be the key dose-limiting organ in the proposed pretargeting scheme, and that blood data alone was not sufficient for predicting its absorbed dose., Conclusions: To attain a favorable distribution of activity and avoid major toxicities, at least 1.0 MBq of (211)At-B-PL(suc) can be administered 24 hours after an i.p. injection of avidin-MX35. These results provide a basis for future i.p. therapy studies in mice of microscopic ovarian cancer.

Background: The aim of this study was to investigate the therapeutic efficacy of advanced ovarian cancer in mice, using α-radioimmunotherapy with different high specific activities. The study was performed using the monoclonal antibody (mAb) MX35 F(ab')2 labeled with the α-particle emitter 211 At., Methods: Animals were intraperitoneally inoculated with ≥1 × 10 7 cells of the ovarian cancer cell line NIH:OVCAR-3. Four weeks later 9 groups of animals were given 25, 50, or 400 kBq 211 At-MX35 F(ab')2 with specific activities equal to 1/80, 1/500, or 1/1200 ( 211 At atom/number of mAbs) for every activity level respectively ( n = 10 in each group). As controls, animals were given PBS or unlabeled MX35 F(ab')2 in PBS ( n = 10 in each group). Eight weeks after treatment the animals were sacrificed and the presence of macroscopic tumors was determined by meticulous ocular examination of the abdominal cavity. Cumulated activity and absorbed dose calculations on tumor cells and tumors were performed using in house developed program. Specimens for scanning electron-microscopy analysis were collected from the peritoneum at the time of dissection., Results: Summing over the different activity levels (25, 50, and 400 kBq 211 At-MX35 F(ab')2) the number of animals with macroscopic tumors was 13, 17, and 22 ( n = 30 for each group) for the specific activities equal to 1/80, 1/500, or 1/1200, respectively. Logistic-regression analysis showed a significant trend that higher specific activity means less probability for macroscopic tumors ( P = 0.02)., Conclusions: Increasing the specific activity indicates a way to enhance the therapeutic outcome of advanced ovarian cancer, regarding macroscopic tumors. Further studies of the role of the specific activity are therefore justified., Competing Interests: The authors have no conflict of interest.

Background: Pretargeting is an approach for enhancing the therapeutic index of radioimmunotherapy by separating the administrations of tumor-targeting substance and radiolabel. In this study, a pretargeting model system of avidin-conjugated monoclonal antibody trastuzumab and biotinylated, (211)At-labeled poly-L-lysine was constructed and analyzed in vitro., Methods: Avidin activated by 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) and thiolated trastuzumab were incubated overnight at 4 degrees C. The monomeric fraction was extracted using size exclusion fast protein liquid chromatography (FPLC) and further purified on an iminobiotin affinity column. Poly-L-lysine was biotinylated with succinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin), followed by direct (211)At-labeling with N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE), and succinylation with succinic anhydride. The avidin-trastuzumab conjugate was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and FPLC, together with cell-binding and biotin-binding analyses. The labeled poly-L-lysine conjugate was assessed in terms of radiochemical purity and avidin binding. Furthermore, the full pretargeting system was evaluated in a tumor cell binding assay., Results: The estimated size of the pretargeting molecule was 220 kDa, which corresponds to that of the expected avidin-trastuzumab monomer. Neither cell-binding ability (64%) nor biotin-binding ability (85%-95%) indicated any severe adverse effects from the chemical modifications. The radiochemical purity of the effector molecule was 92%-97%, and the avidin binding capacity was 91%-93%. The complete pretargeting assay resulted in a binding of 75.3 +/- 6.2% of added effector molecules to cells., Conclusions: The high binding of effector molecules to cells demonstrates a proof of concept for the synthesized molecules and pretargeting system, which will be further evaluated in vivo in future studies., ((c) 2010 American Cancer Society.)

The aim of this study was to investigate the therapeutic efficacy of alpha-radioimmunotherapy of ovarian cancer in mice using different fractionated treatment regimens. The study was performed using the monoclonal antibody MX35 F(ab')(2) labeled with the alpha-particle emitter (211)At. Methods. Nude mice were intraperitoneally inoculated with ~1 x 10(7) cells of the cell line NIH:OVCAR-3. Four weeks later 6 groups of animals were given 400 kBq (211)At-MX35 F(ab')(2) as a single or as a repeated treatment of up to 6 times (n = 18 in each group). The fractionated treatments were given every seventh day. Control animals were treated with unlabeled MX35 F(ab')(2) (n = 12). Eight weeks posttreatment the animals were sacrificed and the presence of macro- and microscopic tumors and ascites was determined. Results. The tumor-free fractions (TFFs) of the animals, defined as the fraction of animals with no macro- and microtumors and no ascites, were 0.17, 0.11, 0.39, 0.44, 0.44, and 0.67 when treated with 400 kBq (211)At-MX35 F(ab')(2) once or 2, 3, 4, 5, or 6 times, respectively. Repeated treatment 3 times or more resulted in a significantly higher (P < .05) TFF than compared to treatment once or twice. The presence of ascites decreased from 15 out of 18 animals in the group given only one treatment to zero for the 2 groups given 5 or 6 fractions. Treatment with unlabeled MX35 F(ab')(2) resulted in a TFF of zero. Conclusion. Weekly repeated intraperitoneal injections of tolerable amounts of activity of (211)At-MX35 F(ab')(2) of up to 6 times produced increased therapeutic efficacy without observed toxicity, indicating a potential increase of the therapeutic index.

Purpose: The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At., Methods: Animals were intraperitoneally inoculated with approximately 1 x 10(7) cells of the cell line, NIH:OVCAR-3. Four (4) weeks later, five groups of animals were given 400 kBq of 211At-MX35 F(ab')(2) with specific activities equal to 130, 65, 32, 16, or 4 kBq/microg, respectively (n = 18 in each group). As controls, animals were given unlabeled MX35 F(ab')(2) (n = 12). Eight (8) weeks after treatment, the animals were sacrificed and the presence of macro- and microscopic tumors and ascites was determined., Results: The tumor-free fractions (TFFs) of the animals, defined as the fraction of animals with no macro- and microtumors and no ascites, were 0.67, 0.73, 0.50, 0.50, and 0.17 when treated as above. Only the TFF of 0.17, for the specific activity of 4 kBq/microg, was significantly less, compared to that of the specific activity of 130 kBq/microg. The TFF for the specific activity of 4 kBq/microg showed a significant lowering, compared to the specific activity of 130 kBq/microg (p < 0.05). Treatment with unlabeled MX35 F(ab')(2) resulted in a TFF of zero., Conclusions: A specific activity-dependent therapeutic outcome could not be shown in the interval of 130- to 16 kBq/mug. For lower specific activities (i.e., 4 kBq/microg), the therapeutic efficacy was significantly lowered.

Unlabelled: The alpha-emitter (211)At labeled to a monoclonal antibody has proven safe and effective in treating microscopic ovarian cancer in the abdominal cavity of mice. Women in complete clinical remission after second-line chemotherapy for recurrent ovarian carcinoma were enrolled in a phase I study. The aim was to determine the pharmacokinetics for assessing absorbed dose to normal tissues and investigating toxicity., Methods: Nine patients underwent laparoscopy 2-5 d before the therapy; a peritoneal catheter was inserted, and the abdominal cavity was inspected to exclude the presence of macroscopic tumor growth or major adhesions. (211)At was labeled to MX35 F(ab')(2) using the reagent N-succinimidyl-3-(trimethylstannyl)-benzoate. Patients were infused with (211)At-MX35 F(ab')(2) (22.4-101 MBq/L) in dialysis solution via the peritoneal catheter. gamma-Camera scans were acquired on 3-5 occasions after infusion, and a SPECT scan was acquired at 6 h. Samples of blood, urine, and peritoneal fluid were collected at 1-48 h. Hematology and renal and thyroid function were followed for a median of 23 mo., Results: Pharmacokinetics and dosimetric results were related to the initial activity concentration (IC) of the infused solution. The decay-corrected activity concentration decreased with time in the peritoneal fluid to 50% IC at 24 h, increased in serum to 6% IC at 45 h, and increased in the thyroid to 127% +/- 63% IC at 20 h without blocking and less than 20% IC with blocking. No other organ uptakes could be detected. The cumulative urinary excretion was 40 kBq/(MBq/L) at 24 h. The estimated absorbed dose to the peritoneum was 15.6 +/- 1.0 mGy/(MBq/L), to red bone marrow it was 0.14 +/- 0.04 mGy/(MBq/L), to the urinary bladder wall it was 0.77 +/- 0.19 mGy/(MBq/L), to the unblocked thyroid it was 24.7 +/- 11.1 mGy/(MBq/L), and to the blocked thyroid it was 1.4 +/- 1.6 mGy/(MBq/L) (mean +/- SD). No adverse effects were observed either subjectively or in laboratory parameters., Conclusion: This study indicates that by intraperitoneal administration of (211)At-MX35 F(ab')(2) it is possible to achieve therapeutic absorbed doses in microscopic tumor clusters without significant toxicity.

Unlabelled: (211)At-labeled tumor-specific antibodies have long been considered for the treatment of disseminated cancer. However, the limited availability of the nuclide and the poor efficacy of labeling procedures at clinical activity levels present major obstacles to their use. This study evaluated a procedure for the direct astatination of antibodies for the production of clinical activity levels., Methods: The monoclonal antibody trastuzumab was conjugated with the reagent N-succinimidyl-3-(trimethylstannyl)benzoate, and the immunoconjugate was labeled with astatine. Before astatination of the conjugated antibody, the nuclide was activated with N-iodosuccinimide. The labeling reaction was evaluated in terms of reaction time, volume of reaction solvent, immunoconjugate concentration, and applied activity. The quality of the astatinated antibodies was determined by in vitro analysis and biodistribution studies in nude mice., Results: The reaction proceeded almost instantaneously, and the results indicated a low dependence on immunoconjugate concentration and applied activity. Radiochemical labeling yields were in the range of 68%-81%, and a specific radioactivity of up to 1 GBq/mg could be achieved. Stability and radiochemical purity were equal to or better than those attained with a conventional 2-step procedure. Dissociation constants for directly astatinated, conventionally astatinated, and radioiodinated trastuzumab were 1.0+/-0.06 (mean+/-SD), 0.44+/-0.06, and 0.29+/-0.02 nM, respectively. The tissue distribution in non-tumor-bearing nude mice revealed only minor differences in organ uptake relative to that obtained with the conventional method., Conclusion: The direct astatination procedure enables the high-yield production of astatinated antibodies with radioactivity in the amounts required for clinical applications.

Radioimmunotherapy (RIT) could be a possible adjuvant treatment method for patients with colorectal carcinoma. The A33 antigen is a promising RIT target, as it is highly and homogenously expressed in 95% of all colorectal carcinomas. In this study, the humanized monoclonal antibody A33 (huA33), targeting the A33 antigen, was labeled with the therapeutic nuclide 211At, and the biodistribution and in vivo targeting ability of the conjugate was investigated in an athymic mouse xenograft model. There was an accumulation of 211At in tumor tissue over time, but no substantial accumulation was seen in any organ apart from the skin and thyroid, indicating no major release of free 211At in vivo. At all time points, the uptake of 211At-huA33 was higher in tumor tissue than in most organs, and at 8 hours postinjection (p.i.), no organ had a higher uptake than tumor tissue. The tumor-to-blood ratio of 211At-huA33 increased with time, reaching 2.5 after 21 hours p.i. The highest absorbed dose was found in the blood, but the tumor received a higher dose than any organ other than the thyroid. An in vivo blocking experiment showed that 211At-huA33 binds specifically to human tumor xenografts in athymic mice. In conclusion, the favorable biodistribution and specific in vivo targeting ability of 211At-huA33 makes it a potential therapeutic agent for the RIT of metastatic colorectal carcinoma.

Objectives: In advanced head and neck squamous cell carcinoma (HNSCC), there is a need for an adjuvant treatment. We aim to evaluate the biodistribution and therapeutic effect of radioimmunotherapy using the alpha emitting, astatine-211-labeled, chimeric monoclonal antibody U36 (U36) on the HNSCC cell line UT-SCC7 in vivo., Study Design: Xenograft tumors were inoculated subcutaneously in nude mice. Astatine-211-labeled U36 was injected intravenously with or without blocking of target with nonlabeled U36., Methods: In the biodistribution experiments, radioactivity was measured in tumors and various organs at set time points. In the therapeutic experiments, two groups (with or without blocking) received therapy, and the tumor growth was compared with that of controls. In addition, one group received nonlabeled U36 only., Results: The biodistribution experiments demonstrated that astatine-211-labeled U36 could target UT-SCC7 xenografts in nude mice. With time, uptake increased in tumors and decreased in normal organs. Nonlabeled U36 did not influence tumor growth. In the two therapy groups, 18 of 20 tumors responded to therapy by decreasing or stabilizing their volumes. Significant difference was seen between the treated groups and the controls (P < .05)., Conclusion: The study illustrates the specific binding of astatine-211-labeled U36 to HNSCC and suggests radioimmunotherapy with the alpha emitting radionuclide to be a useful treatment modality.

Objective: The aim of this study was to investigate the therapeutic efficacy and myelotoxicity during fractionated radioimmunotherapy of ovarian cancer in mice. The study was performed using the monoclonal antibody MX35 F(ab')(2) labeled with the alpha-particle emitter (211)At., Methods: Animals were intraperitoneally inoculated with approximately 1x10(7) cells of the cell line NIH:OVCAR-3. Four weeks later, the mice were given the first treatment. Six groups of animals were intraperitoneally injected with approximately 800, 3x approximately 267, approximately 400, 3x approximately 133, approximately 50 or 3x approximately 17 kBq (211)At-MX35 F(ab')(2) (n=18 in each group). The second and third injections for Groups 2, 4 and 6 were given 4 and 8 days after the first injection, respectively. As controls, animals were treated with unlabeled MX35 F(ab')(2) (n=12). Eight weeks after the last injection, the animals were sacrificed and the presence of macro- and microscopic tumors and ascites was determined. Blood counts were determined for each mouse in Groups 1 and 2 before the first injection and 3, 7, 11, 15 and 23 days after the first injection. The calculation of the mean absorbed dose to the bone marrow was based on the ratio between the (211)At-activity concentration in bone and blood [i.e., the bone-to-blood ratio (BBLR)] as well as that between the (211)At-activity concentration in bone marrow and blood [i.e., the bone-marrow-to-blood ratio (BMBLR)] and the cumulated activity and absorbed fraction of the alpha-particles emitted by (211)At in the bone marrow., Results: The tumor-free fractions of animals were 56% and 41% when treated with approximately 800 kBq and 3x approximately 267 kBq (211)At-MX35 F(ab')(2), respectively; 39% and 28% when treated with approximately 400 kBq and 3x approximately 133 kBq (211)At-MX35 F(ab')(2), respectively; and 17% and 22% when treated with approximately 50 kBq or 3x approximately 17 kBq (211)At-MX35 F(ab')(2), respectively. The nadir of the white blood cell (WBC) counts was decreased (from 46% to 19%, compared with the baseline WBC counts) and delayed (from Day 4 to Day 11 after the first injection) during the fractionated treatment compared with the single-dose treatment. The percentage of injected activity per gram (%IA/g) for blood, bone and bone marrow all peaked 6 h after injection at 13.80+/-1.34%IA/g, 4.00+/-0.69%IA/g and 8.28+/-1.38%IA/g, respectively. The BBLR and BMBLR were 0.20+/-0.04 and 0.58+/-0.01, respectively. The mean absorbed dose to bone marrow was approximately 0.4 Gy after intraperitoneally injecting approximately 800 kBq (211)At-MX35 F(ab')(2)., Conclusion: No advantage was observed in the therapeutic efficacy of using a fractionated regimen compared with a single administration, with the same total amount of administered activity. Alleviation of the myelotoxicity was observed during the fractionated regimen in terms of decreased suppression and delayed nadir of the WBC counts. No thrombocytopenia was observed during either regimen.

Unlabelled: The purpose of this work was to (a) investigate the efficacy of radioimmunotherapy using 211At-MX35 F(ab')2 or 211At-Rituximab F(ab')2 (nonspecific antibody) against differently advanced ovarian cancer in mice; (b) image the tumor growth on the peritoneum; and (c) calculate the specific energy and mean absorbed dose to tumors and critical organs., Methods: Two experiments with 5-wk-old nude mice (n = 100 + 93), intraperitoneally inoculated with approximately 1 x 10(7) NIH:OVCAR-3 cells, were done. At either 1, 3, 4, 5, or 7 wk after inoculation animals were intraperitoneally treated with approximately 400 kBq 211At-MX35 F(ab')2 (n = 50 + 45), approximately 400 kBq 211At-Rituximab F(ab')2 (n = 25 + 24), or unlabeled Rituximab F(ab')2 (n = 25 + 24). At the time of treatment 29 animals were sacrificed and biopsies were taken for determination of tumor sizes using scanning electron microscopy (SEM). Eight weeks after each treatment the animals were sacrificed and the presence of macro- and microscopic tumors and ascites was determined. The specific energy and mean absorbed dose to tumors were calculated. The activity concentration was measured in critical organs and abdominal fluid., Results: When given treatment 1, 3, 4, 5, or 7 wk after cell inoculation the tumor-free fraction (TFF) was 95%, 68%, 58%, 47%, 26%, and 100%, 80%, 20%, 20%, and 0% when treated with 211At-MX35 F(ab')2 or 211At-Rituximab F(ab')2, respectively. The SEM images revealed maximum tumor radius of approximately 30 mum 1 wk after cell inoculation, increasing to approximately 340 mum at 7 wk. Specific energy to cell nuclei varied between 0 and approximately 540 Gy, depending on assumptions regarding activity distribution and tumor size. The mean absorbed dose to thyroid, kidneys, and bone marrow was approximately 35, approximately 4, and approximately 0.3 Gy, respectively., Conclusion: Treatment with 211At-MX35 F(ab')2 or 211At-Rituximab F(ab')2 resulted in a TFF of 95%-100% when the tumor radius was < or =30 microm. The TFF was decreased (TFF < or = 20%) for 211At-Rituximab F(ab')2 when the tumor radius exceeded the range of the alpha-particles. The specific antibody gave for these tumor sizes a significantly better TFF, explained by a high mean absorbed dose (>22 Gy) from the activity bound to the tumor surface and probably some contribution from penetrating activity.

Unlabelled: The use of alpha-particle emitters in radioimmunotherapy (RIT) appears to be promising. We previously obtained convincing results in the treatment of microscopic intraperitoneal ovarian cancer in nude mice by using the alpha-emitter 211At. This study was performed to evaluate the relative biological effectiveness (RBE) of 211At compared with that of 60Co gamma-irradiation in an RIT model. Our endpoint was growth inhibition (GI) of subcutaneous xenografts., Methods: GI after irradiation was studied with subcutaneous xenografts of the human ovarian cancer cell line NIH:OVCAR-3 implanted in nude mice. The animals received an intravenous injection of 211At-labeled monoclonal antibody MX35 F(ab')2 at different levels of radioactivity (0.33, 0.65, and 0.90 MBq). Control mice received unlabeled MX35 F(ab')2 only. To calculate the mean absorbed dose to tumor, a separate biodistribution study established the uptake of 211At in tumors and organs at different times after injection. External irradiation of the tumors was performed with 60Co. Tumor growth was monitored, and the normalized tumor volume (NTV) was calculated for each tumor. GI was defined by dividing the NTV values by the fitted NTV curve obtained from the corresponding control mice. To compare the biologic effects of the 2 radiation qualities, the mean value for GI (from day 8 to day 23) was plotted for each tumor as a function of its corresponding absorbed dose. From exponential fits of these curves, the doses required for a GI of 0.37 (D37) were derived, and the RBE of 211At was calculated., Results: The biodistribution study showed the uptake of the immunoconjugate by the tumor (amount of injected radioactivity per gram) to be 14% after 7 h. At 40 h, the ratio of uptake in tumors to uptake in blood reached a maximum value of 6.2. The administered activities of 211At corresponded to doses absorbed by tumors of 1.35, 2.65, and 3.70 Gy. The value (mean+/-SEM) for D37 was 1.59+/-0.08 Gy. Tumor growth after 60Co external irradiation showed a value for D37 of 7.65+/-1.0 Gy. The corresponding RBE of 211At irradiation was 4.8+/-0.7., Conclusion: Using a tumor GI model in nude mice, we were able to derive an RBE of alpha-particle RIT with 211At. The RBE was found to be 4.8+/-0.7.

Unlabelled: The purpose of this study was to investigate the therapeutic efficacy of-and to estimate the absorbed dose to-tumor cells from radioimmunotherapy (RIT) in an ovarian cancer model using the alpha-particle-emitting nuclide (211)At labeled to monoclonal antibody (mAb) MX35. Previous studies on mAb MOv18 did not allow for dosimetry because of antigen shedding in vitro., Methods: Five-week-old female nude BALB/c nu/nu mice were inoculated intraperitoneally with 1 x 10(7) cells of the human tumor cell line OVCAR-3. Three weeks later, the animals were given approximately 400, 800, or 1,200 kBq of (211)At-labeled mAb MX35 intraperitoneally. As controls, one group of animals was injected with unlabeled mAb and another group was injected with phosphate-buffered saline (PBS). Another group was given approximately 400 kBq of (211)At labeled to the previously investigated mAb MOv18 for efficacy comparison. Two months after treatment, the animals were sacrificed and the presence of macroscopic and microscopic tumors, as well as ascites, was determined. The absorbed dose to tumor cells on the peritoneal surface was estimated in terms of the sum of a specific and a nonspecific contribution. The specific contribution, arising from mAbs binding to the antigenic sites on the cell membrane, was calculated using a dynamic compartment model developed in-house and Monte Carlo software. The model used as input values the number of mAbs injected into the abdominal cavity, N(mAb), the specific activity, A(sp), the association rate constant, k(on), and the maximal number of mAbs bound per cell, B(max)-all determined by in vitro experiments. This specific component of the absorbed dose was calculated for assumed cell cluster sizes with radii of 25, 50, and 100 microm. The nonspecific contribution to the absorbed dose was derived from unbound mAbs freely circulating in the abdominal cavity, also using the Monte Carlo software., Results: In the control groups given unlabeled MX35 or PBS, all 18 animals had ascites, 6 of 9 animals in each group had macroscopic tumors, and all animals had microscopic growth. In the 3 groups given different amounts of (211)At-MX35, only 3 of 25 animals developed ascites. None of these animals had any sign of macroscopic tumors, but 8 had microscopic growth. In the group given (211)At-MOv18, no animals had ascites or macroscopic tumors, but 3 of 10 animals had microscopic tumors. After injecting 400 kBq of (211)At-MX35, the absorbed dose due to specific binding, for a cell cluster with a radius of 50 microm, ranged from 413 to 223 Gy between 0- and 45-microm distance from the cluster center, assuming a homogeneous distribution of (211)At-MX35 in the cluster. The contribution from unbound (211)At-MX35 and (211)At-MX35 only distributed on the cluster surface, for this cluster size, ranged from 7 to 14 Gy and from 29 to 94 Gy, between 0- and 45-microm distance from the cluster center, respectively. The calculated total absorbed doses are in a clinically relevant range and were effective as verified in the nude mice with subclinical intraperitoneal growth of OVCAR-3 cells., Conclusion: (211)At-MX35 injected intraperitoneally exhibits a high efficacy when treating micrometastatic growth of the ovarian cancer cell line OVCAR-3 on the peritoneum of nude mice.

The humanized antibody A33 binds to the A33 antigen, expressed in 95% of primary and metastatic colorectal carcinomas. The restricted pattern of expression in normal tissue makes this antigen a possible target for radioimmunotherapy of colorectal micrometastases. In this study, the A33 antibody was labeled with the therapeutic nuclide (211)At using N-succinimidyl para-(tri-methylstannyl)benzoate (SPMB). The in vitro characteristics of the (211)At-benzoate-A33 conjugate ((211)At-A33) were investigated and found to be similar to those of (125)I-benzoate-A33 ((125)I-A33) in different assays. Both conjugates bound with high affinity to SW1222 cells (K(d) = 1.7 +/- 0.2 nM, and 1.8 +/- 0.1 nM for (211)At-A33 and (125)I-A33, respectively), and both showed good intracellular retention (70% of the radioactivity was still cell associated after 20 hours). The cytotoxic effect of (211)At-A33 was also confirmed. After incubation with (211)At-A33, SW1222 cells had a survival of approximately 0.3% when exposed to some 150 decays per cell (DPC). The cytotoxic effect was found to be dose-dependent, as cells exposed to only 56 DPC had a survival of approximately 5%. The (211)At-A33 conjugate shows promise as a potential radioimmunotherapy agent for treatment of micrometastases originating from colorectal carcinoma.

Unlabelled: The rationale of this study was to determine the myelotoxicity in nude mice of the alpha-emitter 211At conjugated to monoclonal antibodies (mAbs) and to compare the effect with an electron emitter, (99m)Tc, and external irradiation from a 60Co source, for estimation of the relative biological effectiveness (RBE)., Methods: 211At and (99m)Tc were conjugated to the IgG1 mAbs MX35 and 88BV59. Nude female BALB/c mice, 8- to 12-wk old, were injected intraperitoneally or intravenously. The biodistribution was determined 3, 6, and 18 h after injection. The bone-to-blood and bone marrow-to-blood activity concentration ratios (BBLR and BMBLR, respectively) were determined for simultaneously injected 211At- and (99m)Tc-mAbs. Bone marrow samples were taken from the femur. For each mouse, the whole-body retention was measured as well as the blood activity by repeated blood samples from the tail vein (0), 1, 3, 6, 12, and 18 h after injection. External-beam irradiation from a 60Co source was also performed at 3 different dose levels. White blood cell (WBC) counts, red blood cell counts, platelet counts, and hemoglobin were determined for each mouse initially and on days 1, 4, 5, 7, 15, 22, and 27 after injection. The calculations of the absorbed dose to the bone marrow were based on the BBLR, BMBLR, the cumulated activities, and the absorbed fractions. The absorbed fractions, phi, for alpha-particles and electrons in the bone marrow were calculated using Monte Carlo simulations based on a bone marrow dosimetry model., Results: The BMBLR was 0.58 +/- 0.06 and 0.56 +/- 0.06 for the 211At- and (99m)Tc-mAbs, respectively. No significant variation in BMBLR with time was found. The absorbed fractions for alpha-particles and electrons in the bone marrow were 0.88 and 0.75, respectively. The mean absorbed fractions of the photons from (99m)Tc were 0.033 and 0.52 for 140 and 18.3 keV, respectively. When different amounts of 211At- and (99m)Tc-mAbs (0.09-1.3 and 250-1,300 MBq, respectively) were administered intraperitoneally or intravenously, corresponding to absorbed doses to the bone marrow of 0.01-0.60 and 0.39-1.92 Gy, respectively, the WBC counts was suppressed by 1%-90% and 23%-89%, respectively. When external-beam irradiation with a 60Co source was performed to absorbed doses of 1.4, 1.9, and 2.4 Gy, the WBC counts was suppressed by 47%-90%. These results indicate a myelotoxic in vivo RBE of 3.4 +/- 0.6 for alpha-particles compared with (99m)Tc and 5.0 +/- 0.9 compared with 60Co irradiation., Conclusion: The effect on the WBC counts from bone marrow irradiation with 211At-mAbs indicates an in vivo RBE of 3.4 +/- 0.6 in comparison with (99m)Tc-mAbs. The RBE value compared with external irradiation is 5.0 +/- 0.9.

Purpose: The aim of the study was to establish and refine a preclinical model to alpha-immunoradiotherapy of ovarian cancer., Experimental Design: At-211 was produced by cyclotron irradiation of a bismuth-209 target and isolated using a novel dry distillation procedure. Monoclonal antibodies were radiohalogenated with the intermediate reagent N-succinimidyl 3-(trimethylstannyl)benzoate and characterized in terms of radiochemical yield and in vitro binding properties. In vitro OVCAR-3 cells were irradiated using an external Cobalt-60 beam, as reference, or At-211-albumin and labeled antibody. Growth assays were used to establish cell survival. A Monte Carlo program was developed to simulate the energy imparted and the track length distribution. Nude mice were used for studies of WBC depression, with various activities of Tc-99m antibodies, as reference, and At-211 antibodies. In efficacy studies, OVCAR-3 cells were inoculated i.p., and animals were treated 2 weeks later. The animals were either dissected 6 weeks later or followed-up for long-term survival., Results: A rapid distillation procedure, as well as a rapid and high-yield, single-pot labeling procedure, was achieved. From growth inhibition data, the relative biological effectiveness of the alpha-emission for OVCAR-3 cells was estimated to be approximately 5, which is in the same range as found in vivo for hematological toxicity. At-211 MOv18 was found to effectively inhibit the development of tumors and ascites, also resulting in long-term survival without significant toxic effect., Conclusions: Use of the short-range, high-linear energy transfer alpha-emitter At-211 conjugated to a surface epitope-recognizing monoclonal antibody appears to be highly efficient without significant toxicity in a mouse peritoneal tumor model, urging a Phase I clinical trial.

Tsalen- and tsalpn-based nickel complexes with aldehyde functionalities have been prepared (tsalen = N,N'-ethylenebis(thiosalicylideneaminato)(2-) and tsalpn = N,N'-propane-1,3-diyl(thiosalicylideneaminato)(2-)). One of the complexes has been structurally characterized: Ni(pftp) (pftp = N,N-ethylene(6-formyl-4-methyl-2-methyliminatothiophenolato)(2-)) crystallizes in the monoclinic space group C2/c with a = 28.761(7), b = 8.582(2), c = 7.841(4) Å; beta = 98.92(3) degrees, and Z = 4. Ni(pftp) and Ni(eftp) (efpt = N,N-ethylene(6-formyl-4-methyl-2-methyliminatothiophenolato)(2-)) were used as synthons for acyclic and macrocyclic complexes and a prototype of each has been structurally characterized. The Schiff base derivative of Ni(pftp) and 2-(2-aminoethyl)pyridine gives the acyclic complex, Ni(peptp) (peptp =N,N-propane-1,3-diyl(6-(N-(2-ethylpyridyl)iminomethyl)-4-methyl-2-methyliminatothiophenolato)(-)) with the two ethylpyridyl arms furnishing an "open site", which in this complex is vacant, and a "closed site", in which the Ni(2+) is located. Ni(peptp) crystallizes in the monoclinic space group P2(1)/n with a = 10.247(7), b = 21.886(8), c = 15.662(9) Å; beta = 92.58(6) degrees, and Z = 4. Schiff base condensation of Ni(pftp) with diaminopropane gave two different macrocyclic dinickel complexes [L(1)Ni(2)](ClO(4))(2) and L(2)Ni(2). L(1) and L(2) are the 2 + 2 and 4 + 4 macrocycles, respectively, comprising two propylenediamine and two thiocresol units and four propylenediamine and four thiocresol units, respectively. Crystals of L(2)Ni(2) (L(2) = (10,23,36,49-tetramethyl-13,26,39,52-tetramercapto-2,6,15,19,28,32,41,45-octaaza-1,7,14,20,27,33,40,46-octaene[7.7.7.7]metacyclophane) are of poor quality and crystallize in the C2/c space group with a = 31.517(14), b =8.980(2), c = 26.822(12) Å; beta = 117.17(2) degrees, and Z = 4.