Introduction Targeted alpha therapy (TAT), where alpha emitters are used for internal radiotherapy, is a rapidly expanding field with a promise, if successful, to cure disseminated cancers. A sense of urgency to develop new alpha-emitting compounds is therefore shared by both industry and academia.Attempts to provide clinical alpha-particle dosimetry have included the full range from measuring whole-body retention using simple probes to microdosimetry on individual cells imaged by quantitative alpha-cameras. Typically for radiopharmaceuticals, distribution is determined by gamma-camera scintigraphy. For alpha emitters, however, the abundance of gammas or characteristic X-rays suitable for gamma-camera imaging are typically low. Any scintigraphy must therefore be complemented with tissue sampling. This can include blood, urine and feces. For intraperitoneal (i.p.) therapy, sampling i.p. liquid can provide additional information on the biokinetics needed for proper dosimetry. If possible, biopsies can be analyzed ex vivo for uptake and microdistribution. Objectives To investigate the feasability of providing dosimetry for patients treated with alpha-emitter astatine-211. Materials and Methods A retrospective dosimetric study has previously been presented [PMID: 26460999] for patients treated intraperitoneally with 211At-MX35 F(ab’)2. Mean absorbed doses to normal tissues were calculated from single photon emission computed tomography/computed tomography (SPECT/CT) images. In addition, blood, urine and and i.p. fluid were measured for activity. Extrapolation of pre-clinical biodistribution data combined with clinical blood activity data allowed an estimate of absorbed doses in additional tissues.Disseminated tumors are often too small to be imaged. Dose to various micrometastases can, however, be estimated by modelling uptake and release of targeted alphas on single cancer cells and small cell clusters [PMID: 29175984]. Results For patients treated intraperitoneally with 211At-MX35 F(ab’)2, relatively high absorbed doses (approximately 10 Gy) were calculated for microtumors with diameters up to ∼200 μm [PMID: 30683761]. For the normal organs, the urinary bladder, thyroid, and kidneys received the highest absorbed dose. All organ doses were less than 10% of the estimated tolerance dose. Conclusion Dosimetry can and should be used to predict therapeutic effect, but also for estimating possible risks. Such benefit/risk estimates can then be used to further optimize the various alpha-particle therapies that are now being introduced in the clinic. Funding Acknowledgements This work was supported by the Swedish Research Council, the Swedish Cancer Society, the King Gustav V Jubilee Clinic Research Foundation, the Swedish Radiation Safety Authority, and grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement.

Introduction Alpha-emitter astatine-211 (211At; t½=7.2h) is currently used for targeted alpha therapy (TAT) in clinical trials, but expansion is hindered partly by radiation safety concerns. These concerns include transport; management of daughter 207Bi (t½=33a); co-production of 210At (t½=8.1h) and its daughter 210Po (t½=138d).Different regulations and local rules for work with 211At have been reported. Here, we refer to international standard documents such as the IAEA basic safety standards (1); the IAEA regulations for transport (2); and the EU directive on radiation protection (3). Objectives Our aim was to provide some practical guidelines for interpreting and addressing the risks involved with 211At using the exemption levels listed in (1-3). Our aim was also to raise awareness of the inappropriate regulations and local guidelines that requi Materials and Methods All work is based on the 20+ years production of 211At in Copenhagen, Denmark and subsequent transport to and pre-clinical and clinical use in Gothenburg, Sweden. Shipped amount was nearly always <2 GBq 211At. Patients received up to 300 MBq 211At (coupled to an antibody). Both countries have national legislation that adhere to (3) that, in turn, was harmonized with (1).To increase the amount of 211At produced, typically 0.04% of 210At is allowed to be co-produced. All regulations (1-3) list the same exemption limit for the mentioned radionuclides. Briefly, normal exposure to the exemption limit results in 10 uSv effective dose.Cross-border transport of the solid target (<2 GBq 211At) is done in a type A container classified as UN2915. With lead shielding, the transport index is below 0.05 and therefore marked as zero. Packages are thus assigned to category I-WHITE. Results The exemption limit for 211At is 10 MBq. Therefore, gloves and other waste are kept for 72h and then discarded and incinerated as regular hospital waste.Complete decay of 2 GBq 211At results in 21 kBq 207Bi (exemption limit 1 MBq). Co-production of 210At (0.04% of 2 GBq 211At activity) and subsequent decay eventually results in 2 kBq 210Po (exemption limit 10 kBq). Another 6 kBq 210Po can be directly produced in the bombarded target (4). Conclusion Exemption limits for 211At, 207Bi, 210At and 210Po can be used for determining clearance. Daughters 207Bi and 210Po are not a safety concern for <2 GBq 211At. Funding Acknowledgements This work was supported by the Swedish Research Council, the Swedish Cancer Society, the King Gustav V Jubilee Clinic Research Foundation, the Swedish Radiation Safety Authority, and grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement.

Introduction Targeted Alpha Therapy (TAT) is an emerging approach for treating disseminated cancers. A major challenge is eliminating small, clinically undetectable tumor clusters. As tumor size decreases, alpha-particles offer a strong therapeutic advantage due to their high linear energy transfer and short range. Among available alpha-emitters, Astatine-211 is particularly favorable because of its 7.2-hour half-life and 100% alpha decay. Objectives Radioimmunoconjugates can selectively target cancer cells, but their clinical use is often limited by slow pharmacokinetics and extended radiation exposure to healthy tissues. Pretargeting strategies address these issues by separating the tumor-targeting Materials and Methods In this work, we used a bioorthogonal inverse electron-demand Diels–Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz) to generate the radiolabeled compound in vivo. Results Three poly-L-lysine–based effector molecules of different sizes were first evaluated in vitro. An initial in vivo study testing one effector size across three dosing levels showed superior tumor uptake in %IA/g comparing the click-system with the pretargeting agent alone, already 1 h post-effector injection, supporting further optimization of this pretargeting strategy. Conclusion Taken as a whole, the study provides a basis for continued evaluation and refinement of this pretargeting method. Funding Acknowledgements This research was funded by the King Gustaf V Jubilee Clinic Cancer Research Foundation in Göteborg, Sweden, and the Swedish Cancer Society.

Introduction As the number of targeted alpha therapies (TAT) are rapidly growing, there is in increased interest in evaluating long-term effects. Here, we present an update on the survival of 12 patients with recurrent ovarian cancer that has received intraperitoneal TAT. Objectives The aim was to provide an update on long-term survival following intraperitoneal TAT with alpha-emitter astatine-211. Materials and Methods Between February 2005 and February 2011, twelve women with recurrent ovarian cancer received intraperitoneal targeted alpha therapy (TAT) by infusion of 211At-MX35 F(ab’)2 fragments (1). At a long-term follow-up in 2017 three patients were still alive (2). With long-term survivors, it was deemed necessary to estimate possible long-term risks for this therapy . The results were published 2023 (3). Results To date (November 2025) two patients, both treated with i.p. TAT in 2005, are still alive. More than 20 years have passed since they received treatment . Another presented as alive (2) lived 10 years after treatment. Conclusion Long-term risks with intraperitoneal therapy with astatine-211-labelled antibodies appear acceptable, however additional data are needed to confirm this. A new clinical trial with astatine-211-labelled trastuzumab is planned. Funding Acknowledgements This work was supported by the Swedish Research Council, the Swedish Cancer Society, the King Gustav V Jubilee Clinic Research Foundation, the Swedish Radiation Safety Authority, and grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement.

The separation between astatine-211 and polonium-210 during dry distillation with a commercially available equipment was investigated using two different analytical methods. Astatine targets were allowed to completely decay, and dry distillation was performed on the decayed target material, containing polonium-210. Analysis of distillation eluate using alpha spectrometry and alpha camera analysis showed a polonium co-distillation of < 0.1

Introduction Targeted alpha therapies (TAT) are currently evaluated in several clinical trials, but the requirement of metrological traceability for the activity measurements of alpha-emitters are typically not met. For 211At (t½=7.2h) reference sources are not available. In anticipation of a new clinical trial involving 211At, a measurement using absolute 4pi-alpha liquid scintillation counting was performed [1] to establish metrological traceability to the equipment used in the hospital. The resulting activity measurements were then compared with those derived for a previous clinical trial [2] (without metrological traceability). Objectives The aim was to evaluate the impact of metrological traceability when measuring 211At for three radionuclide calibrators and three HPGe detectors. Materials and Methods A sample from an aqueous solution of 211At was measured using absolute 4pi-alpha liquid scintillation counting [1]. Samples from the same solution were then measured in three radionuclide calibrators and one (A) of three (A, B, C) HPGe detectors. Another comparison was made (using an identical 211At solution) between HPGe detector (A) and HPGe detectors (B, C) in different labs. HPGe detectors (A, B) used decay data from DDEP and detector (C) used data from NuDat3. Results For the three radionuclide calibrators, the difference in activity between the setting used for the previous clinical trial and the traceable calibration was 3.9%, 3.9% and -6.9%, respectively. The HPGe detectors differed compared to the traceable calibration 6.6%, -0.1% and 1.7%, resepectively. Had site (C) also used decay data from DDEP the difference would have been -4.5% (instead of 1.7%). Conclusion Before establishing metrological traceability, the radionuclide calibrators underestimated the activity of 211At by up to 7%. HPGe measurements rely on accurate data on the intensity of the 687 keV gamma associated with 211At decay. DDEP lists an uncertainty of 5% for this intensity, which is the largest contributor to the uncertainty of the measurements. There is a 6.5% difference in listed gamma emission intensity between DDEP and NuDat3. This highlights the need for better decay data for 211At if HPGe detectors are used for activity measurements. Funding Acknowledgements This work was supported by the Swedish Research Council, the Swedish Cancer Society, the King Gustav V Jubilee Clinic Research Foundation, the Swedish Radiation Safety Authority, and grants from the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement.

Introduction Targeted Alpha Therapy (TAT) uses alpha emitting radionuclides to treat small tumors. The short range and high LET of the alpha-particle provides high local absorbed dose to small metastases, while sparing normal tissue. For systemic treatment the administered activity is limited by the absorbed dose to the kidney and the bone marrow. Studies have indicated that the activity distribution in the kidney is non-homogeneous. In previous studies we have shown that the alpha-emitter astatine-211 labeled to antibody molecules with different sizes (IgG, M.W. 150 kDa; and F(ab’)2, 95 kDa) have different intra-renal distribution. When predicting kidney toxicity for TAT, it is therefore more suitable to use sub-organ dosimetry than mean absorbed dose to the whole organ. Objectives We are developing methods for image-based small-scale kidney dosimetry and are currently conducting a conceptual study to compare whole-organ based dosimetry with sub-organ dosimetry. The aim is to assess prediction of kidney toxicity after TAT using anti Materials and Methods Mice were injected with astatine-labeled IgG and F(ab’)2. The activity distributions were quantified using an Alpha Camera. Absorbed doses were calculated to the whole kidney and to kidney cortex and medulla separately. Results Preliminary data shows molecule-size dependent activity distributions with higher uptake in cortex for F(ab’)2. Registration of histological and alpha images will be conducted to identify if observed differences are related to morphological structures like glomeruli and proximal tubules. Furthermore, in an on-going long-term study, the dose distribution of astatine-labeled IgG and F(ab’)2 will be related to renal toxicity. The injected activity was chosen so that the mean absorbed dose to the whole kidneys would be similar for both molecule sizes. Renal toxicity will be studied by GFR, histopathology and biomarkers in blood and urine. Conclusion Our preliminary results shows that the activity distribution in the kidney is non-homogeneous and that the different molecule sizes have different intra-renal distribution that in turn will affects the sub-organ dosimetry. Funding Acknowledgements Swedish Research CouncilThe Swedish Cancer SocietyJubileumsklinikens cancerfond

AIM/INTRODUCTION:Astatine-211 is one of the alpha-particle emitting nuclides investigated for use within Targeted Alpha Therapy (TAT) of disseminated cancer. In previous studies, a difference in blood clearance has been observed when comparing astatinated compounds with their iodinated counterparts. This has been explained by a potentially prolonged retention in the blood by present free astatine. This study examined the spontaneous binding of non-conjugated astatine to albumin under physiological conditions in vitro and compared it to that of iodine. Additionally, the in vivo blood circulation patterns of free astatine were assessed and contrasted with those of iodine. MATERIALS AND METHODS:Astatine solutions were formulated following dry distillation and evaporation to dryness of astatine solvated in CHCl3. In vitro evaluation of astatine and iodine association to albumin was mainly performed using size exclusion chromatography applying both disposable columns (PD10 and NAP10) as well as an FPLC system (ÄKTA) with online UV detection and activity fraction collection. Also methanol precipitation and radio-TLC methods were used for analysis. In vivo evaluation was performed using furry BALB/C mice (3/group) with both i.v. and i.p. injection of astatine, followed by sequential blood sampling from the tail vein and biodistribution. RESULTS:Oxidized and unmodified forms of free astatine display a significantly higher and more rapid association to albumin in vitro compared to reduced forms, with >97% compared to <25% associated after 10 min. Both the corresponding oxidized and reduced forms of iodine display a very low and slow association to albumin with <5% associated after 40 min. Oxidized, unmodified and reduced astatine show very similar blood profiles over time as well as a similar uptake in biodistribution after 20-22 h following i.p. injection. Upon i.v. injection a larger difference in blood profiles between the species could be observed, which in turn was different compared to the curve obtained after i.p. injection. In addition, an unexpected uptake of astatide in stomach was found. In all cases the blood profile and biodistribution of astatine was significantly different compared to iodine, which displayed a greater and more rapid blood clearance and specific accumulation in thyroid. CONCLUSION:Different forms of unbound astatine differ in their association to albumin. However, all investigated forms of free astatine associates to albumin to a much higher degree than iodine. This behavior could explain the prolonged blood circulation of free astatine compared to iodine.

Abstract Background Pretargeted radioimmunotherapy of cancer has the potential to increase tumor specific uptake of activity when compared with conventional radioimmunotherapy. This is especially true in radioimmunotherapy with nuclides that exhibit a relatively short half-life. When administering antibody-based pretargeting molecules systemically, the antibodies often show a relatively slow clearance from the blood. Therefore, the use of a clearing agent is advantageous to remove unbound pretargeting molecules from the circulation, facilitating a reduction in the nonspecific radiation exposure to normal tissue while maximizing the dose delivered to the tumors. Results In the current study, two types of poly-L-lysine based clearing agents were produced for two different pretargeting systems: (strept)avidin/biotin and Tetrazine/Transcyclooctene. Poly-L-lysine was used as scaffold for production of clearing agents. The polymer is available in multiple sizes and can readily be modified with several functional groups, allowing different pretargeting strategies to be used. In vivo evaluation of the biotin-functionalized poly-L-lysine clearing agent, 110 repeating units, resulted in a decrease in blood concentration of the Iodine-125 labeled pretargeting agent of 50%, circa 23 h after injection, compared to controls. Two sizes, 68 and 143 repeating units, of the tetrazine-functionalized poly-L-lysine clearing agent were also evaluated, which at 23 h after injection decreased the blood concentration of the Iodine-125 labeled pretargeting agent to 58 and 38% respectively. Conclusion The straightforward synthesis of poly-L-lysine based clearing agents makes kit preparation possible and these agents show good potential for further evaluation, especially within the Tetrazine/Transcyclooctene pretargeting system where no liver or kidney accumulation was observed.

Several studies have illustrated the significant increase in the production yield of cyclotron produced At-211 by irradiating targets at higher beam energies, highlighting that this approach warrants consideration. Recently published data also confirms the importance of the Bi-209(alpha,x)Po-210 reaction in addition to the increasing At-210 production at higher energies, stressing the importance of determining the total Po-210 activity on irradiated targets and the validation of radiochemical separation of Po-210 from astatine as an impurity. In this work, two experiments were performed: (i) the dissolution of irradiated Bi-targets to validate the calculated At-211, At-210 and Po-210 activities on target and (ii) the radiochemical separation of astatine by extraction chromatography to determine the activity balance of At-211, At-210 and Po-210 after target processing. The calculated At-211, At-210 and Po-210 activities were validated on targets irradiated at the Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. At-211 and At-210 activities were determined by gamma spectrometry, whereas the Po-210 activity was measured by liquid scintillation counting (LSC) after target dissolution with concentrated nitric acid. The activity balance during target processing and extraction chromatography was established using targets irradiated at Arronax, Nantes, France. Targets were characterised after the dissolution with an in-house developed dissolution unit. Dissolution yield, extraction yield and separation capability were assessed by determining At-211, At-210 and Po-210 activities at every step. The results show that more than 88% of the total Po-210 activity in targets irradiated at approximate to 28.8 MeV is attributable to the direct production of Po-210. This amounts to about 53% at energies of 29.8 MeV. A maximal discrepancy of 21% was found when comparing the total measured activity to the total calculated Po-210. Activity balance calculations show a mean decay-corrected dissolution yield of 88.9% +/- 9.5% and an extraction yield of 87.56% +/- 11.4% (n = 5) of At-211/At-210 resulting in an overall yield of 77.8% +/- 10.5% (n = 5). In addition, the radiochemical separability was confirmed by removing >95% of the previously formed Po-210 before extraction. This work confirms the important underestimation of the total Po-210 component on irradiated targets at 28.8 and 29.8 MeV when calculating solely from At-210 measurements. In addition, the validation measurements indicate that calculations can be used to estimate the total Po-210 activity. The experimental setup of target dissolution and extraction chromatography presented a very good yield. It allowed the safe isolation and management of Po-210, leaving At-210 as the only source for future ingrowth of Po-210. This finding is an important step in moving beyond the conventional approach of irradiating Bi-targets at limited incident energies.

BackgroundA 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, 211At 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 211At 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 211At, and the in vitro binding of the most favorable polymer size, in order to optimize the pretargeted radioimmunotherapy with 211At.ResultsIn 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.ConclusionsOverall, 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.

The α-emitter 211At deposits a high amount of energy within a few cell diameters, resulting in irreparable DNA double-strand breaks while minimizing off-target toxicity. We investigated the use of the 211At-labeled anti-CD45 monoclonal antibody (mAb) 211At-CD45-B10 as a nonmyeloablative conditioning regimen for dog-leukocyte-antigen-haploidentical hematopoietic cell transplantation. Methods: Seventeen healthy dogs were injected with either a 0.50 (n = 14) or 0.75 (n = 3) mg/kg dose of anti-CD45 mAb labeled with 211At (8.436-23.199 MBq [0.228-0.627 mCi/kg]) on day -3. Peripheral blood stem cells from dog-leukocyte-antigen-haploidentical donors were given on day 0. Peripheral blood chimerism was calculated by polymerase chain reaction assays, and blood clearance of the radioimmunoconjugate was studied using enzyme-linked immunosorbent assay and radioactivity measurements of serial blood samples. Results: All dogs achieved donor chimerism by day 28 (range, 27%-100%). The hematopoietic engraftment rate was 100%, though engraftment durability was variable. No difference in absorbed dose to blood was seen for the 2 mAb dosing levels studied. Neutropenia (0-29 cells/μL), lymphocytopenia (36-130 cells/μL), and thrombocytopenia (1.5-9 × 103/μL) with prompt recovery were observed. The main adverse nonhematologic event related to 211At-CD45-B10 was mild reversible transaminitis. Graft-versus-host disease was not seen. Twelve of the 17 dogs survived over 30 d, with donor chimerism ranging from 3% to 99%. Conclusion: The results suggest that nonmyeloablative conditioning with 211At-CD45-B10 could be used in haploidentical hematopoietic cell transplantation though with variable engraftment.

Intraperitoneal 211At-based targeted α-therapy (TAT) may hold great promise as an adjuvant therapy after surgery and chemotherapy in epithelial ovarian cancer to eradicate any remaining undetectable disease. This implies that it will also be delivered to patients possibly already cured by the primary treatment. An estimate of long-term risks is therefore sought to determine whether the treatment is justified. Methods: Baseline data for risk estimates of α-particle irradiation were collected from published studies on excess cancer induction and mortality for subjects exposed to either 224Ra treatments or Thorotrast contrast agent (25% ThO2 colloid, containing 232Th). Organ dosimetry for 224Ra and Thorotrast irradiation were taken from the literature. These organ-specific risks were then applied to our previously reported dosimetry for intraperitoneal 211At-TAT patients. Results: Risk could be estimated for 10 different organ or organ groups. The calculated excess relative risk per gray (ERR/Gy) could be sorted into 2 groups. The lower-ERR/Gy group, ranging up to a value of approximately 5, included trachea, bronchus, and lung, at 0.52 (95% CI, 0.21-0.82); stomach, at 1.4 (95% CI, -5.0-7.9); lymphoid and hematopoietic system, at 2.17 (95% CI, 1.7-2.7); bone and articular cartilage, at 2.6 (95% CI, 2.0-3.3); breast, at 3.45 (95% CI, -10-17); and colon, at 4.5 (95% CI, -3.5-13). The higher-ERR/Gy group, ranging from approximately 10 to 15, included urinary bladder, at 10.1 (95% CI, 1.4-23); liver, at 14.2 (95% CI, 13-16); kidney, at 14.9 (95% CI, 3.9-26); and lip, oral cavity, and pharynx, at 15.20 (95% CI, 2.73-27.63). Applying a typical candidate patient (female, age 65 y) and correcting for the reference population mortality rate, the total estimated excess mortality for an intraperitoneal 211At-monoclonal antibody treatment amounted to 1.13 per 100 treated. More than half this excess originated from urinary bladder and kidney, 0.29 and 0.34, respectively. Depending on various adjustments in calculation and assumptions on competing risks, excess mortality could range from 0.11 to 1.84 per 100 treated. Conclusion: Published epidemiologic data on lifelong detriment after α-particle irradiation and its dosimetry allowed calculations to estimate the risk for secondary cancer after 211At-based intraperitoneal TAT. Measures to reduce dose to the urinary organs may further decrease the estimated relative low risk for secondary cancer from 211At-monoclonal antibody-based intraperitoneal TAT.

Supplementary Figure S1. Estimating regional tumor volume within transverse sections. Table S1. Tumor dimensions of the human breast cancer cells inoculated into tibiae Supplementary Figure S2. Clonogenic survival of alpha-particle irradiated MDA-MB-231 (solid line) and MCF-7 (dashed line) human breast cancer cells. Table S2. Statistical significance within treatment groups and timepoints forpercentage ofcells withone or more y-H2AX foci Table S3. Statistical significance within treatment groups and timepoints for percentage of bystander cancer cells with 0, 1-2, 3-5 and 5+ y-H2AX foci Table S4. Statistical significance within treatment groups and timepoints for percentage of irradiated cancer cells with 0, 1-2, 3-5 and 5+ y-H2AX foci Table S5. Statistical significance within treatment groups and timepoints for percentage of osteocyte cells with 0, 1-2, 3-5 and 5+ y-H2AX foci Table S6. Statistical significance within treatment groups and timepoints for percentage of cells that are TUNEL+.

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 211At for targeted alpha therapy and 125I as a surrogate for the imaging radionuclides 123, 124I. 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 211At.

Astatine-211 (211At) 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 211At. 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 (125I) and then adapted for 211At, enabling the use of both elements as a potential theranostic pair. PMs that were core-radiolabeled with both 125I or 211At 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 211At- labeled PMs, 4-5 % ID/g of the 211At could still be detected in the blood, showing a promising in vivo stability of the PMs. Further, 211At-labeled PMs accumulated in the spleen (20-30 %ID/g) and the liver (2.5- 5.5 %ID/g), along with some detection of 211At 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 211At core-radiolabel was observed in the blood.

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 211At-farletuzumab in invitro 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 211At-farletuzumab (n = 16); or with a combination of 125I-farletuzumab and 211At-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 2x10(6) NIH:OVCAR-3 cells. Twelve days later, the treatments were initiated by i.p.-administration. Specific treatment was given by 211At-labeled farletuzumab (group A; n = 22, 170 kBq/mL) which is specific for OVCAR-3 cells. Control treatments were given by either 211Atlabeled 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 At-211-farletuzumab was similar to that with 125I as radiolabel, and also to that of At-211-labeled MX35 antibody. The tumor-free fraction (TFF) of the three control groups were all low (PBS 12%, unlabeled specific farletuzumab 9% and unspecific 211At-rituximab 14%). TFF following treatment with 211At-farletuzumab was 91%. Conclusion: The current investigation of intraperitoneal therapy with 211At-farletuzumab, delivered at clinically relevant At-211-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.

Targeted α-therapy (TAT) can eradicate tumor metastases while limiting overall toxicity. One of the most promising α-particle emitters is astatine-211 (211At). However, 211At-carbon bonds are notoriously unstable in vivo and no chelators are available. This hampers its adoption in TAT. In this study, the stability of 211At on the surface of gold nanoparticles (AuNPs) was investigated. The employed AuNPs had sizes in the 25–50 nm range. Radiolabeling by non-specific surface-adsorption in >99% radiochemical yield was achieved by mixing 211At and AuNPs both before and after polyethylene glycol (PEG) coating. The resulting 211At-AuNPs were first challenged by harsh oxidation with sodium hypochlorite, removing roughly 50% of the attached 211At. Second, incubation in mouse serum followed by a customized stability test, showed a stability of >95% after 4 h in serum. This high stability was further confirmed in an in vivo study, with comparison to a control group of free 211At. The AuNP-associated 211At showed low uptake in stomach and thyroid, which are hallmark organs of uptake of free 211At, combined with long circulation and high liver and spleen uptake, consistent with nanoparticle biodistribution. These results support that gold surface-adsorbed 211At has high biological stability and is a potentially useful delivery system in TAT.

Abstract Radiation-induced bystander effects have been implicated in contributing to the growth delay of disseminated tumor cells (DTC) caused by 223RaCl2, an alpha particle–emitting radiopharmaceutical. To understand how 223RaCl2 affects the growth, we have quantified biological changes caused by direct effects of radiation and bystander effects caused by the emitted radiations on DTC and osteocytes. Characterizing these effects contribute to understanding the efficacy of alpha particle–emitting radiopharmaceuticals and guide expansion of their use clinically. MDA-MB-231 or MCF-7 human breast cancer cells were inoculated intratibially into nude mice that were previously injected intravenously with 50 or 600 kBq/kg 223RaCl2. At 1-day and 3-days postinoculation, tibiae were harvested and examined for DNA damage (γ-H2AX foci) and apoptosis in osteocytes and cancer cells located within and beyond the range (70 μm) of alpha particles emitted from the bone surface. Irradiated and bystander MDA-MB-231 and MCF-7 cells harbored DNA damage. Bystander MDA-MB-231 cells expressed DNA damage at both treatment levels while bystander MCF-7 cells required the higher administered activity. Osteocytes also had DNA damage regardless of inoculated cancer cell line. The extent of DNA damage was quantified by increases in low (1–2 foci), medium (3–5 foci), and high (5+ foci) damage. MDA-MB-231 but not MCF-7 bystander cells showed increases in apoptosis in 223RaCl2-treated animals, as did irradiated osteocytes. In summary, radiation-induced bystander effects contribute to DTC cytotoxicity caused by 223RaCl2. Implications: This observation supports clinical investigation of the efficacy of 223RaCl2 to prevent breast cancer DTC from progressing to oligometastases.

Rationale The role of radiation-induced bystander effects in cancer therapy with alpha-particle emitting radiopharmaceuticals remains unclear. With renewed interest in using alpha-particle emitters to sterilize disseminated tumor cells, micrometastases, and tumors, a better understanding of the direct effects of alpha particles and the contribution of the bystander responses they induce is needed to refine dosimetric models that help predict clinical benefit. Accordingly, this work models and quantifies the relative importance of direct effects (DE) and bystander effects (BE) in the growth delay of human breast cancer xenografts observed previously in the tibiae of mice treated with (RaCl2)-Ra-223. Methods A computational model of MDA-MB-231 and MCF-7 human breast cancer xenografts in the tibial bone marrow of mice administered (RaCl2)-Ra-223 was created. A Monte Carlo radiation transport simulation was performed to assess individual cell absorbed doses. The responses of the breast cancer cells to direct alpha particle irradiation and gamma irradiation were needed as input data for the model and were determined experimentally using a colony-forming assay and compared to the responses of preosteoblast MC3T3-E1 and osteocyte-like MLO-Y4 bone cells. Using these data, a scheme was devised to simulate the dynamic proliferation of the tumors in vivo, including DE and BE propagated from the irradiated cells. The parameters of the scheme were estimated semi-empirically to fit experimental tumor growth. Results A robust BE component, in addition to a much smaller DE component, was required to simulate the in vivo tumor proliferation. We also found that the relative biological effectiveness (RBE) for cell killing by alpha particle radiation was greater for the bone cells than the tumor cells. Conclusion This modeling study demonstrates that DE of radiation alone cannot explain experimental observations of (RaCl2)-Ra-223-induced growth delay of human breast cancer xenografts. Furthermore, while the mechanisms underlying BE remain unclear, the addition of a BE component to the model is necessary to provide an accurate prediction of the growth delay. More complex models are needed to further comprehend the extent and complexity of (RaCl2)-Ra-223-induced BE.