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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.

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+.

Astatine-211 (211At) has physical properties that make it one of the top candidates for use as a radiation source for alpha particle-based radionuclide therapy, also referred to as targeted alpha therapy (TAT). Here, we summarize the main results of the completed clinical trials, further describe ongoing trials, and discuss future prospects.

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.

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 (223)RaCl(2). METHODS. A computational model of the MDA-MB-231 and MCF-7 human breast cancer xenografts in the tibial bone marrow of mice administered (223)RaCl(2) 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 thus 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 as well as 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 direct effects of radiation alone cannot explain experimental observations of (223)RaCl(2)-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 (223)RaCl(2)-induced BE.

Astatine-211 (

Intraperitoneal

The role of radiation-induced bystander effects in radiation therapy remains unclear. With renewed interest in therapy with α-particle emitters, and their potential for sterilizing disseminated tumor cells (DTCs), it is critical to determine the contribution of bystander effects to the overall response so they can be leveraged for maximum clinical benefit. Methods: Female Foxn1(nu) athymic nude mice were administered 0, 50, or 600 kBq/kg (223)RaCl(2) to create bystander conditions. At 24 hours after administration, MDA-MB-231 or MCF-7 human breast cancer cells expressing luciferase were injected into the tibial marrow compartment. Tumor burden was tracked weekly via bioluminescence. Results: The MDA-MB-231 xenografts were observed to have a 10-day growth delay in the 600 kBq/kg treatment group only. In contrast, MCF-7 cells had 7- and 65-day growth delays in the 50 and 600 kBq/kg groups, respectively. Histologic imaging of the tibial marrow compartment, α-camera imaging, and Monte Carlo dosimetry modeling revealed DTCs both within and beyond the range of the α-particles emitted from (223)Ra in bone for both MCF-7 and MDA-MB-231 cells. Conclusion: Taken together, these results support the participation of (223)Ra-induced antiproliferative/cytotoxic bystander effects in delayed growth of DTC xenografts. They indicate that the delay depends on the injected activity and therefore is dose-dependent. They suggest using (223)RaCl(2) as an adjuvant treatment for select patients at early stages of breast cancer.

1073 Objectives: Kikuchi-Fujimoto disease (KFD), also known as histiocytic necrotizing lymphadenitis, is a rare cause of lymphadenopathy with a highe prevalence in Asia. It can involve systemic lymph nodes, with cervical lymph nodes most commonly affected, and is easily misdiagnosed as lymphoma. This study investigated 18F-FDG PET/CT findings in KFD, and its usefulness for differential diagnosis from non-Hodgkin lymphoma (NHL).Methods: Three groups of patients [10 KFD, 12 indolent lymphoma (IL) and 15 aggressive lymphoma (AL)] underwent 18F-FDG PET/CT scan. Two experienced nuclear medicine physician independently interpreted the images and recorded the areas, size, and SUVmax and SULmax of lymph nodes involved. Possible organ involvements or other hypermetabolic lesions were also analyzed. Some serological indicators were collected.Results: The median age of KFD group was 23 (13-56) years old, with 8 women included. There were 9 cases with fever, 10 with lymph node enlargement, 3 with lymph node pain, 5 with skin rash, 5 with pharyngalgia, 3 with muscle pain, 5 with arthralgia and 1 with subcutaneous nodules of lower limbs. The course of disease was 31 (10-185) days. Leukocyte count was 8.4 (1.3-13.8)×109/L, neutrophil count 5.2 (0.8-12.6)×109/L, LDH 463.5 (188-1123) U/L, ESR 45 (4-101) mm/H, CRP 21.6 (11.5-101.8) mg/L, ALT 27 (11-273) U/L, AST 39.5 (21-276) U/L. At least two lymph node regions were involved in all participates, including neck in 10 cases, axilla in 9 cases, mediastinum in 8 cases, abdomen in 7 cases, pelvic in 6 cases and groin in 6 cases. The largest lymph node was 22 (13-36) mm, lymph node SUVmax 8.4 (6-19.2), highest SULmax 9.9 (4.9-15.2), liver SUVmax 2.8 (1.8-3.3), spleen size 9 (6-10) costal units, spleen SUVmax 3.4 (2.1-4.7), and bone marrow SUVmax 4.3 (3.1-5.4). High metabolic focis were found in 1 case of subcutaneous nodule with SUVmax 3.5, 2 cases of skin lesion with SUVmax 2.1 and 3.2, and 1 case of muscle lesion with SUVmax 2.1. The density of lymph nodes was uneven in 2 cases and blurred in 2 cases, which were confirmed by ultrasound. The correlation analysis showed that SUVmax of lymph nodes was correlated with LDH (r=-0.927, p

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 ofFor biodistribution, using intravenous injections and mice carrying subcutaneous (s.c.) tumors, the animals were administered eitherThe biodistribution ofThe current investigation of intraperitoneal therapy with

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 mm3, NIH:OVCAR-3) were injected repeatedly (1-3 intravenous injections 7-10 d apart, allowing bone marrow recovery) with 211At-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 211At-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.

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 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 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 2 × 106 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 211At-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 211At-farletuzumab was similar to that with 125I as radiolabel, and also to that of 211At-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 211At-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.

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.

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.

A biokinetic model was constructed to evaluate and optimize various intraperitoneal radioimmunotherapies for micrometastatic tumors. The model was used to calculate the absorbed dose to both anticipated microtumors and critical healthy organs and demonstrated how intraperitoneal targeted radiotherapy can be optimized to maximize the ratio between them. Methods: The various transport mechanisms responsible for the biokinetics of intraperitoneally infused radiolabeled monoclonal antibodies (mAbs) were modeled using a software package. Data from the literature were complemented by pharmacokinetic data derived from our clinical phase I study to set parameter values. Results using the β-emitters 188Re, 177Lu, and 90Y and the α-emitters 211At, 213Bi, and 212Pb were compared. The effects of improving the specific activity, prolonging residence time by introducing an osmotic agent, and varying the activity concentration of the infused agent were investigated. Results: According to the model, a 1.7-L infused saline volume will decrease by 0.3 mL/min because of lymphatic drainage and by 0.7 mL/min because of the transcapillary convective component. The addition of an osmotic agent serves to lower the radiation dose to the bone marrow. Clinically relevant radioactivity concentrations of α- and β-emitters bound to mAbs were compared. For α-emitters, microtumors receive high doses (>20 Gy or 100 Sv [relative biological effect = 5]). Since most of the tumor dose originates from cell-bound radionuclides, an increase in the specific activity would further increase the tumor dose without affecting the dose to peritoneal fluid or bone marrow. For β-emitters, tumors will receive almost entirely nonspecific irradiation. The dose from cell-bound radiolabeled mAbs will be negligible by comparison. For the long-lived 90Y, tumor doses are expected to be low at the maximum activity concentration delivered in clinical studies. Conclusion: According to the presented model, α-emitters are needed to achieve radiation doses high enough to eradicate microscopic tumors.

α-radioimmunotherapy targeting CD45 may substitute for total-body irradiation in hematopoietic cell transplantation (HCT) preparative regimens for lymphoma. Our goal was to optimize the anti-CD45 monoclonal antibody (mAb; CA12.10C12) protein dose for 211At-radioimmunotherapy, extending the analysis to include intraorgan 211At activity distribution and α-imaging–based small-scale dosimetry, along with immunohistochemical staining. Methods: Eight normal dogs were injected with either a 0.75 (n = 5) or 1.00 (n = 3) mg/kg dose of 211At-B10-CA12.10C12 (11.5–27.6 MBq/kg). Two were euthanized and necropsied 19–22 h after injection, and 6 received autologous HCT 3 d after 211At-radioimmunotherapy, after lymph node and bone marrow biopsies at 2–4 and/or 19 h after injection. Blood was sampled to study toxicity and clearance; CD45 targeting was evaluated by flow cytometry. 211At localization and small-scale dosimetry were assessed using two α-imaging systems: an α-camera and an ionizing-radiation quantum imaging detector (iQID) camera. Results:211At uptake was highest in the spleen (0.31–0.61% injected activity [%IA]/g), lymph nodes (0.02–0.16 %IA/g), liver (0.11–0.12 %IA/g), and marrow (0.06–0.08 %IA/g). Lymphocytes in blood and marrow were efficiently targeted using either mAb dose. Lymph nodes remained unsaturated but displayed targeted 211At localization in T lymphocyte–rich areas. Absorbed doses to blood, marrow, and lymph nodes were estimated at 3.1, 2.4, and 3.4 Gy/166 MBq, respectively. All transplanted dogs experienced transient hepatic toxicity. Liver enzyme levels were temporarily elevated in 5 of 6 dogs; one treated with 1.00 mg mAb/kg developed ascites and was euthanized 136 d after HCT. Conclusion:211At-anti-CD45 radioimmunotherapy with 0.75 mg mAb/kg efficiently targeted blood and marrow without severe toxicity. Dosimetry calculations and observed radiation-induced effects indicated that sufficient 211At-B10-CA12.10C12 localization was achieved for efficient conditioning for HCT.

α-Emitting radionuclides deposit a large amount of energy within a few cell diameters and may be particularly effective for radioimmunotherapy targeting minimal residual disease (MRD). To evaluate this hypothesis, (211)At-labeled 1F5 monoclonal antibody (mAb) (anti-CD20) was studied in both bulky lymphoma tumor xenograft and MRD animal models. Superior treatment responses to (211)At-labeled 1F5 mAb were evident in the MRD setting. Lymphoma xenograft tumor-bearing animals treated with doses of up to 48 µCi of (211)At-labeled anti-CD20 mAb ([(211)At]1F5-B10) experienced modest responses (0% cures but two- to threefold prolongation of survival compared with negative controls). In contrast, 70% of animals in the MRD lymphoma model demonstrated complete eradication of disease when treated with (211)At-B10-1F5 at a radiation dose that was less than one-third (15 µCi) of the highest dose given to xenograft animals. Tumor progression among untreated control animals in both models was uniformly lethal. After 130 days, no significant renal or hepatic toxicity was observed in the cured animals receiving 15 µCi of [(211)At]1F5-B10. These findings suggest that α-emitters are highly efficacious in MRD settings, where isolated cells and small tumor clusters prevail.

Intraperitoneally administered radiolabeled monoclonal antibodies (mAbs) have been tested in several clinical trials, often with promising results, but have never proven curative. Methods: We have previously presented simulations of clinically relevant amounts of intraperitoneal 90Y-mAbs for treatment of minimal disease and shown that such treatments are unlikely to eradicate microtumors. Our previous model simulated the kinetics of intraperitoneally infused radiolabeled mAbs in humans and showed the benefit of instead using α-emitters such as 211At. In the current work, we introduce penetration of mAbs into microtumors with radii of up to 400 μm. Calculations were performed using dynamic simulation software. To determine the radiation dose distribution in nonvascularized microtumors of various sizes after intraperitoneal 211At-radioimmunotherapy, we used an in-house-developed Monte Carlo program for microdosimetry. Our aim was to find methods that optimize the therapy for as wide a tumor size range as possible. Results: Our results show that high-specific-activity radiolabeled mAbs that are bound to a tumor surface will penetrate slowly compared with the half-lives of 211At and shorter-lived radionuclides. The inner-core cells of tumors with radii exceeding 100 μm may therefore not be sufficiently irradiated. For lower specific activities, the penetration rate and dose distribution will be more favorable for such tumors, but the dose to smaller microtumors and single cells will be low. Conclusion: Our calculations show that the addition of a boost with unlabeled mAb 1-5 h after therapy results in sufficient absorbed doses both to single cells and throughout microtumors up to approximately 300 μm in radius. This finding should also hold for other high-affinity mAbs and short-lived α-emitters.

Background Studies combining immune checkpoint inhibitors with external beam radiation have shown a therapeutic advantage over each modality alone. The purpose of these works is to evaluate the potential of targeted delivery of high LET radiation to the tumor microenvironment via an immune checkpoint inhibitor. Methods The impact of protein concentration on the distribution of 111In-DTPA-anti-PD-L1-BC, an 111In-antibody conjugate targeted to PD-L1, was evaluated in an immunocompetent mouse model of breast cancer. 225Ac-DOTA-anti-PD-L1-BC was evaluated by both macroscale (ex vivo biodistribution) and microscale (alpha-camera images at a protein concentration determined by the 111In data. Results The evaluation of 111In-DTPA-anti-PD-L1-BC at 1, 3, and 10 mg/kg highlighted the impact of protein concentration on the distribution of the labeled antibody, particularly in the blood, spleen, thymus, and tumor. Alpha-camera images for the microscale distribution of 225Ac-DOTA-anti-PD-L1-BC showed a uniform distribution in the liver while highly non-uniform distributions were obtained in the thymus, spleen, kidney, and tumor. At an antibody dose of 3 mg/kg, the liver was dose-limiting with an absorbed dose of 738 mGy/kBq; based upon blood activity concentration measurements, the marrow absorbed dose was 29 mGy/kBq. Conclusions These studies demonstrate that 225Ac-DOTA-anti-PD-L1-BC is capable of delivering high LET radiation to PD-L1 tumors. The use of a surrogate SPECT agent, 111In-DTPA-anti-PD-L1-BC, is beneficial in optimizing the dose delivered to the tumor sites. Furthermore, an accounting of the microscale distribution of the antibody in preclinical studies was essential to the proper interpretation of organ absorbed doses and their likely relation to biologic effect.

Alpha-particle emitters, such as astatine-211 (211At), are generally considered suitable for the treatment of small cell clusters due to their short path length, while beta-particle emitters, for example, Lutetium-177 (177Lu), have a longer path length and are considered better for small, established tumors. A combination of such radionuclides may be successful in regimens of radioimmunotherapy. In this study, rats were treated by sequential administration of first a 177Lu-labeled antibody, followed by a 211At-labeled antibody 25 days later.Rats bearing solid colon carcinoma tumors were treated with 400 MBq/kg body weight 177Lu-BR96. After 25 days, three groups of animals were given either 5 or 10 MBq/kg body weight of 211At-BR96 simultaneously with or without a blocking agent reducing halogen uptake in normal tissues. Control animals were not given any 211At-BR96. Myelotoxicity, body weight, tumor size, and development of metastases were monitored for 120 days.Tumors were undetectable in 90% of the animals on day 25, independent of treatment. Additional treatment with 211At-labeled antibodies did not reduce the proportion of animals developing metastases. The rats suffered from reversible myelotoxicity after treatment.Sequential administration of 177Lu-BR96 and 211At-BR96 resulted in tolerable toxicity providing halogen blocking but did not enhance the therapeutic effect.

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 211At 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.

Despite aggressive chemotherapy combined with hematopoietic stem cell transplantation (HSCT), many patients with acute myeloid leukemia (AML) relapse. Radioimmunotherapy (RIT) using monoclonal antibodies labeled with β-emitting radionuclides has been explored to reduce relapse. β emitters are limited by lower energies and nonspecific cytotoxicity from longer path lengths compared with α emitters such as (211)At, which has a higher energy profile and shorter path length. We evaluated the efficacy and toxicity of anti-CD45 RIT using (211)At in a disseminated murine AML model. Biodistribution studies in leukemic SJL/J mice showed excellent localization of (211)At-anti-murine CD45 mAb (30F11) to marrow and spleen within 24 hours (18% and 79% injected dose per gram of tissue [ID/g], respectively), with lower kidney and lung uptake (8.4% and 14% ID/g, respectively). In syngeneic HSCT studies, (211)At-B10-30F11 RIT improved the median survival of leukemic mice in a dose-dependent fashion (123, 101, 61, and 37 days given 24, 20, 12, and 0 µCi, respectively). This approach had minimal toxicity with nadir white blood cell counts2.7 K/µL 2 weeks after HSCT and recovery by 4 weeks. These data suggest that (211)At-anti-CD45 RIT in conjunction with HSCT may be a promising therapeutic option for AML.

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.

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.

Bioconjugates used in internal radiotherapy exhibit heterogeneous distributions in organs and tumors, implying a risk of nonuniform dose distribution in therapeutic applications using a-particle emitters. Tools are required that provide data on the activity distribution for estimation of absorbed dose on a suborgan level. The a-camera is a quantitative imaging technique developed to detect a-particles in tissues ex vivo. The aim of this study was to evaluate the characteristics of this imaging system and to exemplify its potential use in the development of a-radioimmunotherapy. Methods: The a-camera combines autoradiography with a scintillating technique and optical registration by a charge-coupled device (CCD). The imaging system characteristics were evaluated by measurements of linearity, uniformity, and spatial resolution. The technique was applied for quantitative imaging of 211At activity distribution in cryosections of tumors, kidney, and whole body. Intratumoral activity distributions of tumor-specific 211 At-MX35-F(ab9)2 were studied at various times after injection. The postinjection activity distributions in the renal cortex and whole kidneys were compared for 211 At-F(ab9)2 and 211 At-IgG trastuzumab. Results: Quantitative analysis of a-camera images demonstrated that the pixel intensity increased linearly with activity in the imaged specimen. The spatial resolution was 35 6 11 mm (mean 6 SD) and the uniformity better than 2%. Kidney cryosections revealed a higher cortex–to–whole kidney ratio for 211 At-F(ab9)2 than for 211At-IgG (1.38 6 0.03 and 0.77 6 0.04, respectively) at 2 h after injection. Nonuniform intratumoral activity distributions were found for tumor-specific 211At-MX35-F(ab9)2 at 10 min and 7 h after injection; after 21 h, the distribution was more uniform. Conclusion: The characteristics of the a-camera are promising, suggesting that this bioimaging system can assist the development, evaluation, and refinement of future targeted radiotherapy approaches using a-emitters. The a-camera provides quantitative data on the activity distribution in tissues on a near-cellular scale and can therefore be used for small-scale dosimetry, improving the prediction of biologic outcomes with a-particles with short path length and high linear energy trans

211At-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.

Purpose Ovarian cancer is often diagnosed at an advanced stage with dissemination in the peritoneal cavity. Most patients achieve clinical remission after surgery and chemotherapy, but approximately 70% eventually experience recurrence, usually in the peritoneal cavity. To prevent recurrence, intraperitoneal (i.p.) targeted α therapy has been proposed as an adjuvant treatment for minimal residual disease after successful primary treatment. In the present study, we calculated absorbed and relative biological effect (RBE)-weighted (equivalent) doses in relevant normal tissues and estimated the effective dose associated with i.p. administration of 211 At-MX35 F(ab') 2 . Methods and Materials Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of 211 At-MX35 F(ab') 2 . Potassium perchlorate was given to block unwanted accumulation of 211 At in thyroid and other NIS-containing tissues. Mean absorbed doses to normal tissues were calculated from clinical data, including blood and i.p. fluid samples, urine, γ-camera images, and single-photon emission computed tomography/computed tomography images. Extrapolation of preclinical biodistribution data combined with clinical blood activity data allowed us to estimate absorbed doses in additional tissues. The equivalent dose was calculated using an RBE of 5 and the effective dose using the recommended weight factor of 20. All doses were normalized to the initial activity concentration of the infused therapy solution. Results The urinary bladder, thyroid, and kidneys (1.9, 1.8, and 1.7 mGy per MBq/L) received the 3 highest estimated absorbed doses. When the tissue-weighting factors were applied, the largest contributors to the effective dose were the lungs, stomach, and urinary bladder. Using 100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. Conclusion Intraperitoneal 211 At-MX35 F(ab') 2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective dose potentially corresponds to a risk of treatment-induced carcinogenesis, optimization may still be valuable.

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.

The purpose of the present study was to investigate the therapeutic efficacy of 211At-labelled specific monoclonal antibody MOv18 in nude mice with intraperitoneal growth of the human ovarian cancer cell line OVCAR3. In the first part of the study the antibody was injected intraperitoneally when the cancer growth was microscopic. The injected activity was 485-555 kBq. The median survival for treated mice was 213 days compared to 138 days for untreated mice (p < 0.014, log-rank test). No obvious toxicity was seen. Thirty-three percent of the mice were apparently free of cancer after 7 months and were probably cured. In the second part of the study mice with macroscopic cancer and signs of ascites were injected intraperitoneally with the same 211At-labelled antibody (377-389 kBq). This treatment possibly delayed the production of ascites. Hopefully radioimmunotherapy with regionally administered 211At-labelled antibody will be of value in women with ovarian cancer as well.

BACKGROUND: Evaluation of jaw bone quality at implant placement is mainly based on preoperative radiographic assessments and subjective hand registrations during implant site preparation. An objective technique with cutting torque measurements has been introduced, presenting an objective bone quality or bone hardness value of individual implant sites. PURPOSE: The purpose of this study was to evaluate cutting torque measurements during implant placement and to compare these values in different regions in mandibles and maxillae. The objective was to identify implants at risk for failing at implant placement. MATERIAL AND METHODS: Cutting torque measurements were performed during placement of Mk II self-tapping implants (Branemark System) in 105 patients, comprising 72 edentulous (40 maxillae) and 34 partially edentulous (22 maxillae) jaws. A total of 523 implants were inserted, of which 420 were of the Mk II design and of which 412 were subjected to cutting torque measurements. Statistical analyses were performed by comparing cutting torque values of maxillae and mandibles and of different jaw regions. Cutting torque values were also correlated with radiographically and clinically assessed bone quality scores. Patients were followed clinically for a minimum of 3 years. RESULTS: A statistically significant difference in cutting torque values of maxillae and mandibles was seen, although not when comparing anterior and posterior regions within the same jaws or of different jaws. Significant correlations were found between values of cutting torque and bone quality. The majority of failures were seen in bone of medium to high density, whereas implants inserted in bone of poor density presented a better outcome, perhaps due to an adapted surgical protocol and an extended healing period. The overall implant survival rate at 3 years was 95%, and when analyzing different jaw categories, survival rates of 92.0% and 99.4% were seen for edentulous maxillae and mandibles, respectively. The corresponding figures for partially edentulous jaws were 95.4% and 97.6%. CONCLUSION: It was not possible to identify sites at risk for future implant losses or to determine a lower limit value of cutting torque in order to achieve successful implant integration.

Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ((211)At), concentrated in the thyroid by the same mechanism as (131)I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ((60)Co) and alpha particles from (211)At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to (211)At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1Gy (211)At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative effectiveness of alpha particles.

Objective: The aim of this study was to synthesise and evaluate polylysine-based effectors for pretargeted radioimmunotherapy and imaging. These molecules can readily be size-modified and charge-modified to decrease the renal uptake of radioactivity, which is often a major problem for small radiolabeled molecules. Several chelators and biotin molecules (for antibody-streptavidin-binding in vivo) are also easily incorporated into one structure because of the polylysine.Methods: The effectors were synthesised using poly-L-lysine, NHS-LC-biotin, CHX-A’’-DTPA or p-SCN-Bn-DOTA and succinic anhydride. They were characterised, labelled with 213Bi for targeted α therapy, 68Ga for PET and 111In for SPECT, and evaluated in vitro. A kidney uptake study was performed as well with two different-sized 213Bi-labeled effectors, to evaluate how the difference in size affects the renal filtration.Results: Radiochemical purities between 97.4±0.6 % and 99.6±0.1 % and decay-corrected yields of 80.2±2.4 % after purification were achieved with the radiolabeled molecules, as well as a specific activity of 7.6 × 103GBq/µmol. The avidin binding capacity was 94.4±1.9%. The kidney uptake study demonstrated a reduction of renal absorbed dose by 80% when modifying the molecular size and charge.Conclusion: The synthesised polylysine-based effectors show potential for further in vivo evaluation in pretargeted radioimmunotherapy and imaging.

Purpose: Pretargeted radioimmunotherapy (PRIT) against intraperitoneal (i.p.) ovarian microtumors using avidin-conjugated monoclonal antibody MX35 (avidin-MX35) and (211)At-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PLsuc) was compared with conventional radioimmunotherapy (RIT) using (211)At-labeled MX35 in a nude mouse model.Mice were inoculated i.p. with 1×10(7) NIH:OVCAR-3 cells. After 3 weeks, they received PRIT (1.0 or 1.5 MBq), RIT (0.9 MBq), or no treatment. Concurrently, 10 additional animals were sacrificed and examined to determine disease progression at the start of therapy. Treated animals were analyzed with regard to presence of tumors and ascites (tumor-free fraction; TFF), 8 weeks after therapy.Tumor status at baseline was advanced: 70% of sacrificed animals exhibited ascites. The TFFs were 0.35 (PRIT 1.0 MBq), 0.45 (PRIT 1.5 MBq), and 0.45 (RIT). The 1.5-MBq PRIT group exhibited lower incidence of ascites and fewer tumors1 mm than RIT-treated animals.PRIT was as effective as RIT with regard to TFF; however, the size distribution of tumors and presence of ascites indicated that 1.5-MBq PRIT was more efficient. Despite advanced disease in many animals at the time of treatment, PRIT demonstrated good potential to treat disseminated ovarian cancer.

The introduction of the short-lived α-emitter (211)At to intraperitoneal radioimmunotherapy has raised the issue of the tolerance dose of the peritoneum. The short range of the α-particles (70 μm) and the short half-life (7.21 h) of the nuclide yield a dose distribution in which the peritoneum is highly irradiated compared with other normal tissues. To address this issue, mice were injected with (211)At-trastuzumab to irradiate the peritoneum to absorbed doses ranging between 0 and 50 Gy and followed for up to 34 weeks. The peritoneum-to-plasma clearance of a small tracer, (51)Cr-ethylenediamine tetraacetic acid, was measured for evaluation of the small solute transport capacity of the peritoneal membrane. The macroscopic status of the peritoneum and the mesenteric windows was documented when the mice were sacrificed. Biopsies of the peritoneum were taken for morphology and immunohistochemical staining against plasminogen activator inhibitor-1 and calprotectin. Peritoneum-to-plasma clearance measurements indicated a dose-dependent decrease in peritoneal transport capacity in irradiated mice. However, macroscopic and microscopic evaluations of the peritoneal membrane showed no difference between irradiated mice versus controls. The results imply that the peritoneal membrane tolerates absorbed doses as high as 30-50 Gy from α-particle irradiation with limited response.

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.(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).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.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.

Pretargeted radioimmunotherapy (PRIT) using an anti-CD45 antibody (Ab)–streptavidin (SA) conjugate and DOTA-biotin labeled with β-emitting radionuclides has been explored as a strategy to decrease relapse and toxicity. α-emitting radionuclides exhibit high cytotoxicity coupled with a short path length, potentially increasing the therapeutic index and making them an attractive alternative to β-emitting radionuclides for patients with acute myeloid leukemia. Accordingly, we have used 213Bi in mice with human leukemia xenografts. Results demonstrated excellent localization of 213Bi-DOTA-biotin to tumors with minimal uptake into normal organs. After 10 minutes, 4.5% ± 1.1% of the injected dose of 213Bi was delivered per gram of tumor. α-imaging demonstrated uniform radionuclide distribution within tumor tissue 45 minutes after 213Bi-DOTA-biotin injection. Radiation absorbed doses were similar to those observed using a β-emitting radionuclide (90Y) in the same model. We conducted therapy experiments in a xenograft model using a single-dose of 213Bi-DOTA-biotin given 24 hours after anti-CD45 Ab-SA conjugate. Among mice treated with anti-CD45 Ab-SA conjugate followed by 800 μCi of 213Bi- or 90Y-DOTA-biotin, 80% and 20%, respectively, survived leukemia-free for more than 100 days with minimal toxicity. These data suggest that anti-CD45 PRIT using an α-emitting radionuclide may be highly effective and minimally toxic for treatment of acute myeloid leukemia.

The therapeutic effects of 125 I-labelled (18–97 MBq) monoclonal antibodies (MAb) C-242, C-215 and S-S.1 were studied in nude mice with human colorectal adenocarcinoma tumours. The antibodies were administered 2 or 10–16 days after implantation of the tumour cells. The monoclonal antibody C-242 was internalized into the tumour cells, C-215 was internalized to a lower degree while S-S.1 (unspecific MAb) was not internalized at all. No enhanced therapeutic effect of 125 I-C-242 was observed, as a result of Auger electrons, compared with 125 I-C-215 and 125 I-S-S.1.

Besides bone marrow, the kidneys are often dose-limiting organs in internal radiotherapy. The effects of high-linear energy transfer (LET) radiation on the kidneys after alpha-radioimmunotherapy (alpha-RIT) with the alpha-particle emitter, (211)At, were studied in nude mice by serial measurements of the glomerular filtration rate (GFR). The renal toxicity was evaluated at levels close to the dose limit for the bone marrow and well within the range for therapeutic efficacy on tumors. Astatinated MX35-F(ab')(2) monoclonal antibodies were administered intravenously to nude mice. Both non-tumor-bearing animals and animals bearing subcutaneous xenografts of the human ovarian cancer cell line, OVCAR-3, were used. The animals received approximately 0.4, 0.8, or 1.2 MBq in one, two, or three fractions. The mean absorbed doses to the kidneys ranged from 1.5 to 15 Gy. The renal function was studied by serial GFR measurements, using plasma clearance of (51)Cr-EDTA, up to 67 weeks after the first astatine injection. A dose-dependent effect on GFR was found and at the time interval 8-30 weeks after the first administration of astatine, the absorbed doses causing a 50% decrease in GFR were 16.4 +/- 3.3 and 14.0 +/- 4.1 Gy (mean +/- SEM), tumor- and non-tumor-bearing animals, respectively. The reduction in GFR progressed with time, and at the later time interval, (31-67 weeks) the corresponding absorbed doses were 7.5 +/- 2.4 and 11.3 +/- 2.3 Gy, respectively, suggesting that the effects of radiation on the kidneys were manifested late. Examination of the kidney sections showed histologic changes that were overall subdued. Following alpha-RIT with (211)At-MX35-F(ab')(2) at levels close to the dose limit of severe myelotoxicity, the effects found on renal function were relatively small, with only minor to moderate reductions in GFR. These results suggest that a mean absorbed dose to the kidneys of approximately 10 Gy is acceptable, and that the kidneys would not be the primary dose-limiting organ in systemic alpha-RIT when using (211)At-MX35-F(ab')(2).

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.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.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.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.

PURPOSE: To investigate the potential use of astatine-211 (211At)-labeled trastuzumab for the treatment of HER-2-positive, radioresistant ovarian carcinoma. METHODS AND MATERIALS: Four-week-old nude mice were inoculated intraperitoneally with 5 . 10(6) SKOV-3 cells in 0.4 mL saline on Day 0. The endpoint was the total tumor weight in each mouse on Day 63. Three experiments were performed in which the response to single-dose and fractionated treatment with unlabeled and 211At-labeled antibody was evaluated. RESULTS: Experiment 1 showed, for the same total amount of trastuzumab, a dose-response relationship between 211At activity (0-400 kBq on Day 7) and therapeutic efficacy (p = 0.001). The effect of varying the amount of unlabeled trastuzumab was studied in Experiment 2. All mice, except for the controls, received 400 kBq 211At-trastuzumab, and different groups received 5, 50, or 500 microg trastuzumab on Day 7. The increase from 5 to 50 microg trastuzumab reduced the tumors by 78% in weight. No tumors were present in mice given 500 microg trastuzumab. In Experiment 3, the effect of a fractionated treatment regimen was studied. Mice that received 100 kBq 211At-trastuzumab on Days 7 and 8 had a 42% smaller tumor burden than did controls. Groups of mice injected with 200 + 100 kBq on Days 7 and 21 and mice injected with 100 kBq on Days 7, 8, and 21 both had 24% less tumor weight than the corresponding controls. CONCLUSION: The combination of 500 microg trastuzumab and 400 kBq 211At-trastuzumab had the greatest effect, with complete eradication of the tumors in this nude mouse model.

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.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.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.Treatment with 211At-MX35 F(ab')2 or 211At-Rituximab F(ab')2 resulted in a TFF of 95%-100% when the tumor radius wasor =30 microm. The TFF was decreased (TFFor = 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.

PURPOSE: To elucidate the therapeutic efficacy of alpha-radioimmunotherapy of ovarian cancer in mice. This study: (i) estimated the minimum required activity (MRA), giving a reasonable high therapeutic efficacy; and (ii) calculated the specific energy to tumor cell nuclei and the metastatic cure probability (MCP) using various assumptions regarding monoclonal-antibody (mAb) distribution in measured tumors. The study was performed using the alpha-particle emitter Astatine-211 (211At) labeled to the mAb MX35 F(ab')2. METHODS AND MATERIALS: Animals were inoculated intraperitoneally with approximately 1 x 10(7) cells of the cell line NIH:OVCAR-3. Four weeks later animals were treated with 25, 50, 100, or 200 kBq 211At-MX35 F(ab')2 (n = 74). Another group of animals was treated with a nonspecific mAb: 100 kBq 211At-Rituximab F(ab')2 (n = 18). Eight weeks after treatment the animals were sacrificed and presence of macro- and microscopic tumors and ascites was determined. An MCP model was developed and compared with the experimentally determined tumor-free fraction (TFF). RESULTS: When treatment was given 4 weeks after cell inoculation, the TFFs were 25%, 22%, 50%, and 61% after treatment with 25, 50, 100, or 200 kBq (211)At-MX35 F(ab')2, respectively, the specific energy to irradiated cell nuclei varying between approximately 2 and approximately 400 Gy. CONCLUSION: As a significant increase in the therapeutic efficacy was observed between the activity levels of 50 and 100 kBq (TFF increase from 22% to 50%), the conclusion was that the MRA is approximately 100 kBq (211)At-MX35 F(ab')2. MCP was most consistent with the TFF when assuming a diffusion depth of 30 mum of the mAbs in the tumors.

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.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.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.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.