α-particle therapy for synovial sarcoma in the mouse using an astatine-211-labeled antibody against frizzled homolog 10.

Synovial sarcoma (SS) is a rare yet refractory soft-tissue sarcoma that predominantly affects young adults. We show in a mouse model that radioimmunotherapy (RIT) with an α-particle emitting anti-Frizzled homolog 10 (FZD10) antibody, synthesized using the α-emitter radionuclide astatine-211 (211 At-OTSA101), suppresses the growth of SS xenografts more efficiently than the corresponding ß-particle emitting anti-FZD10 antibody conjugated with the ß-emitter yettrium-90 (90 Y-OTSA101). In biodistribution analysis, 211 At was increased in the SS xenografts but decreased in other tissues up to 1 day after injection as time proceeded, albeit with a relatively higher uptake in the stomach. Single 211 At-OTSA101 doses of 25 and 50 µCi significantly suppressed SS tumor growth in vivo, whereas a 50-µCi dose of 90 Y-OTSA101 was needed to achieve this. Importantly, 50 µCi of 211 At-OTSA101 suppressed tumor growth immediately after injection, whereas this effect required several days in the case of 90 Y-OTSA101. Both radiolabeled antibodies at the 50-µCi dosage level significantly prolonged survival. Histopathologically, severe cellular damage accompanied by massive cell death was evident in the SS xenografts at even 1 day after the 211 At-OTSA101 injection, but these effects were relatively milder with 90 Y-OTSA101 at the same timepoint, even though the absorbed doses were comparable (3.3 and 3.0 Gy, respectively). We conclude that α-particle RIT with 211 At-OTSA101 is a potential new therapeutic option for SS.

Targeted cancer cell ablation in mice by an α-particle-emitting astatine-211-labeled antibody against major histocompatibility complex class I chain-related protein A and B.

Major histocompatibility complex class I chain-related protein A and B (MICA/B) are ligands of the immune receptor, natural-killer group 2 member D. MICA/B expression is often found in several types of cancer but is restricted in normal tissues. Here, we show that an α-particle emitting astatine-211 (211At)-labeled antibody targeting MICA/B (211At-anti MICA/B Ab) efficiently ablates cancer cells in vitro and in vivo. We generated 211At-anti MICA/B Ab, an anti-MICA/B antibody conjugated with a highly cytotoxic α-particle emitting radionuclide 211At. 211At-anti MICA/B Ab binds to human osteosarcoma SaOS2 and U2OS cells that exhibit high levels of MICA/B expression and efficiently kills those cells in vitro. Biodistribution analysis using xenograft mouse models of HCT116 p53-/- positive for MICA/B expression, showed increased 211At in the xenografts for up to 22 h after injection as time proceeded. A single dose of 211At-anti MICA/B Ab (1 MBq) showed significant reduction in the tumor growth rate of HCT116 p53-/- xenografts compared to 211At-labeled mouse IgG (1 MBq) at 21 days after injection. No body weight loss and erythrocytopenia was evident in mice that received 211At-anti MICA/B. Leukocytopenia and thrombocytopenia were observed within a week after 211At-anti MICA/B injection, but counts of red blood cells and platelets were recovered to control levels at about 3-4 weeks after injection. Taken together, these data strongly demonstrate that targeted α-particle therapy using 211At-anti-MICA/B Ab emitting highly cytotoxic α-particles is a potential new therapeutic option for several types of cancer.

Locoregional therapy with α-emitting trastuzumab against peritoneal metastasis of human epidermal growth factor receptor 2-positive gastric cancer in mice.

Peritoneal metastasis of gastric cancer (PMGC) is incurable and thus has an extremely poor prognosis. We have found, however, that locoregionally administered trastuzumab armed with astatine-211 (211 At-trastuzumab) is effective against human epidermal growth factor receptor 2 (HER2)-positive PMGC in a xenograft mouse model. We first observed that 211 At-trastuzumab can specifically bind and effectively kill NCI-N87 (N87) cells, which are HER2-positive human metastatic GC cells, both in vitro and in s.c. tumors. We established a PMGC mouse model using N87 xenografts stably expressing luciferase to test α-particle radioimmunotherapy with 211 At-trastuzumab against PMGC. Biodistribution analysis in this PMGC mouse model revealed that the i.p. administration of 211 At-trastuzumab (1 MBq) was a more efficient means of delivery of 211 At into metastatic tumors than i.v. injection; the maximum tumor uptake with i.p. administration was over 60% injected dose per gram of tissue (%ID/g) compared to approximately 18%ID/g with i.v. injection. Surprisingly, a single i.p. injection of 211 At-trastuzumab (1 MBq) was sufficient to completely eradicate intraperitoneally disseminated HER2-positive GC xenografts in two of six treated mice by inducing DNA double-strand breaks, and to drastically reduce the tumor burden in another three mice. No bodyweight loss, leukocytopenia, or significant biochemical changes in liver or kidney function were observed in the treatment group. Accordingly, locoregionally administered 211 At-trastuzumab significantly prolonged the survival time of HER2-positive PMGC mice compared with control treatments. Our results provide a proof-of-concept demonstration that locoregional therapy with 211 At-trastuzumab may offer a new treatment option for HER2-positive PMGC.

Utility of 211At-Trastuzumab for the Treatment of Metastatic Gastric Cancer in the Liver: Evaluation of a Preclinical α-Radioimmunotherapy Approach in a Clinically Relevant Mouse Model.

A liver metastasis from a primary gastric cancer (LMGC) is relatively common and results in an extremely poor prognosis due to a lack of effective therapeutics. We here demonstrate in a clinically relevant mouse model that an α-particle radioimmunotherapy approach with 211At-labeled trastuzumab has efficacy against LMGCs that are positive for human epidermal growth factor receptor 2 (HER2). Methods:211At was produced in a cyclotron via a 209Bi (α,2n) 211At reaction. 211At-trastuzumab was subsequently generated using a single-step labeling method. NCI-N87 cells (HER2-positive human gastric cancer cells) carrying a luciferase gene were intrasplenically transplanted into severe combined immunodeficiency mice to generate an HER2-positive LMGC model. A biodistribution study was then conducted through the intravenous injection of 211At-trastuzumab (1 MBq) into these LMGC xenograft mice. In parallel with this experimental therapy, phosphate-buffered saline, intact trastuzumab, or 211At-nonspecific human IgG (1 MBq) was injected into control groups. The therapeutic efficacy was evaluated by monitoring tumor changes by chemiluminescence imaging. Body weights, white blood cell counts, and serum markers of tissue damage were monitored at regular intervals. Microdosimetry using a CR-39 plastic detector was also performed. Results: The biodistribution analysis revealed an increased uptake of 211At-trastuzumab in the metastasized tumors that reached approximately 12% of the injected dose per gram of tissue (%ID/g) at 24 h. In contrast, its uptake to the surrounding liver was about 4 %ID/g. The LMGCs in the mouse model reduced dramatically at 1 wk after the single systemic injection of 211At-trastuzumab. No recurrences were observed in 6 of 8 mice treated with this single injection, and their survival time was significantly prolonged compared with the control groups, including the animals treated with 211At-nonspecific antibodies. No severe toxicities or abnormalities in terms of body weight, white blood cell number, liver function, or kidney parameters were observed in the 211At-trastuzumab group. Microdosimetric studies further revealed that 211At-trastuzumab had been delivered at an 11.5-fold higher dose to the LMGC lesions than to the normal liver. Conclusion: α-radioimmunotherapy with 211At-trastuzumab has considerable potential as an effective and safe therapeutic option for LMGC.

131I-IITM and 211At-AITM: Two Novel Small-Molecule Radiopharmaceuticals Targeting Oncoprotein Metabotropic Glutamate Receptor 1.

Targeted radionuclide therapy (TRT) targeting oncoproteins facilitates the delivery of therapeutic radionuclides to tumor tissues with high precision. Herein, we developed 2 new radiopharmaceuticals, 4-131I-iodo- and 4-211At-astato-N-[4-(6-(isopropylamino)pyridine-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide (131I-IITM and 211At-AITM), targeting the ectopic metabotropic glutamate receptor 1 (mGluR1) in melanomas for TRT studies. Methods:131I-IITM and 211At-AITM were synthesized by reacting a stannyl precursor with 131I-NaI and 211At in the presence of an oxidizing agent. The therapeutic efficacy and safety of the 2 radiopharmaceuticals were investigated using mGluR1-expressing B16F10 melanoma cells and melanoma-bearing mice. Results:131I-IITM and 211At-AITM were obtained with a radiochemical purity of greater than 99% and radiochemical yields of 42.7% ± 10.4% and 45.7% ± 6.5%, respectively, based on the total radioactivity of used radionuclides. 131I-IITM and 211At-AITM exhibited a maximum uptake of 4.66% ± 0.70 and 7.68% ± 0.71 percentage injected dose per gram (%ID/g) in the targeted melanomas, respectively, and were rapidly cleared from nontarget organs after intravenous injection. Both agents markedly inhibited melanoma growth compared with the controls (61.00% and 95.68%, respectively). In the melanoma model, considerably greater therapeutic efficacy with negligible toxicity was observed using 211At-AITM. Conclusion: The nontoxic radiopharmaceuticals 131I-IITM and 211At-AITM are useful high-precision TRT agents that can be used to target the oncoprotein mGluR1 for melanoma therapy.

Evidence of Local Concentration of α-Particles from 211At-Labeled Antibodies in Liver Metastasis Tissue.

We investigated the local concentration of α-particles from 211At-labeled trastuzumab antibodies against human epidermal growth factor receptor type 2 antigens in liver metastasis tissue of mice. Methods: Mice carrying metastatic cancer in their liver were injected with 211At-agent. After 12 h, the liver was removed and sliced, and 2 tissue samples of liver tissues without lesions and one containing metastatic lesions were mounted on the CR-39 plastic nuclear track detector. Microscope images of the tissues on the CR-39 were acquired. After irradiation for 31 h, the tissues were removed from the CR-39. A microscope image of α-particle tracks on the CR-39 was acquired after chemical etching. The positions of each tissue sample and the emitted α-particle tracks were adjusted to the same coordinates. Results: The positional distribution of α-particle tracks emitted from 211At was consistent within the tissue. The α-particle tracks were mainly allocated in the tumor region of the tissue. The absorbed dose in individual cells segmented by 10-µm intervals was obtained by the spectroscopic analysis of the linear-energy-transfer spectrum. The concentration efficiency-the track density ratio of α-particle tracks in the necrotized tissue, which was the tumor region, to the normal tissue-was found to be 6.0 ± 0.2. In the tumor region, the high-linear-energy-transfer α-particles deposited a large enough dose to cause lethal damage to the cancer cells. Conclusion: The total absorbed dose ranged from 1 to 7 Gy with a peak at around 2 Gy, which would correspond to a 2-3 times higher biologically equivalent dose because of the high relative biological effectiveness of the α-particles emitted from 211At.

Validating α-particle emission from 211At-labeled antibodies in single cells for cancer radioimmunotherapy using CR-39 plastic nuclear track detectors.

Recently, 211At has received increasing attention as a potential radionuclide for cancer radioimmunotherapy. It is a α-particle emitter, which is extremely effective against malignant cells. We demonstrate a method to verify the efficiency of 211At-labeled trastuzumab antibodies (211At-trastuzumab) against HER2 antigens, which has not been determined for radioimmunotherapy. A CR-39 plastic nuclear detector is used for measuring the position and the linear energy transfer (LET) of individual 211At α- particle tracks. The tracks and 211At-trastuzumab-binding cells were co-visualized by using the geometric information recorded on the CR-39. HER2-positive human gastric cancer cells (NCI-N87), labelled with 211At-trastuzumab, were dropped on the centre of the CR-39 plate. Microscope images of the cells and the corresponding α-tracks acquired by position matching were obtained. In addition, 3.5 cm × 3.5 cm macroscopic images of the whole plate were acquired. The distribution of number of α-particles emitted from single cells suggests that 80% of the 211At-trastuzumab-binding cells emitted α-particles. It also indicates that the α-particles may strike the cells several times along their path. The track-averaged LET of the α-particles is evaluated to be 131 keV/µm. These results will enable quantitative evaluation of delivered doses to target cells, and will be useful for the in vitro assessment of 211At-based radioimmunotherapeutic agents.

PU-H71, a novel Hsp90 inhibitor, as a potential cancer-specific sensitizer to carbon-ion beam therapy.

PU-H71, a heat shock protein 90 (Hsp90) inhibitor, has yielded therapeutic efficacy in many preclinical models and is currently in clinical trials. Carbon-ion radiotherapy (CIRT) has provided successful tumor control; however, there is still room for improvement, particularly in terms of tumor-specific radiosensitization. The Hsp90 inhibitor PU-H71 has been shown to sensitize tumor cells to X-ray radiation. A murine osteosarcoma cell line (LM8) and a normal human fibroblast cell line (AG01522) were treated with PU-H71 before X-ray, 14- or 50-keV/µm carbon-ion beam (C-ion) irradiation. Cell survival and protein expression were evaluated with colony formation and western blot, respectively. Treatment with PU-H71 alone was shown to be non-toxic to both cell lines; however, PU-H71 was shown to significantly sensitize LM8 cells to not only X-ray, but also to C-ion irradiation, while only a minimal sensitizing effect was observed in AG01522 cells. PU-H71 treatment was found to suppress the protein expression levels of Rad51 and Ku70, which are associated with the homologous recombination pathway and the non-homologous end-joining pathway of double-strand break repair. The findings reported here suggest that PU-H71 could be a promising radiosensitizer for CIRT.

The purine scaffold Hsp90 inhibitor PU-H71 sensitizes cancer cells to heavy ion radiation by inhibiting DNA repair by homologous recombination and non-homologous end joining.

BACKGROUND AND PURPOSE: PU-H71 is a purine-scaffold Hsp90 inhibitor developed to overcome limitations of conventional Hsp90 inhibitors. This study was designed to investigate the combined effect of PU-H71 and heavy ion irradiation on human tumor and normal cells. MATERIALS AND METHODS: The effects of PU-H71 were determined by monitoring cell survival by colony formation, and DNA double-strand break (DSB) repair by γ-H2AX foci and immuno-blotting DSB repair proteins. The mode of cell death was evaluated by sub-G1 DNA content (as an indicator for apoptosis), and mitotic catastrophe. RESULTS: PU-H71 enhanced heavy ion irradiation-induced cell death in three human cancer cell lines, but the drug did not radiosensitize normal human fibroblasts. In irradiated tumor cells, PU-H71 increased the persistence of γ-H2AX foci, and it reduced RAD51 foci and phosphorylated DNA-PKcs, key DSB repair proteins involved in homologous recombination (HR) and non-homologous end joining (NHEJ). In some tumor cell lines, PU-H71 altered the sub-G1 cell fraction and mitotic catastrophe following carbon ion irradiation. CONCLUSION: Our results demonstrate that PU-H71 sensitizes human cancer cells to heavy ion irradiation by inhibiting both HR and NHEJ DSB repair pathways. PU-H71 holds promise as a radiosensitizer for enhancing the efficacy of heavy ion radiotherapy.

Transcriptomic Signatures of Auger Electron Radioimmunotherapy Using Nuclear Targeting (111)In-Trastuzumab for Potential Combination Therapies.

(111)In-labeled trastuzumab modified with nuclear localizing signal (NLS) peptides ((111)In-trastuzumab-NLS) efficiently delivers an Auger electron (AE) emitter (111)In into the cell nucleus and is thus a promising radiopharmaceutical in AE radioimmunotherapy (AE-RIT) for targeted killing of HER2-positive cancer. However, further improvement of its therapeutic efficacy is required. In this study, the authors show a transcriptomic approach to identify potential targets for enhancing the cytotoxic effects of (111)In-trastuzumab-NLS. They generated two types of (111)In-trastuzumab-NLS harboring different numbers of NLS peptides, (111)In-trastuzumab-NLS-S and -L. These radioimmunoconjugates (230 and 460 kBq) showed a significant higher cytotoxicity to SKBR3 human breast cancer cells overexpressing HER2 compared to (111)In-trastuzumab. Microarray analysis revealed that NF-kB-related genes (38 genes) were significantly changed in transcription by (111)In trastuzumab-NLS-L (230 kBq) treatment. Quantitative reverse transcription polymerase chain reaction confirmed the microarray data by showing transcriptional alternation of selected NF-κB target genes in cells treated with (111)In-trastuzumab-NLS-L. Interestingly, bortezomib, a drug known as a NF-κB modulator, significantly enhanced the cytotoxicity of (111)In-trastuzumab-NLS-L in SKBR3 cells. Taken together, the transcriptome data suggest the possibility that the modulation of NF-kB signaling activity is a molecular signature of (111)In-trastuzumab-NLS and coadministration of bortezomib may be efficacious in enhancement of AE-RIT with (111)In-trastuzumab-NLS.