Jostein Dahle

Experimental α-particle radioimmunotherapy of breast cancer using 227Th-labeled p-benzyl-DOTA-trastuzumab

BackgroundThe aim of the present study was to explore the biodistribution, normal tissue toxicity, and therapeutic efficacy of the internalizing low-dose rate alpha-particle-emitting radioimmunoconjugate 227Th-trastuzumab in mice with HER2-expressing breast cancer xenografts.MethodsBiodistribution of 227Th-trastuzumab and 227Th-rituximab in nude mice bearing SKBR-3 xenografts were determined at different time points after injection. Tumor growth was measured after administration of 227Th-trastuzumab, 227Th-rituximab, cold trastuzumab, and saline. The toxicity of 227Th-trastuzumab was evaluated by measurements of body weight, blood cell, and clinical chemistry parameters, as well as histological examination of tissue specimens.ResultsThe tumor uptake reached peak levels of 34% ID/g (4.6 kBq/g) 3 days after injection of 400 kBq/kg of 227Th-trastuzumab. The absorbed radiation dose to tumor was 2.9 Gy, while it was 2.4 Gy to femur due to uptake of the daughter nuclide 223Ra in bone; the latter already explored in clinical phases I and II trials without serious toxicity. A significant dose-dependent antitumor effect was observed for dosages of 200, 400, and 600 kBq/kg of 227Th-trastuzumab but no effect of 400 and 600 kBq/kg 227Th-rituximab (non-tumor binding). No serious delayed bone marrow or normal organ toxicity was observed, but there was a statistical significant reduction in blood cell parameters for the highest-dose group of 227Th-trastuzumab treatment.ConclusionInternalizing 227Th-trastuzumab therapy was well tolerated and resulted in a dose-dependent inhibition of breast cancer xenograft growth. These results warrant further preclinical studies aiming at a clinical trial in breast cancer patients with metastases to bone.

Fractionated therapy of HER2-expressing breast and ovarian cancer xenografts in mice with targeted alpha emitting 227Th-DOTA-p-benzyl-trastuzumab

Background The aim of this study was to investigate therapeutic efficacy and normal tissue toxicity of single dosage and fractionated targeted alpha therapy (TAT) in mice with HER2-expressing breast and ovarian cancer xenografts using the low dose rate radioimmunoconjugate 227Th-DOTA-p-benzyl-trastuzumab. Methodology/Principal Findings Nude mice carrying HER2-overexpressing subcutaneous SKOV-3 or SKBR-3 xenografts were treated with 1000 kBq/kg 227Th-trastuzumab as single injection or four injections of 250 kBq/kg with intervals of 4–5 days, 2 weeks, or 4 weeks. Control animals were treated with normal saline or unlabeled trastuzumab. In SKOV-3 xenografts tumor growth to 10-fold size was delayed (p<0.01) and survival with tumor diameter less than 16 mm was prolonged (p<0.05) in all TAT groups compared to the control groups. No statistically significant differences were seen among the treated groups. In SKBR-3 xenografts tumor growth to 10-fold size was delayed in the single injection and 4–5 days interval groups (p<0.001) and all except the 4 weeks interval TAT group showed improved survival to the control groups (p<0.05). Toxicity was assessed by blood cell counts, clinical chemistry measurements and body weight. Transient reduction in white blood cells was seen for the single injection and 4–5 days interval groups (p<0.05). No significant changes were seen in red blood cells, platelets or clinical chemistry parameters. Survival without life threatening loss of body weight was significantly prolonged in 4 weeks interval group compared to single injection group (p<0.05) for SKOV-3 animals and in 2 weeks interval group compared with the 4–5 days interval groups (p<0.05) for SKBR-3 animals. Conclusions/Significance The same concentration of radioactivity split into several fractions may improve toxicity of 227Th-radioimmunotherapy while the therapeutic effect is maintained. Thus, it might be possible to increase the cumulative absorbed radiation dose to tumor with acceptable toxicity by fractionation of the dosage.

Biodistribution and Dosimetry of 177Lu-tetulomab, a New Radioimmunoconjugate for Treatment of Non-Hodgkin Lymphoma

The biodistribution of the anti-CD37 radioimmunoconjugate 177Lu-tetraxetan-tetulomab (177Lu-DOTA-HH1) was evaluated. Biodistribution of 177Lu-tetraxetan-tetulomab was compared with 177Lu-tetraxetan-rituximab and free 177Lu in nude mice implanted with Daudi lymphoma xenografts. The data showed that 177Lu-tetulomab had a relevant stability and tumor targeting properties in the human lymphoma model. The half-life of 177Lu allowed significant tumor to normal tissue ratios to be obtained indicating that 177Lu-tetraxetan-tetulomab could be suitable for clinical testing. The biological and effective half-life in blood was higher for 177Lu-tetraxetan-tetulomab than for 177Lu-tetraxetan-rituximab. The biodistribution of 177Lu-tetraxetan-tetulomab did not change significantly when the protein dose was varied from 0.01 to 1 mg/kg. Dosimetry calculations showed that the absorbed radiation doses to normal tissues and tumor in mice were not significantly different for 177Lu-tetraxetan-tetuloma b and 177Lu-tetraxetan-rituximab. The absorbed radiation doses were extrapolated to human absorbed radiation doses. These extrapolated absorbed radiation doses to normal tissues for 177Lu-tetraxetan-tetulomab at an injection of 40 MBq/kg were significantly lower than the absorbed radiation doses for 15 MBq/kg Zevalin, suggesting that higher tumor radiation dose can be reached with 177Lu-tetraxetan-tetulomab in the clinic.

Relative Biologic Effects of Low-Dose-Rate α-Emitting 227Th-Rituximab and β-Emitting 90Y-Tiuexetan-Ibritumomab Versus External Beam X-Radiation

PURPOSE To determine the relative biologic effects (RBE) of alpha-particle radiation from 227Th-rituximab and of beta-radiation from 90Y-tiuexetan-ibritumomab (Zevalin) compared with external beam X-radiation in the Raji lymphoma xenograft model. METHODS AND MATERIALS Radioimmunoconjugates were administered intravenously in nude mice with Raji lymphoma xenografts at different levels of activity. Absorbed dose to tumor was estimated by separate biodistribution experiments for 227Th-rituximab and Zevalin. Tumor growth was measured two to three times per week after injection or X-radiation. Treatment-induced increase in growth delay to reach tumor volumes of 500 and 1,000 mm3, respectively, was used as an end point. RESULTS The absorbed radiation dose-rate in tumor was slightly more than 0.1 Gy/d for the first week following injection of 227Th-rituximab, and thereafter gradually decreased to 0.03 Gy/d at 21 days after injection. For treatment with Zevalin the maximum dose-rate in tumor was achieved already 6 h after injection (0.2 Gy/d), and thereafter decreased to 0.01 Gy/d after 7 days. The relative biologic effect was between 2.5 and 7.2 for 227Th-rituximab and between 1 and 1.3 for Zevalin. CONCLUSIONS Both at low doses and low-dose-rates, the 227Th-rituximab treatment was more effective per absorbed radiation dose unit than the two other treatments. The considerable effect at low doses suggests that the best way to administer low-dose-rates, alpha-emitting radioimmunoconjugates is via multiple injections.

Overexpression of human OGG1 in mammalian cells decreases ultraviolet A induced mutagenesis

7,8-dihydro-8-oxo-guanine (8-oxoG) is a mutagenic DNA lesion that is induced by ultraviolet A (UVA) radiation. 8-oxoG results in increased frequency of GC-->TA transversion mutations. UVA-induced mutant frequency was measured in the guanine phosphoribosyl transferase (gpt) gene of Chinese hamster ovary cells (AS52) that were stably transfected to overexpress the hOGG1 protein, the human DNA repair glycosylase for 8-oxoG. This mutant frequency was compared with ultraviolet A-induced mutant frequency in AS52 cells stably transfected with the same vector without the hOGG1 gene. The mutant frequency was significantly decreased in the hOGG1 overexpressing cells irradiated with 300 and 400 kJ/m2 ultraviolet A radiation, corresponding to 25% and 10% cell survival, respectively. The hOGG1 overexpressing cells repaired oxidative DNA lesions three times faster than the vector only cells as measured by a semi-automated alkaline elution assay using FPG enzyme, the bacterial OGG1 analogue, to cut DNA at oxidative base modifications. Thus, the lower mutation frequency in UVA-induced mutations of the hOGG1 overexpressing cells may be related to the increased repair of 8-oxoG. No GC-->TA mutations were detected in the UVA-irradiated hOGG1 overexpressing cells. The results suggest a link between the 8-oxoG lesion and UVA-induced mutagenesis. We propose that hOGG1 has a role in maintaining genomic stability in mammalian cells after oxidative stress.

Genome-wide microarray analysis of human fibroblasts in response to γ radiation and the radiation-induced bystander effect

Radiation-induced bystander effects have been studied extensively due to their potential implications for cancer therapy and radiation protection; however, a complete understanding of the molecular mechanisms remains to be elucidated. In this study, we monitored transcriptional responses to γ radiation in irradiated and bystander fibroblasts simultaneously employing a genome-wide microarray approach to determine factors that may be modulated in the generation or propagation of the bystander effect. For the microarray data we employed analysis at both the single-gene and gene-set level to place the findings in a biological context. Unirradiated bystander fibroblasts that were recipients of growth medium harvested from irradiated cultures 2 h after exposure to 2 Gy displayed transient enrichment in gene sets belonging to ribosome, oxidative phosphorylation and neurodegenerative disease pathways associated with mitochondrial dysfunctions. The response to direct irradiation was characterized by induction of signaling and apoptosis genes and the gradual formation of a cellular immune response. A set of 14 genes, many of which were regulated by p53, were found to be induced early after irradiation (prior to medium transfer) and may be important in the generation or propagation of the bystander effect.

Treatment of HER2-Expressing Breast Cancer and Ovarian Cancer Cells With Alpha Particle-Emitting 227Th-Trastuzumab

PURPOSE To evaluate the cytotoxic effects of low-dose-rate alpha particle-emitting radioimmunoconjugate (227)Th-p-isothiocyanato-benzyl-DOTA-trastuzumab ((227)Th-trastuzumab [where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]) internalized by breast and ovarian cancer cell lines in order to assess the potential of (227)Th-trastuzumab as a therapeutic agent against metastatic cancers that overexpress the HER2 oncogene. METHODS AND MATERIALS Clonogenic survival and cell growth rates of breast cancer cells treated with (227)Th-trastuzumab were compared with rates of cells treated with nonbinding (227)Th-rituximab, cold trastuzumab, and X-radiation. Cell growth experiments were also performed with ovarian cancer cells. Cell-associated radioactivity was measured at several time points, and the mean radiation dose to cells was calculated. RESULTS SKBR-3 cells got 50% of the mean absorbed radiation dose from internalized activity and 50% from cell surface-bound activity, while BT-474 and SKOV-3 cells got 75% radiation dose from internalized activity and 25% from cell surface-bound activity. Incubation of breast cancer cells with 2.5 kBq/ml (227)Th-trastuzumab for 1 h at 4°C, followed by washing, resulted in mean absorbed radiation doses of 2 to 2.5 Gy. A dose-dependent inhibition of cell growth and an increase in apoptosis were induced in all cell lines. CONCLUSIONS Clinically relevant activity concentrations of (227)Th-trastuzumab induced a specific cytotoxic effect in three HER2-expressing cell lines. The cytotoxic effect of (227)Th-trastuzumab was higher than that of single-dose X-radiation (relative biological effectiveness = 1.2). These results warrant further studies of treatment of breast cancer and ovarian cancer with (227)Th-trastuzumab.

Targeted Alpha Therapy with 227Th-trastuzumab of Intraperitoneal Ovarian Cancer in Nude Mice

UNLABELLED The aim of the current study was to investigate the therapeutic effect of 227Th-radioimmunotherapy on intraperitoneally growing human bioluminescent HER2 positive ovarian cancer cells. METHODS In vitro toxicity of 227Th-trastuzumab in bioluminescent SKOV3-luc-D3 ovarian cancer cells was assessed in a growth assay. The biodistribution of intraperitoneally administrated 227Th-trastuzumab in athymic nude mice without tumor cells was determined. For in vivo therapy, seventy female athymic nude mice were intraperitoneally inoculated with tumor cells 17 days prior to injection of single 227Th-trastuzumab doses of 1000 kBq/kg, 600 kBq/kg or 400 kBq/kg, or three injections with 400 kBq/kg 227Th-trastuzumab separated by 4 weeks. Two control groups were given either 20 µg unlabeled trastuzumab or 0.9% NaCl. In vivo bioluminescence imaging was performed weekly before and after onset of therapy. Tumor growth, survival and toxicity were compared. RESULTS There was a statistically significant therapeutic effect of the 227Th-trastuzumab treatment both with respect to survival and tumor growth. The maximum tolerated dosage was 600 kBq/kg 227Th-trastuzumab. In the in vitro study, two hours incubation with 20 kBq/ml of 227Th-trastuzumab, followed by washing, and subsequent culture of the cells resulted in an average absorbed radiation dose of 6 Gy after 11 days and complete growth inhibition. CONCLUSION Targeted alpha therapy with 227Th-trastuzumab of human SKOV3-luc-D3 cells growing intraperitoneally in nude mice was clearly superior to unlabeled trastuzumab therapy. The results warrant further studies of 227Th-radioimmunotherapy used as adjuvant treatment and for metastatic cancer.

Preclinical evaluation of 227Th-labeled and 177Lu-labeled trastuzumab in mice with HER-2-positive ovarian cancer xenografts.

ObjectiveThe aim of the present study was to compare the biodistribution, normal tissue toxicity, and therapeutic effect of two low-dose rate radioimmunoconjugates (RICs) in mice with HER2-expressing ovarian cancer xenografts: the &agr;-particle-emitting 227Th-trastuzumab and the &bgr;-particle-emitting 177Lu-trastuzumab. Materials and methodsTrastuzumab (Herceptin), conjugated to DOTA and radiolabeled with 227Th or 177Lu, was injected intravenously into mice bearing SKOV-3 xenografts. The biodistribution was determined at different time points after injection. The organs were collected and measured for radioactivity content using a gamma spectrometer. Inhibition of tumor growth was measured after a single injection of 227Th-trastuzumab, 227Th-rituximab, 177Lu-trastuzumab, trastuzumab alone, and NaCl. The toxicity of 227Th-trastuzumab and 177Lu-trastuzumab was evaluated by measurement of body weight, determination of blood cell counts, analysis of clinical chemistry parameters, and histological examination of tissue specimens. ResultsThe absorbed radiation dose to the tumor was 4 Gy after administration of 400 kBq/kg 227Th-trastuzumab and 72 MBq/kg 177Lu-trastuzumab. A significantly better antitumor effect of 227Th-trastuzumab (8 and 30 days’ growth delay for 400 and 600 kBq/kg, respectively) was observed as compared with untreated control, trastuzumab alone, 600 kBq/kg 227Th-rituximab (nonspecific targeting), and 72 MBq/kg 177Lu-trastuzumab. Mean survival of mice after treatment with 227Th-trastuzumab (107±9 and 129±12 days for 400 and 600 kBq/kg 227Th-trastuzumab, respectively) was significantly improved compared with control (88±11 days) and other RICs (85±8 and 66±6 days for 72 MBq/kg 177Lu-trastuzumab and 600 kBq/kg 227Th-rituximab, respectively) (P<0.05, Kaplan–Meier). Treatment-related toxicity was not observed in any group except for a transient decrease in white blood cells between 3 and 9 weeks after treatment with 400 and 600 kBq/kg 227Th-trastuzumab. ConclusionThe &agr;-particle-emitting RIC 227Th-trastuzumab effectively delayed tumor growth and prolonged survival of mice compared with &bgr;-emitting 177Lu-trastuzumab administered at the same absorbed radiation dose to tumor. This new therapeutic approach warrants further studies aiming at clinical testing in patients with micrometastatic ovarian cancer.

Toxicity and Relative Biological Effectiveness of Alpha Emitting Radioimmunoconjugates

Radioimmunotherapy based on α-particle emitters has excellent properties as a treatment against micrometastatic and disseminated cancers because of the short path length (50 - 80 μm) and high linear energy transfer (∼ 100 keV/ μm). Alpha-particles produce clustered DNA double-strand breaks and highly reactive hydroxyl radicals when hitting biological tissue. Hence, targeted α-particle therapy offers the potential of selective tumor cell killing with low damage to surrounding normal tissue. The ideal applications for targeted α-therapy are in treating neoplastic cells in circulation or when cancer cells are present as free-floating cells or spread along compartment walls. This review will provide a brief overview of the most promising radionuclides for targeted α-therapy and compare their relative biological effectiveness (RBE) and normal tissue toxicity.