α-Imaging Confirmed Efficient Targeting of CD45-Positive Cells After 211At-Radioimmunotherapy for Hematopoietic Cell Transplantation.

UNLABELLED: 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 (211)At-radioimmunotherapy, extending the analysis to include intraorgan (211)At 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 (211)At-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 (211)At-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. (211)At 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: (211)At 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 (211)At 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: (211)At-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 (211)At-B10-CA12.10C12 localization was achieved for efficient conditioning for HCT.

Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status.

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.

Comparative analysis of transcriptional gene regulation indicates similar physiologic response in mouse tissues at low absorbed doses from intravenously administered 211At.

UNLABELLED: (211)At is a promising therapeutic radionuclide because of the nearly optimal biological effectiveness of emitted α-particles. Unbound (211)At accumulates in the thyroid gland and in other vital normal tissues. However, few studies have been performed that assess the (211)At-induced normal-tissue damage in vivo. Knowledge about the extent and quality of resulting responses in various organs offers a new venue for reducing risks and side effects and increasing the overall well-being of the patient during and after therapy. METHODS: Female BALB/c nude mice were injected intravenously with 0.064-42 kBq of (211)At or mock-treated, and the kidneys, liver, lungs, and spleen were excised 24 h after injection. A transcriptional gene expression analysis was performed in triplicate using RNA microarray technology. Biological processes associated with regulated transcripts were grouped into 8 main categories with 31 subcategories according to gene ontology terms for comparison of regulatory profiles. RESULTS: A substantial decrease in the total number of regulated transcripts was observed between 0.64 and 1.8 kBq of (211)At for all investigated tissues. Few genes were differentially regulated in each tissue at all absorbed doses. In all tissues, most of these genes showed a nonmonotonous dependence on absorbed dose. However, the direction of regulation generally remained uniform for a given gene. Few known radiation-associated genes were regulated on the transcriptional level, and their expression profile generally appeared to be dose-independent and tissue-specific. The regulatory profiles of categorized biological processes were tissue-specific and reflected the shift in regulatory intensity between 0.64 and 1.8 kBq of (211)At. The profiles revealed strongly regulated and nonregulated subcategories. CONCLUSION: The strong regulatory change observed between 0.64 and 1.8 kBq is hypothesized to result not only from low-dose effects in each tissue but also from physiologic responses to ionizing radiation-induced damage to, for example, the (211)At-accumulating thyroid gland. The presented results demonstrate the complexity of responses to radionuclides in vivo and highlight the need for further research to also consider physiology in ionizing radiation-induced responses.

Differential gene expression in human fibroblasts after alpha-particle emitter (211)At compared with (60)Co irradiation.

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

Effective cancer therapy with the alpha-particle emitter [211At]astatine in a mouse model of genetically modified sodium/iodide symporter-expressing tumors.

PURPOSE: The sodium/iodide symporter (NIS) gene is currently explored in several trials to eradicate experimental cancer with radiodine ((131)I) by its beta-emission. We recently characterized NIS-specific cellular uptake of an alternative halide, radioastatine ((211)At), which emits high-energy alpha-particles. The aim of this study was to investigate in vivo effects of the high linear energy transfer (LET) emitter (211)At on tumor growth and outcome in nude mice. EXPERIMENTAL DESIGN: We administered radioastatide in a fractionated therapy scheme to NMRI nude mice harboring rapidly growing solid tumors established from a papillary thyroid carcinoma cell line genetically modified to express NIS (K1-NIS). Animals were observed over 1 year. Tumor growth, body weight, blood counts, survival, and side effects were measured compared with control groups without therapy and/or lack of NIS expression. RESULTS: Within 3 months, radioastatide caused complete primary tumor eradication in all cases of K1-NIS tumor-bearing nude mice (n = 25) with no tumor recurrence during 1 year follow-up. Survival rates of the K1-NIS/(211)At group were 96% after 6 months and 60% after 1 year, in contrast to those of control groups (maximum survival 40 days). CONCLUSION: Our study indicates that (211)At represents a promising substrate for NIS-mediated therapy of various cancers either with endogenous or gene transfer-mediated NIS expression.

Myelotoxicity and RBE of 211At-conjugated monoclonal antibodies compared with 99mTc-conjugated monoclonal antibodies and 60Co irradiation in nude mice.

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

The development of a [211AT]-astatinated endoradiotherapeutic drug: part IV--late radiation effects.

PURPOSE: 6-[211At]-astato-MNDP is a high-LET endoradiotherapeutic drug that selectively targets to an oncogenically associated alkaline phosphatase isoenzyme expressed by certain tumors. A detailed histopathological study of the late tissue effects of its endogenous alpha-particle emissions has been carried out in a murine tumor model. MATERIALS AND METHODS: Thyroid-blocked male C57BI/10 mice bearing a s.c. transplanted rectal adenocarcinoma were treated with a single i.p. injection of 10-750 kBq 6-[211At]-astato-MNDP. Cured mice (131) were studied. Detailed autopsies and histological examinations were performed on all mice. The study was concluded after 756 days. RESULTS: Lymphoma, plasmacytoma, and intercurrent infections secondary to chronic pulmonary fibrosis were the most commonly found late manifestations of alpha-radiation exposure. Low grade B-cell non-Hodgkin's lymphoma occurred in 19 (24.7%) of 77 mice, 13-17 months after receiving 3.5-185 kBq 6-[211At]-astato-MNDP. The incidence of lymphoma alone and its latency was similar to that of the control population (23.3%). Treatment doses exceeding 200 kBq 6-[211At]-astato-MNDP, were associated with the development of soft tissue plasmacytoma in 7 (13%) of 54 mice, after 17-22 months. Generalized debilitation and nonspecific infections supervening pulmonary fibrosis significantly contributed to the late morbidity and mortality observed in mice treated with 300-750 kBq 6-[211At]-astato-MNDP. Dosimetry has afforded LD50/360 and LD50/420 estimates of 12-14 and 10-12 Cobalt-Gray equivalent (CGyE), respectively, for chronic lung damage. There was no histological evidence of chronic radiation damage to other critical healthy tissues. Normal thyroid morphology was preserved. CONCLUSIONS: Dose activities of 6-[211At]-astato-MNDP exceeding 300 kBq, were associated an increased risk of tumor induction and development of varying degrees of chronic pulmonary fibrosis implicated in the onset of terminal intercurrent infections. Within the therapeutic dose range 55-300 kBq 6-[211At]-astato-MNDP, mortality associated with the incidence of significant late radiation damage in critical normal tissues and latent carcinogenesis was less than 15%. Data from this murine model suggest that clinically relevant activities of 6-[211At]-astato-MNDP may be given without unacceptable toxicity.

211At-methylene blue in targeted radiotherapy of disseminated melanoma.

Targeted radiotherapy with 211At-methylene blue (211At-MTB) is a systemic treatment selectively directed at melanoma due to a high affinity of MTB to melanin synthesized in the tumor cells. Since MTB forms a strong complex with melanin, it is an effective carrier for a number of radioisotopes to be addressed to the tumor deposits of any size including individually dispersed melanoma cells. Thus, appropriately radiolabeled MTB can be used for either diagnosis or therapy of the neoplasm. As predicted and found in animal experiments, 211At-MTB is most effective therapeutically. Histopathological investigations showed that the highly pigmented 211At-MTB-treated tumors were characterized initially by perivascular oedema and hydropic degeneration of tumor cells followed by gradual development of extensive areas of coagulative necrosis. The necrotic tumor areas contained microvessels occluded by thrombi and tended to undergo microfocal calcification. Although melanoma-bearing animals successfully treated with 211At-MTB did not reveal any adverse effects of the therapy, detailed toxicological studies were undertaken. No serious macro- or microscopic lesions were observed in normal organs of 211At-MTB treated mice. Only the relative number of small lymphocytes in the groin lymph nodes in a minority of animals was variably reduced, most often in conjunction with the treatment of highly, but not poorly, pigmented tumors.