(1/23) Preparation of alpha-emitting 213Bi-labeled antibody constructs for clinical use.

Preclinical evaluation of alpha particle-emitting 213Bi-labeled antibody constructs have demonstrated the specificity and potency of these agents in a variety of cancer systems. The transition of a 213Bi-radiolabeled antibody from a preclinical construct to a clinical drug represented a difficult task that involved development of reliable and validated methods to provide multiple MBq quantities of a pure, immunoreactive agent that met pharmaceutical standards to treat patients. METHODS: The methods used for the preparation of (213Bi)CHX-A-diethylenetriamine pentaacetic acid (DTPA)-HuM195, an alpha particle-emitting anti-CD33 antibody construct for therapy of myeloid leukemias, is used as a specific example. This article describes methods for reagent purification, drug labeling, radioprotection and chromatographic purification. Quality of the drug is evaluated using radiochemical incorporation and purity assays with instant thin-layer chromatography (ITLC) and high-performance liquid chromatography (HPLC), determination of cell-based antibody total immunereactivity, small animal safety, pyrogen level, sterility and radionuclidic purity. RESULTS: Sixty-seven doses were prepared. Individual doses ranged from 148 to 814 MBq. Specific activities ranged from 329 to 766 MBq/mg. The radiolabeling efficiency (median +/- SD) of CHX-A-DTPA-HuM195 with 213Bi was 81% +/- 9% (n = 67) after 9 min. The construct was purified by size-exclusion chromatography and was found to be 99% +/- 2% pure (n = 67) by either ITLC or HPLC methods. The immunoreactivity of (213Bi)CHX-A-DTPA-HuM195 was 89% +/- 9% (n = 44) and was independent of the specific activity. The formulated pharmaceutical was found to contain < or =4 +/- 1 EU/mL pyrogens (n = 66); all samples examined were sterile. An 225Ac radionuclidic impurity was present at a level of 0.04 +/- 0.03 x 10(-6)/mL (n = 10) in a product volume of 7.4 +/- 0.5 mL (n = 67). Each of the 67 doses was injected intravenously into patients without complication as part of a phase I clinical trial. CONCLUSION: These data show that 213Bi-labeled antibody constructs can be prepared and administered safely to humans at a wide range of therapeutic levels.  (+info)

(2/23) Tumor therapy with targeted atomic nanogenerators.

A single, high linear energy transfer alpha particle can kill a target cell. We have developed methods to target molecular-sized generators of alpha-emitting isotope cascades to the inside of cancer cells using actinium-225 coupled to internalizing monoclonal antibodies. In vitro, these constructs specifically killed leukemia, lymphoma, breast, ovarian, neuroblastoma, and prostate cancer cells at becquerel (picocurie) levels. Injection of single doses of the constructs at kilobecquerel (nanocurie) levels into mice bearing solid prostate carcinoma or disseminated human lymphoma induced tumor regression and prolonged survival, without toxicity, in a substantial fraction of animals. Nanogenerators targeting a wide variety of cancers may be possible.  (+info)

(3/23) Targeted actinium-225 in vivo generators for therapy of ovarian cancer.

Advanced ovarian cancer is largely incurable, but initially it is frequently confined to the i.p. space. We explored i.p. radioimmunotherapy in a mouse model of human ovarian cancer. Use of a targeted actinium-225 ((225)Ac) in vivo generator of alpha particles exploits the extreme, selective cytotoxicity of alpha particles, while providing a feasible half-life to enable delivery to tumor. (225)Ac chelated with 2-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid was conjugated to trastuzumab, an anti-HER-2/neu antibody. The radioimmunoconjugate was tested for immunoreactivity, internalization, and cytotoxicity using a human ovarian carcinoma cell line, SKOV3. (225)Ac-labeled trastuzumab retained immunoreactivity (50-90%), rapidly internalized into cells (50% at 2 h), and had an ED(50) of 1.3 nCi/ml after 4 days of incubation in vitro. i.p. administered (225)Ac- or (111)In-labeled trastuzumab behaved similarly with high tumor uptake [56-60% injected dose per gram (% ID/g) at 4 h, which increased to 65-70% ID/g at 24 h]. Tumor uptake was 3-5-fold higher than liver and spleen, the normal organs with the highest uptake. i.v. administration of (111)In-labeled trastuzumab produced slightly higher normal organ uptake compared with i.p.-administered (111)In-labeled trastuzumab. However, tumor uptake was low, 5%-26% ID/g. Therapy was examined with native trastuzumab and 220, 330, and 450 nCi of (225)Ac-labeled trastuzumab or (225)Ac-labeled control antibody at different dosing schedules. Therapy was initiated 9 days after tumor seeding. Groups of control mice and those administered native trastuzumab had median survivals of 33 and 37 or 44 days, respectively. Median survival was 52-126 days with (225)Ac-labeled trastuzumab at various doses and schedules, and 48-64 days for (225)Ac-labeled control the same schedules. Deaths from toxicity occurred with the highest activity levels. In conclusion, i.p. administration with a (225)Ac-labeled internalizing anti-HER-2/neu antibody can extend survival significantly in a nude mouse model of human ovarian cancer at levels that produce no apparent gross toxicity.  (+info)

(4/23) Pharmacokinetics, dosimetry, and toxicity of the targetable atomic generator, 225Ac-HuM195, in nonhuman primates.

Short-lived alpha-emitting isotopes individually conjugated to monoclonal antibodies have now reached human use, but little is still known about their toxicity. Use of antibody targetable (225)Ac nanogenerators is a new approach in the field of alpha-immunotherapy offering the advantage of a 10-d half-life (t(1/2)) and increased potency due to generation of 3 new atoms, yielding a total of 4 alpha-particles. However, the 3 alpha-emitting daughter elements generated have the potential for significant toxicity as these nuclides are no longer bound to the carrier IgG. METHODS: Cynomolgus monkeys were used to evaluate the toxicity of prototype (225)Ac nanogenerators. Monoclonal antibody HuM195 (anti-CD33) is the carrier for planned human clinical trials of (225)Ac; there are no CD33 sites in cynomolgus monkeys. In one experiment, 2 monkeys received a single intravenous dose of (225)Ac-HuM195 at 28 kBq/kg. This dose level is approximately the planned initial human dose. In another experiment, 2 animals received a dose escalation schedule of 3 increasing (225)Ac-HuM195 doses with a cumulative activity of 377 kBq/kg. The whole-blood t(1/2) of (225)Ac, ratios of (225)Ac to its ultimate alpha-emitting daughter nuclide (213)Bi, generation of monkey anti-HuM195 antibodies (MAHA), hematologic indices, serum biochemistries, and clinical parameters were measured. Monkeys were euthanized and examined histopathologically when the dose escalation reached toxicity. RESULTS: The blood t(1/2) of (225)Ac-HuM195 was 12 d, and 45% of generated (213)Bi daughters were cleared from the blood. MAHA production was not detected. Approximately 28 kBq/kg of (225)Ac caused no toxicity at 6 mo, whereas a cumulative dose of approximately 377 kBq/kg caused severe toxicity. In the cumulative dosing schedule, single doses of approximately 37 kBq/kg resulted in no toxicity at 6 wk. After approximately 130 kBq/kg were administered, no toxicity was observed for 13 wk. However, 28 wk after this second dose administration, mild anemia and increases of blood urea nitrogen and creatinine were detected. After administration of an additional 185 kBq/kg, toxicity became clinically apparent. Monkeys were euthanized 13 and 19 wk after the third dose administration (cumulative dose was 377 kBq/kg). Histopathologic evaluation revealed mainly renal tubular damage associated with interstitial fibrosis. CONCLUSION: (225)Ac nanogenerators may result in renal toxicity and anemia at high doses. The longer blood t(1/2) and the lack of target cell antigens in cynomolgus monkeys may increase toxicity compared with human application. Therefore, a dose level of at least 28 kBq/kg may be a safe starting dose in humans. Hematologic and renal function will require close surveillance during clinical trials.  (+info)

(5/23) Engineered liposomes for potential alpha-particle therapy of metastatic cancer.

Disseminated, metastatic cancer is frequently incurable. Targeted alpha-particle emitters hold great promise as therapeutic agents for disseminated disease. (225)Ac is a radionuclide generator that has a 10-d half-life and results in alpha-emitting daughter elements ((221)Fr, (217)At, (213)Bi) that lead to the emission of a total of 4 alpha-particles. The aim of this study was to develop approaches for stable and controlled targeting of (225)Ac to sites of disseminated tumor metastases. Liposomes with encapsulated (225)Ac were developed to retain the potentially toxic daughters at the tumor site. METHODS: (225)Ac was passively entrapped in liposomes. To experimentally test the retention of actinium and its daughters by the liposomes, the gamma-emissions of (213)Bi were measured in liposome fractions, which were separated from the parent liposome population and the free radionuclides, at different times. Under equilibrium conditions the decay rate of (213)Bi was used to determine the concentration of (225)Ac. Measurements of the kinetics of (213)Bi activity were performed to estimate the entrapment of (213)Bi, the last alpha-emitting daughter in the decay chain. RESULTS: Stable pegylated phosphatidylcholine-cholesterol liposomes of different sizes and charge were prepared. Multiple (more than 2) (225)Ac atoms were successfully entrapped per liposome. (225)Ac retention by zwitterionic liposomes was more than 88% over 30 d. Retention by cationic liposomes was lower. A theoretical calculation showed that for satisfactory (213)Bi retention (>50%), liposomes of relatively large sizes (>650 nm in diameter) are required. (213)Bi retention was experimentally verified to be liposome-size dependent. For large liposomes, the measured (213)Bi retention was lower than theoretically predicted (less than 10%). CONCLUSION: This work supports the hypothesis that it may be possible to develop (225)Ac-based therapies by delivering multiple (225)Ac atoms in liposomes. Improvements in the retention of (225)Ac daughters will likely be necessary to fulfill this potential. Because of the size of the liposomal structures required to contain the daughters, the approach is ideally suited for locoregional therapy (e.g., intraperitoneal, intrahepatic artery, or intrathecal).  (+info)

(6/23) Thorium and actinium polyphosphonate compounds as bone-seeking alpha particle-emitting agents.

The present study explores the use of alpha-particle-emitting, bone-seeking agents as candidates for targeted radiotherapy. Actinium and thorium 1,4,7,10 tetraazacyclododecane N,N',N'',N''' 1,4,7,10-tetra(methylene) phosphonic acid (DOTMP) and thorium-diethylene triamine N,N',N'' penta(methylene) phosphonic acid (DTMP) were prepared and their biodistribution evaluated in conventional Balb/C mice at four hours after injection. All three bone-seeking agents showed a high uptake in bone and a low uptake in soft tissues. Among the soft tissue organs, only kidney had a relatively high uptake. The femur/kidney ratios for 227Th-DTMP, 228-Ac-DOTMP and 227Th-DOTMP were 14.2, 7.6 and 6.0, respectively. A higher liver uptake of 228Ac-DOTMP was seen than for 227Th-DTMP and 227Th-DOTMP. This suggests that some demetallation of the 228Ac-DOTMP complex had occurred. The results indicate that 225Ac-DOTMP, 227Th-DOTMP and 227Th-DTMP have promising properties as potential therapeutic bone-seeking agents.  (+info)

(7/23) Alpha-particle emitting atomic generator (Actinium-225)-labeled trastuzumab (herceptin) targeting of breast cancer spheroids: efficacy versus HER2/neu expression.

PURPOSE: The humanized monoclonal antibody, trastuzumab (Herceptin), directed against HER2/neu, has been effective in the treatment of breast cancer malignancies. However, clinical activity has depended on HER2/neu expression. Radiolabeled trastuzumab has been considered previously as a potential agent for radioimmunotherapy. The objective of this study was to investigate the efficacy of trastuzumab labeled with the alpha-particle emitting atomic generator, actinium-225 ((225)Ac), against breast cancer spheroids with different HER2/neu expression levels. (225)Ac has a 10-day half-life and a decay scheme yielding four alpha-particles. EXPERIMENTAL DESIGN: The breast carcinoma cell lines MCF7, MDA-MB-361 (MDA), and BT-474 (BT) with relative HER2/neu expression (by flow cytometry) of 1:4:18 were used. Spheroids of these cell lines were incubated with different concentrations of (225)Ac-trastuzumab, and spheroid growth was measured by light microscopy over a 50-day period. RESULTS: The activity concentration required to yield a 50% reduction in spheroid volume at day 35 was 18.1, 1.9, and 0.6 kBq/ml (490, 52, 14 nCi/ml) for MCF7, MDA, and BT spheroids, respectively. MCF7 spheroids continued growing but with a 20-30 day growth delay at 18.5 kBq/ml. MDA spheroid growth was delayed by 30-40 days at 3.7 kBq/ml; at 18.5 kBq/ml, 12 of 12 spheroids disaggregated after 70, days and cells remaining from each spheroid failed to form colonies within 2 weeks of being transferred to adherent dishes. Eight of 10 BT spheroids failed to regrow at 1.85 kBq/ml. All of the BT spheroids at activity concentrations 3.7 kBq/ml failed to regrow and to form colonies. The radiosensitivity of these three lines as spheroids was evaluated as the activity concentration required to reduce the treated to untreated spheroid volume ratio to 0.37, denoted DVR(37). An external beam radiosensitivity of 2 Gy was found for spheroids of all three of the cell lines. After alpha-particle irradiation a DVR(37) of 1.5, 3.0, and 2.0 kBq/ml was determined for MCF7, MDA, and BT, respectively. CONCLUSION: These studies suggest that (225)Ac-labeled trastuzumab may be a potent therapeutic agent against metastatic breast cancer cells exhibiting intermediate to high HER2/neu expression.  (+info)

(8/23) Treatment of neuroblastoma meningeal carcinomatosis with intrathecal application of alpha-emitting atomic nanogenerators targeting disialo-ganglioside GD2.

Labeling of specific antibodies with bifunctional chelated Actinium-225 ((225)Ac; an alpha generator) allows the formation of new, highly potent and selective alpha-emitting anticancer drugs. We synthesized and evaluated a radioimmunoconjugate based on 3F8, an IgG(3) antibody that specifically binds to ganglioside GD2, which is overexpressed by many neuroectodermal tumors including neuroblastoma. The (225)Ac-1,4,7,10-tetra-azacylododecane (DOTA)-3F8 construct was evaluated for radiochemical purity and sterility, immunoreactivity, cytotoxicity in vitro, induction of apoptosis on GD2-positive cells, as well as for pharmacological biodistribution and metabolism of the (225)Ac generator and its daughters in a nude mouse xenograft model of neuroblastoma. The (225)Ac-3F8 showed an IC(50) of 3 Bq/ml (80 pCi/ml) on the neuroblastoma cell line, NMB7, in vitro. Apoptosis of these cells was not observed. Biodistribution in mice showed specific targeting of a subcutaneous tumor; there was redistribution of the (225)Ac daughter nuclides mainly from blood to kidneys and to small intestine. Toxicity was examined in cynomolgus monkeys. Monkeys injected with 1 to 3 doses of intrathecal (225)Ac-3F8 radioimmunoconjugate (80 to 150 kBq/kg total dose) did not show signs of toxicity based on blood chemistry, complete blood counts, or by clinical evaluations. Therapeutic efficacy of intrathecal (225)Ac-3F8 was studied in a nude rat xenograft model of meningeal carcinomatosis. The (225)Ac-3F8 treatment improved survival 2-fold from 16 to 34 days (P = 0.01). In conclusion, in vivo alpha generators targeted by 3F8 warrant additional study as a possible new approach to the treatment of carcinomatous meningitis.  (+info)

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