Combination of 177 Lu-lilotomab with rituximab significantly improves the therapeutic outcome in preclinical models of non-Hodgkin's lymphoma.

OBJECTIVES: To investigate the therapeutic potential of the next-generation anti-CD37 radioimmunoconjugate 177 Lu-lilotomab satetraxetan (177 Lu-lilotomab) in combination with the anti-CD20 antibody rituximab for treatment of mice with non-Hodgkin's lymphoma (NHL) xenografts. METHODS: Nude mice with subcutaneous (s.c.) Burkitt's lymphoma Daudi xenografts and SCID mice intravenously (i.v.) injected with Mantle cell lymphoma Rec-1 cells were treated with either 177 Lu-lilotomab or rituximab alone or with the combination of both treatments. Tumour volume, body weight, blood counts and clinical status were monitored. CD20 expression was measured using flow cytometry with fluorescence-labelled rituximab. RESULTS: The combination of 177 Lu-lilotomab and rituximab was synergistic for treatment of nude mice with s.c. Daudi xenografts while it was additive for treatment of SCID mice with i.v. injected Rec-1 cells. Binding of rituximab to NHL cells in-vitro was increased by pretreatment with 177 Lu-lilotomab. CONCLUSIONS: Treatment of mice with NHL xenografts with 177 Lu-lilotomab synergistically increased tumour suppression of subsequent anti-CD20 immunotherapy and improved survival. If the same effect is confirmed in a recently started clinical study, it could change the way radioimmunotherapy and CD20 immunotherapy would be used in the future.

Ra-224 labeling of calcium carbonate microparticles for internal α-therapy: Preparation, stability, and biodistribution in mice.

Internal therapy with α-emitters should be well suited for micrometastatic disease. Radium-224 emits multiple α-particles through its decay and has a convenient 3.6 days of half-life. Despite its attractive properties, the use of 224 Ra has been limited to bone-seeking applications because it cannot be stably bound to a targeting molecule. Alternative delivery systems for 224 Ra are therefore of considerable interest. In this study, calcium carbonate microparticles are proposed as carriers for 224 Ra, designed for local therapy of disseminated cancers in cavitary regions, such as peritoneal carcinomatosis. Calcium carbonate microparticles were radiolabeled by precipitation of 224 Ra on the particle surface, resulting in high labeling efficiencies for both 224 Ra and daughter 212 Pb and retention of more than 95% of these nuclides for up to 1 week in vitro. The biodistribution after intraperitoneal administration of the 224 Ra-labeled CaCO3 microparticles in immunodeficient mice revealed that the radioactivity mainly remained in the peritoneal cavity. In addition, the systemic distribution of 224 Ra was found to be strongly dependent on the amount of administered microparticles, with a reduced skeletal uptake of 224 Ra with increasing dose. The results altogether suggest that the 224 Ra-labeled CaCO3 microparticles have promising properties for use as a localized internal α-therapy of cavitary cancers.

Radon-220 diffusion from 224Ra-labeled calcium carbonate microparticles: Some implications for radiotherapeutic use.

Alpha-particle emitting radionuclides continue to be the subject of medical research because of their high energy and short range of action that facilitate effective cancer therapies. Radium-224 (224Ra) is one such candidate that has been considered for use in combating micrometastatic disease. In our prior studies, a suspension of 224Ra-labeled calcium carbonate (CaCO3) microparticles was designed as a local therapy for disseminated cancers in the peritoneal cavity. The progenies of 224Ra, of which radon-220 (220Rn) is the first, together contribute three of the four alpha particles in the decay chain. The proximity of the progenies to the delivery site at the time of decay of the 224Ra-CaCO3 microparticles can impact its therapeutic efficacy. In this study, we show that the diffusion of 220Rn was reduced in labeled CaCO3 suspensions as compared with cationic 224Ra solutions, both in air and liquid volumes. Furthermore, free-floating lead-212 (212Pb), which is generated from released 220Rn, had the potential to be re-adsorbed onto CaCO3 microparticles. Under conditions mimicking an in vivo environment, more than 70% of the 212Pb was adsorbed onto the CaCO3 at microparticle concentrations above 1 mg/mL. Further, the diffusion of 220Rn seemed to occur whether the microparticles were labeled by the surface adsorption of 224Ra or if the 224Ra was incorporated into the bulk of the microparticles. The therapeutic benefit of differently labeled 224Ra-CaCO3 microparticles after intraperitoneal administration was similar when examined in mice bearing intraperitoneal ovarian cancer xenografts. In conclusion, both the release of 220Rn and re-adsorption of 212Pb are features that have implications for the radiotherapeutic use of 224Ra-labeled CaCO3 microparticles. The release of 220Rn through diffusion may extend the effective range of alpha-particle dose deposition, and the re-adsorption of the longer lived 212Pb onto the CaCO3 microparticles may enhance the retention of this nuclide in the peritoneal cavity.

Assessment of long-term radiotoxicity after treatment with the low-dose-rate alpha-particle-emitting radioimmunoconjugate (227)Th-rituximab.

PURPOSE: The anti-CD20 antibody rituximab labelled with the alpha-particle-emitting radionuclide (227)Th is of interest as a radiotherapeutic agent for treatment of lymphoma. Complete regression of human lymphoma Raji xenografts in 60% of mice treated with 200 kBq/kg (227)Th-rituximab has been observed. To evaluate possible late side effects of (227)Th-rituximab, the long-term radiotoxicity of this potential radiopharmaceutical was investigated. METHODS: BALB/c mice were injected with saline, cold rituximab or 50, 200 or 1,000 kBq/kg (227)Th-rituximab and followed for up to 1 year. In addition, nude mice with Raji xenografts treated with various doses of (227)Th-rituximab were also included in the study. Toxicity was evaluated by measurements of mouse body weight, white blood cell (WBC) and platelet counts, serum clinical chemistry parameters and histological examination of tissues. RESULTS: Only the 1,000 kBq/kg dosage resulted in decreased body weight of the BALB/c mice. There was a significant but temporary decrease in WBC and platelet count in mice treated with 400 and 1,000 kBq/kg (227)Th-rituximab. Therefore, the no-observed-adverse-effect level (NOAEL) was 200 kBq/kg. The maximum tolerated activity was between 600 and 1,000 kBq/kg. No significant signs of toxicity were observed in histological sections in any examined tissue. There were significantly (p < 0.05), but transiently, higher concentrations of serum bile acids and aspartate aminotransferase in mice treated with either (227)Th-rituximab or non-labelled antibody when compared with control mice. The maximum tolerated dose to bone marrow was between 2.1 and 3.5 Gy. CONCLUSION: Therapeutically relevant dose levels of (227)Th-rituximab were well tolerated in mice. Bone marrow suppression, as indicated by decrease in WBC count, was the dose-limiting radiotoxicity. These toxicity data together with anti-tumour activity data in a CD20-positive xenograft mouse model indicate that therapeutic effects could be obtained with relatively safe dosage levels of the radioimmunoconjugate.

In situ Generated 212Pb-PSMA Ligand in a 224Ra-Solution for Dual Targeting of Prostate Cancer Sclerotic Stroma and PSMA-positive Cells.

BACKGROUND: New treatments combating bone and extraskeletal metastases are needed for patients with metastatic castration-resistant prostate cancer. The majority of metastases overexpress prostate-specific membrane antigen (PSMA), making it an ideal candidate for targeted radionuclide therapy. OBJECTIVE: The aim of this study was to test a novel liquid 224Ra/212Pb-generator for the rapid preparation of a dual-alpha targeting solution. Here, PSMA-targeting ligands are labelled with 212Pb in the 224Ra-solution in transient equilibrium with daughter nuclides. Thus, natural bone-seeking 224Ra targeting sclerotic bone metastases and 212Pb-chelated PSMA ligands targeting PSMA-expressing tumour cells are obtained. METHODS: Two PSMA-targeting ligands, the p-SCN-Bn-TCMC-PSMA ligand (NG001), specifically developed for chelating 212Pb, and the most clinically used DOTA-based PSMA-617 were labelled with 212Pb. Radiolabelling and targeting potential were investigated in situ, in vitro (PSMA-positive C4-2 human prostate cancer cells) and in vivo (athymic mice bearing C4-2 xenografts). RESULTS: NG001 was rapidly labelled with 212Pb (radiochemical purity >94% at concentrations of ≥15 µg/ml) using the liquid 224Ra/212Pb-generator. The high radiochemical purity and stability of [212Pb]Pb- NG001 were demonstrated over 48 hours in the presence of ascorbic acid and albumin. Similar binding abilities of the 212Pb-labelled ligands were observed in C4-2 cells. The PSMA ligands displayed comparable tumour uptake after 2 hours, but NG001 showed a 3.5-fold lower kidney uptake than PSMA- 617. Radium-224 was not chelated and, hence, showed high uptake in bones. CONCLUSION: A fast method for the labelling of PSMA ligands with 212Pb in the 224Ra/212Pb-solution was developed. Thus, further in vivo studies with dual tumour targeting by alpha-particles are warranted.

Calibration of sodium iodide detectors and reentrant ionization chambers for 212Pb activity in different geometries by HPGe activity determined samples.

Lead-212 is a promising radionuclide for cancer therapy, but no primary 212Pb activity standardization has been published. A need therefore exists for accurate estimation of injected doses of 212Pb activity in equilibrium with progeny, when it comes to preclinical and clinical trials. In this study, 212Pb activity was determined using a high purity germanium (HPGe) detector, which allowed the determination of geometry-specific calibration factors for commercially available reentrant ionization chambers (ICs) and sodium iodide (NaI) detectors.

203/212Pb Theranostic Radiopharmaceuticals for Image-guided Radionuclide Therapy for Cancer.

Receptor-targeted image-guided Radionuclide Therapy (TRT) is increasingly recognized as a promising approach to cancer treatment. In particular, the potential for clinical translation of receptor-targeted alpha-particle therapy is receiving considerable attention as an approach that can improve outcomes for cancer patients. Higher Linear-energy Transfer (LET) of alpha-particles (compared to beta particles) for this purpose results in an increased incidence of double-strand DNA breaks and improved-localized cancer-cell damage. Recent clinical studies provide compelling evidence that alpha-TRT has the potential to deliver a significantly more potent anti-cancer effect compared with beta-TRT. Generator-produced 212Pb (which decays to alpha emitters 212Bi and 212Po) is a particularly promising radionuclide for receptor-targeted alpha-particle therapy. A second attractive feature that distinguishes 212Pb alpha-TRT from other available radionuclides is the possibility to employ elementallymatched isotope 203Pb as an imaging surrogate in place of the therapeutic radionuclide. As direct non-invasive measurement of alpha-particle emissions cannot be conducted using current medical scanner technology, the imaging surrogate allows for a pharmacologically-inactive determination of the pharmacokinetics and biodistribution of TRT candidate ligands in advance of treatment. Thus, elementally-matched 203Pb labeled radiopharmaceuticals can be used to identify patients who may benefit from 212Pb alpha-TRT and apply appropriate dosimetry and treatment planning in advance of the therapy. In this review, we provide a brief history on the use of these isotopes for cancer therapy; describe the decay and chemical characteristics of 203/212Pb for their use in cancer theranostics and methodologies applied for production and purification of these isotopes for radiopharmaceutical production. In addition, a medical physics and dosimetry perspective is provided that highlights the potential of 212Pb for alpha-TRT and the expected safety for 203Pb surrogate imaging. Recent and current preclinical and clinical studies are presented. The sum of the findings herein and observations presented provide evidence that the 203Pb/212Pb theranostic pair has a promising future for use in radiopharmaceutical theranostic therapies for cancer.

Relative biologic effects of low-dose-rate alpha-emitting 227Th-rituximab and beta-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.

Therapeutic Effect of α-Emitting 224Ra-Labeled Calcium Carbonate Microparticles in Mice with Intraperitoneal Ovarian Cancer.

BACKGROUND: Ovarian cancer patients with chemotherapy-resistant residual microscopic disease in the peritoneal cavity have a considerable need for new treatment options. Alpha-emitting radionuclides injected intraperitoneally may be an attractive therapeutic option in this situation as they are highly cytotoxic, while their short range in tissues can spare surrounding radiosensitive organs in the abdomen. Herein we evaluate the therapeutic efficacy of a novel α-emitting compound specifically designed for intracavitary radiation therapy. METHODS: The α-emitter 224Ra was absorbed on calcium carbonate microparticles. Immunodeficient, athymic nude mice with human ovarian cancer cells growing intraperitoneally were treated with different activity levels of 224Ra-microparticles. Tumor growth, survival, and tolerance of the treatment were assessed. Two tumor models based on the cell lines, ES-2 and SKOV3-luc, with different growth patterns were studied. RESULTS: In both models, intraperitoneal treatment with 224Ra-microparticles gave significant antitumor effect with either considerably reduced tumor volume or a survival benefit. An advantageous discovery was that only a few kilobecquerels per mouse were needed to yield therapeutic effects. The treatment was well tolerated up to a dose of 1000 kBq/kg with no signs of acute or subacute toxicity observed. CONCLUSIONS: Intraperitoneal α-therapy with 224Ra-microparticles demonstrated a significant potential for treatment of peritoneal micrometastases in ovarian carcinoma.

High-linear energy transfer irradiation targeted to skeletal metastases by the alpha-emitter 223Ra: adjuvant or alternative to conventional modalities?

The bone-seeking, alpha-particle-emitting radiopharmaceutical Alpharadin, 223RaCl2 (half-life=11.4 days), is under clinical development as a novel treatment for skeletal metastases from breast and prostate cancer. This article summarizes the current status of preclinical and clinical research on 223RaCl2. Potential advantages of 223Ra to that of external beam irradiation and registered beta-emitting bone seekers are discussed. Published data of 223Ra dosimetry in mice and a therapeutic study in a skeletal metastases model in nude rats have indicated significant therapeutic potential of bone-seeking alpha-emitters. This article provides short-term and long-term results from the first clinical single dosage trial. We also present data from a repeated dosage study of five consecutive injections of 50 kBq/kg body weight, once every 3rd week, or two injections of 125 kBq/kg body weight, 6 weeks apart. Furthermore, interim results are described for a randomized phase 2 trial involving 64 patients with hormone-refractory prostate cancer and painful skeletal metastases who received four monthly injections of 223Ra or saline as an adjuvant to external beam radiotherapy. Lastly, we present preliminary dose estimates for 223Ra in humans. Results indicate that repeated dosing is feasible and toxicity is low, and that opportunities are available for combined treatment strategies.

Preparation of TH227-labeled radioimmunoconjugates, assessment of serum stability and antigen binding ability.

In this study, the feasibility of constructing radioimmunoconjugates by using the novel therapeutic candidate alpha-emitter, (227)Th, was evaluated. By use of the bifunctional chelator, p-SCN-benzyl-DOTA, (227)Th was conjugated to the two monoclonal antibodies, rituximab and trastuzumab. Their stability in 80% fetal bovine serum at 37 degrees C was measured. The immunoreactive fractions were determined by using CD20- and HER/2-positive cells, respectively. The overall labeling yield spanned from 6% to 17%. The radioimmunoconjugates demonstrated a relevant stability in serum and showed appropriate antigen-binding abilities.

Evaluation of the binding of radiolabeled rituximab to CD20-positive lymphoma cells: an in vitro feasibility study concerning low-dose-rate radioimmunotherapy with the alpha-emitter 227Th.

Radioimmunotherapy (RIT) with the alpha-emitter 227Th is currently under evaluation. 227Th is conjugated to the chimeric anti-CD20 monoclonal antibody rituximab, using the chelator p-isothiocyanato-benzyl-DOTA. In this study, the binding of 227Th-DOTA-p-benzyl-rituximab to three different CD-20-positive lymphoma cell lines, Raji, Rael, and Daudi, were evaluated. Equilibrium and kinetic binding experiments were used to determine binding parameters, including the association and dissociation rate constants, the equilibrium dissociation constants, and the total number of antigens for Raji, Rael, and Daudi cells. There were significant differences between the cell lines with respect to both Kd and the total number of antigens. Rael cells had more than three times as many antigens as the other two cell lines, and the functional Kd found for Rael cells was significantly higher than that found for Raji and Daudi cells. These results were confirmed using flow cytometry. Rituximab was found to be localized in patches on the cell membrane. The findings indicated that 227Th-labeled rituximab has relevant antigen-targeting properties for radioimmunotherapy.

A one-step method for determining the maximum number of bound antibodies, and the affinity and association rate constants for antibody binding.

OBJECTIVE: A reliable analysis of antibody binding may lead to more successful selection of the optimal antibodies. The most important parameters are affinity (equilibrium dissociation constant, Kd), the number of antigen sites on the cells (Bmax) and the on (ka) and off (kd) rate constants of binding. The affinity and the number of cellular binding sites are usually determined by equilibrium binding experiments and subsequent Scatchard analysis. The on and off rate constants are determined by kinetic binding experiments. However, it is necessary to perform two to three different types of experiment in order to determine these parameters. METHODS: We have developed an alternative one-step method based on a kinetic binding experiment and a mathematical description of antibody binding to antigen. The method was compared with kinetic and equilibrium binding methods. RESULTS: The results obtained using two different cell lines were in good agreement with results obtained with Scatchard analysis and kinetic binding experiments. CONCLUSION: An alternative one-step method for determination of parameters describing binding of antibodies to antigens on cells has been developed. The method gives reliable estimates of affinity and number of antigens and in addition gives information on the kinetics of binding.

Preparation of 212Pb-labeled monoclonal antibody using a novel 224Ra-based generator solution.

INTRODUCTION: Alpha-emitting radionuclides have gained considerable attention as payloads for cancer targeting molecules due to their high cytotoxicity. One attractive radionuclide for this purpose is 212Pb, which by itself is a ß-emitter, but acts as an in vivo generator for its short-lived α-emitting daughters. The standard method of preparing 212Pb-labeled antibodies requires handling and evaporation of strong acids containing high radioactivity levels by the end user. An operationally easier and more rapid process could be useful since the 10.6h half-life of 212Pb puts time constraints on the preparation protocol. In this study, an in situ procedure for antibody labeling with 212Pb, using a solution of the generator nuclide 224Ra, is proposed as an alternative protocol for preparing 212Pb-radioimmunoconjugates. METHODS: Radium-224, the generator radionuclide of 212Pb, was extracted from its parent nuclide, 228Th. Lead-212-labeling of the TCMC-chelator conjugated monoclonal antibody trastuzumab was carried out in a solution containing 224Ra in equilibrium with progeny. Subsequently, the efficiency of separating the 212Pb-radioimmunoconjugate from 224Ra and other unconjugated daughter nuclides in the solution using either centrifugal separation or a PD-10 desalting size exclusion column was evaluated and compared. RESULTS: Radiolabeling with 212Pb in 224Ra-solutions was more than 90% efficient after only 30min reaction time at TCMC-trastuzumab concentrations from 0.15mg/mL and higher. Separation of 212Pb-labeled trastuzumab from 224Ra using a PD-10 column was clearly superior to centrifugal separation. This method allowed recovery of approximately 75% of the 212Pb-antibody-conjugate in the eluate, and the remaining amount of 224Ra was only 0.9±0.8% (n=7). CONCLUSIONS: The current work demonstrates a novel method of producing 212Pb-based radioimmunoconjugates from a 224Ra-solution, which may be simpler and less time-consuming for the end user compared with the method established for use in clinical trials of 212Pb-TCMC-trastuzumab.

Initial evaluation of (227)Th-p-benzyl-DOTA-rituximab for low-dose rate alpha-particle radioimmunotherapy.

Radioimmunotherapy has proven clinically effective in patients with non-Hodgkin's lymphoma. Radioimmunotherapy trials have so far been performed with beta-emitting isotopes. In contrast to beta-emitters, the shorter range and high linear energy transfer (LET) of alpha particles allow for more efficient and selective killing of individually targeted tumor cells. However, there are several obstacles to the use of alpha-particle immunotherapy, including problems with chelation chemistry and nontarget tissue toxicity. The alpha-emitting radioimmunoconjugate (227)Th-DOTA-p-benzyl-rituximab is a new potential anti-lymphoma agent that might overcome some of these difficulties. The present study explores the immunoreactivity, in vivo stability and biodistribution, as well as the effect on in vitro cell growth, of this novel radioimmunoconjugate. To evaluate in vivo stability, uptake in balb/c mice of the alpha-particle-emitting nuclide (227)Th alone, the chelated form, (227)Th-p-nitrobenzyl-DOTA and the radioimmunoconjugate (227)Th-DOTA-p-benzyl-rituximab was compared in a range of organs at increasing time points after injection. The immunoreactive fraction of (227)Th-DOTA-p-benzyl-rituximab was 56-65%. During the 28 days after injection of radioimmunoconjugate only, very modest amounts of the (227)Th had detached from DOTA-p-benzyl-rituximab, indicating a relevant stability in vivo. The half-life of (227)Th-DOTA-p-benzyl-rituximab in blood was 7.4 days. Incubation of lymphoma cells with (227)Th-DOTA-p-benzyl-rituximab resulted in a significant antigen-dependent inhibition of cell growth. The data presented here warrant further studies of (227)Th-DOTA-p-benzyl-rituximab.

First in vivo evaluation of liposome-encapsulated 223Ra as a potential alpha-particle-emitting cancer therapeutic agent.

BACKGROUND: Liposomes carrying chemotherapeutics have had some success in cancer treatment and may also be suitable carriers for therapeutic radionuclides. This study was designed to evaluate the biodistribution and to estimate the radiation doses of the alpha emitter 223Ra loaded into pegylated liposomes in selected tissues. MATERIALS AND METHODS: 223Ra was encapsulated in pegylated liposomal doxorubicin (PLD) by ionophore-mediated loading. The biodistribution of liposomal 223Ra was compared to free cationic 223Ra in Balb/C mice. RESULTS: Liposomal 223Ra circulated in the blood with an initial half-life in excess of 24 hours, which agreed well with that reported for PLD in rodents, while the blood half-life of cationic 223Ra was considerably less than an hour. When liposomal 223Ra was catabolized, the released 223Ra was either excreted or taken up in the skeleton. This skeletal uptake increased up to 14 days after treatment, but did not reach the level seen with free 223Ra. Pre-treatment with non-radioactive PLD 4 days in advance lessened the liver uptake of liposomal 223Ra. Dose estimates showed that the spleen, followed by bone surfaces, received the highest absorbed doses. CONCLUSION: Liposomal 223Ra was relatively stable in vivo and may have potential for radionuclide therapy and combination therapy with chemotherapeutic agents.

First clinical experience with alpha-emitting radium-223 in the treatment of skeletal metastases.

PURPOSE: The main goals were to study the safety and tolerability of the alpha-emitter radium-223 (223Ra) in breast and prostate cancer patients with skeletal metastases. In addition, pain palliation was evaluated. EXPERIMENTAL DESIGN: Fifteen prostate and 10 breast cancer patients enrolled in a phase I trial received a single i.v. injection of 223Ra. Five patients were included at each of the dosages: 46, 93, 163, 213, or 250 kBq/kg and followed for 8 weeks. Palliative response was evaluated according to the pain scale of the European Organization for Research and Treatment of Cancer QLQ C30 questionnaire at baseline and at 1, 4, and 8 weeks after injection. RESULTS: Weekly blood sampling during follow-up revealed mild and reversible myelosuppression with nadir 2 to 4 weeks after the injection. Importantly, for thrombocytes only grade 1 toxicity was reported. Grade 3 neutropenia and leucopenia occurred in two and three patients, respectively. Mild, transient diarrhea was observed in 10 of the 25 patients. Nausea and vomiting was more frequently observed in the highest dosage group. Serum alkaline phosphatase decreased with nadir averages of 29.5% in females and 52.1% in males. Pain relief was reported by 52%, 60%, and 56% of the patients after 7 days, 4, and 8 weeks, respectively. 223Ra cleared rapidly from blood and was below 1% of initial level at 24 hours. Gamma camera images indicated, in accordance with pretreatment (99m)Tc-MDP scans, accumulation of 223Ra in skeletal lesions. Elimination was mainly intestinal. Median survival exceeded 20 months. CONCLUSIONS: 223Ra was well tolerated at therapeutically relevant dosages. Phase II studies have therefore been initiated.

Radiotoxicity of the alpha-emitting bone-seeker 223Ra injected intravenously into mice: histology, clinical chemistry and hematology.

BACKGROUND: The alpha-emitter 223Ra, which localizes in osteoblastic active zones, including on skeletal surfaces and in osteoblastic metastases, has recently been introduced as a potential therapeutic agent against skeletal metastases. Here, the adverse effects of high dosages in animals were investigated. MATERIALS AND METHODS: Balb/c mice received intravenously (i.v.) either 1250, 2500, or 3750 kBq/kg of dissolved 223RaCl2 and were followed in the initial toxicity phase. At the 4-week end-point, the animals were sacrificed and blood samples were collected to study the effects on clinical chemistry and hematological parameters. Selected organs were weighed and tissue samples examined by microscopy. RESULTS: Treatment with 223Ra caused a dose-related minimal to moderate depletion of osteocytes and osteoblasts in the bones. Furthermore, a dose-related minimal to marked depletion of the hematopoietic cells in the bone marrow, and a minimal to slight extramedullary hematopoiesis in the spleen and in the mandibular and mesenteric lymph nodes were observed. The LD50 for acute toxicity, defined as death within 4 weeks of receiving the substance, was not reached. CONCLUSION: This study demonstrated that high doses of the bone-seeker 223Ra did not completely inactivate the blood-producing cells. The relatively high tolerance to skeletal alpha doses was probably caused by the surviving pockets of red bone marrow cells beyond the range of alpha particles from the bone surfaces, and the recruitment of peripheral stems cells.

Significant antitumor effect from bone-seeking, alpha-particle-emitting (223)Ra demonstrated in an experimental skeletal metastases model.

The therapeutic efficacy of the alpha-particle-emitting radionuclide (223)Ra (t(1/2) = 11.4 days) in the treatment against experimental skeletal metastases in rats was addressed. Biodistribution studies, involving measurement of (223)Ra in bone marrow samples, were performed in rats after i.v. injection. To study the therapeutic effect of (223)Ra, an experimental skeletal metastases model in nude rats was used. Animals that had received 10(6) MT-1 human breast cancer cells were treated with (223)Ra doses in the range of 6-30 kBq after 7 days. The biodistribution experiment demonstrated that (223)Ra was selectively concentrated in bone as compared with soft tissues. The femur content of (223)Ra was 800 +/- 56% of injected dose per gram tissue times gram body weight (b.w.; mean +/- SD) 1 day after the injection and 413 +/- 23% of injected dose per gram tissue times gram b.w. at 14 days. The femur:kidney ratio increased from (5.9 +/- 2.0).10(2) at 1 day to (7.2 +/- 3.0).10(2) at 14 days, whereas the femur:liver ratio increased from (6.2 +/- 0.2).10(2) to (9.1 +/- 6.6).10(2). Femur:spleen ratio increased from (8.1 +/- 0.3).10(2) at 1 day to (6.4 2.2).10(3) at 14 days. The femoral bone:marrow ratio was 6.5 +/- 2.1 after day 1 and larger than 15 at day 14. All of the tumor-bearing control animals had to be sacrificed because of tumor-induced paralysis 20-30 days after injection with tumor cells, whereas the rats treated with > or =10 kBq of (223)Ra had a significantly increased symptom-free survival (P < 0.05). Also 36% (5 of 14) of rats treated with 11 kBq and 40% (2 of 5) of rats treated with 10 kBq were alive beyond the 67-day follow-up period. No signs of bone marrow toxicity or b.w. loss were observed in the groups of treated animals. The significant antitumor effect of (223)Ra at doses that are tolerated by the bone marrow is most likely linked to the intense and highly localized radiation dose from alpha-particles at the bone surfaces. The results of this study indicate that (223)Ra should be additionally studied as a potential bone marrow-sparing treatment of cancers involving the skeleton.

Evaluation of CD146 as Target for Radioimmunotherapy against Osteosarcoma.

BACKGROUND: Osteosarcoma is a rare form of cancer but with a substantial need for new active drugs. There is a particular need for targeted therapies to combat metastatic disease. One possible approach is to use an antibody drug conjugate or an antibody radionuclide conjugate to target the osteosarcoma metastases and circulating tumor cells. Herein we have evaluated a radiolabeled monoclonal antibody targeting CD146 both in vitro and in vivo. METHODS AND RESULTS: A murine monoclonal anti-CD146 IgG1 isotype antibody, named OI-3, was developed along with recombinant chimeric versions with human IgG1 or human IgG3 Fc sequences. Using flow cytometry, selective binding of OI-3 to human osteosarcoma cell lines OHS, KPDX and Saos-2 was confirmed. The results confirm a higher expression level of CD146 on human osteosarcoma cells than HER2 and EGFR; antigens targeted by commercially available therapeutic antibodies. The biodistribution of 125I-labeled OI-3 antibody variants was compared with 125I-labeled chimeric anti-EGFR antibody cetuximab in nude mice with subcutaneous OHS osteosarcoma xenografts. OI-3 was able to target CD146 expressing tumors in vivo and showed improved tumor to tissue targeting ratios compared with cetuximab. Subsequently, the three OI-3 variants were conjugated with p-SCN-Bn-DOTA and labeled with a more therapeutically relevant radionuclide, 177Lu, and their biodistributions were studied in the nude mouse model. The 177Lu-labeled OI-3 variants were stable and had therapeutically relevant biodistribution profiles. Dosimetry estimates showed higher absorbed radiation dose to tumor than all other tissues after administration of the chimeric IgG1 OI-3 variant. CONCLUSION: Our results indicate that CD146 can be targeted in vivo by the radiolabeled OI-3 antibodies.