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.

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.

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.

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.

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.

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.

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.

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.

Targeted Cancer Therapy with a Novel Anti-CD37 Beta-Particle Emitting Radioimmunoconjugate for Treatment of Non-Hodgkin Lymphoma.

177Lu-DOTA-HH1 (177Lu-HH1) is a novel anti-CD37 radioimmunoconjugate developed to treat non-Hodgkin lymphoma. Mice with subcutaneous Ramos xenografts were treated with different activities of 177Lu-HH1, 177Lu-DOTA-rituximab (177Lu-rituximab) and non-specific 177Lu-DOTA-IgG1 (177Lu-IgG1) and therapeutic effect and toxicity of the treatment were monitored. Significant tumor growth delay and increased survival of mice were observed in mice treated with 530 MBq/kg 177Lu-HH1 as compared with mice treated with similar activities of 177Lu-rituximab or non-specific 177Lu-IgG1, 0.9% NaCl or unlabeled HH1. All mice injected with 530 MBq/kg of 177Lu-HH1 tolerated the treatment well. In contrast, 6 out of 10 mice treated with 530 MBq/kg 177Lu-rituximab experienced severe radiation toxicity. The retention of 177Lu-rituximab in organs of the mononuclear phagocyte system was longer than for 177Lu-HH1, which explains the higher toxicity observed in mice treated with 177Lu-rituximab. In vitro internalization studies showed that 177Lu-HH1 internalizes faster and to a higher extent than 177Lu-rituximab which might be the reason for the better therapeutic effect of 177Lu-HH1.

177Lu-DOTA-HH1, a novel anti-CD37 radio-immunoconjugate: a study of toxicity in nude mice.

BACKGROUND: CD37 is an internalizing B-cell antigen expressed on Non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia cells (CLL). The anti-CD37 monoclonal antibody HH1 was conjugated to the bifunctional chelator p-SCN-Bn-DOTA and labelled with the beta-particle emitting radionuclide 177Lu creating the radio-immunoconjugate (RIC) 177Lu-DOTA-HH1 (177Lu-HH1, trade name Betalutin). The present toxicity study was performed prior to initiation of clinical studies with 177Lu-HH1. METHODOLOGY/PRINCIPAL FINDINGS: Nude mice with or without tumor xenografts were treated with 50 to 1000 MBq/kg 177Lu- HH1 and followed for clinical signs of toxicity up to ten months. Acute, life threatening bone marrow toxicity was observed in animals receiving 800 and 1000 MBq/kg 177Lu-HH1. Significant changes in serum concentrations of liver enzymes were evident for treatment with 1000 MBq/kg 177Lu-HH1. Lymphoid depletion, liver necrosis and atrophy, and interstitial cell hyperplasia of the ovaries were also observed for mice in this dose group. CONCLUSIONS/SIGNIFICANCE: 177Lu-DOTA-HH1 was well tolerated at dosages about 10 times above those considered relevant for radioimmunotherapy in patients with B-cell derived malignancies.The toxicity profile was as expected for RICs. Our experimental results have paved the way for clinical evaluation of 177Lu-HH1 in NHL patients.

Advantage of lutetium-177 versus radioiodine immunoconjugate in targeted radionuclide therapy of b-cell tumors.

BACKGROUND: We herein report a comparison of the radiolabels 177Lu and 125I bound to the monoclonal antibody HH1 that targets the CD37 antigen expressed on non-Hodgkin B-cell lymphomas. MATERIALS AND METHODS: Mixtures of 177Lu and 125I-labeled HH1 antibody were co-injected into nude mice carrying Ramos xenografts and the biodistribution using the paired label format allowing tracer comparisons in each individual mouse. RESULTS: Products of the two radionuclides had very similar immunoractivity in vitro but showed different properties in vivo. Both products had relevant stability in blood and most normal tissues in nude mice carrying subcutaneous Ramos xenografts. However, both the tumor uptake and retention were significantly higher for 177Lu vs. 125I labeled HH1. The tumor to normal tissue ratios were several-fold improved for 177Lu compared to radioiodine labeled antibodies. CONCLUSION: The data presented herein support the evaluation of CD37 as a target for clinical 177Lu-based radioimmunotherapy against b-cell malignancies.

Evaluating antigen targeting and anti-tumor activity of a new anti-CD37 radioimmunoconjugate against non-Hodgkin's lymphoma.

The monoclonal antibody against CD20, rituximab, alone, or as part of combination therapies, is standard therapy for non-Hodgkin's B-cell lymphoma. Despite significantly better clinical results obtained for beta-emitting radioimmunoconjugates (RICs), RICs targeting CD20 are not commonly used in medical practice, partly because of competition for the CD20 target. Therefore, novel therapeutic approaches against other antigens are intriguing. Here, the binding properties of a novel antibody against CD37 (tetulomab) were compared with those of rituximab. The therapeutic effect of (177)Lu-tetulomab was compared with (177)Lu-rituximab on Daudi cells in vitro. The biodistribution, therapeutic and toxic effects of (177)Lu-tetulomab and unlabeled tetulomab were determined in SCID mice injected with Daudi cells. The affinity of tetulomab to CD37 was similar to the affinity of rituximab to CD20, but the CD37-tetulomab complex was internalized 10-times faster than the CD20-rituximab complex. At the same concentration of antibody, (177)Lu-tetulomab was significantly more efficient in inhibiting cell growth than was (177)Lu-rituximab, even though the cell-bound activity of (177)Lu-rituximab was higher. Treatment with 50 and 100 MBq/kg (177)Lu-tetulomab resulted in significantly increased survival of mice, compared with control groups treated with tetulomab or saline. The CD37 epitope recognized by tetulomab was highly expressed in 216 out of 217 tumor biopsies from patients with B-cell lymphoma. This work warrants further pre-clinical and clinical studies of (177)Lu-tetulomab.

Biodistribution and dosimetry of (177)Lu-tetulomab, a new radioimmunoconjugate for treatment of non-Hodgkin lymphoma.

The biodistribution of the anti-CD37 radioimmunoconjugate (177)Lu-tetraxetan-tetulomab ((177)Lu-DOTA-HH1) was evaluated. Biodistribution of (177)Lu-tetraxetan-tetulomab was compared with (177)Lu-tetraxetan-rituximab and free (177)Lu in nude mice implanted with Daudi lymphoma xenografts. The data showed that (177)Lu-tetulomab had a relevant stability and tumor targeting properties in the human lymphoma model. The half-life of (177)Lu allowed significant tumor to normal tissue ratios to be obtained indicating that (177)Lu-tetraxetan-tetulomab could be suitable for clinical testing. The biological and effective half-life in blood was higher for (177)Lu-tetraxetan-tetulomab than for (177)Lu-tetraxetan-rituximab. The biodistribution of (177)Lu-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 (177)Lu-tetraxetan-tetuloma b and (177)Lu-tetraxetan-rituximab. The absorbed radiation doses were extrapolated to human absorbed radiation doses. These extrapolated absorbed radiation doses to normal tissues for (177)Lu-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 (177)Lu-tetraxetan-tetulomab in the clinic.

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.

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 vitro cytotoxicity of low-dose-rate radioimmunotherapy by the alpha-emitting radioimmunoconjugate Thorium-227-DOTA-rituximab.

PURPOSE: To determine whether the low-dose-rate alpha-particle-emitting radioimmunoconjugate (227)Th-1,4,7,10-p-isothiocyanato-benzyl-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-rituximab can be used to inactivate lymphoma cells growing as single cells and small colonies. METHODS AND MATERIALS: CD20-positive lymphoma cell lines were treated with (227)Th-DOTA-rituximab for 1-5 weeks. To simulate the in vivo situation with continuous but decreasing supply of radioimmunoconjugates from the blood pool, the cells were not washed after incubation with (227)Th-DOTA-rituximab, but half of the medium was replaced with fresh medium, and cell concentration and cell-bound activity were determined every other day after start of incubation. A microdosimetric model was established to estimate the average number of hits in the nucleus for different localizations of activity. RESULTS: There was a specific targeted effect on cell growth of the (227)Th-DOTA-rituximab treatment. Although the cells were not washed after incubation with (227)Th-DOTA-rituximab, the average contribution of activity in the medium to the mean dose was only 6%, whereas the average contribution from activity on the cells' own surface was 78%. The mean dose rates after incubation with 800 Bq/mL (227)Th-DOTA-rituximab varied from 0.01 to 0.03 cGy/min. The average delay in growing from 10(5) to 10(7) cells/mL was 15 days when the cells were treated with a mean absorbed radiation dose of 2 Gy alpha-particle radiation from (227)Th-DOTA-rituximab, whereas it was 11 days when the cells were irradiated with 6 Gy of X-radiation. The relative biologic effect of the treatment was estimated to be 2.9-3.4. CONCLUSIONS: The low-dose-rate radioimmunoconjugate (227)Th-DOTA-rituximab is suitable for inactivation of single lymphoma cells and small colonies of lymphoma cells.

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.

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.