Jean-Pierre Pouget

Clinical radioimmunotherapy—the role of radiobiology

Conventional external-beam radiation therapy is dedicated to the treatment of localized disease, whereas radioimmunotherapy represents an innovative tool for the treatment of local or diffuse tumors. Radioimmunotherapy involves the administration of radiolabeled monoclonal antibodies that are directed specifically against tumor-associated antigens or against the tumor microenvironment. Although many tumor-associated antigens have been identified as possible targets for radioimmunotherapy of patients with hematological or solid tumors, clinical success has so far been achieved mostly with radiolabeled antibodies against CD20 (131I-tositumomab and 90Y-ibritumomab tiuxetan) for the treatment of lymphoma. In this Review, we provide an update on the current challenges aimed to improve the efficacy of radioimmunotherapy and discuss the main radiobiological issues associated with clinical radioimmunotherapy.

Cell membrane is a more sensitive target than cytoplasm to dense ionization produced by auger electrons.

Abstract Pouget, J-P., Santoro, L., Raymond, L., Chouin, N., Bardiès, M., Bascoul-Mollevi, C., Huguet, H., Azria, D., Kotzki, P-O., Pè legrin, M., Vivès, E. and Pèlegrin, A. Cell Membrane is a More Sensitive Target than Cytoplasm to Dense Ionization Produced by Auger Electrons. Radiat. Res. 170, 192–200 (2008). To improve radioimmunotherapy with Auger electron emitters, we assessed whether the biological efficiency of 125I varied according to its localization. A-431 and SK-OV-3 carcinoma cells were incubated with increasing activities (0–4 MBq/ml) of 125I-labeled vectors targeting the cell membrane, the cytoplasm or the nucleus. We then measured cell survival by clonogenic assay and the mean radiation dose to the nucleus by assessing the cellular medical internal radiation dose (MIRD). The relationship between survival and the radiation dose delivered was investigated with a linear mixed regression model. For each cell line, we obtained dose–response curves for the three targets and the reference values (i.e., the dose leading to 75, 50 or 37% survival). When cell survival was expressed as a function of the total cumulative decays, nuclear 125I disintegrations were more harmful than disintegrations in the cytoplasm or at the cell membrane. However, when survival was expressed as a function of the mean radiation dose to the nucleus, toxicity was significantly higher when 125I was targeted to the cell membrane than to the cytoplasm. These findings indicate that the membrane is a more sensitive target than the cytoplasm for the dense ionization produced by Auger electrons. Moreover, cell membrane targeting is as cytotoxic as nuclear targeting in SK-OV-3 cells. We suggest that targeting the membrane rather than the cytoplasm may contribute to the development of more efficient radioimmunotherapies based on Auger electron radiation, also because most of the available vectors are directed against cell surface antigens.

Noninternalizing Monoclonal Antibodies Are Suitable Candidates for 125I Radioimmunotherapy of Small-Volume Peritoneal Carcinomatosis

We have previously shown that, in vitro, monoclonal antibodies (mAbs) labeled with the Auger electron emitter 125I are more cytotoxic if they remain at the cell surface and do not internalize in the cytoplasm. Here, we assessed the in vivo biologic efficiency of internalizing and noninternalizing 125I-labeled mAbs for the treatment of small solid tumors. Methods: Swiss nude mice bearing intraperitoneal tumor cell xenografts were injected with 37 MBq (370 MBq/mg) of internalizing (anti-HER1) 125I-m225 or noninternalizing (anti-CEA) 125I-35A7 mAbs at days 4 and 7 after tumor cell grafting. Nonspecific toxicity was assessed using the irrelevant 125I-PX mAb, and untreated controls were injected with NaCl. Tumor growth was followed by bioluminescence imaging. Mice were sacrificed when the bioluminescence signal reached 4.5 × 107 photons/s. Biodistribution analysis was performed to determine the activity contained in healthy organs and tumor nodules, and total cumulative decays were calculated. These values were used to calculate the irradiation dose by the MIRD formalism. Results: Median survival (MS) was 19 d in the NaCl-treated group. Similar values were obtained in mice treated with unlabeled PX (MS, 24 d) and 35A7 (MS, 24 d) or with 125I-PX mAbs (MS, 17 d). Conversely, mice treated with unlabeled or labeled internalizing m225 mAb (MS, 76 and 77 d, respectively) and mice injected with 125I-35A7 mAb (MS, 59 d) showed a significant increase in survival. Irradiation doses were comparable in all healthy organs, independently from the mAb used, whereas in tumors the irradiation dose was 7.4-fold higher with 125I-labeled noninternalizing than with internalizing mAbs. This discrepancy might be due to iodotyrosine moiety release occurring during the catabolism of internalizing mAbs associated with high turnover rate. Conclusion: This study indicates that 125I-labeled noninternalizing mAbs could be suitable for radioimmunotherapy of small solid tumors and that the use of internalizing mAbs should not be considered as a requirement for the success of treatments with 125I Auger electrons.

Modulation of DNA Damage by Pentoxifylline and α-Tocopherol in Skin Fibroblasts Exposed to Gamma Rays

Abstract Laurent, C., Pouget, J-P. and Voisin, P. Modulation of DNA Damage by Pentoxifylline and α-Tocopherol in Skin Fibroblasts Exposed to Gamma Rays. Radiat. Res. 164, 63–72 (2005). Previous in vivo studies showed the combination pentoxifylline (PTX) and α-tocopherol was highly efficient in reducing late radiation-induced skin damage. The present work aimed at investigating the molecular and cellular mechanisms involved in the effects of this combination. Primary cultures of confluent dermal fibroblasts were γ-irradiated in the presence of PTX and trolox (Tx), the water-soluble analogue of α-tocopherol. Drugs were added either before or after radiation exposure and were maintained over time. Their antioxidant capacity and their effect on radiation-induced ROS production was assessed together with cell viability and clonogenicity. DNA damage formation was assessed by the alkaline comet assay and by the micronucleus (MN) test. Cell cycle distribution was also determined. The combination of PTX/ Tx was shown to reduce both immediate and late ROS formation observed in cells after irradiation. Surprisingly, decrease in DNA strand breaks measured by the comet assay was observed any time drugs were added. In addition, the micronucleus test revealed that for cells irradiated with 10 Gy, a late significant increase in MN formation occurred. The combination of PTX/Tx was shown to be antioxidant and to decrease radiation-induced ROS production. The observed effects on DNA damage at any time the drugs were added suggest that PTX/Tx could interfere with the DNA repair process.

Introduction to Radiobiology of Targeted Radionuclide Therapy

During the last decades, new radionuclide-based targeted therapies have emerged as efficient tools for cancer treatment. Targeted radionuclide therapies (TRTs) are based on a multidisciplinary approach that involves the cooperation of specialists in several research fields. Among them, radiobiologists investigate the biological effects of ionizing radiation, specifically the molecular and cellular mechanisms involved in the radiation response. Most of the knowledge about radiation effects concerns external beam radiation therapy (EBRT) and radiobiology has then strongly contributed to the development of this therapeutic approach. Similarly, radiobiology and dosimetry are also assumed to be ways for improving TRT, in particular in the therapy of solid tumors, which are radioresistant. However, extrapolation of EBRT radiobiology to TRT is not straightforward. Indeed, the specific physical characteristics of TRT (heterogeneous and mixed irradiation, protracted exposure, and low absorbed dose rate) differ from those of conventional EBRT (homogeneous irradiation, short exposure, and high absorbed dose rate), and consequently the response of irradiated tissues might be different. Therefore, specific TRT radiobiology needs to be explored. Determining dose–effect correlation is also a prerequisite for rigorous preclinical radiobiology studies because dosimetry provides the necessary referential to all TRT situations. It is required too for developing patient-tailored TRT in the clinic in order to estimate the best dose for tumor control, while protecting the healthy tissues, thereby improving therapeutic efficacy. Finally, it will allow to determine the relative contribution of targeted effects (assumed to be dose-related) and non-targeted effects (assumed to be non-dose-related) of ionizing radiation. However, conversely to EBRT where it is routinely used, dosimetry is still challenging in TRT. Therefore, it constitutes with radiobiology, one of the main challenges of TRT in the future.

General overview of radioimmunotherapy of solid tumors.

Radioimmunotherapy (RIT) represents an attractive tool for the treatment of local and/or diffuse tumors with radiation. In RIT, cytotoxic radionuclides are delivered by monoclonal antibodies that specifically target tumor-associated antigens or the tumor microenvironment. While RIT has been successfully employed for the treatment of lymphoma, mostly with radiolabeled antibodies against CD20 (Bexxar(®); Corixa Corp., WA, USA and Zevalin(®); Biogen Idec Inc., CA, USA and Schering AG, Berlin, Germany), its use in solid tumors is more challenging and, so far, few trials have progressed beyond Phase II. This review provides an update on antibody-radionuclide conjugates and their use in RIT. It also discusses possible optimization strategies to improve the clinical response by considering biological, radiobiological and physical features.

DNA damage in cultured skin microvascular endothelial cells exposed to gamma rays and treated by the combination pentoxifylline and α-tocopherol

Purpose: This in vitro study aims at evaluating the effect of the combination of pentoxifylline (PTX) and trolox (Tx), the water-soluble analogue of α-tocopherol, on the oxidative state and DNA damage in dermal microvascular endothelial cells exposed to doses up to 10 Gy of ionizing radiation. Materials and methods: Confluent primary cultures of dermal endothelial cells were gamma irradiated at 3 and 10 Gy, and 0.5 mM of both drugs, PTX and Tx, was added either before (15 min) or after (30 min or 24 h) irradiation. Reactive oxygen species (ROS), measured by the dichlorodihydrofluorescein diacetate assay, and DNA damage, assessed by the comet and micronucleus assays, were measured at different times after exposure (0 – 21 days). Results: The PTX/Tx treatment decreased the early and delayed peak of ROS production by a factor of 2.8 in 10 Gy-irradiated cells immediately after irradiation and the basal level by a factor of 2 in non-irradiated control cells. Moreover, the level of DNA strand breaks, as measured by the comet assay, was shown to be reduced by half immediately after irradiation when the PTX/Tx treatment was added 15 min before irradiation. However, unexpectedly, it was decreased to a similar extent when the drugs were added 30 min after radiation exposure. This reduction was accompanied by a 2.2- and 3.6-fold higher yield in the micronuclei (MN) frequency observed on days 10 and 14 post-irradiation, respectively. Conclusion: These results suggest that oxidative stress and DNA damage induced in dermal microvascular endothelial cells by radiation can be modulated by early PTX/Tx treatment. These drugs acted not only as radical scavengers, but they were also responsible for the increased MN frequency in 10 Gy-irradiated cells. Thus, these drugs may cause a possible interference with DNA repair processes.

Brief Intraperitoneal Radioimmunotherapy of Small Peritoneal Carcinomatosis Using High Activities of Noninternalizing 125I-Labeled Monoclonal Antibodies

We assessed the efficiency and toxicity of brief intraperitoneal radioimmunotherapy using high activities of 125I-labeled monoclonal antibody (mAb) in the treatment of small-volume peritoneal carcinomatosis. Methods: Brief intraperitoneal radioimmunotherapy consisted of a 185-MBq (740 MBq/mg) intraperitoneal injection of 125I-35A7 (an anti–carcinoembryonic antigen mAb) into athymic nude mice 4 d after peritoneal tumor xenografting and, after 1 h, abundant washing of the peritoneal cavity with saline solution to remove unbound radioactivity. Another group of mice received this treatment plus a 37-MBq intravenous injection of 125I-35A7 on day 7 or 11 after grafting. Control groups received a brief treatment followed by an additional intravenous injection on day 7 of either saline solution or irrelevant 125I-PX. Tumor growth was monitored by bioluminescence imaging and SPECT/CT, and hematologic toxicity was evaluated by complete blood counts. Survival time was reported, and the mice were sacrificed when the bioluminescence signal reached 4.5 × 107 photons/s. The biodistribution of 125I-35A7 mAb after intravenous or brief treatment was assessed, and the mean absorbed irradiation dose by organs and tumors was calculated using the MIRD formalism. Results: Mild, transient hematologic toxicity was observed after the brief treatment plus intravenous 125I-mAb, with no weight loss. Median survival increased from 32 d in the control groups, to 46 d in the brief treatment group, to 66 d in the group additionally receiving intravenous treatment on day 11, to 73 d in the group additionally receiving intravenous treatment on day 7. The brief treatment alone resulted in a 3-fold higher tumor-to-blood uptake ratio than did the standard intravenous treatment, and the mean absorbed irradiation doses by tumors were 11.6 Gy for the brief treatment and 16.7 Gy for the additional intravenous treatment. For healthy tissues other than blood, the mean absorbed irradiation dose did not exceed 1 Gy after brief treatment and 4.2 Gy after intravenous treatment. Conclusion: The efficiency, low toxicity, and high tumor–to–healthy tissue uptake ratio associated with brief intraperitoneal 125I-35A7 radioimmunotherapy suggest that this method can be used in combination with radiation-synergistic drugs in the therapy of small-volume peritoneal carcinomatosis after cytoreductive surgery.

Peptides in receptor-mediated radiotherapy: from design to the clinical application in cancers

Short peptides can show high affinity for specific receptors overexpressed on tumor cells. Some of these are already used in cancerology as diagnostic tools and others are in clinical trials for therapeutic applications. Therefore, peptides exhibit great potential as a diagnostic tool but also as an alternative or an additional antitumoral approach upon the covalent attachment of a therapeutic moiety such as a radionuclide or a cytotoxic drug. The chemistry offers flexibility to graft onto the targeting-peptide either fluorine or iodine directly, or metallic radionuclides through appropriate chelating agent. Since short peptides are straightforward to synthesize, there is an opportunity to further improve existing peptides or to design new ones for clinical applications. However, several considerations have to be taken into account to optimize the recognition properties of the targeting-peptide to its receptor, to improve its stability in the biological fluids and its residence in the body, or to increase its overall therapeutic effect. In this review, we highlight the different aspects which need to be considered for the development of an efficient peptide receptor-mediated radionuclide therapy in different neoplasms.

DNA damage-centered signaling pathways are effectively activated during low dose-rate Auger radioimmunotherapy

INTRODUCTION Low dose-rate radioimmunotherapy (RIT) using (125)I-labelled monoclonal antibodies ((125)I-mAbs) is associated with unexpected high cytotoxicity per Gy. METHODS We investigated whether this hypersensitivity was due to lack of detection of DNA damage by the targeted cells. DNA damage was measured with the alkaline comet assay, gamma-H2AX foci and the micronucleus test in p53(-/-) and p53(+/+) HCT116 cells exposed to increasing activities of internalizing anti-HER1 (125)I-mAbs or non-internalizing anti-CEA (125)I-mAbs. The expression of proteins involved in radiation response and progression of cells through the cycle were determined. RESULTS Cell hypersensitivity to low absorbed doses of anti-CEA (125)I-mAbs was not due to defect in DNA damage detection, since ATM (ataxia telangiectasia mutated gene), gamma-H2AX, p53 and p21 were activated in RIT-treated HCT116 cells and G2/M cell cycle arrest was observed. Moreover, the alkaline comet assay showed that DNA breaks accumulated when cells were placed at 4°C during exposure but were repaired under standard RIT conditions (37°C), suggesting that lesions detected under alkaline conditions (mostly DNA single strand breaks and alkali-labile sites) are efficiently repaired in treated cells. The level of gamma-H2AX protein corroborated by the level of foci measured in nuclei of treated cells was shown to accumulate with time thereby suggesting the continuous presence of DNA double strand breaks. This was accompanied by the formation of micronuclei. CONCLUSION Hypersensitivity to non-internalizing (125)I-mAbs is not due to lack of detection of DNA damage after low absorbed dose-rates. However, DNA double strand breaks accumulate in cells exposed both to internalizing and non-internalizing (125)I-mAbs and lead to micronuclei formation. These results suggest impairment in DNA double strand breaks repair after low absorbed doses of (125)I-mAbs.