Alpha-particles induce autophagy in multiple myeloma cells

Objectives: Radiations emitted by the radionuclides in radioimmunotherapy (RIT) approaches induce direct killing of the targeted cells as well as indirect killing through bystander effect. Our research group is dedicated to the development of α-RIT, i.e RIT using α-particles especially for the treatment of multiple myeloma (MM). γ-irradiation and β-irradiation have been shown to trigger apoptosis in tumor cells. Cell death mode induced by 213Bi α-irradiation appears more controversial. We therefore decided to investigate the effects of 213Bi on MM cell radiobiology, notably cell death mechanisms as well as tumor cell immunogenicity after irradiation.Methods: Murine 5T33 and human LP-1 multiple myeloma (MM) cell lines were used to study the effects of such α-particles. We first examined the effects of 213Bi on proliferation rate, double strand DNA breaks, cell cycle and cell death. Then, we investigated autophagy after 213Bi irradiation. Finally, a co-culture of dendritic cells (DC) with irradiated tumour cells or their culture media was performed to test whether it would induce DC activation.Results: We showed that 213Bi induces DNA double strand breaks, cell cycle arrest and autophagy in both cell lines but we detected only slight levels of early apoptosis within the 120 hours following irradiation in 5T33 and LP-1. Inhibition of autophagy prevented 213Bi induced inhibition of proliferation in LP-1 suggesting that this mechanism is involved in cell death after irradiation. We then assessed the immunogenicity of irradiated cells and found that irradiated LP-1 can activate DC through the secretion of soluble factor(s), however no increase in membrane or extracellular expression of danger associated molecular patterns (DAMPs) was observed after irradiation.Conclusion: This study demonstrates that 213Bi induces mainly necrosis in MM cells, low levels of apoptosis and also autophagy that might be involved in tumor cell death

Therapeutic efficacy of alpha-RIT using a 213Bi-anti-hCD138 antibody in a mouse model of ovarian peritoneal carcinomatosis

Purpose: Ovarian peritoneal carcinomatosis is a pathology for which effective cures are currently lacking. New research protocols seek to eradicate residual micrometastases following cytoreductive surgery by using Hyperthermic Intraperitoneal Chemotherapy (HIPEC), or Radioimmunotherapy (RIT). This study aims to firstly develop alpha-RIT using an anti-CD138 mAb radiolabeled with an alpha-emitter, bismuth 213 (213Bi-B-B4) and HIPEC in a nude mouse model, and secondly to compare and combine these techniques.Material and Methods: A murine model of postoperative ovarian peritoneal carcinomatosis was established. A pilot group of six mice received an intraperitoneal injection of luciferase-tagged SHIN-3 cells and bioluminescence was measured every day. Cytoreductive surgery was performed at day 14 (n=4) and 29 (n=2). Because the residual bioluminescence signal measured after surgery was equivalent to that obtained 3 days after the graft, HIPEC or alpha-RIT treatments were applied 3 days after the graft. Ten mice were treated by HIPEC with cisplatine (37.5 mg/mL), 11 with 7.4 MBq of 213Bi-B-B4, 7 with 11.1 MBq of 213Bi-B-B4 and 10 mice were treated with the combined therapy (HIPEC + 7.4 MBq of 213Bi-B-B4). Eleven mice received no treatment. Bioluminescence imaging and survival were assessed.Results: Alpha-RIT 7.4 MBq and 11.1 MBq significantly improved survival (p=0.0303 and p=0.0070 respectively) whereas HIPEC and HIPEC + alpha-RIT treatments did not significantly ameliorate survival as compared to the control group.Conclusions: Survival was significantly increased by alpha-RIT treatment in mice with peritoneal carcinomatosis of ovarian origin, however HIPEC alone or in combination with alpha-RIT had no significant effect

Improvement of the Targeting of Radiolabeled and Functionalized Liposomes with a Two-Step System Using a Bispecific Monoclonal Antibody (Anti-CEA × Anti-DTPA–In)

This study proposes liposomes as a new tool for pretargeted radioimmunotherapy (RIT) in solid tumors. Tumor pretargeting is obtained by using a bispecific monoclonal antibody (BsmAb, anti-CEA x anti-DTPA-In) and pegylated radioactive liposomes containing a lipid-hapten conjugate (DSPE-PEG-DTPA-In). In this work, the immunospecificity of tumor targeting is demonstrated both in vitro by fluorescence microscopy and in vivo by biodistribution studies.Methods: Carcinoembryonic antigen (CEA)-expressing cells (LS174T) were used either in cell culture or as xenografts in nude mice. Doubly fluorescent liposomes or doubly radiolabeled liposomes were respectively used for in vitro and in vivo studies. In each case, a tracer of the lipid bilayer (rhodamine or indium-111 (111In)) and a tracer of the aqueous phase (fluorescein or iodine-125 (125I)) were present. The targeting of liposomes was assessed with BsmAb for active targeting or without for passive targeting.Results: Data obtained with the lipid bilayer tracer showed a fluorescent signal on cell membranes two to three times higher for active than for passive targeting. This immunospecificity was confirmed in vivo with tumor uptake of 7.5 ± 2.4 % ID/g (percentage of injected dose per gram of tissue) for active targeting versus 4.5 ± 0.45 % ID/g for passive targeting (p = 0.03). Regarding the aqueous phase tracer, results are slightly more contrasted. In vitro, the fluorescent tracer seems to be released in the extracellular matrix, which can be correlated with the in vivo data. Indeed, the tumor uptake of 125I is lower than that of 111In: 5.1 ± 2.5 % ID/g for active targeting and 2.7 ± 0.6 % ID/g for passive targeting, but resulted in more favorable tumor/organs ratios.Conclusion: This work demonstrated the tumor targeting immunospecificity of DSPE-PEG-DTPA-In liposomes by two different methods. This original and new approach suggests the potential of immunospecific targeting liposomes for the RIT of solid tumors

Single-dose anti-CD138 radioimmunotherapy: bismuth-213 is more efficient than lutetium-177 for treatment of multiple myeloma in a preclinical model

Objectives: Radioimmunotherapy (RIT) has emerged as a potential treatment option for multiple myeloma (MM). In humans, a dosimetry study recently showed the relevance of RIT using an antibody targeting the CD138 antigen. The therapeutic efficacy of RIT using an anti-CD138 antibody coupled to 213Bi, an α-emitter, was also demonstrated in a preclinical MM model. Since then, RIT with β-emitters has shown efficacy in treating hematologic cancer. In this paper, we investigate the therapeutic efficacy of RIT in the 5T33 murine MM model using a new anti-CD138 monoclonal antibody labeled either with 213Bi for α-RIT or 177Lu for β-RIT.Methods: A new monoclonal anti-CD138 antibody, 9E7.4, was generated by immunizing a rat with a murine CD138-derived peptide. Antibody specificity was validated by flow cytometry, biodistribution and α-RIT studies. Then, a β-RIT dose-escalation assay with the 177Lu-radiolabeled 9E7.4 mAb was performed in KalwRij C57/BL6 mice 10 days after i.v. engraftment with 5T33 MM cells. Animal survival and toxicological parameters were assessed to define the optimal activity.Results: α-RIT performed with 3.7 MBq of 213Bi-labeled 9E7.4 anti-CD138 mAb increased median survival to 80 days compared to 37 days for the untreated control and effected cure in 45% of animals. β-RIT performed with 18.5 MBq of 177Lu-labeled 9E7.4 mAb was well tolerated and significantly increased mouse survival (54 versus 37 days in the control group); however, no mice were cured with this treatment.Conclusion: This study revealed the advantages of α-RIT in the treatment of MM in a preclinical model where β-RIT shows almost no efficacy

Pharmacokinetics and Dosimetry Studies for Optimization of Pretargeted Radioimmunotherapy in CEA-Expressing Advanced Lung Cancer Patients

Objectives. A phase I pretargeted radioimmunotherapy trial (EudractCT 200800603096) was designed in patients with metastatic lung cancer expressing carcinoembryonic antigen (CEA) to optimize bispecific antibody and labelled peptide doses, as well as the delay between their injections.Methods. Three cohorts of 3 patients received the anti-CEA x anti-histamine-succinyl-glycine (HSG) humanized trivalent bispecific antibody (TF2) and the IMP288 bivalent HSG-peptide. Patients underwent a pre-therapeutic imaging session S1 (44 or 88 nmol/m2 of TF2 followed by 4.4 nmol/m2, 185 MBq, of 111In-labelled IMP288), and, 1-2 weeks later, a therapy session S2 (240 or 480 nmol/m2 of TF2 followed by 24 nmol/m2, 1.1 GBq/m2, 177Lu-labeled IMP288). The pretargeting delay was 24 or 48 hours. The dose schedule was defined based on pre-clinical TF2 pharmacokinetic studies, on our previous clinical data using the previous anti-CEA pretargeting system and on clinical results observed in the first patients injected using the same system in the Netherlands.Results. TF2 pharmacokinetics (PK) was represented by a two-compartment model in which the central compartment volume was linearly dependent on the patient's surface area. PK were remarkably similar, with a clearance of 0.33 +/- 0.03 L/h per m2. 111In- and 177Lu-IMP288 PK were also well represented by a two-compartment model. IMP288 PK were faster (clearance 1.4 to 3.3 l/h). The central compartment volume was proportional to body surface area and IMP288clearance depended on the molar ratio of injected IMP288 to circulating TF2 at the time of IMP288 injection. Modelling of image quantification confirmed the dependence of IMP288 kinetics on circulating TF2, but tumour activity PK were variable. Organ absorbed doses were not significantly different in the 3 cohorts, but the tumour dose was significantly higher with the higher molar doses of TF2 (p < 0.002). S1 imaging predicted absorbed doses calculated in S2. Conclusion. The best dosing parameters corresponded to the shorter pretargeting delay and to the highest TF2 molar doses. S1 imaging session accurately predicted PK as well as absorbed doses of S2, thus potentially allowing for patient selection and dose optimization

A pretargeting system for tumor PET imaging and radioimmunotherapy

International audienceLabeled antibodies, as well as their fragments and antibody-derived recombinant constructs, have long been proposed as general vectors to target radionuclides to tumor lesions for imaging and therapy. They have indeed shown promise in both imaging and therapeutic applications, but they have not fulfilled the original expectations of achieving sufficient image contrast for tumor detection or sufficient radiation dose delivered to tumors for therapy. Pretargeting was originally developed for tumor immunoscintigraphy. It was assumed that directly-radiolabled antibodies could be replaced by an unlabeled immunoconjugate capable of binding both a tumor-specific antigen and a small molecular weight molecule. The small molecular weight molecule would carry the radioactive payload and would be injected after the bispecific immunoconjugate. It has been demonstrated that this approach does allow for both antibody-specific recognition and fast clearance of the radioactive molecule, thus resulting in improved tumor-to-normal tissue contrast ratios. It was subsequently shown that pretargeting also held promise for tumor therapy, translating improved tumor-to-normal tissue contrast ratios into more specific delivery of absorbed radiation doses. Many technical approaches have been proposed to implement pretargeting, and two have been extensively documented. One is based on the avidin-biotin system, and the other on bispecific antibodies binding a tumor-specific antigen and a hapten. Both have been studied in preclinical models, as well as in several clinical studies, and have shown improved targeting efficiency. This article reviews the historical and recent preclinical and clinical advances in the use of bispecific-antibody-based pretargeting for radioimmunodetection and radioimmunotherapy of cancer. The results of recent evaluation of pretargeting in PET imaging also are discussed