Therapeutic efficacy of intravesical -radioimmunotherapy with Bi-213-anti-EGFR-MAb in a human bladder cancer nude mouse model

Objectives: Due to a high recurrence rate after transurethral resection of urothelial cancer new therapeutic strategies are urgently needed. The aim of this study was to establish an orthotopic human bladder cancer mouse model using an EGFR-overexpressing cancer cell line and to compare the therapeutic efficacy of intravesically instilled Bi-213-anti-EGFR-MAb with Mitomycin C. Methods: Female swiss nu/nu mice were catheterized and urothelial lesions were set by electrocautery. 2x106 luciferase transfected EJ28 cells were inoculated intravesically in 7 groups of 10 mice each. 1h, 7d and 14d after cancer cell inoculation 0.925 MBq of Bi-213-MAb were instilled into the bladder. 2 groups received 40µg Mitomycin C at 1h and 7d, 1 group received unconjugated MAb, the control only PBS. Tumor development and therapy response were imaged by bioluminescence imaging and survival observed up to 300d. Results: Mice of the control group and those treated with the unconjugated MAb reached a median survival of 41d and 69d, respectively. Mice that underwent Bi-213-MAb therapy 1h, 7d and 14d after cell instillation survived >300d in 90%, 80% and 40%, respectively. Mitomycin C treatment after 1h and 7d resulted in a survival >300d in 40% and 50%, respectively. Conclusions: Intravesically instilled Bi-213-MAb significantly prolonged survival without toxicity whereas Mitomycin C induced nephrotoxicity. Thus, therapy using Bi-213-anti-EGFR-MAb is a very promising approach to reduce the high recurrence rate of urothelial carcinoma.JRC.E.5-Nuclear chemistr

Analysis of DNA double-strand breaks in gastric cancer cells after treatment with 213Bi-immunoconjugates

Objectives: To facilitate dosimetric calculations in -emitter therapy, new concepts, preferably based on biological models, are needed. Since cytotoxicity of -particles is due to induction of DNA double-strand breaks (DSBs), detection of DSBs should be a straightforward concept. At sites of DSBs histones H2AX are phosphorylated, resulting in -H2AX. Using an antibody that specifically binds to -H2AX, the numbers of DSBs can be correlated to the -emitter activity applied. The aim of this study was to quantify DNA-DSBs in gastric cancer cells after incubation with 213Bi-immunoconjugates. Methods: The monoclonal antibody d9MAb specifically binds to HSC45-M2 gastric cancer cells expressing mutant d9-E-cadherin. HSC45-M2 cells were incubated with different activity concentrations of tumor-specific 213Bi-d9MAb conjugates for 3 h (t = 46 min). At different time points after incubation -H2AX was detected using immunofluorescence and Western blotting. Results: Incubation of HSC45-M2 cells seeded in chamber slides with 1.48 GBq/ml caused massive formation of -H2AX foci in the nuclei of treated cells that were not observed in the neighboring chamber incubated with PBS only. Thus, -emission during 213Bi decay is unable to induce DSBs while -particles triggered massive DSBs. Phosphorylation of histone H2AX in HSC45-M2 cells could also be demonstrated via Western blotting. Induction of -H2AX foci was dependent on 213Bi-d9MAb activity concentration. Conclusions: Detection of DSBs via -H2AX foci is a promising concept to evaluate cytotoxicity and to estimate doses in 213Bi-immunotherapy.JRC.E.5-Nuclear chemistr

213Bi-radioimmunotherapy Defeats Early-stage Disseminated Gastric Cancer in Nude Mice

The a-emitter 213Bi is characterized by a high relative biological effectiveness. 213Bi-immunoconjugates targeting tumor-specific d9-E-cadherin have been proven to effectively kill tumor cells in a murine peritoneal carcinomatosis model. The aim of the present study was to optimize the efficacy of 213Bi-radioimmunotherapy for disseminated gastric cancer in a mouse model of early- and advanced-stage disease and to evaluate the long-term toxicity of 213Bi-immunoconjugates. For that purpose, nude mice were treated with different activities of 213Bi-d9 monoclonal antibody (MAb) targeting d9-E-cadherin or unspecific 213Bi-d8MAb at days 1 or 8 after inoculation of HSC45-M2 gastric cancer cells expressing mutant d9-E-cadherin. Therapeutic efficacy was evaluated by monitoring survival for up to 300 days. Long-term toxicity was evaluated by the survival of tumor-free mice injected with 213Bi-immunoconjugates, kidney function parameters and histopathological examination of kidneys. We showed that survival was significantly prolonged following treatment of mice with 213Bi-immunoconjugates (0.37¿22.2 MBq) at day 1 after tumor cell inoculation (P < 0.002). Therapy with 1.85 MBq of 213Bi-d9MAb was most successful, defeating early-stage disease in 87% of all cases. Treatment at day 8 after tumor cell inoculation was less efficient. Long-term nephrotoxicity could only be observed following application of 22.2 MBq of 213Bi-d9MAb, the highest activity applied in the therapy trials. As treatment with 1.85 MBq 213Bi-d9MAb showed excellent therapeutic efficacy without any signs of acute or chronic toxicity, radioimmunotherapy with the a-emitter 213Bi is a promising concept for treatment of early peritoneal carcinomatosis. (Cancer Sci 2007; 98: 1215¿1222)JRC.E.5-Nuclear chemistr

Radioimmunotherapy of human bladder cancer in a nude mouse model comparing Bi-213-anti-EGFR-MAb and Th-226-anti-EGFR-MAb

Objectives: Transurethral resection of urothelial cancer still results in high recurrence rates. In new concepts for therapy of disseminated tumor cells -emitter immunoconjugates are applied. Therefore the aim of this study was to compare the therapeutic efficacy of Bi-213- and Th-226-anti-EGFR-MAb in an orthotopic EGFR-overexpressing bladder carcinoma model. Methods: 2x106 luciferase transfected EJ28 cancer cells were instilled into the bladders of female swiss nu/nu mice following urothelial electrocautery. 10 tumor-bearing mice each were intravesically instilled with 0.925 MBq of Bi-213-MAb or 0.37 MBq Th-226-MAb 1h, 7d and 14d after cell inoculation; controls received PBS. Tumor development and therapy response was imaged via bioluminescence imaging and survival observed up to 300d. Results: Mice of the control group reached a median survival of 41d. Bi-213-anti-EGFR therapy prolonged survival >300 d in 90%, 80% and 40% of animals treated 1h, 7d and 14d after cell instillation, respectively. Therapy with Th-226-MAb starting 10/2008 turned out to be as effective as Bi-213 therapy: 90% of animals of each group treated 1h, 7d and 14 d after cell inoculation are still alive without signs of toxicity. Conclusions: Both -emitter-anti-EGFR conjugates effectively eradicated disseminated tumor cells.Thus, therapy using Th-226-anti-EGFR might be a very promising supplement and/or alternative to Bi-213-anti-EGFR in treatment of urothelial cancer.JRC.E.5-Nuclear chemistr

In vitro and in vivo comparison of 226Th and 213Bi-labelled radioconjugates for target alpha therapy

The novel short-lived alpha emitter 226Th (t1/2 = 31 min) decays via a rapid cascade of four alpha particles with a cumulative energy of 27.7 MeV. The single alpha emitter 213Bi (t1/2 = 31 min) emits one alpha particle of 8.4 MeV per decay. We compare the cytotoxicity of 226Th- vs. 213Bi-labelled radioconjugates towards sensitive and chemo- and radioresistant myeloid leukemia cells in vitro and in a bladder carcinoma model in vivo.JRC.DDG.I.5-Nanobioscience

Treatment of Bladder Cancer with Bi-213-anti-EGFR Immunoconjugates in a Nude Mouse Model

Therapy of non-muscle-invasive bladder cancer (carcinoma in situ) comprises transurethral resection of the tumor and subsequent instillation of the chemotherapeutic drug mitomycin C in order to eradicate remaining tumor cells. Yet 15 – 40% of treated patients relapse within 5 years. Therefore, new therapeutic strategies to combat tumor recurrence are needed. Alpha-particle emitting radionuclides efficiently kill single tumor cells or small tumor cell clusters. Because the epidermal growth factor receptor (EGFR) is overexpressed on bladder cancer cells conjugates composed of the alpha-emitter Bi-213 and the anti-EGFR antibody matuzumab should provide a powerful drug to eliminate disseminated bladder cancer cells. Therefore, the aims of our study were (i) to analyse the cytotoxic effects of Bi-213-anti-EGFR radioimmunoconjugates at the cellular level, (ii) to evaluate therapeutic efficacy of intravesically applied Bi-213-anti-EGFR-Mab in a nude mouse model with intravesical human bladder cancer xenografts, (iii) to compare Bi-213-anti-EGFR-Mab efficacy with chemotherapy using mitomycin C and (iv) to demonstrate that radioimmunotherapy does not damage the bladder wall.JRC.E.5-Nuclear chemistr