Alpha-particle emitting 213Bi-anti-EGFR immunoconjugates eradicate tumor cells independent of oxygenation

Hypoxia is a central problem in tumor treatment because hypoxic cells are less sensitive to chemo- and radiotherapy than normoxic cells. Radioresistance of hypoxic tumor cells is due to reduced sensitivity towards low Linear Energy Transfer (LET) radiation. High LET α-emitters are thought to eradicate tumor cells independent of cellular oxygenation. Therefore, the aim of this study was to demonstrate that the cell-bound α-particle emitting 213Bi immunoconjugates efficiently kill hypoxic just like normoxic CAL33 tumor cells. For that purpose CAL33 cells were incubated with 213Bianti- EGFR-MAb or irradiated with photons with a nominal energy of 6 MeV both under hypoxic and normoxic conditions. Oxygenation of cells was checked via the hypoxia-associated marker HIF-1α. Survival of cells was analysed using the clonogenic assay. Cell viability was monitored with the WST colorimetric assay. Results were evaluated statistically using a t-test and a Generalized Linear Mixed Model (GLMM). Survival and viability of CAL33 cells decreased both after incubation with increasing 213Bi-anti-EGFR-MAb activity concentrations (9.25 kBq/ml – 1.48 MBq/ml) and irradiation with increasing doses of photons (0.5 – 12 Gy). Following photon irradiation survival and viability of normoxic cells were significantly lower than those of hypoxic cells at all doses analysed. In contrast, cell death induced by 213Bianti- EGFR-MAb turned out to be independent of cellular oxygenation. These results demonstrate for the first time that α-particle emitting 213Bi-immunoconjugates eradicate hypoxic tumor cells as effective as normoxic cells. Therefore, 213Biradioimmunotherapy seems to be an appropriate strategy for treatment of hypoxic tumors.JRC.E.5-Nuclear chemistr

Treatment of Peritoneal Carcinomatosis by Targeted Delivery of the Radio-Labeled Tumor Homing Peptide 213Bi-DTPA-[F3]2 into the Nucleus of Tumor Cells

BACKGROUND: Alpha-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for alpha-particle emitting isotopes facilitating selective tumor therapies. PRINCIPAL FINDINGS: A dimer of the vascular tumor homing peptide F3 was chemically coupled to the alpha-emitter (213)Bi ((213)Bi-DTPA-[F3](2)). We found (213)Bi-DTPA-[F3](2) to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of (213)Bi-DTPA-[F3](2) we treated mice bearing intraperitoneally growing xenograft tumors with (213)Bi-DTPA-[F3](2). In a tumor prevention study between the days 4-14 after inoculation of tumor cells 6x1.85 MBq (50 microCi) of (213)Bi-DTPA-[F3](2) were injected. In a tumor reduction study between the days 16-26 after inoculation of tumor cells 6x1.85 MBq of (213)Bi-DTPA-[F3](2) were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found (213)Bi-DTPA-[F3](2) to accumulate in tumors but only low activities were found in control organs except for the kidneys, where (213)Bi-DTPA-[F3](2) is found due to renal excretion. CONCLUSIONS/SIGNIFICANCE: In conclusion we report that (213)Bi-DTPA-[F3](2) is a novel tool for the targeted delivery of alpha-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology

Das Klosterland des Athos /

Mode of access: Internet

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

Fractionated Locoregional Low-Dose Radioimmunotherapy Improves Survival in a Mouse Model of Diffuse Type Gastric Cancer Using A 213BI-conjugated Monoclonal Antibody

Locoregional radioimmunotherapy of intraperitoneal tumor cell dissemination of diffuse type gastric cancer using the Alpha-emitter 213Bi displayed good therapeutic results after single application depending on the time interval between tumor cell inoculation and injection of the 213Bi-immunoconjugate. The aim of the present study was to compare single versus double intraperitoneal injection of a tumor specific antibody (d9MAb) conjugated with low activities of 213Bi in terms of therapeutic efficacy and toxicity.JRC.E.5-Nuclear chemistr

Immuno- and constitutive proteasomes do not differ in their abilities to degrade ubiquitinated proteins

Immunoproteasomes are alternative forms of proteasomes that have an enhanced ability to generate antigenic peptides. Recently, Seifert and colleagues reported surprising observations concerning the functions of immunoproteasomes and cellular responses to interferon-γ: (1) that immunoproteasomes degrade ubiquitinated proteins faster than the constitutive proteasomes, (2) that polyubiquitin conjugates accumulate after interferon-γ treatment but then are preferentially degraded by immunoproteasomes, and (3) that immunoproteasome deficiency causes the formation of inclusions and more severe experimental autoimmune encephalomyelitis (EAE). In contrast, we find that polyubiquitin conjugates do not transiently accumulate following IFNγ-treatment and that immunoproteasomes do not prevent the formation of intracellular inclusions or protect against EAE. Furthermore, purified 26S constitutive and immunoproteasomes bind ubiquitin conjugates similarly and degrade them at similar rates. We conclude that, although immunoproteasomes can increase the generation of peptides appropriate for MHC class I presentation, they do not degrade ubiquitinated proteins more efficiently than constitutive particles

High molecular mass radioimmunoconjugates are promising for intraperitoneal α-emitter immunotherapy due to prolonged retention in the peritoneum

Abstract Aim. Therapeutic efficacy of intraperitoneal radioimmunotherapy is dependent on the time of retention of the radioimmunoconjugates within the peritoneal cavity. Therefore, the aim of this study was to investigate intraperitoneal retention of Fab, IgG and IgM radioimmunoconjugates. Methods. Female Balb/c mice were injected with 213Bi- or 111In-labeled IgM, IgG and recombinant Fab conjugates intraperitoneally or intravenously. At different time points after injection, whole body distribution of radionuclides was imaged using a gamma camera. Distribution of radionuclides in selected organs was determined via γ-counting after sacrifice. Biological half-lives of the conjugates were calculated from whole body activities. Results. After i.p. injection 213Bi-Fab rapidly accumulated in the kidneys indicative of glomerular filtration and reabsorption. Accumulation of 213Bi-IgG in the kidneys was significantly lower. 213Bi-IgM showed a striking accumulation in the liver 180 min after i.p. injection. 111In-IgG persisted in the circulation up to 72 h both after i.p. and i.v. injection. 111In-IgM showed a continuous accumulation in the liver. Moreover, 111In-IgM was significantly higher 24 h after i.v. injection than i.p. injection both in liver and spleen. These differences could be confirmed via scintigraphy. After injection of 111In-IgG differences in scintigraphic images between i.v. and i.p. were clearly visible only at 3 h. Biological half lives were 24 h, 45 h and 165 h for 111In-IgM, 111In-Fab and 111In-IgG, respectively. Conclusion. Retention of radioimmunoconjugates in the peritoneal cavity positively correlates with the molecular mass of the antibody. Therefore, IgM radioimmunoconjugates should be preferably used in intraperitoneal radioimmunotherapy.JRC.E.5-Nuclear chemistr