Mutagenesis induced by targeted alpha therapy using 213Bi-cDTPA-9.2.27 in lacZ transgenic mice.

Targeted alpha therapy utilizes alpha-emitting radionuclides conjugated to monoclonal antibodies to allow specific irradiation of cancer cells whilst sparing normal, healthy tissues. The mutagenic potential of (213)Bi conjugated to a human melanoma antigen-specific antibody (9.2.27) was examined using an in vivo transgenic mouse model containing multiple copies of a lacZ target gene in every cell, allowing the quantification and comparison of mutagenesis in different organs. Mice received an ip injection of 16.65 MBq of (213)Bi-cDTPA-9.2.27, and were sacrificed at 24 h, 1 w and 4 w post-injection. Pharmacokinetic studies gave the absorbed and effective doses for each organ. The mutant frequency and mutant spectra were analysed for the brain, spleen and kidneys. The brain and spleen did not show significant increases in induced mutation frequencies compared to spontaneous background levels or changes in mutant spectra, these results being independent of p53 status. However, elevated mutation frequencies and persistent size change mutations were observed in the kidneys, but are not significant at the p = 0.05 level. The effect of p53 status was also evident, as p53 heterozygotes displayed higher mutation frequencies than their wild-type counterparts, suggesting a reduction in the p53 gene may lead to an increased susceptibility to mutagenesis. These effects were time dependent and levels returned to those of the controls at 4 w post-irradiation, albeit with a predominant residue of size mutations. These effects were observed at activities very much higher than those expected for the therapy of human patients. As such, the induction of secondary cancer with the (213)Bi-cDTPA-9.2.27 alpha immunoconjugate is not expected to be a significant problem in the clinic.

Inhibition of micrometastatic prostate cancer cell spread in animal models by 213Bilabeled multiple targeted alpha radioimmunoconjugates.

PURPOSE: To investigate the therapeutic potential of 213Bilabeled multiple targeted alpha-radioimmunoconjugates for treating prostate cancer (CaP) micrometastases in mouse models. EXPERIMENTAL DESIGN: PC-3 CaP cells were implanted s.c., in the prostate, and intratibially in NODSCID mice. The expression of multiple tumor-associated antigens on tumor xenografts and micrometastases was detected by immunohistochemistry. Targeting vectors were two monoclonal antibodies, and a plasminogen activator inhibitor type 2 that binds to cell surface urokinase plasminogen activator, labeled with 213Bi using standard methodology. In vivo efficacy of multiple alpha conjugates (MTAT) at different activities was evaluated in these mouse models. Tumor growth was monitored during observations and local regional lymph node metastases were assessed at the end of experiments. RESULTS: The take rate of PC-3 cells was 100% for each route of injection. The tumor-associated antigens (MUC1, urokinase plasminogen activator, and BLCA-38) were heterogeneously expressed on primary tumors and metastatic cancer clusters at transit. A single i.p. injection of MTAT (test) at high and low doses caused regression of the growth of primary tumors and prevented local lymph node metastases in a concentration-dependent fashion; it also caused cancer cells to undergo necrosis and apoptosis. CONCLUSIONS: Our results suggest that MTAT can impede primary PC-3 CaP growth at three different sites in vivo through induction of apoptosis, and can prevent the spread of cancer cells and target lymph node micrometastases in a concentration-dependent manner. MTAT, by targeting multiple antigens, can overcome heterogeneous antigen expression to kill small CaP cell clusters, thus providing a potent therapy for micrometastases.

Preparation and testing of bevacizumab radioimmunoconjugates with Bismuth-213 and Bismuth-205/Bismuth-206.

Bevacizumab, a humanized anti-VEGF monoclonal antibody has shown promise in various clinical trials. We report the development and testing of Bi-213 (an alpha-emitting radionuclide) labeled bevacizumab for in vitro and in vivo studies using two different chelators viz cDTPA and CHX-A''. The developed labeling method showed high labeling yields of 93.6% and 89.7% for cDTPA and CHX-A'' respectively and the results were reproducible. The in vitro and in vivo stability tests were carried out using Bi-213 and long half-life Bismuth isotope (Bi-205/Bi-206) for pharmacokinetics. The in vitro results showed remarkable stability of the radiolabeled bevacizumab regardless of the chelator. The in vivo pharmacokinetics studies however, showed that the uptake and retention of cDTPA-bevacizumab was significantly higher in kidneys (p-value 0.02) and lower in liver and spleen (p-value <0.0001 and 0.0009 respectively). The values for the two conjugates compared well in blood but the longer term data for CHX-A'' conjugate showed slow clearance resulting in a significantly longer blood half-life of the product (211 hours compared to 4 hours for cDTPA-bevacizumab). Preliminary in vivo results showed increased efficacy of the combination therapy compared to bevacizumab only. The tumor area (mm(2)) decreased from 24.8 +/- 3.6 and 12.8 +/- 1.7 for 1 and 3 mg/kg cold bevacizumab only to 6.5 +/- 0.7 and 7.5 +/- 4.8 when single dose of 333 MBq/kg of (213)Bi-bevacizumab was administered as combination therapy. In conclusion it can be said that stable radiolabeled bevacizumab conjugates can be prepared with Bi-213 with either chelators used. The shorter blood half-life with cDTPA-bevacizumab may not be a major concern with Bi-213 as its own half-life is 46 minutes only. The combination therapy proved superior to bevacizumab alone therapy, a phenomenon that can be particularly useful in cancers where bevacizumab alone has shown limited success like prostate cancer.

Pharmacokinetics and toxicity of (213)Bi-labeled PAI2 in preclinical targeted alpha therapy for cancer.

OBJECTIVES: The plasminogen activator inhibitor type 2 (PAI2) when labelled with (213)Bi forms the (213)Bi-PAI2 alpha conjugate (AC). This AC has been shown to be efficacious in preclinical studies with breast, ovarian, prostate and pancreatic cancers. The objectives of this study were to investigate the pharmacokinetics and in vivo stability of (213)Bi-PAI2 in mice, its toxicity in mice and rabbits; and to determine whether a prior injection of a metal chelator (Ca-DTPA) or lysine can reduce toxicity by decreasing renal uptake. METHODS: Two chelators (CHX-A"-DTPA and cDTPA) were used for preparation of the (213)Bi-PAI2 conjugate, for intraperitoneal administration in mice and ear vein injection in rabbits. The mice were sacrificed at different time points for pharmacokinetic studies. Blood and organs were collected for toxicity studies for all groups. RESULTS: Both chelators were found to have similar %ID/g in the kidneys over four hours. Mice and rabbits did not show any short term toxicity over 13 weeks at 1420 MBq/kg and 120 MBq/kg (213)Bi-PAI2 respectively. Kidney uptake was decreased three fold by lysine. Radiation nephropathy was observed at 20-30 weeks in mice, leading to severe weight loss, whereas severe and widespread renal tubular necrosis was observed at 13 weeks in rabbits. CONCLUSIONS: Radiation nephropathy is the dose limiting toxicity observed in mice and rabbits. Lysine can reduce kidney uptake by three fold. Based on long-term monitoring, the maximum tolerance doses (MTD) are 350 and 120 MBq/kg for mice and rabbits respectively.

Interim analysis of toxicity and response in phase 1 trial of systemic targeted alpha therapy for metastatic melanoma.

PURPOSE: The aim is to assess toxicity and response of systemic alpha therapy for metastatic melanoma. EXPERIMENTAL DESIGN: This is an open-labelled Phase 1 dose escalation study to establish the effective dose of the alpha-immunoconjugate (213)Bi-cDTPA-9.2.27 mAb (AIC). Tools used to investigate the effects were physical examination; imaging of tumors; pathology; GFR; CT and changes in tumor marker. Responses were assessed using RECIST criteria. RESULTS AND DISCUSSION: Twenty-two patients with stage IV melanoma/in-transit metastasis were treated with activities of 55-947 MBq. Using RECIST criteria 50% showed stable disease and 14% showed partial response. One patient (6%) showed near complete response and was retreated because of an excellent response to the initial treatment. Another patient showed response in his tumor on mandible and reduction in lung lesions. Overall 30% showed progressive disease. The tumor marker melanoma inhibitory activity protein (MIA) showed reductions over eight weeks in most of the patients. The disparity of dose with responders is discussed. No toxicity was observed over the range of administered activities. CONCLUSION: Observation of responses without any toxicity indicates that targeted alpha therapy has the potential to be a safe and effective therapeutic approach for metastatic melanoma.

Control of prostate cancer spheroid growth using 213Bi-labeled multiple targeted alpha radioimmunoconjugates.

BACKGROUND: Micrometastasis is a major problem for prostate cancer (CaP) patients. Our study investigated the therapeutic potential of multiple targeted alpha-therapy (MTAT) in the treatment of CaP micrometastases (spheroids) using (213)Bi-labeled multiple targeted alpha-radioimmunoconjugates. METHODS: The expression of multiple tumor-associated antigens (TAAs) on frozen sections of human fresh CaP tissues and spheroids cultured from DU 145 and LNCaP-LN3 CaP cell lines was detected by immunohistochemistry and flow cytometry. Targeting vectors were two monoclonal antibodies (MAbs), and plasminogen activator inhibitor type 2 (PAI2) that binds to cell surface urokinase plasminogen activator (uPA). These vectors were labeled with (213)Bi using standard methodology. DU 145 and LNCaP-LN3 spheroids were incubated with different activities of test and control alpha-conjugates (ACs), and spheroid growth was measured for volume change and growth delay over a 50-day period using light microscopy. RESULTS: TAAs were expressed heterogeneously on frozen sections from human CaP tissues and CaP spheroids. MTAT combining three ACs (one-third dose of each) with an activity of 6.4 MBq/ml completely targeted small DU 145 and LNCaP-LN3 spheroids (diameter <100 microm) and slightly regressed the growth of medium spheroids (180-200 microm); MTAT with 2.2 or 4.8 MBq/ml activities delayed the growth of tumor spheroids. CONCLUSIONS: Our results suggest that the cytotoxicity of MTAT to CaP spheroids is highly dependent on antigenic expression, concentration of radioactivity and spheroid size. MTAT may be a potent therapeutic agent for micrometastases, effectively targeting small CaP cell clusters, and overcoming the heterogeneous expression of targeted antigens.

Preclinical studies of bismuth-213 labeled plasminogen activator inhibitor type 2 (PAI2) in a prostate cancer nude mouse xenograft model.

OBJECTIVES: The key objective of the study was to determine the single and multiple dose toxicity and efficacy of Bismuth-213 labeled PAI2 based targeted alpha therapy by selectively targeting uPA and uPAR in regressing prostate cancer in a nude mouse model. Targeted alpha therapy (TAT) is an experimental therapeutic modality for cancer. Another objective was to compare the in vivo pharmacokinetics using two different chelators to form the radioisotope-protein construct. METHODS: The single and multiple intra-peritoneal (IP) dose toxicity and efficacy of 213BiPAI2 was investigated in nude mice and its toxicity in rabbits. CD 31 staining for vasculature and uPA expression were measured at different stages of tumor growth. The pharmacokinetics of the chelators cDTPA and CHX-A'' were measured. RESULTS: All TAT regimes were well tolerated in mice and rabbits on biochemical and haematological examination. Capillaries became evident at six days post-cell inoculation. uPA expression was positive at all stages of tumour growth. No significant differences were observed between cDTPA and CHX-A. '' Inhibition of tumour growth was observed at 947 and 1421 MBq/kg single dose injection at three days post-PC3 cell inoculation. The three day post-inoculation multiple dose regime gave complete tumour growth inhibition at a total dose of 947 MBq/kg given on five successive days. Mice treated at 6, 12 and 18 days post-inoculation showed significantly slower tumour growth compared to controls. CONCLUSIONS: The efficacy of single and multiple dose TAT in mice was demonstrated within tolerance limits, the multiple dose regime being no more toxic than the single dose. Either of the two chelators could be used for 213Bi studies.

Antigenic expression of human metastatic prostate cancer cell lines for in vitro multiple-targeted alpha-therapy with 213Bi-conjugates.

PURPOSE: Control of metastatic prostate cancer (CaP) is an elusive objective. Some 30% of patients with clinically localized CaP will develop micrometastatic disease. Defining the expression of tumor-associated antigens on CaP will enable appropriate selection of therapeutic targets. METHODS AND MATERIALS: The expression of tumor-associated antigens on CaP cell lines (PC-3, DU 145, and LNCaP-LN3) was detected by immunohistochemistry and flow cytometry. Test and control alpha-conjugates were prepared using monoclonal antibodies, an inhibitor, plasminogen activator inhibitor type 2, that binds to the cell-membrane-bound protease, urokinase plasminogen activator, and a control protein labeled with (213)Bi using standard methods. These were used singly or together against three different CaP cell lines in vitro. The cytotoxicity of the alpha-conjugates was assessed using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) assay. RESULTS: The PC-3 and DU 145 cancer cell lines expressed antigens that bind monoclonal antibodies BLCA-38 and #394 (mouse anti-human urokinase plasminogen activator B-chain) but not J591. The LNCaP-LN3 cells bound J591 but not #394 or BLCA-38. For the PC-3, DU 145, and LNCaP-LN3 cell lines, multiple-targeted alpha-therapy combining four alpha-conjugates (one-quarter doses of each) gave D(0) (37% cell survival) values of 15, 17, and 27 microCi/mL compared with those of the controls of 272, 289, and 281 microCi/mL, respectively. CONCLUSION: Metastatic prostate cancer-associated antigens recognized by multiple monoclonal antibodies are potential targets for alpha-therapy. Multiple-targeted alpha-therapy produced cytotoxicity specific to three CaP cell lines and may form the basis of treatment for micrometastatic CaP, overcoming the heterogeneity of expression of the targeted antigens.

Cytotoxicity of human prostate cancer cell lines in vitro and induction of apoptosis using 213Bi-Herceptin alpha-conjugate.

HER-2 has been implicated in the oncogenesis of human prostate cancer (CaP) and is the target of a new treatment for metastatic breast cancer using the humanised monoclonal antibody (MAb) trastuzumab (Herceptin). In this study, a novel alpha-particle emitting [213Bi]Herceptin construct, which targets the HER-2 extracellular domain on CaP cells, was prepared and evaluated in vitro. We used immunocytochemistry, flow cytometry and Western blot analysis to examine the expression of HER-2 in a panel of established human CaP cell lines, used the MTS assay to evaluate the cytotoxicity of 213Bi-Herceptin on these cell lines and the TUNEL assay to document apoptosis. The results indicate that LNCaP-LN3 cells express high levels of HER-2 protein, in contrast, DU 145 cells express low to medium levels, and PC-3 cells express an undetectable level of HER-2 protein. 213Bi-Herceptin was specifically cytotoxic to LNCaP-LN3 cells in a concentration-dependent fashion, cause the cells to undergo apoptosis, whereas DU 145 showed an HER-2 level-dependent response to 213Bi-Herceptin cytotoxicity. In contrast, PC-3 cells were resistant to 213Bi-Herceptin-induced cytotoxicity. The 213Bi-Herceptin induced apoptosis in LNCaP-LN3 cells could be inhibited by incubation with unlabeled Herceptin. Our results suggest that 213Bi-Herceptin alpha-conjugate might be a promising new agent for the treatment of preangiogenic cancer cell clusters or micro-metastases with high levels of HER-2 expression.

In vitro testing of the leukaemia monoclonal antibody WM-53 labeled with alpha and beta emitting radioisotopes.

We report the preparation and testing of a new alpha emitting radio-immunoconjugate (RIC) against acute myeloid leukaemia (AML) using CD33 positive monoclonal antibody WM-53 (specific for HL-60 cell line). Using cyclic anhydride of diethylenetriaminepentacetic acid (cDTPAa) as chelator, antibody was labeled with 213Bi (alpha), 149Tb (alpha), 153Sm (beta) and 152Tb (positron). In vitro testing showed high labeling efficiency (90-95%) and stability (11-19% leaching) with immunoreactivity virtually the same before and after labeling. DNA synthesis data and MTS cell survival were compared for all RICs. Only the alpha emitter was found to be capable of inhibiting DNA synthesis and had selective cell kill with activity as low as 2-3 microCi. The high stability and outstanding cytotoxicity of the 213Bi conjugate provides the basis for targeted alpha therapy for the control of metastatic and disseminated cancer such as AML.