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.

Bismuth-213 radioimmunotherapy with C595 anti-MUC1 monoclonal antibody in an ovarian cancer ascites model.

PURPOSE: Control of ovarian cancer (OC) ascites remains a major objective in post-surgical treatment. The aim of this study was to investigate the effect of targeted alpha therapy (TAT) for the control of ascites in an OC ascites mouse model; the biodistribution of (213)Bi-C595 and its long term toxicity. METHODS: The expression of tumor-associated antigen mucin-1 (MUC-1) in OVCAR3 ascites cells in mice and OC cancer tissues in patients was detected by indirect immmunostaining. The monoclonal antibody (MAb) C595 was labeled with (213)Bi using the chelator cDTPA to form the alpha-immunoconjugate (AIC). Mice were injected with different concentrations of AIC by i.p administration. Changes in tumor progression were assessed by measurement of the circumference of the abdomen. RESULTS: MUC-1 is strongly expressed in 73% of OC tissues. At 9 days post-cell inoculation in mice, a single injection of 355 MBq/kg of (213)Bi-C595 can prolong survival by 25 days. A high tumor: blood ratio (5.8) was found in biodistribution study. The maximum tolerance dose (MTD) was more than 1180 MBq/kg up to 21 weeks. CONCLUSIONS: C595 is a specific targeting vector for ovarian cancer cells, which show a high percentage of expression of MUC1. (213)Bi-C595 can effectively target and kill ovarian cancer cells in vitro and in vivo. (213)Bi-C595 is the recommended alpha conjugate for a Phase I clinical trial for ovarian cancer.

The cytokinesis-block micronucleus assay as a biological dosimeter for targeted alpha therapy.

Ionizing radiation causes structural chromosomal aberrations, a proportion of which give rise to chromosome fragments without spindle attachment organelles. When a cell divides, some of these fragments are excluded from the main daughter nuclei and form small nuclei within the cytoplasm. The cytokinesis-block micronucleus assay allows these micronuclei (MN) to be counted, providing an in situ biological dosimeter. In this study, we evaluated the micronucleus frequency in peripheral blood lymphocytes after in vitro incubation with the alpha conjugates (213)BiI(3) and (213)Bi-9.2.27 (AIC). Lymphocytes were inoculated in vitro AIC for 3 h. Further, we report the first MN measurements in melanoma patients after targeted alpha therapy (TAT) with (213)Bi-9.2.27. Patients were injected with 260-360 MBq of AIC, and blood samples taken at 3 h, 2 weeks and 4 weeks post-treatment. Absorbed dose (MIRD) and effective total body dose (PED) were calculated. The MN frequency in lymphocytes was similar for equal in vitro incubation activities of (213)BiI(3) and (213)Bi-9.2.27 (P=0.5), indicating that there is no selective targeting of lymphocytes by the alpha conjugates. After inoculation with 10-1200 kBq mL-1 of AIC, there was a substantial activity-related increase in MN. The number of MN in the blood of treated patients peaked at 3 h post-TAT, slowly returning to baseline levels by 4 weeks. The mean photon equivalent dose (PED) is 0.43 Gy (SD 0.15) and the mean MIRD calculated absorbed dose is 0.11 Gy (SD 0.03), giving an RBE=4+/-0.4 for this study.

Tumour anti-vascular alpha therapy: a mechanism for the regression of solid tumours in metastatic cancer.

Targeted alpha therapy (TAT) is an emerging therapeutic modality, thought to be best suited to cancer micrometastases, leukaemia and lymphoma. TAT has not been indicated for solid tumours. However, several melanoma patients in a phase 1 clinical trial of systemic targeted alpha therapy for melanoma experienced marked regression of subcutaneous and internal tumours. This response cannot be ascribed to killing of all cancer cells in the tumours by targeted alpha therapy. These new observations support the original hypothesis that tumours can be regressed by a mechanism called tumour anti-vascular alpha therapy. This effect depends on the expression of targeted receptors by capillary pericytes and contiguous cancer cells, and on the short-range and high-energy transfer of alpha radiation.

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.

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.

Cytotoxicity of PAI2, C595 and Herceptin vectors labeled with the alpha-emitting radioisotope Bismuth-213 for ovarian cancer cell monolayers and clusters.

The vectors PAI2, C595 and Herceptin target the membrane-bound uPA, MUC1 and HER2 antigens expressed by cancer cells, respectively. The expression of these receptors was tested in the ovarian cancer cell line OVCAR-3; MUC-1 was strongly expressed (3+), uPA moderately expressed (2+), but HER2 was negative (-). The alpha-emitting radionuclide Bismuth-213 was chelated with these targeting vectors to form alpha conjugates (ACs), the cytotoxicity of which were tested with OVCAR-3 cells. The PAI2 and C595 ACs are highly cytotoxic to the ovarian monolayer cancer cells and cell clusters in a concentration-dependent fashion and cause morphological changes of treated cancer cells, inducing apoptosis. These ACs are potential candidates for the control of ovarian cancer at the minimum residual disease (MRD) stage.

Intralesional targeted alpha therapy for metastatic melanoma.

UNLABELLED: This paper reports the development and application of intralesional targeted alpha therapy (TAT) for melanoma, being the first part of a program to establish a new systemic therapy. RATIONALE: Labelling the benign targeting vector 9.2.27 with 213Bi forms the alpha-immunoconjugate (AIC), which is highly cytotoxic to targeted melanoma cells. OBJECTIVE: To investigate the safety and efficacy of intralesional AIC in patients with metastatic skin melanoma. FINDINGS: 16 melanoma patients were recruited. All the patients were positive to the monoclonal antibody 9.2.27. AIC doses from 50 to 450 mCi injected into lesions of different sizes resulted in massive cell death, as observed by the presence of tumour debris. The AIC was very effective in delivering a high dose to the tumour while sparing other tissues. There were no significant changes in blood proteins and electrolytes. There was no evidence of a human-antimouse-antibody reaction. Evidence of significant decline in serum marker melanoma-inhibitory-activity protein (MIA) at 2 weeks post-TAT was observed. CONCLUSIONS: Intralesional TAT for melanoma was found to be quite safe up to 450 mCi, and efficacious at a dose of 200 mCi. MIA, apoptosis and ki67 proliferation marker tests all indicated that TAT is a promising therapy for the control of inoperable secondary melanoma or primary ocular melanoma.

In vivo studies of pharmacokinetics and efficacy of Bismuth-213 labeled antimelanoma monoclonal antibody 9.2.27.

OBJECTIVES: Key objectives of the study were to determine the pharmacokinetics and efficacy of the alpha emitting Bismuth-213 labeled 9.2.27 alpha-immunoconjugate (AIC). METHODS: Balb/c nude mice were injected with varying doses of AIC to determine the pharmacokinetics of the AIC. The results were normalized to percent counts per minute (CPM) per gram per 3.7 MBq of the AIC (%CPM/g/3.7MBq) for each organ. Efficacy was determined by injected varying doses of AIC to different stages of tumor growth for intra-lesional, systemic and multiple dose TAT. RESULTS: Biodistribution studies showed similar pharmacokinetics for blood and brain, liver, kidneys, spleen, gut, heart, lungs and bone marrow, indicating that there was no retention of AIC. This is particularly important for brain (due to the presence of NG2+ cells) as the antibody 9.2.27 may reach NG2 positive cells. Tumor growth at 2-days post-inoculation was completely inhibited by TAT. The response to TAT was inversely proportional to tumor growth, i.e., a reduction in response was observed with increased tumor burden. A multiple dose regime was found to be more effective than single dose. CONCLUSIONS: TAT is effective for the treatment of micrometastatic melanoma, when the tumor is preangiogenic in the form of isolated cells or cell clusters. There is no evidence of retention of AIC in brain, kidneys and other vital organs.

Pre-clinical study of 213Bi labeled PAI2 for the control of micrometastatic pancreatic cancer.

PURPOSE: The urokinase plasminogen activator (uPA) and its receptor (uPAR) are expressed by pancreatic cancer cells and can be targeted by the plasminogen activator inhibitor type 2 (PAI2). We have labeled PAI2 with (213)Bi to form the alpha conjugate (AC), and have studied its in vitro cytotoxicity and in vivo efficacy. METHODS AND MATERIALS: The expression of uPA/uPAR on pancreatic cell lines, human pancreatic cancer tissues, lymph node metastases, and mouse xenografts were detected by immunohistochemistry, confocal microscopy, and flow cytometry. Cytotoxicity was assessed by the MTS and TUNEL assay. At 2 days post-cancer cell subcutaneous inoculation, mice were injected with AC by local or systemic injection. RESULTS: uPA/uPAR is strongly expressed on pancreatic cancer cell lines and cancer tissues. The AC can target and kill cancer cells in vitro in a concentration-dependent fashion. Some 90% of TUNEL positive cells were found after incubation with 1.2 MBq/ml of AC. A single local injection of approximately 222 MBq/kg 2 days post-cell inoculation can completely inhibit tumor growth over 12 weeks, and an intraperitoneal injection of 111 MBq/kg causes significant tumor growth delay. CONCLUSIONS: (213)Bi-PAI2 can specifically target pancreatic cancer cells in vitro and inhibit tumor growth in vivo. (213)Bi-PAI2 may be a useful agent for the treatment of post-surgical pancreatic cancer patients with minimum residual disease.

Targeted alpha therapy for cancer.

Targeted alpha therapy (TAT) offers the potential to inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The practicality and efficacy of TAT is tested by in vitro and in vivo studies in melanoma, leukaemia, colorectal, breast and prostate cancers, and by a phase 1 trial of intralesional TAT for melanoma. The alpha-emitting radioisotope used is Bi-213, which is eluted from the Ac-225 generator and chelated to a cancer specific monoclonal antibody (mab) or protein (e.g. plasminogen activator inhibitor-2 PAI2) to form the alpha-conjugate (AC). Stable alpha-ACs have been produced which have been tested for specificity and cytotoxicity in vitro against melanoma (9.2.27 mab), leukaemia (WM60), colorectal (C30.6), breast (PAI2, herceptin), ovarian (PAI2, herceptin, C595), prostate (PAI2, J591) and pancreatic (PAI2, C595) cancers. Subcutaneous inoculation of 1-1.5 million human cancer cells into the flanks of nude mice causes tumours to grow in all mice. Tumour growth is compared for untreated controls, nonspecific AC and specific AC, for local (subcutaneous) and systemic (tail vein or intraperitoneal) injection models. The 213Bi-9.2.27 AC is injected into secondary skin melanomas in stage 4 patients in a dose escalation study to determine the effective tolerance dose, and to measure kinematics to obtain the equivalent dose to organs. In vitro studies show that TAT is one to two orders of magnitude more cytotoxic to targeted cells than non-specific ACs, specific beta emitting conjugates or free isotopes. In vivo local TAT at 2 days post-inoculation completely prevents tumour formation for all cancers tested so far. Intra-lesional TAT can completely regress advanced sc melanoma but is less successful for breast and prostate cancers. Systemic TAT inhibits the growth of sc melanoma xenografts and gives almost complete control of breast and prostate cancer tumour growth. Intralesional doses up to 450 microCi in human patients are effective in regressing melanomas, with no concomitant complications. These results point to the application of local and systemic TAT in the management of secondary cancer. Results of the phase 1 clinical trial of TAT of subcutaneous, secondary melanoma indicate proof of the principle that TAT can make tumours in patients regress.

Hip girth as a predictor of abdominal adiposity in postmenopausal women.

OBJECTIVES: Our primary objective was to determine whether hip girth could accurately predict abdominal fat and total body fat in postmenopausal women. A secondary objective was to investigate the relation between body mass index (BMI) and hip girth in postmenopausal women. METHODS: Body weight, height, girths, skinfolds, and whole-body dual-energy x-ray absorptiometric scans were obtained in 75 postmenopausal women ages 45 to 76 y. Total body fat, abdominal fat, and the ratio of abdominal fat to lean mass (F:L) were calculated from the dual-energy x-ray absorptiometric scans. Equations predicting total and abdominal fat were developed for 50 subjects by using stepwise multiple regression analysis. These equations were then cross-validated in the remaining 25 subjects. RESULTS: Hip girth was a strong predictor of total fat (r = 0.86, P < 0.0001, standard error of the estimate = 4.9 kg) and abdominal fat (r = 0.92, P < 0.0001, standard error of estimate = 0.84 kg); with age added, the standard errors of the estimate were 3.8 and 0.8 kg, respectively. Hip girth correlated with BMI (r = 0.89, P < 0.0001). The "healthy" BMI cutoff value lower than 25 kg/m2 equated to a hip girth smaller than 100 cm and an abdominal F:L lower than 1.0. Waist girth (r = 0.83, P < 0.0001) and hip girth (r = 0.76, P < 0.0001) were good predictors of abdominal F:L. CONCLUSIONS: Hip girth is a practical and useful predictor of abdominal and total adiposity in postmenopausal women. A hip girth of 100 cm or an abdominal F:L of 1.0 can be used as an equivalent cutoff value for the "healthy" BMI value of 25 kg/m2 in this group of women.

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.