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Evidence for the efficacy of targeted alpha therapy for the control of pancreatic cancer in preclinical models is reviewed. Results are given for in vitro pancreatic cancer cells and clusters and micro-metastatic cancer lesions in vivo. Two complementary targeting vectors are examined. These are the C595 monoclonal antibody that targets the MUC1 antigen and the PAI2 ligand that targets the uPA receptor. The expression of the tumor-associated antigen MUC-1 and the uPA receptor on three pancreatic cancer cell lines is reported for cell clusters, human mouse xenografts and lymph node metastases, as well as for human pancreatic cancer tissues, using immuno-histochemistry, confocal microscopy and flow cytometry. The targeting vectors C595 and PAI2 were labeled with the alpha emitting radioisotope 213Bi using the chelators cDTPA and CHX-A″ to form the alpha-conjugates (AC). Cell clusters were incubated with the AC and examined at 48 hours. Apoptosis was documented using the TUNEL assay. In vivo, the anti-proliferative effect for tumors was tested at two days post-subcutaneous cell inoculation. Mice were injected with different concentrations of AC by local or systemic administration. Changes in tumor progression were assessed by tumor size. MUC-1 and uPA are strongly expressed on CFPAC-1, PANC-1 and moderate expression was found CAPAN-1 cell clusters and tumor xenografts. The ACs can target pancreatic cells and regress cell clusters (~100 µm diameter), causing apoptosis in some 70–90 % of cells. At two days post-cell inoculation in mice, a single local injection of 74 MBq/kg of AC causes complete inhibition of tumor growth. Systemic injections of 111, 222 and 333 MBq/kg of alpha-conjugate caused significant tumor growth delay in a dose dependent manner after 16 weeks, compared with the non-specific control at 333 MBq/kg. Cytotoxicity was assessed by the MTS and TUNEL assays. The C595 and PAI2-alpha conjugates are indicated for the treatment of micro-metastatic pancreatic cancer with over-expression of MUC1 and uPA receptors in post-surgical patients with minimal residual disease. The observation of tumor regression in a Phase I clinical trial of targeted alpha therapy for metastatic melanoma indicates that alpha therapy can regress tumors by a process called tumor anti-vascular alpha therapy (TAVAT). As a consequence, this therapy could be indicated for the management of non-surgical pancreatic cancer tumors.

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.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.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.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.

BACKGROUND. Micrometastasis is a major problem for prostate cancer (CaP) patients. Our study investigated the therapeutic potential of multiple targeted α-therapy (MTAT) in the treatment of CaP micrometastases (spheroids) using Bi-labeled multiple targeted α-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-LNS 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 Bi using standard methodology. DU 145 and LNCaP-LN3 spheroids were incubated with different activities of test and control α-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

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.

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.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.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 (diameter100 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.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.