PaCa-Ag1: A Candidate Pancreatic Cancer-specific Tumor Marker

Abstract

There is an urgent need to identify novel pancreatic carcinoma-specific markers to improve the early diagnosis and treatment of this lethal disease. The present investigation describes the purification of a new mouse monoclonal antibody, mAb3C4, its use in the characterization of the pancreatic carcinoma–specific PaCa-Ag1 and the preclinical exploration of mAb3C4 and PaCa-Ag1 in the context of pancreatic carcinoma. MAb3C4 (an IgG1) was generated by Immuno-substractive Hyperimmunization (ISHIP) in the context of a model of in vitro pancreatic carcinogenesis. Fluorescence staining and ultrastructural analyses of intact rat pancreatic carcinoma cells showed the expression of PaCa-Ag1 on the cells’ plasma membrane. Most importantly, mAb3C4 showed strong cross reactivity with the cells of six human pancreatic carcinoma cell lines examined. PaCa-Ag1 was absent from untransformed, normal BMRPA1 cells, normal rodent and human pancreatic and other normal tissues. Traces of PaCa-Ag1 were seen in lysates of renal, colorectal and prostate adenocarcinomas. By immunoblotting with mAb3C4, PaCa-Ag1 was identified as a poorly glycosylated polypeptide of Mr ~ 43.5kD with a pI of 3.67 for both the human and rat species. Subcellular fractionation and membrane extraction from pancreatic carcinoma cells confirmed PaCa-Ag1 as an integral membrane protein. Cytofluorimetric quantitation showed the presence of up to 14.8x104 PaCa-Ag1 sites on rodent and up to 10.5 times (up to 8.14x105) PaCa-Ag1 sites on human pancreatic carcinoma cells. A fast ka of 7.26x106M-1s-1 and a slow Kd of 1.9x10-6s-1 indicated a strong affinity km of 3.89x1011M-1 of mAb3C4 for PaCa-Ag1. During chemical carcinogenesis, low levels of PaCa-Ag1 were first seen at the 2nd passage of the transforming cells (47%) suggesting PaCa-Ag1 as an early marker of transformation. An Ab (mAb3C4) -capture ELISA identified PaCa-Ag1 in spent media of pancreatic cancer cell cultures, in ascites and sera of mice transplanted with pancreatic cancer. Since mAb3C4 displayed potent complement-dependent cytotoxicity against rodent and human pancreatic carcinoma cells in vitro, the immunotherapeutic potential was evaluated by continuous infusion of mAb3C4 into athymic mice xeno transplanted with pancreatic carcinomas. The results showed a significant inhibition of tumor growth (p<0.0001) with a log of tumor-cell-kill of 2.34. Infusion of a non-specific but class identical mAb had no effect. Taken together, these results support the notion that the PaCa-Ag1 is a potentially valuable early pancreatic cancer-specific marker that together with mAb3C4 will allow the introduction of a novel diagnostic tool(s) and therapeutic approach(s) for this disease.