Development and evaluation of monoclonal antibodies against phosphatidylethanolamine binding protein 1 in pancreatic cancer patients

Abstract

Phosphatidylethanolamine binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), has been considered as a suppressor of metastasis and a prognostic marker in prostate cancer, breast cancer, gastrointestinal stromal tumors, melanoma, and epithelial ovarian cancer. In this report, recombinant PEBP1 was successfully expressed in an Escherichia coli system. A panel of monoclonal antibodies (mAbs) against PEBP1 with high specificity and affinity was generated and characterized using ELISA, western blot analysis, immunofluorescent staining and immunohistochemical staining. PEBP1 expression in normal 293 cells and a few pancreatic cancer cell lines was detected with mAb 7F12 in western blot analysis. To screen for a pair of mAbs with optimal binding affinity to soluble PEBP1, ForteBio's Octet system was used. Sandwich ELISA with mAb pair 4F10 and 8E2 showed a linear correlation between absorbance and PEBP1 protein concentration over a range of 7 to 100ng/ml. MAb 4A11 detected a high level expression of PEBP1 in normal pancreatic tissue, and cancer adjacent normal pancreatic tissue in a pancreatic tissue microarray (TMA) comprising 80 human tissue cores. Pancreatic cancer tissues show a no or very weak staining intensity of PEBP1. In 69 valid cases, PEBP1 expression was significantly lower in tumor than in normal pancreas (p=8.40E-14) and adjacent normal tissue (p=8.46E-17). PEBP1 expression in pancreatic cancer was not associated with pTMN stage, differentiation grade and pathologic diagnosis. In conclusion, our results suggest that PEBP1 overexpresses in normal pancreas but significantly decreases its expression in pancreatic cancer tissues. Anti-PEBP1 mAbs 4A11, 4F10, 7F12, and 8E2 are potential clinical diagnostic agents for pancreatic cancer.