A Basic Study for Tumor Vaccine Development in Human Endometrial Adenocarcinoma-development of tumor vaccination with plasmid DNA containing fusion gene encoding c-erbB2 protein

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We have previously reported that a 185 kDa transmenbrane glycoprotein (p185) coded by c-erbB2 is involved in the invasion and metastasis of human endometrial adenocarcinoma. In this study, we investigated the immunogen activity of plasmid DNAs encoding human c-erbB2 protein in order to evaluate the possibility of using the c-erbB2 gene as an immunogen for tumor vaccination in endometrial adenocarcinoma. The plasmid coding the full-length (pCMV-ferbB2) or the several lengths of the promoter regions of human c-erbB2 was constructed by initially cloning with pCR Bluescript SK(+) vector, and then transferring it into pcDNA3 vector. Expression of c-erbB2 protein was tested by in vitro transfection of 3T3 NIH cells and immunoblot analysis of cells lysate. Female BALB/c mice were boosted in both quadriceps muscles with 20μg of each plasmid threetimes and were bled by tetro-orbital puncture. Serum was stored at -20℃ until assay. Serum antibody responses to human p185 were determined in solid-phase enzyme-linked immunosorbent (ELISA) assay by using a cell lysate of 3T3 NIH cells transfected with pCMV-ferbB2. ELISA assay showed that the injections of all tested plasmids induced lower antibody responses, whereas these plasmids expressed p185. Thus we tried to produce the constructs coding the other regions of c-erbB2, however we have not yet obtained the plasmids which induce higher antibody response.On the other hand, we have recently reported that ezrin, a membrane-cytoskeletal linking protein, is involved in invasive ability of endometrial cancer cells. Ezrin is also identified as tumor-associated transplantation antigen in methylcholanthen-induced murine sarcoma. Thus we now investigate the possibility of using the ezrin gene as an immunogen.