Preparation Of Anti-he4 Monoclonal Antibodies For Detection Of Ovarian Cancer

Ovarian cancer is the leading cause of death from gynecologic malignancy among women worldwide. Early stage ovarian cancer has an excellent prognosis if properly evaluated. Unfortunately, 70~80% of patients could not be found until the cancer progressed to the late stages, and their 5-year survival rate is lower than 20%. In contrast, if we can detect and treat the cancer at early stage, when tumor is found to be only in the ovary, the 5-year survival rate is up to 70~90%. Therefore, a sensitive and specific early detection method is no doubt appealing.In the present, the only clinically accepted serum marker for ovarian cancer is CA125. Numerous studies showed that the sensitivity and specificity of CA125 is not high enough for a screening test. HE4 (human epididymis protein 4) is one of the most promising new serum biomarkers for ovarian cancer, which is a glycoprotein and first detected in human epididymal epithelium. The biological function of HE4 is still unknown although some research suggested it may play a role of protease. HE4 is highly expressed by ovarian carcinomas and serum HE4 has been shown to elevate in both early and advanced stage endometrial cancers. Clinical studies also confirmed that the combination of serum HE4 and CA125 raised the sensitivity of detection compared to that of CA125 alone. Therefore, the objectives of this study were to obtain recombinant HE4 and its antibodies for developing serum HE4 assay methods, which would provide more powerful tool for early detection and prognosis supervision of ovarian cancer.During the antibodies preparation methods, animals were commonly immunized with purified protein. But sometimes it is difficult to obtain a quantity of purified proteins enough for immunization, and the recombinant proteins might be deficient in immunogenicity to initiate immune reaction, such as inclusion body proteins expressed by E.coli. DNA immunization via electroporation could make a way to produce antibodies without proteins preparation. As we known, the proteins expressed in vivo have structures more similar to its natural structure. Electroporation could help the DNA taken up by the cells, prolong its expression, and the continually expressed protein could provoke and boost the memory lymphocyte responses. In addition, electroporation causing local tissue inflammation and large numbers of inflammatory cell infiltration improved the antigen presentation. Therefore, we will try to obtain high titer antibodies by DNA immunization via electroporation in vivo.We obtained the cDNA encoding HE4 from ovarian cancer tissues by RT-PCR. The amplified product was inserted into PMD19T vector via T-A complement and then subcloned into pPICZαA and pcDNA3.1(+), pichia pastoris and mammalian cell expression vectors, respectively. Recombinant plasmids were identified by PCR, restriction enzyme digestion and sequencing. The indentified pPICZαA-HE4 plasmid was then transformed into pichia pastoris GS115 by electroporation and positive recombinant strains were screened by PCR and induced expression. The expressed recombinant protein was identified by ELISA and Western-blot with commercial anti-HE4 monoclonal antibody. The expression conditions were also optimized for obtaining a quantity of recombinant HE4 to develop a quantitative method.On the other hand, the plasmids constructed were also used to immune BALB/c mice via electroporation in vivo. The hybridomas were achieved by fusing the immunized spleen cells with Sp2/0 myeloma cell line and the positive clones secreting anti-human HE4 antibodies were screened by further subcloning. The secreted antibodies properties were determined by Western blot, Ig subtyping and epitope analysis.The results showed that we obtained the cDNA encoding HE4 from ovarian cancer tissues and successfully constructed the pcDNA3.1-HE4 and pPICZαA-HE4 eukaryotic expression vector. The secretory expression of HE4 protein could be detected from, the recombinant pichia strains transformed with pPICZαA-HE4 and the recombinant HE4 was glycosylated with special confirmations; The expression level of recombinant HE4 reached the highest after 12 hours induced by 2% concentration of methanol, at pH 4.0 and 26℃. After purification, 0.413 mg/mL recombinant HE4 protein was obtained from 1 liter culture medium. At the same time, we obtained two hybridoma cell lines, named 1-7-C and 3-12-C respectively, which could continuously and stably secrete specific anti-HE4 monoclonal antibodies. The antibodies titers were 1:160 and 1: 320 respectively. ELISA and Western-blot assay showed that the monoclonal antibodies recognized the HE4 protein with natural conformation. Subgroup assay showed that they are both IgM antibodies, and their light chains were typeλ.Therefore, the research successfully developed the engineered pichia pastoris strains, which could produce a quantity recombinant HE4 with special conformation. It is valuable in the future studies for its diagnostic and therapeutic application. In addition, two mouse anti-human HE4 hybridomas were obtained by DNA immunization and they secreted antibodies specifically recognize the recombinant HE4 which made it possible to develop a quantitative method for detection of serum or urinary HE4 level.