Expression Of Death Receptor 5 In Colon Cancer Tissues And Cloning Of A Novel Human Anti-death Receptor 5 Antibody

[Background]For recent years, the incidence of colon cancer has been increasing. Although an obvious progress on clinical diagnosis, operational skills, and comprehensive adjunctive therapies such as chemotherapy, radiotherapy etc. has been improved constantly, the five-year survival rate and cure rate of colon cancer have no impressive improvement. In recent years, tumor biological immunotherapy with the advantages of precise targeting, high efficiency and hypo-toxicity has been gained more attention and may become a promising therapy method against tumor. Death Receptor 5 (DR5) is a member of Tumor Necrosis Factor receptor (TNF-receptor) family. It mainly distributes on the surface of different tumor cells, while less or not expressed in normal cells. Binding of TNF-Related Apoptosis Inducing Ligand (TRAIL) and DR5 induces the activation of caspase-3, which can specifically induce the apoptosis of sensitive tumor cells. All these characteristics make DR5 as a significant target in tumor biological therapy.Phage antibody library (pAb library) built in 1990s is a new technology to prepare human antibodies. This technology bypassed the traditional hybridoma method to prepare monoclonal antibodies (mAb), even without immunization procedure. Along with the approval and clinical use of a humanized antibody "Humira", developed by Abbott Laboratories Co., Ltd. using phage display technique, on treating rheumatic arthritis pAb library technology has become an important platform on the research and development of therapeutic antibodies.[Aims]Using immunohistochemistry method to determine the difference of DR5 expression between normal colon, colon tubular adenoma and colon tubular adenocarcinoma and to explore the relationship of DR5 expression in colon tubular adenocarcinoma with gender, tumor size, depth of invasion, lymph node metastasis, pathologic type, histological type and clinical prognosis of TNM, in order to make a preliminary judgment of whether DR5 can become a potential target in tumor biological immunotherapy. Then we screened human single chain antibodies against DR5 from a natural phage antibody library and tested their binding activity with the antigen.[Methods]HE staining was used to confirm the pathological diagnosis of test samples and immunohistochemistry was used to detect the expression of DR5 in normal colon, colon tubular adenoma, colon tubular adenocarcinoma and mucinous adenocarcinoma of colon. Using plastic tubes coated with purified eukaryotic DR5-Fc protein, specific phage antibodies were cloned from a large phage antibody library by four rounds of screening. Then soluble antibodies were obtained by isopropyl-beta-D-thiogalactopyranoside (IPTG) induced expression and the binding activity of antibodies were identified by ELISA, immunocytochemistry and immunohistochemistry and the obtained antibodies were analyzed by DNA fingerprinting and sequencing.[Results]1. The expression of DR5 in normal colon, colon tubular adenoma and adenocarcinoma of colonThe expression of DR5 in normal colon tissues were lower than in colon tubular adenoma and colon tubular adenocarcinoma, the tissues of colon tubular adenocarcinoma and colon tubular adenoma had a relatively high expression of DR5, but the expression between them had no statistic difference.2. The relationship between DR5 expression and clinical pathological features of colon tubular adenocarcinomaThe expression of DR5 in colon tubular adenocarcinoma was related to the tumor TNM clinical prognostic stages, but had no significant relationship with the patients’ gender, tumor sizes, types, histological types, invasive stages and lymph node metastasis. There was a clear downward trend in the expression of DR5 in the late and terminal stages(Ⅲ, Ⅳ) of colorectal tubular adenocarcinoma compared with the early and middle stages(Ⅰ, Ⅱ).3. Screening of anti-DR5 human antibody using large natural pAb libraryAfter 4 rounds of screening, pAb was significantly enriched. Through ELISA detection,59 out of the 80 colonies had binding activity with DR5-Fc,46 out of which were specific binding colonies, with a positive rate of 73.6%. The 46 phage positive colonies were then used to prepare soluble scFv, and 26 out of them showed specific binding with DR5-Fc.4. Further analysis of the positive clonesThe 26 positive clones were analyzed by fingerprinting and 4 different fingerprinting patterns were obtained. Sequence analysis showed that the obtained four antibodies stem from different embryonal system, the variable regions of light chain belong to VK3, VL1, VL2 subgroup, and the variable regions of heavy chain belong to VH1, VH3 subgroup.5. Identification and selection of antibody binding activity by antigen ELISA cells, immunocytochemistry and immunohistochemistryThe phage antibodies and soluble antibodies derived from the 4 positive clones specifically bind with DR5 protein expressed in cells.[Conclusion]1. The expression of DR5 in normal colon tissues is lower than that in colon cancer tissues with significant difference, suggesting that DR5 may be a promising target for cancer biological immunotherapy.2. The expression of DR5 in colon tubular adenoma is significantly increased comparing with in normal colon tissue, suggesting that DR5 may play certain role in precancerous lesion formation.3. The expressions of DR5 are higher at the early and middle stages of colon tubular adenocarcinoma than those at late and terminal stages of colon tubular adenocarcinoma providing a preliminary clue for the individualized use of DR5 antibody in future clinical trials.4. Four specific human scFvs against DR5 were obtained from a large natural phage antibody library.5. The phage antibody and the soluble antibody derived from the four genotypes of positive clones possess well cell binding activity.