Characterization of a Novel Anti-DR5 Monoclonal Antibody WD1 with the Potential to Induce Tumor Cell Apoptosis

• Published in: Cellular & molecular immunology 2008-02, Vol.5 (1), p.55-60
• Main Authors: Wang, Jing, Lin, Zhou, Qiao, Chunxia, Lv, Ming, Yu, Ming, Xiao, He, Wang, Qingyang, Wang, Liyan, Feng, Jiannan, Shen, Beifen, Ma, Yangfang, Li, Yan
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Animals; Annexin V; Antibodies; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antibodies, Monoclonal - metabolism; Apoptosis; Biomedical and Life Sciences; Biomedicine; Cell Line, Tumor; Complementary DNA; Enzyme-linked immunosorbent assay; Escherichia coli; Humans; Immunofluorescence; Immunology; Jurkat Cells; Medical Microbiology; Mice; Mice, Inbred BALB C; Microbiology; Molting; Monoclonal antibodies; Nickel; Polymerase chain reaction; Receptors, TNF-Related Apoptosis-Inducing Ligand - immunology; Receptors, TNF-Related Apoptosis-Inducing Ligand - isolation & purification; Receptors, TNF-Related Apoptosis-Inducing Ligand - metabolism; RNA-directed DNA polymerase; TNF-Related Apoptosis-Inducing Ligand - immunology; TNF-Related Apoptosis-Inducing Ligand - metabolism; TRAIL protein; Transfection; Tumor necrosis factor; Tumors; Vaccine; 免疫系统; 单克隆抗体
• ISSN: 1672-7681; 2042-0226
• DOI: 10.1038/cmi.2008.7

TNF-related apoptosis-inducing ligand （TRAIL） is a TNF family member capable of inducing apoptosis. Death receptor 5 （DR 5） is a key receptor of TRAIL and plays an important role in TRAIL-induced apoptosis. To prepare monoclonal antibodies （mAbs） against DR5, cDNA encoding soluble DR5 （sDR5） was firstly amplified by reverse transcriptase-polymerase chain reaction （RT-PCR） with specific primers, and then inserted into a prokaryotic expression vector pET-30a. The recombinant plasmid was expressed in Escherichia coli strain BL21 （DE3）, and sDR5 was purified by nickel affinity chromatography. As an antigen, sDR5 was used to immunize mice. Hybridomas secreting antibodies against sDR5 were identified. One positive done was selected to produce antibody, WD1. ELISA and immunofluorescence demonstrated that WD1 could bind recombinant sDR5 and membranebound DR5 （mDR5） on Jurkat and Molt-4 cells. ATPLite assays showed that Jurkat and Molt-4 cells were sensitive to the antibody in a dose dependent manner. The Annexin V/PI assays and Giemsa＇s staining both showed that WD1 could induce Jurkat cell apoptosis efficiently. Transient transfection of 293T cells and indirect immunofluorescence assay demonstrated that mAb （WD1） couldn＇t cross-react with DR4. Our findings indicated that the novel antibody, WD1 could act as a direct agonist, bind DR5 characteristically, and initiate efficient apoptotic signaling and tumor regression. Thus, WD1 would be a leading candidate for potential cancer therapeutics. Cellular ＆ Molecular Immunology.