Preparation And Antitumor Effect Of Anti-DR5 Monoclonal Antibody

BackgroundTNF-related apoptosis-inducing ligand (TRAIL) is a TNF family member capable of inducing apoptosis. As a type II membrane protein, it has the ability of inducing apoptosis, which was happened rapidly in susceptible cells including both malignant and some transformed cells. But there is report that the different recombinant versions TRAIL has different effect in anti-tumor and cytoxicity. And death receptor 5 (DR5) is a key receptor of TRAIL and it plays an important role in TRAIL-induced apoptosis. Specific targeting of DR5 by agonistic antibodies might be advantageos over rTRAIL administration if tumor cells are protected from rTRAIL-induced apoptosis by the expression of cell surface decoy receptors. Focus on the development of the anti- DR5 monoclonal antibody with apoptosis inducing ability can be the ideal candidate was a good direction to overcome the side effects of TRAIL. Although some anti-DR5 antibodies were prepared, and an anti-DR5 mAb has been used in phase ? clinic trials, and it has been considered a promising approach, so far, there are is still no antibody against DR5 was available on the medicine market.ObjectiveTo study the monoclonal antibody of DR5 biological effect and the effective mechanism; then analyze the interrelation of the ability of inducing apoptosis of anti-DR5 mAb(WD1) and DR5 activation in cells.MethodsE.coli BL21 was transfected with recombinant vector pET30a/DR5 and induced with 0.1mM IPTG, The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE,Western blot and mass spectrum of protein. sDR5 was used as antigen to immunize Balb/c mice. The spleen cells of the immunized mice were used to prepare the mAb by hybridoma techniques. Western Blot and immunofluorescence demonstrated that the mAb could bind to sDR5 and mDR5. MTT results showed that WD1 could suppress the cells growth. Giemsa's staining,AnnexinⅤ/PI results showed the apoptosis effect of Jurkat cells that WD1 affected. The cDNA sequences of human DR5 intracellular were amplified by RT-PCR techmique from Jurkat and Daudi cells, the validity of the sequences was confirmed. Immunoprecipitation of DISC showed that the relationship of the formation of DISC and the ability of DR5 activation by WD1.ResultsE.coli BL21 was transfected with recombinant vector pET30a/DR5 and induced with 0.1mM IPTG, then sDR5 was purified by nickel affinity chromatography. SDS-PAGE and mass spectrum of protein sDR5 analysis demonstrated that there existed a recombinant protein, and its relative molecular weight was accordance with that of our expected. Western-blot analysis indicated that the recombinant protein can be recognized by anti-DR5 mAb.One positive clone was selected to produce antibody, WD1.Western Blot and immunofluorescence demonstrated that WD1 could bind recombinant sDR5 and membrane-bound DR5 (mDR5) on Jurkat, Molt-4, and Daudi cells. MTT assays showed that Jurkat and Molt-4 cells were sensitive to the antibody in a dose dependent manner, but Daudi cells were resistant to WD1. 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.The cDNA sequences of human DR5 intracellular were amplified by RT-PCR techmique from Jurkat and Daudi cells, the validity of the sequences was confirmed by automatic DNA sequencing and the sequences were correct completely. It demonstrated that the human DR5 intracellular protein in Jurkat and Daudi cells were not mutation. Immunoprecipitation of DISC showed that using WD1, FADD and capase-8 were detected in DR5 receptor complex of Jurkat cells, while the Daudi cells that were resistant to WD1–induced cell death were unable to recruit FADD and caspase-8 to DISC.Conclusions(1)gain the DR5 extracellular fragment proteins with bioactivity.(2)acquire an anti-DR5 monoclonal antibody (mAb)-WD1 that could induce cell apoptosis.(3)the cause of inducing Jurkat cell apoptosis is the formation of DISC.