Lung Cancer Cell Apoptosis Sensitivity Of Anti-human Dr5 Monoclonal Antibody Ad5-10 And Its Molecular Mechanism

Lung cancer is the leading cause of cancer-related mortality in the world.Non-small cell lung cancers (NSCLCs) account for about 75-80% of all types of lungcancers. Surgery offers substantial cure rates for those NSCLC patients with earlystage disease (stagesⅠandⅡ). However, 70% of the lung cancer patients present atstageⅢ/Ⅳ, and most of these patients continue to die of disease progression becauseof resistance to chemotherapeutic drugs or radiation therapy. Clearly, new treatmentstrategies are necessary to improve lung cancer therapy.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is amember of the structurally related TNF family. Up to date, five TRAIL receptors havebeen identified, such as TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1,TRID), TRAIL-R4 (DcR2, TRUNDD) and OPG (osteoprotegerin). Agonisticantibodies against death receptor 4 and 5 are cytotoxic to various cancer cells. In thepresent study, the sensitivity and molecular mechanism of five human lung cancer celllines to previously reported agonistic antibody against DR5, AD5-10, wereinvestigated. Of these cell lines, A549 and SCLC showed a moderate sensitivity toAD5-10 and other three cell lines were resistant. We showed that the varioussensitivities of lung cancer cell lines to AD5-10 were not related to the expressionlevel of DR5, but the expression and cleavage of c-FLIP_L in these cells. Inhibition ofendogenous c-FLIP_L expression by siRNA significantly enhanced AD5-10 inducedcell death in these lung cancer cells. And this sensitizing effect was associated withdecreased expression of Bid and Bcl-X_L, change of mitochondrial membrane potential,release of cytochrome c from mitochondria, and caspase activation. Therefore, thesedata provided evidences that c-FLIP_L expression and cleavage are involved in thesensitivity to DR5-mediated lung cancer cell apoptosis. Moreover, we demonstratedthat c-FLIP was expressed in 87.9% (29 of 33) of the tissues from lung carcinoma patients, but little in the normal controls, suggesting that inhibition of c-FLIPexpression might be a potential strategy for lung cancer therapy.In the present study, we have successfully constructed the single chain fragmentvariable (scFv) and human/mouse chimeric antibody of AD5-10, a functionalanti-DR5 MAb. The scFv gene of anti-DR5 MAb was amplified and cloned into thepET-15b, then expressed in E. coli. Then detect the tumoricidal activity of the purifiedscFv protein by MTS-PMS assay. The results showed that purified anti-DR5-ScFvinduces tumor cell cell death in vitro. And the VL and VH genes of anti-DR5 MAbwere amplified and cloned into the light and heavy chain expression vectorsrespectively, then the recombinant plasmids were co-transfected into CHO-dhfr~- cellsfor expression. Screen for the positive clone by the two selective genes (neo and dhfr).Then identify the humanization and specificity of chimeric antibody by ELISA andweatern blot. And detect the tumoricidal activity of the expressed chimeric antibodyby MTS-PMS assay. The expression vectors can stably express chimeric antibody inCHO-dhfr~- cells. In the cell supernatant of the F4'clone, we identified the human IgGheavy constant region Cγl and light constant region Cκ. Moreover, the secretedchimeric antibody retained the binding capacity to the antigen (DR5) and decreasedthe cell viability of Jurkat cells by 26.85% in vitro. The human/mouse anti-DR5chimeric antibody has been expressed successfully in eukaryotic cells, which has thehuman IgG constant region, antigen binding specificity and tumoricidal activity. Thisstudy provides the evidences that gene engineering antibody against TRAIL receptorDR5 has potential application in the future of tumor antibody therapy.