摘要
AD5-10是一株新的鼠抗人死亡受体5(death receptor 5,DR5)单克隆抗体,为本实验室在前期研究中获得,能够强烈诱导多种肿瘤细胞系发生凋亡,抑制小鼠体内移植瘤的生长,对人正常肝细胞和人外周血淋巴细胞没有明显的诱导毒性,已成为研发生物类药物的候选者,具有抗肿瘤作用的应用前景。但是研究发现,某些肿瘤细胞对AD5-10的诱导却表现为明显的抗性,其抗性是否具有一定的表现规律,其内在的调控机制又是什么,仍然是目前一个有待阐明的科学问题。本论文拟选用肺癌细胞系A549、SCLC、H460、H1299和Calu-3,分析细胞表面DR5表达水平与细胞对AD5-10诱导凋亡敏感性之间的关系;并进一步寻找凋亡相关调节分子,研究其调节细胞对AD5-10诱导凋亡敏感性的内在分子机制。本研究所选的五株肺癌细胞中,A549和SCLC细胞对AD5-10中度敏感,其他三株细胞对AD5-10不太敏感。研究表明,这五株肺癌细胞对AD5-10的敏感性与细胞中DR5的表达水平无关。但发现,使用AD5-10处理,敏感或中度敏感细胞中,c-FLIP_L发生明显的切割,c-FLIP_L的表达量显著下降;而不太敏感或抗性的细胞中的c-FLIP_L的表达水平没有明显变化。表明c-FLIP_L是参与调节AD5-10诱导细胞凋亡的关键分子。
进一步研究证明,c-FLIP_L过量表达可以部分抑制人T淋巴细胞白血病细胞SVT35、人肺癌细胞A549对AD5-10的敏感性;而亚毒性浓度的化疗药物环磷酰胺(CHX)、放线菌素D(ActD)能通过下调细胞中c-FLIP_L蛋白表达水平,增强AD5-10对肺癌细胞的杀伤活性。此外,通过c-FLIP_L siRNA特异抑制细胞中的c-FLIP_L的表达,可以显著增强多种肺癌细胞对AD5-10诱导凋亡的敏感性。其调节机制是c-FLIP_L siRNA和AD5-10能激活细胞中caspase依赖的信号通路,下调Bcl-2家族蛋白Bid和Bcl-X_L的表达,进而促进线粒体膜电位的降低以及细胞色素C的释放。并且,研究证明c-FLIP_L siRNA和AD5-10联合作用对正常人细胞没有毒性。
最后,我们检测了人肺癌组织中c-FLIP_L的表达,结果显示c-FLIP_L特异高水平表达于人肺癌纽织中的肿瘤细胞中,而在正常组织和肿瘤细胞周围间质细胞中表达量很低,两者在统计学上具有显著差异。以上研究提示特异下调c-FLIP_L可能是一种非常有潜力的治疗肺癌的策略,特别是针对那些对功能性单抗单独作用产生抗性的肿瘤病人。
第二部分研究中,本文还利用AD5-10可变区基因,构建了表达鼠单链抗体(ScFv)的原核表达载体,并获成功表达。初步证明了它们的肿瘤杀伤活性。同时,我们将AD5-10的可变区基因和人抗体恒定区基因,分别克隆入表达载体pKN100VL和pG1D105VH,通过筛选获得了稳定表达和分泌人-鼠嵌合抗体(anti-DR5 mV-hH)的重组细胞;分泌表达的嵌合抗体与人DR5蛋白具有特异性结合活性,并且能明显杀伤体外培养的人T淋巴细胞白血病Jurkat细胞,为发展人源化抗体药物的研究奠定了基础。
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.
引文
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