【作者】 王伟；

【导师】 高斌；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2012， 博士

【摘要】 单链抗体(Single chain variable fragment)具有分子量小,容易穿透组织等优点。但是,单链抗体亲和力较低,在血清中容易被清除,这些不足限制了单链抗体在临床上的应用。本研究利用人软骨多聚基质蛋白的超卷曲螺旋结构域(coiled coil domain, COMP48)可以形成五聚体的特点,将TRAIL死亡受体4(death receptor4, DR4)和死亡受体5(death receptor5, DR5)的单链抗体进行五聚化,以期延长单链抗体在体内的半衰期,提高单链抗体的亲合力和生物学效应,诱导肿瘤细胞发生凋亡,达到治疗肿瘤的目的。单链抗体可以利用COMP48结构域进行多聚化。单链抗体转化成五聚体后,亲和力得到加大增强。通过BIAcore仪器测量,单链抗体五聚体的亲合力常数KD值可达10-10M,而对应单链抗体的约为10-7M,亲和力提高近一千倍。ELISA分析结果表明,五聚体抗体分别特异识别其对应的受体,未见交叉反应。体外诱导凋亡和细胞抑制实验结果均表明,五聚体抗体与单链抗体相比具有更明显的肿瘤细胞(A54、colo205、HCT-116)杀伤效果,但是对正常细胞如PBMC及人慢性白血病细胞系K562没有杀伤作用,表明正常细胞及部分肿瘤细胞对抗体具有不敏感性。裸鼠荷瘤模型(人结肠癌colo205)结果显示,单链抗体和五聚体抗体均有不同程度的抑瘤效果。针对DR5的五聚体抗体8fcomp治疗效果最好,与其单链抗体形式相比具有显著性差异,但针对DR4的单链抗体4cmono的抑瘤效果好于其五聚体4ccomp,其原因还有待于进一步探讨。更多还原

【Abstract】 Single chain variable fragment (scFv) antibody has relativly small molecular size and is easy to penetrate into solid tumor. However, due to its low affinity and rapid clearance in blood, the usage of scFv is limited. In this study, we utilized COMP48, a coiled coil domain of human cartilage oligomerization protein, to pentamerize a group of agonistic scFv to TRAIL receptors DR4and DR5. As expected, the affinity of scFv increased, half life extended, and finally, efficacy to kill tumor greatly improved.After fusion with COMP48, pentavelant scFv has been successfully formed. The pentavalent scFvs (combody),4ccomp and8fcomp have great improvement in affinity in BIAcore assay. The KD value of4ccomp and8fcomp is about10-10M, while that of the monomeric scFv,4cmono and8fmono is about10-M. ELISA data showed that4ccomp and8fcomp specifically bind to respective receptor without showing apparent cross reactivity. In apoptosis and cell inhibition assays it is indicated that pentavalent scFvs have stronger toxicity on tumor cell lines, such as A549, colo205and HCT-116. In the xenograft tumor model,8fcomp showed best efficacy, but4cmono was better than4ccomp in therapy, which demonstrates that both affinity and penetration efficiency are important for a protein agonist drug against tumor.更多还原