高亲和力的人源化抗CD20抗体的构建及生物学功能研究

发布时间：2018-03-22 06:31

本文选题：CD20分子　切入点：抗体　出处：《聊城大学》2017年硕士论文　论文类型：学位论文

【摘要】：非霍奇金淋巴瘤是一种常见的B细胞型淋巴瘤。现代医疗技术中的放化疗技术已经不能满足人类迫切治疗该类疾病的渴求,而具有靶向治疗机制的单克隆抗体方法开始被人们广泛研究和应用。研究发现CD20分子表达于NHL细胞表面,则此分子是靶向治疗的理想靶点。利妥昔单抗(Rituxiamb)是第一个被美国食品药品监督管理局(FDA)批准上市并用于治疗NHL的单抗药物。但因Rituximab分子中含有30%的鼠源序列,患者在长期用药以后,易引起人抗鼠抗体免疫应答(HAMA)而产生严重的不良反应。同时,Rituximab的临床反应率只有50%,完全缓解率仅有10%。因此,严重制约了Rituximab在临床中的长期应用。针对这一现象,人们开始对Rituximab进行改造,有的用与鼠源序列相似的人骨架区来替换Rituximab中的鼠源框架区,有的用提高Rituximab的亲和力来加强抗肿瘤活性。如将两者同时进行改造,则杀伤肿瘤细胞的效果有可能比只是改造其中一方面的效果要好。目的:改造新型的抗体可以从亲和力和免疫原性两方面进行,可通过提高亲和力来增加抗原与抗体的识别和结合,对于免疫原性主要是通过降低抗体中的鼠源序列来减少在人体内的抗性。本文旨在构建较高亲和力和较低免疫原性的CD20抗体,并验证是否具有相应的生物学活性。方法:1.通过专利分析,进行高亲和力和人源化的设计改造,将CD20H和CD20L分别克隆到真核表达载体pc DNA3.1(+)和pc DNA3.1/ZEO(+)中去,利用转染试剂jet PEI将轻重链表达载体共转染中国仓鼠卵巢细胞CHO-K1,通过有限稀释法进行单克隆细胞株的筛选,通过逆转录PCR检测m RNA,ELISA检测表达量,并将贴壁细胞驯化成悬浮培养。2.利用流式细胞术检测细胞上清与CD20阳性细胞Raji的结合活性,并对其生物学功能进行检测,如细胞杀伤活性、ADCC效应以及CDC效应。结果:1.获得了目的抗体轻链和重链的基因,构建重组表达载体pc DNA3.1/ZEO(+)-CD20L和pc DNA3.1(+)-CD20H,可在CHO-K1细胞中稳定表达,并筛选了17株单克隆细胞株,RT-PCR证明目的基因在CHO细胞中成功的表达,ELISA法测得培养上清的含量在0.45-4.72μg/m L之间,单抗细胞株在无血清培养基中能够悬浮生长。流式检测表明上清中的抗体可与Raji细胞结合,同时培养上清具有较好的ADCC与CDC杀伤活性,但直接的细胞杀伤活性的结果并不明显。
[Abstract]:Non-Hodgkin 's lymphoma is a common type of B-cell lymphoma. However, monoclonal antibody methods with targeted therapeutic mechanism have been widely studied and applied. It has been found that CD20 molecules are expressed on the surface of NHL cells. Rituxiamb was the first drug to be approved by the U.S. Food and Drug Administration for use in the treatment of NHL. But because the Rituximab molecule contains 30% of the mouse origin sequence, After a long period of administration, patients are prone to serious adverse reactions caused by human anti-mouse antibody immune response (Hama). Meanwhile, the clinical reaction rate of Rituximab is only 50, and the complete remission rate is only 10. The long-term application of Rituximab in clinical practice has been seriously restricted. In response to this phenomenon, people began to modify Rituximab, and some replaced the rodent frame region in Rituximab with a human skeleton region similar to the murine origin sequence. Some increase the affinity of Rituximab to enhance its antitumor activity. The effect of killing tumor cells is likely to be better than that of just modifying one of them. Objective: the modification of new antibodies can be carried out both in terms of affinity and immunogenicity. The recognition and binding of antigens and antibodies can be enhanced by increasing affinity. The aim of this study is to construct CD20 antibodies with high affinity and low immunogenicity. Method: 1.Through patent analysis, the design of high affinity and humanization was modified, and CD20H and CD20L were cloned into eukaryotic expression vectors pcDNA3.1 () and pcDNA3.1 / ZEO (), respectively. Chinese hamster ovarian cell line CHO-K1 was co-transfected with jet PEI. The monoclonal cell lines were screened by limited dilution method, and the expression levels of CHO-K1 were detected by reverse transcription PCR (RT-PCR) and mRNA-ELISA. The adherent cells were domesticated into suspension culture. 2. Flow cytometry was used to detect the binding activity of the supernatant to Raji and its biological function. Results: 1. The gene of light chain and heavy chain of target antibody was obtained, and the recombinant expression vector pcDNA3.1% ZEO (pc-CD20L and pc-CD20H) was constructed, which could be expressed stably in CHO-K1 cells. In addition, 17 monoclonal cell lines were screened by RT-PCR. The successful expression of the target gene in CHO cells was determined by Elisa. The supernatant of culture was between 0.45-4.72 渭 g / mL. Flow cytometry showed that the antibody in the supernatant could bind to Raji cells, and the supernatant had better ADCC and CDC killing activity, but the results of direct cytotoxicity were not obvious.
【学位授予单位】：聊城大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.1

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