重组抗CD20单克隆抗体工程细胞珠的构建

发布时间：2018-12-11 20:14

【摘要】：恶性淋巴瘤是淋巴结和结外部位淋巴组织的免疫细胞肿瘤,来源于淋巴细胞或组织细胞的恶变,其中非霍奇金淋巴瘤(Non-Hodgkin′s lymphoma,NHL)较难治愈。经过放化疗目前该病的治愈率仅有25%,因此对于治疗复发性、顽固性低度或滤泡性非霍奇金淋巴瘤的治疗急需新的治疗方案。在治疗B细胞淋巴瘤时,CD20分子是理想的靶抗原,表达于95%以上正常或恶化的B细胞表面。它起始表达于前B淋巴细胞(pre-B)阶段,到B细胞终端分化成浆细胞前结束,一直被认为是B系细胞表面特有的标识。抗CD20单抗与B细胞上的CD20结合,并引发B细胞溶解的免疫反应,细胞溶解的可能机制包括补体依赖的细胞毒作用(CDC)和抗体依赖的细胞介导的细胞毒作用(ADCC)[2]。使用针对B细胞淋巴瘤抗原CD20分子的单克隆抗体是治疗非霍奇金淋巴瘤的重要方案。经过全球的多中心临床试验证实具有明显提高肿瘤患者生存率、显著提高患者生命质量、延长生命周期的疗效,与目前国际上采用的标准肿瘤治疗药物比较,具有毒副反应小的特点。迄今为止全球至少50万非霍奇金淋巴瘤患者接受了抗CD20单克隆抗体的治疗,而作为全球最成功的抗体药物之一,由罗氏公司生产的用于治疗非霍奇金淋巴瘤的利妥昔单抗注射液(中国进口注册商品名“美罗华”)[[I]],在2011年的全球销量达到约60亿美元,由此可见,开发此类药物将会为肿瘤患者带来极大的益处,并会为开发此类药物的生物技术公司带来巨大的的经济和社会收益,具有广阔的市场前景。本项目旨在构建一种工程细胞珠,使其表达的活性成分即:重组抗CD20单克隆抗体具有与利妥昔单抗相同的氨基酸序列。根据文献资料参照美罗华合成了包含信号肽在内的重组抗CD20单克隆抗体的轻链和重链基因片段,并将其分别插入到p REA202哺乳动物细胞表达载体中,构建表达抗体轻链和重链的载体p REA202/REA202L和p REA202/REA202H。将构建的表达抗体轻链和重链的载体经脂质体共转染CHO-DG44细胞,经G418抗性筛选阳性克隆,再通过ELISA筛选高表达抗体的的克隆,之后用MTX逐步加压筛选、克隆,最终获得一株高表达抗体的细胞株CHO-REA202。对该工程细胞株的初步鉴定表明,表达产物为完整的抗CD20单抗,抗体表达能力可以达到20至23PCD(pg/cell/day)。以该细胞株建立细胞库,工作细胞库细胞复苏后,进行连续传代培养,考察工程细胞的稳定性。根据研究结果,将种子细胞培养阶段传一次作为一代,生产培养阶段每培养48小时作为一代。细胞传代试验共传代60代,分别取传代0、10、20、40、60代的细胞进行各项考察。结果表明在CHO-REA202的传代过程中:REA202蛋白轻重链基因的DNA序列未发上改变;REA202蛋白轻重链基因的拷贝数水平未发生明显改变;细胞的生长特性、倍增时间和形态未发生明显改变;REA202蛋白的表达水平未发生明显改变。该结果表明工程细胞CHO-REA202具有良好的传代稳定性,符合《中国药典》中对于细胞的传代稳定性的要求。
[Abstract]:Malignant lymphoma is an immune cell tumor of the lymphoid tissue of the lymph node and the junction, which is derived from the malignant transformation of lymphocytes or histiocytes, of which the non-Hodgkin's lymphoma (NHL) is more difficult to cure. The cure rate of the disease is only 25% after radiotherapy and chemotherapy, and therefore, a new treatment scheme is needed for the treatment of recurrent, intractable low-grade or follicular non-Hodgkin's lymphoma. In the treatment of B-cell lymphoma, the CD20 molecule is an ideal target antigen, expressed above 95% of the normal or deteriorated B-cell surface. It is initially expressed in the pre-B-lymphocyte (pre-B) stage, and the end of the differentiation of the B-cell terminal into the plasma cell has been considered to be a unique identification of the surface of the B-cell. Anti-CD20 monoclonal antibodies bind to CD20 on B-cells and elicit an immune response to B-cell lysis, which may include complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)[2]. The use of monoclonal antibodies against B-cell lymphoma antigen CD20 molecules is an important protocol for the treatment of non-Hodgkin's lymphoma. Through the global multi-center clinical trial, the survival rate of the tumor patients is obviously improved, the quality of life of the patients is obviously improved, the curative effect of the life cycle is prolonged, and the drug has the characteristics of small toxic and side reaction, compared with the standard tumor treatment drugs adopted at present. To date, at least 500,000 non-Hodgkin's lymphoma patients have received treatment with anti-CD20 monoclonal antibodies, one of the world's most successful antibody drugs, Rituximab for treatment of non-Hodgkin's lymphoma, produced by Roche,[[I]], has a global sales of around $6 billion in 2011, and it is clear that the development of such a drug will provide significant benefits for patients with tumors, and can bring huge economic and social benefits to the biotechnology company for the development of such drugs, and has a wide market prospect. The purpose of this project is to construct an engineering cell bead to express the active ingredient, that is, the recombinant anti-CD20 monoclonal antibody has the same amino acid sequence as the rituximab. A light chain and a heavy chain gene fragment of a recombinant anti-CD20 monoclonal antibody, including a signal peptide, was synthesized according to the literature, and inserted into a pREA202 mammalian cell expression vector, respectively, to construct a vector p REA202/ REA202L and a p REA202/ REA202H expressing an antibody light chain and a heavy chain, respectively. CHO-DG44 cells are co-transfected with the vector of the constructed expression antibody light chain and the heavy chain through the liposome, the positive clone is screened by the G418 resistance, the clone of the high-expression antibody is screened by ELISA, and then the cell strain CHO-REA 202 with a high expression antibody is finally obtained by using the MTX step-by-step pressure screening and cloning. The preliminary identification of the engineering cell line shows that the expression product is intact anti-CD20 monoclonal antibody, and the expression ability of the antibody can reach 20 to 23PCD (pg/ cell/ day). the cell bank is established by the cell line, and after the cell bank of the working cell is recovered, the cell bank is continuously transmitted and cultured, and the stability of the engineering cell is examined. According to the results of the study, the seed cell culture stage was transferred to one generation, and the production and culture stages were each incubated for 48 hours as a generation. The cells were passaged for 60 passages, and the cells of 0, 10, 20, 40 and 60 passages were collected. The results showed that in the course of the passage of CHO-REA202, the DNA sequence of the heavy chain gene of the REA202 protein was not changed; the level of the copy number of the heavy chain gene of the REA202 protein did not change significantly; the growth characteristics, the doubling time and the morphology of the cells did not change significantly; The expression level of the REA202 protein did not change significantly. The results show that the CHO-REA202 of the engineering cell has good passage stability and is in accordance with the requirements for the passage stability of the cell in the Chinese Pharmacopoeia.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.1

【相似文献】