鼠抗人CD40突变体单克隆抗体的研制及其生物学功能的研究

发布时间：2018-06-18 20:30

本文选题：突变体 + CD40　；参考：《苏州大学》2007年硕士论文

【摘要】： CD40是分子量为45～50KD的Ⅰ型跨膜糖蛋白,属于肿瘤坏死因子受体(TNFR)超家族成员,表达于多种抗原递呈细胞、内皮细胞、上皮细胞、造血前体细胞、成纤维细胞以及某些肿瘤细胞。通过单克隆抗体激发CD40分子不仅可以直接作用于表达CD40分子的肿瘤细胞,其介导的信号还可在多个环节影响肿瘤发生、发展,如调控细胞增殖/凋亡,增强其它治疗手段的敏感性,此外还能通过调节适应性和固有免疫应答而起到抗肿瘤作用。然而由于CD40在体内正常组织的广泛表达,三株抗人CD40单抗虽然已进入美国临床前期试验,但其存在的副作用和安全性问题仍引起高度关注。本科室以往的研究发现一些肿瘤细胞株和新鲜肿瘤细胞表达CD40突变体(CD40mu)(CAC→CAA,78His→78Gln) (NCBI Assay Id: ss23134804,Reference SNP Id:rs17177493),该突变位点位于CD40胞外段第二个富含半胱氨酸的区域,这一区域是CD40与CD40L(CD154)相互作用的重要区域。基因转染细胞的构建进一步证实了该突变体与抗体的结合能力与野生型CD40截然不同,CD40mu胞外段一个氨基酸的突变可导致局部结构改变而形成新的抗原表位,该抗原表位为CD40mu所特有,CD40mu在肿瘤中的表达特点和生物学意义以及单抗激发CD40mu对肿瘤细胞生物学行为的影响有待进一步研究。 1.分泌鼠抗人CD40mu单抗的杂交瘤细胞株的制备利用已成功构建的基因转染细胞L929/CD40mu为免疫原,免疫BALB/c小鼠,采用B淋巴细胞融合技术,将反复免疫后的小鼠脾脏细胞与小鼠骨髓瘤细胞SP2/0进行细胞融合,以突变体基因转染细胞L929/CD40mu为阳性筛选细胞,以野生型CD40基因转染细胞L929/CD40wt为阴性筛选细胞,通过间接免疫荧光标记法对杂交瘤细胞株进行筛选,经多次克隆化培养,最终获得四株持续、稳定分泌鼠抗人CD40mu单克隆抗体的杂交瘤细胞株,分别命名为3G5、5H6、3B9和2B6,前三株抗体的重链为IgG1,2B6的重链为IgG2a,轻链均为κ链。四株抗体均可用于免疫组织化学分析,其中3G5和2B6可用于Western-blot检测。杂交瘤细胞株经体外连续传代(40代)培养,液氮冻存半年后复苏,仍生长良好,稳定分泌抗体。 2.鼠抗人CD40mu单克隆抗体的体外生物学特性研究运用成功制备的单抗检测发现,部分恶性B细胞株及上皮性肿瘤细胞株表达CD40mu,其在新鲜肿瘤组织中也有高水平的表达。四株抗体识别的抗原位点不同,对细胞株的识别谱也不相同。继而采用抗CD40mu单抗对表达CD40mu的人卵巢癌细胞株HO8910和人白血病细胞株K562细胞的体外实验初步表明,单抗2B6能够促进HO8910细胞和K562细胞的生长,促使HO8910细胞进入S期的比例增加和细胞因子IL-6的分泌,而另一株单抗3G5则能够诱导表达CD40mu分子的细胞株HO8910细胞凋亡。综上所述,本研究成功制备了四株特异性鼠抗人CD40mu单克隆抗体,并初步探讨了CD40mu在肿瘤中的表达及其生物学意义,证实了肿瘤细胞表达突变的CD40分子;不同的单抗激发表达该突变体的肿瘤细胞后引起的生物学效应也不同,或促进细胞增殖或诱导其凋亡。这些特异性抗体的获得为深入探讨肿瘤细胞表达CD40mu分子的生物学意义提供了物质基础,也为靶向CD40mu的肿瘤生物治疗提供了新思路。
[Abstract]:CD40 is a type I transmembrane glycoprotein with a molecular weight of 45 to 50KD. It belongs to the member of the tumor necrosis factor receptor (TNFR) superfamily. It is expressed in a variety of antigen presenting cells, endothelial cells, epithelial cells, hematopoietic progenitor cells, fibroblasts and some tumor cells. Using monoclonal antibodies to stimulate CD40 molecules can not only directly affect the expression of CD40. Molecular tumor cells, its mediated signal can also affect the occurrence and development of tumors in many links, such as regulating cell proliferation / apoptosis, enhancing the sensitivity of other therapies, in addition to regulating adaptive and inherent immune responses. However, three anti human CD strains are widely expressed in normal tissues in the body. 40 McAbs have entered preclinical trials in the United States, but their side effects and safety problems still attract high attention. Previous studies in the undergraduate laboratory found that some tumor cell lines and fresh tumor cells expressed CD40 mutants (CD40mu) (CAC to CAA, 78His to 78Gln) (NCBI Assay Id: ss23134804, Reference SNP). The mutation loci is located in second cysteine rich regions of the CD40 extracellular segment. This region is an important region of the interaction between CD40 and CD40L (CD154). The construction of gene transfected cells further confirms that the binding ability of the mutant to the antibody is very different from that of the wild type CD40. The mutation of one amino acid in the extracellular segment of CD40mu can lead to the local structure. A new epitope is formed, the epitope of the antigen is unique to CD40mu. The expression and biological significance of CD40mu in the tumor and the effect of CD40mu on the biological behavior of the tumor cells need to be further studied.
Preparation of 1. hybridoma cell lines secreting mouse anti human CD40mu monoclonal antibody
Using the successfully constructed gene transfected cell L929/CD40mu as immunogen, immunized BALB/c mice and using B lymphocyte fusion technique, the spleen cells after repeated immunization were fused with the murine myeloma cells SP2/0, and the mutant gene transfected L929/CD40mu was the positive screening cell, and the transfection of the wild type CD40 gene was fine. The cell L929/CD40wt was negative screening cells, and the hybridoma cell lines were screened by indirect immunofluorescence. After multiple cloning and culture, four hybridoma cell lines that secreted the anti human CD40mu monoclonal antibody were obtained, which were named 3G5,5H6,3B9 and 2B6 respectively. The heavy chain of the first three antibodies was IgG1,2B6 to Ig. G2a, light chain is kappa chain. Four antibodies can be used in immunohistochemical analysis, in which 3G5 and 2B6 can be used for Western-blot detection. Hybridoma cell lines are cultured in vitro (40 generations), and the liquid nitrogen is resuscitated for half a year after the cryopreservation. It still grows well and secretes antibodies steadily.
In vitro biological characteristics of 2. mouse anti human CD40mu monoclonal antibody
Using the monoclonal antibody test prepared successfully, some malignant B cell lines and epithelial tumor cell lines expressed CD40mu, and they also expressed high levels in the fresh tumor tissues. The antigen loci identified by the four antibodies were different and the identification spectrum of the cell lines were different. Then, the anti CD40mu monoclonal antibody was used for the human ovarian cancer cell line HO expressing CD40mu. In vitro experiments of 8910 and human leukemia cell line K562 cells showed that McAb 2B6 could promote the growth of HO8910 cells and K562 cells, increase the proportion of HO8910 cells into S phase and the secretion of cytokine IL-6, while another monoclonal antibody 3G5 can induce apoptosis of HO8910 cells that express CD40mu molecules.
To sum up, four monoclonal anti human CD40mu monoclonal antibodies were successfully prepared, and the expression and biological significance of CD40mu in the tumor were preliminarily discussed, and the CD40 molecules expressed in the tumor cells were confirmed, and the biological effects caused by different monoclonal antibodies to express the mutant tumor cells were different, or promoted. The acquisition of these specific antibodies provides a material basis for exploring the biological significance of the expression of CD40mu molecules in tumor cells, and also provides a new way of thinking for the biological treatment of tumor targeting CD40mu.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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