抗Aβ人源性抗体的制备

发布时间：2018-08-28 16:07

【摘要】： 阿尔茨海默病( Alzheimer’s disease, AD)是一种多发于老年人群的中枢神经系统退行性疾病,主要临床特征为记忆衰退、认知及运动功能产生障碍等。随着全球人口老龄化的加剧,阿尔茨海默病的发病几率正在不断上升,由此引发的一系列社会问题正在日益加剧。目前对于AD的发病尚缺乏有效的治疗手段,因此研制能够治疗AD的特效药物具有重要的意义。现阶段关于AD的发病机理还不十分清楚,其中β样淀粉蛋白(β-amyloid protein,Aβ)由正常生理状态下的可溶性单体形式聚集为具有神经细胞毒性的淀粉样蛋白沉积被认为是在AD发病过程中的关键事件。有研究表明:Aβ是各种因素引起阿尔茨海默病的共同途径,是AD发病和发展的关键因素,因此Aβ被普遍认为是治疗AD的有效靶分子。Aβ是由主要存在于脑组织中的β-淀粉样前体蛋白(β-amyloid precursor protein ,APP)经过β代谢途径形成的。一般由39～43个氨基酸组成,具有一个β折叠片层的二级结构,分子整体呈现疏水性,容易聚集,形成不溶性沉积。1999年,Schenk等首次应用Aβ1-42对AD模型小鼠进行主动免疫取得了突破性进展。此后陆续有研究报道证实Aβ肽段及其抗体可应用于AD的免疫治疗,并能够得到很好的治疗效果。由Elan公司研制的Aβ1-42疫苗Betabloc(AN1792)经过I期临床试验后,发现Betabloc能有效的激发免疫应答,产生与Aβ淀粉样沉积结合的抗体,能够有效的激活小胶质细胞清除Aβ淀粉样沉积形成的斑块,并且显著改善患者的认知功能。但遗憾的是,在Ⅱ期临床实验过程中,部分患者脑部出现炎症反应,试验被迫终止。与此同时对于AD的被动免疫治疗方面取得了巨大的进展,研究证实应用抗AβN端抗体可以有效地改善AD的相关症状,并且不会引发Th1细胞的免疫反应。Elan和惠氏公司联合研制的抗AβN端的人源化抗体Bapineuzumab是第一个开发用于治疗AD的抗体,目前正在进行Ⅲ期临床试验。本研究中,我们希望以Aβ1-42及其N端(Aβ1-12、Aβ1-15)为靶标分子通过采用细菌筛选结合固相筛选等多种筛选方案对大容量人源性噬菌体抗体库进行筛选以获得特异性抗体,为开发AD的治疗性抗体奠定基础。首先,本研究中为了针对Aβ1-12进行抗体筛选以及为筛选获得的特异性抗体进行初步的表位鉴定提供表位肽段,我们根据Aβ1-42的分子结构及细菌鞭毛表面展示系统的特点,将Aβ1-42分为Aβ1-12、Aβ12-23、Aβ21-32、Aβ31-42四种表位肽段,分别构建可表达四种肽段与细菌鞭毛蛋白融合表达的重组质粒,并利用细菌鞭毛表面展示系统将其分别表达于E.coli GI826鞭毛上。SDS-PAGE电泳、Western blot及免疫荧光鉴定结果证明了四种Aβ抗原表位肽段与鞭毛蛋白融合表达并成功的展示于E.coli GI826表面。其次,我们对Aβ1-42及其N端(Aβ1-12、Aβ1-15)进行了人源性抗体的高通量筛选。目前AD被动免疫研究所应用的抗体多为鼠源性抗体,限制了其在人体中的应用。而从人源性抗体库中获得的抗体则避免了这一问题,可不经人源化改造直接应用于人体。本研究中我们分别采用了针对Aβ1-12的细菌扣除筛选、针对Aβ1-42的固相筛选、针对Aβ1-12的细菌固相结合筛选、以及针对Aβ1-15的固相筛选四个筛选方案对库容量可达1.35×1010的大容量全合成人源性噬菌体单链抗体库进行了特异性抗体的筛选,实现了抗体的高通量筛选。在筛选过程中共随机挑选、鉴定了约2000个克隆,其中细菌筛选克隆阳性率较低,固相筛选克隆阳性率可达50%,但是由于固相筛选是针对Aβ1-42全长进行筛选,获得的特异性抗体可能与Aβ1-42的任意区域结合。为了针对AβN端进行高质量的抗体筛选,我们采用了先针对Aβ1-12进行细菌扣除筛选再应用Aβ1-42进行固相筛选的方案,针对Aβ1-12进行了细菌固相结合筛选,克隆阳性率可达40%。与此同时我们也针对Aβ1-15进行了固相筛选,但克隆阳性率较低。最终我们在细菌筛选过程中,获得了一株特异性噬菌体抗体G10。在Aβ1-42固相筛选过程中,获得了两株特异性噬菌体抗体18、26。在细菌固相结合筛选过程中我们获得了两株特异性噬菌体抗体H9、B5。在Aβ1-15固相筛选过程中获得了一株特异性抗体87。其中18与H9为同一株抗体,这一结果证明了我们所采用的方案可靠且有效。再次,我们将获得的特异性单链抗体改造为全抗体形式并对获得瞬时表达的全抗体进行了特异性鉴定。为了获得稳定性更好的抗体形式,我们对筛选得到的5株特异性噬菌体单链抗体进行了全抗体形式的改造,并对这5株全抗体在293-F细胞中进行了瞬时表达。结果18及87号抗体未能得到表达,G10、26、B5获得了表达。特异性鉴定结果发现G10失去了与Aβ1-42的结合能力,26、B5能与Aβ1-42特异性结合,最终我们获得了26和B5两株特异性的全抗体。最后,我们对26和B5这两株全抗体进行了表位鉴定,并对能与AβN端(Aβ1-12、Aβ1-15)结合的抗体进行了亲和力检测。利用细菌鞭毛表面展示的Aβ分段肽段对26和B5进行了表位鉴定,结果显示26与Aβ31-42特异性结合,B5与Aβ1-12特异性结合,因此初步认为26的抗原表位位于Aβ31-42,B5的抗原表位位于Aβ1-12。非竞争ELISA结果显示B5亲和力为KD=1.4×10-8mol/L 综上所述,我们建成了Aβ1-42分段表位肽段的细菌鞭毛展示系统,为今后对Aβ的表位研究工作打下了基础,并且成功通过抗体库的高通量筛选从大容量人源性噬菌体抗体库中,获得了两株特异性单链抗体26和B5,在全抗体水平上进行表位鉴定,可初步认为26的表位位于Aβ31-42内,B5的表位位于Aβ1-12内,同时测得B5全抗体亲和力为KD=1.4×10-8mol/L,为进一步深入研究AD的治疗性抗体奠定基础。
[Abstract]:Alzheimer's disease (AD) is a degenerative disease of the central nervous system that occurs frequently in the elderly. Its main clinical features are memory loss, cognitive and motor dysfunction. At present, the pathogenesis of AD is not very clear. Among them, amyloid protein (Abeta) is polymerized in the form of soluble monomer under normal physiological conditions. The accumulation of amyloid proteins with neurocytotoxicity is considered to be a key event in the pathogenesis of AD. Studies have shown that Abeta is a common pathway of various factors causing Alzheimer's disease and a key factor in the pathogenesis and development of AD. Therefore, Abeta is generally regarded as an effective target molecule for the treatment of AD. The beta-amyloid precursor protein (APP) is formed by the beta-metabolic pathway. It is generally composed of 39-43 amino acids and has a secondary structure of a beta-folded lamella. The molecule as a whole is hydrophobic, easy to aggregate and form insoluble deposits. In 1999, Schenk et al. first used Abeta 1-42 to host AD model mice. A breakthrough has been made in animal immunity. Since then, reports have confirmed that A-beta peptide and its antibody can be used in immunotherapy of AD and can achieve good therapeutic effect. Betabloc (AN1792), a vaccine of A-beta 1-42 developed by Elan Company, has been found to be effective in eliciting immune response and producing amyloid A-beta after phase I clinical trials. Sedimentation-bound antibodies can effectively activate microglia to clear plaques formed by amyloid A beta deposition and significantly improve cognitive function in patients. Unfortunately, during phase II clinical trials, some patients experienced inflammation in the brain and the tests were terminated. Meanwhile, passive immunotherapy for AD has been achieved. Great progress has been made. Studies have shown that anti-A-beta-N-terminal antibodies can effectively improve AD-related symptoms without triggering an immune response in Th1 cells. We hope that a large number of human phage antibody libraries will be screened by bacterial screening combined with solid phase screening to obtain specific antibodies, and lay a foundation for the development of therapeutic antibodies against AD.
Firstly, in order to screen antibodies against Abeta 1-12 and provide epitope peptides for preliminary epitope identification of specific antibodies, we divided Abeta 1-42 into four epitope peptides: Abeta 1-12, Abeta 12-23, Abeta 21-32 and Abeta 31-42 according to the molecular structure of Abeta 1-42 and the characteristics of bacterial flagella display system. The recombinant plasmids expressing four peptides fused with bacterial flagellin were expressed on E.coli GI826 flagella by bacterial flagella surface display system. SDS-PAGE electrophoresis, Western blot and immunofluorescence assay showed that the four peptides fused with flagellin and were successfully displayed on E.coli GI. The 826 surface.
Secondly, high throughput screening of human antibodies against A-beta-42 and its N-terminal (A-beta-12, A-beta-15) has been carried out. At present, most of the antibodies used in AD passive immunity research institute are mouse-derived antibodies, which limit their application in human body. The antibodies obtained from human antibody library avoid this problem and can be directly applied without humanization modification. In this study, we used deduction screening for A-beta-12, solid-phase screening for A-beta-42, solid-phase binding screening for A-beta-12, and solid-phase screening for A-beta-15 to screen a large-capacity phage single-chain antibody library with a capacity of 1.35 *1010. During the screening process, about 2000 clones were selected randomly, of which the positive rate of bacterial screening clones was low, and the positive rate of solid-phase screening clones could reach 50%. However, because solid-phase screening was aimed at the full-length screening of A-beta 1-42, the specific antibodies could be obtained with any region of A-beta 1-42. Domain binding. In order to screen high-quality antibodies against Abeta 1-12, we used the method of deduction screening for Abeta 1-12 and then solid-phase screening for Abeta 1-12. The positive rate of bacterial solid-phase binding screening for Abeta 1-12 was up to 40%. At the same time, we also carried out solid-phase screening for Abeta 1-15, but clone positive. Finally, we obtained a specific phage antibody G10 in the process of bacterial screening. Two specific phage antibodies 18,26 were obtained during the solid phase screening of A-beta 1-42. Two specific phage antibodies H9 and B5 were obtained during the solid phase screening of A-beta 1-15. A specific antibody 87.18 of which is the same as H9. This result proves that our scheme is reliable and effective.
Thirdly, we transformed the specific single-chain antibody into full-antibody form and identified the specificity of the instantaneous expression of the whole antibody. In order to obtain a more stable form of antibody, we modified the full-antibody form of the five strains of phage-specific single-chain antibodies and the five strains of antibodies were fine in 293-F. Results No. 18 and No. 87 antibodies were not expressed, and G10, 26 and B5 were expressed. Specificity identification showed that G10 lost its binding ability to AB1-42, 26 and B5 could specifically bind to AB1-42. Finally, we obtained two specific antibodies, 26 and B5.
Finally, we identified the epitopes of 26 and B5, and detected the affinity of the antibodies binding to the N terminal of A beta (A beta 1-12, A beta 1-15). The epitopes of 26 and B5 were identified by the A beta fragment peptides displayed on the flagella surface of bacteria. The results showed that 26 specifically binds to A beta 31-42 and B5 specifically binds to A beta 1-12. The antigenic epitope of 26 was located at A beta 31-42 and the antigenic epitope of B5 was located at A beta 1-12. Non-competitive ELISA showed that the affinity of B5 was KD=1.4 *10-8mol/L.
To sum up, we have constructed a flagella display system for the peptide fragments of Abeta 1-42, which lays a foundation for further study on the epitope of Abeta. Two specific single chain antibodies 26 and B5 were obtained from a large-scale phage antibody library by high-throughput screening of antibody library, and epitopes were carried out at the whole antibody level. The results showed that the epitope of 26 was located in A beta 31-42 and the epitope of B5 was located in A beta 1-12. The affinity of B5 antibody was 1.4 *10-8 mol/L, which laid a foundation for further study of therapeutic antibody of AD.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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