人源性抗核抗体Fab片段抗体库的构建筛选及鉴定

发布时间：2018-01-31 06:37

本文关键词： 肿瘤坏死治疗抗核抗体Fab 噬菌体抗体库筛选噬菌体展示　出处：《南方医科大学》2008年博士论文　论文类型：学位论文

【摘要】： 研究背景与目的: 肿瘤是当前人类健康与生命的最大威胁,发生率有增无减,死亡率已跃居诸病种之首。目前人类对于肿瘤的本质缺乏足够的认识,通常认为肿瘤是多因素、多发病机制的复杂疾病,主要采用手术、放疗、化疗、生物治疗等手段进行综合治疗。手术是早期肿瘤治疗的重要手段,但相对于肿瘤这一全身性疾病而言,具有无法克服的局限性。化疗和放疗一直是人们治疗肿瘤特别是中晚期肿瘤的主要武器,但是这两种疗法缺乏特异性,因此,虽然具有一定的疗效,但患者生活质量差,常常因为无法耐受其毒副作用而停止治疗。此外,肿瘤细胞对化放疗敏感性的逐渐降低及随之而来的高复发率也阻碍了这两种治疗手段成为攻克肿瘤的最终方法。肿瘤的分子靶向治疗是近年来研究的热点和亮点,是根本意义上的肿瘤特异性治疗手段,其中单克隆抗体是这一领域的基石。选择与恶性肿瘤抗原特异性结合的单克隆抗体,利用基因工程手段进行商业化大量生产,然后连接上对肿瘤有杀伤性的负载如放射性核素~(131)I,这种具有靶向性的放疗技术叫做生物导向治疗技术或放射免疫治疗(Radioimmunotherapy,RIA),具有靶向性好、毒性小等优点,非常适合用于肿瘤的内放射治疗。多年来,人们尝试过采用针对肿瘤细胞膜抗原的单克隆抗体进行放射免疫治疗,但是针对肿瘤细胞膜抗原的单克隆抗体存在如下各种问题:①目前还没有发现所有肿瘤共同的抗原,不管是肿瘤特异性抗原(TSA)还是肿瘤相关性抗原(TAA),都只表达于部分肿瘤,因此,对不同的肿瘤需要不同的单克隆抗体;②由于肿瘤的不均质性,只有部分肿瘤细胞表面表达抗原,该类单抗注入体内后,只有少量单抗聚集在肿瘤部位,导致放射性核素积聚较少;③抗原调变:肿瘤细胞表达某抗原一段时间后,不再表达该抗原,而可能表达另一种抗原。因此抗肿瘤细胞膜抗原的单抗用于实体瘤的治疗效果不够理想,寻找新的肿瘤抗原、探索治疗实体瘤的新方法就显得十分必要。研究人员发现,尽管癌细胞和正常细胞之间有许多相似的特性使得我们的治疗手段左右为难,但它们之间仍然存在明显的差异。在不同阶段的细胞变化周期中,癌组织中变性和死亡细胞占据很高的比例,同时胞膜完整性丧失,细胞膜表面渗透性异常。而正常组织只有很少细胞以很慢的速度坏死,且坏死的细胞会快速有序地被组织清除,它们在死亡过程中只有核碎裂,没有异常的膜渗透改变。以往的治疗均着眼于杀死活的癌细胞,忽略了变性坏死细胞。经过测算发现,与正常组织不同,50%左右的肿瘤细胞在分裂后很快出现变性。由于肿瘤中血供不足及巨噬细胞反应异常,使变性细胞越来越多,在肿瘤中心位置形成大片坏死区,成为恶性实体肿瘤的典型特征。利用恶性实体肿瘤的这一病理特征,研究人员提出了与之相对应的一种新的肿瘤治疗模式:肿瘤坏死治疗(Tumor Necrosis Therapy,TNT)。TNT属于肿瘤分子靶向治疗的范畴,其基本原理是利用肿瘤组织的微血管内皮细胞的不连续性,基底膜的不完整性和肿瘤细胞膜的高通透性特点,应用能与细胞内核抗原成份结合的靶向分子携带杀瘤物质,滞留或定位于肿瘤组织内,造成肿瘤的特异性杀伤。这些靶向分子主要是抗细胞核内某些组份的抗体,杀瘤物质为放射性核素、药物、毒素或某些抗瘤细胞因子等。瘤细胞杀伤后释出的靶向分子又可进入相邻的瘤组织造成进一步的杀伤,如此使杀瘤范围不断扩大,直至正常组织细胞。由于TNT靶向分子在正常组织中的高清除、低滞留,因而在保证较好杀瘤效果的同时,对周围正常组织的损伤却很小。肿瘤细胞核人鼠嵌合单抗(chTNT)是Peregrine开发成功的一种针对肿瘤坏死组织的新型抗体,具有明确的抗肿瘤作用,但是,其作为一种鼠/人嵌合单抗必然有其自身的不足。鼠源抗体的人源化是一项比较繁杂的技术,而且仍然存在一定程度的宿主抗体反应。随着生物技术的发展,单克隆抗体必然要经历从鼠源单抗、鼠/人嵌合单抗、人源化单抗到全人单抗的发展阶段。因此,作为临床治疗用抗体,相对于嵌合抗体与人源化抗体而言,全人源抗体无疑是最佳的选择。目前,以抗体库技术和人-人杂交瘤技术为手段的全人源抗体的制备技术已经较为成熟,因此有必要通过此类技术制备以肿瘤坏死区变性坏死细胞核为靶点的可用于TNT的全人源化单克隆抗体。Fab片段拥有重链Fd基因与完整的轻链基因,这种小分子抗体保持了抗体活性,大小为完整IgG的1/3。因其不含有Fc段,分子量小,免疫原性低,组织穿透力相对较强,故在肿瘤治疗上有其优越性,适合用于TNT治疗。本研究拟构建抗核抗体Fab片段噬菌体组合文库,并利用噬菌体表面呈现技术(Phage Display)筛选(Panning)针对双链DNA的人源性抗核抗体Fab片段,为进一步的放免靶向治疗奠定基础。研究方法: 1)从4名间接免疫荧光法检测抗核抗体(均质型)滴度大于1:10000的自身免疫病患者外周血中分离单个核细胞(PBMNC),抽提总RNA,反转录获取cDNA文库。 2)以获取的cDNA文库为模版,PCR法扩增轻链κ、λ基因及免疫球蛋白分子重链Fd基因。 3)将轻链克隆入pComb3Hss载体以构建轻链文库。 4)将重链基因载入κ/λ-pComb3Hss载体,构建完成组合文库。 5)将重组质粒转化大肠杆菌XL1-Blue,用辅助噬菌体M13KO7感染,随机的组合文库表达于丝状噬菌体表面,完成噬菌体表面展示。 6)通过4轮淘筛,富集已构建的人源性抗核抗体Fab噬菌体抗体库,间接ELISA法鉴定4轮淘筛后抗核抗体Fab噬菌体抗体,提取阳性克隆的噬菌粒DNA,切除gⅢ基因片段,自连接后转化大肠杆菌XL1-Blue,以IPTG诱导表达可溶性人源性抗核抗体Fab片段;应用间接ELISA法及荧光免疫法对表达产物进行鉴定。 7)制备用于纯化人IgG Fab片段的亲和层析柱。 8)利用制备的亲和层析柱纯化人源性抗核抗体Fab片段。 9)Western blotting及间接免疫荧光法鉴定纯化产物。实验结果: 1)从外周血单个核细胞中抽提得到总RNA,并成功反转录获得cDNA文库。 2)PCR法扩增了大小均为660bp左右的轻链κ、λ基因及重链Fd基因,并成功构建了库容为2×10~4的轻链基因抗体库(轻链库)和库容为4×10~4的人Fab抗体库(组合文库)。 3)通过噬菌体表面展示技术,在组合文库的基础上获得了噬菌体滴度为2×10~9pfu/ml的富含人源性抗核抗体Fab片段的噬菌体抗体库。 4)通过固相化抗原吸附筛选法筛选获得2个阳性克隆,切除gⅢ基因片段,实现了可溶性人源性抗核抗体Fab片段的表达。 5)间接ELISA法检测结果显示:制备的可溶性人源性抗核抗体Fab片段呈现抗dsDNA阳性,抗核糖核蛋白阴性;间接免疫荧光法结果显示:Hep2细胞和猴肝脏组织细胞核显示均质型荧光,绿蝇短膜虫的动基体显示均质型荧光。证实获得的可溶性人源性抗核抗体Fab片段具有特异性结合双链DNA的免疫学活性。 6)制备完成可用于纯化人IgG Fab片段的亲和层析柱。 7)利用制备的亲和层析柱纯化获得一定浓度和数量的人源性抗核抗体Fab片段,经Western blotting证实为人IgG Fab片段,间接免疫荧光法明确其具有一定的抗dsDNA的免疫学活性。结论: 通过自身免疫性疾病患者的外周血单个核细胞获得了cDNA文库,并据此成功构建了人源性抗核抗体Fab片段组合文库,依靠噬菌体表面展示技术获得了相应的噬菌体抗体库。通过固相化抗原吸附筛选法筛选获得了阳性克隆,并实现了可溶性抗核抗体Fab片段的表达,该抗核抗体Fab片段具有一定的特异性抗dsDNA的免疫学活性,为进一步的肿瘤免疫靶向治疗奠定了基础。
[Abstract]:Research background and purpose:
The tumor is the biggest threat to human health and life. The incidence of the disease mortality increase, has leapt to the first. The human nature of the tumor for lack of sufficient knowledge, usually believed that cancer is a multi factor complex disease pathogenesis, mainly by surgery, radiotherapy, chemotherapy, biological therapy combined therapy. The operation is an important means for the treatment of early stage tumors, but because tumor is a systemic disease, has limitations that cannot be overcome. Chemotherapy and radiotherapy are the main weapon in the treatment of tumors especially advanced cancer, but these two therapies lack of specificity, therefore, has a certain effect, but the quality of life of patients poor, often because they can not tolerate the side effects of stopping treatment. In addition, the high recurrence of tumor cells to radiotherapy sensitivity gradually decreased and the subsequent rate also hindered These two kinds of treatment methods become the ultimate method to overcome the tumor. Tumor molecular targeted therapy is a hot and bright spot of research in recent years, is a tumor specific treatment fundamentally, the monoclonal antibody is the cornerstone of this field.
Monoclonal antibody selection and combination of malignant tumor specific antigen, are produced commercially by means of genetic engineering, and then connect to the tumor killing load such as a radionuclide of ~ (131) I, which has targeted radiotherapy technology called biological treatment technology oriented or radioimmunotherapy (Radioimmunotherapy, RIA). Has the advantages of good targeting, low toxicity, very suitable for radiation therapy of cancer. Over the years, people have tried using monoclonal antibodies to tumor cell membrane antigen of radioimmunotherapy, but is a monoclonal antibody against tumor cell membrane antigen has the following problems: 1 has not found all tumor common antigen whether the tumor specific antigen (TSA) and tumor associated antigen (TAA), are only expressed in some tumors, therefore, for the different needs of different monoclonal tumor Due to the long antibody; tumor heterogeneity, only part of the tumor cell surface antigen expression, the monoclonal antibody injected into the body, only a small amount of monoclonal antibody accumulation at the tumor site, leading to the accumulation of radionuclides less; antigenic modulation: tumor cells express an antigen. After a period of time, no longer express the antigen, while another may express antigens. Therefore anti tumor cell membrane antigen monoclonal antibody for tumor treatment effect is not ideal, looking for new tumor antigens, it is very necessary to explore a new method for the treatment of solid tumors.
The researchers found that although between cancer cells and normal cells have many similar characteristics in a dilemma makes our treatment, but there are obvious differences between them. In the different stages of the cell cycle, cell degeneration and death occupy a very high proportion of cancer tissues, while the loss of cellular membrane integrity, cell membrane surface the abnormal and normal tissue permeability. Only a few cells at a very slow speed and necrosis, necrotic cells will be rapid and orderly organization clear, they in the process of dying only nuclear fragmentation, no abnormal membrane permeability. Previous treatments were focused on killing live cancer cells, ignoring the degeneration and necrosis of cells. After the calculations showed that different from normal tissue degeneration, soon appeared about 50% of the tumor cells in the division. Due to insufficient supply of blood and abnormal tumor cell degeneration of macrophage reaction, make more and more More, forming a large necrotic area in the tumor center, has become a typical feature of malignant solid tumors.
The pathological features with malignant solid tumors, the researchers propose a new approach for cancer treatment and the corresponding tumor necrosis therapy (Tumor Necrosis Therapy, TNT.TNT) belongs to the category of tumor molecular targeted therapy, its basic principle is not continuous use of microvascular endothelial cells in tumor tissues. The basement membrane integrity and cell membrane of the high permeability characteristics, applications can be combined with cell antigens of kernel targeting molecules carrying antitumor substances, stranded or located in tumor tissue destruction caused by tumor specific targeting molecules. These are some components of the anti nuclear antibody, kill the tumor material for radionuclides, drugs, toxins or some antitumor cytokines. Tumor cell killing after the release of the target molecules can enter the adjacent tumor tissue causing further destruction, thus killing tumor continuously Expand, until the normal tissue cells. The TNT targeting molecules in normal tissues with high clearance, low retention, and thus has good antitumor effect at the same time, to the surrounding normal tissue damage is very small.
The tumor cells chimeric monoclonal antibody (chTNT) Peregrine is the successful development of a new antibody against tumor necrosis tissue, with definite anti-tumor effect, but its shortcomings as a mouse / human chimeric antibody will have its own. Mouse antibody humanization is a relatively complicated technology. But there are still host antibody reaction to some extent. With the development of biotechnology, the monoclonal antibody bound to experience from the murine monoclonal antibody, mouse / human chimeric monoclonal antibody, humanized monoclonal antibody to the stage of development of fully human antibodies. Therefore, antibody used as a clinical treatment, compared with chimeric and humanized antibodies for all people antibody is undoubtedly the best choice. At present, the antibody library technology and human human hybridoma technology as a means of human antibody preparation technology has been more mature, so it is necessary to prepare tumor necrosis area through such technology The degeneration and necrosis of nucleus as the target for light chain gene TNT humanized monoclonal antibody.Fab fragment with Fd heavy chain gene and complete, this small molecule antibody maintained antibody activity, 1/3. for full size IgG because it does not contain Fc, small molecular weight, low immunogenicity and tissue penetration power is relatively strong, so it has its superiority in the treatment of tumors, suitable for the treatment of TNT.
This study intends to construct antinuclear antibody Fab fragment phage antibody library, and using phage display technology (Phage Display) (Panning) were derived for double stranded DNA antibody Fab fragment, to lay the foundation for further treatment from the target.
Research methods:
1) antinuclear antibody detection from 4 indirect immunofluorescence (homogeneous) separation of mononuclear cells in patients with autoimmune disease was more than 1:10000 in the peripheral blood (PBMNC), total RNA extraction, reverse transcription for cDNA library.
2) to obtain the cDNA library as template, amplified light chain kappa lambda gene PCR, and immunoglobulin heavy chain molecule Fd gene.
3) light chain was cloned into pComb3Hss vector to construct the light chain library.
4) the heavy chain gene into kappa / lambda -pComb3Hss carrier, to complete the construction of combinatorial libraries.
5) the recombinant plasmid was transformed into Escherichia coli XL1-Blue infection with helper phage M13KO7, random combinatorial library was expressed on the surface of filamentous phage, phage display is completed.
6) after 4 rounds of panning, the enrichment of human anti nuclear antibody Fab phage antibody library, indirect ELISA method for identification of 4 rounds of panning after anti nuclear antibody Fab phage antibody, extract the positive cloned phagemid DNA G gene fragment excision, since after the connection was transformed into Escherichia coli XL1-Blue, induced expression soluble human anti nuclear antibody Fab fragment to IPTG; identification of the expressed product by indirect immunofluorescence method and ELISA method.
7) for the preparation of purified human IgG Fab fragment affinity chromatography column.
8) purification of human antinuclear antibody Fab fragment prepared by affinity chromatography.
9) Western blotting and indirect immunofluorescence identification of purified product.
The experimental results:
1) from peripheral blood mononuclear cells were extracted from the total RNA, and reverse transcription cDNA library.
2) were amplified by PCR size were about 660bp light chain kappa, lambda gene and Fd heavy chain gene, and construct the capacity for light chain gene antibody library was 2 * 10~4 (light chain Library) and the capacity of 4 x 10~4 human Fab antibody library (Library).
3) by phage display technology, based on combinatorial library was obtained on the phage titer of phage antibody library for the rich source of 2 x 10~9pfu/ml antinuclear antibody Fab fragment.
4) antigen adsorption by solid-phase screening, we obtained 2 positive clones, G gene fragment excision, the expression of the antinuclear antibody Fab fragment of human soluble sources.
5) the result of indirect ELISA method showed that the preparation of soluble human anti nuclear antibody Fab fragment showed positive anti dsDNA, anti ribonucleoprotein negative results; indirect immunofluorescence showed that Hep2 cells and monkey liver nuclei showed homogeneous fluorescence, kinetoplast Crithidia luciliae showed homogeneous fluorescence. That soluble human obtain antinuclear antibody Fab fragment has immunological activity of specific binding of double stranded DNA.
6) to complete the preparation can be used for the purification of human IgG fragment Fab affinity chromatography column.
7) obtain the source of concentration and quantity of antinuclear antibody Fab fragment was purified by affinity chromatographic column was prepared by using Western, blotting confirmed human IgG Fab fragment, indirect immunofluorescence with anti dsDNA specific immunological activity.
Conclusion:
The patients with autoimmune diseases of peripheral blood mononuclear cells obtained from cDNA library, and then successfully constructed human anti nuclear antibody Fab fragment library, rely on technology to obtain a phage antibody library by phage display. The immobilized antigen adsorption screening positive clones were obtained, and the expression of soluble antinuclear antibody Fab fragment, the antinuclear antibody Fab fragment has immunological activity specific anti dsDNA, further to the target for tumor immune therapy has laid the foundation.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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