本文關(guān)鍵詞： 腫瘤壞死治療 抗核抗體Fab 噬菌體抗體庫 篩選 噬菌體展示　出處：《南方醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

【摘要】： 研究背景與目的: 腫瘤是當(dāng)前人類健康與生命的最大威脅,發(fā)生率有增無減,死亡率已躍居諸病種之首。目前人類對(duì)于腫瘤的本質(zhì)缺乏足夠的認(rèn)識(shí),通常認(rèn)為腫瘤是多因素、多發(fā)病機(jī)制的復(fù)雜疾病,主要采用手術(shù)、放療、化療、生物治療等手段進(jìn)行綜合治療。手術(shù)是早期腫瘤治療的重要手段,但相對(duì)于腫瘤這一全身性疾病而言,具有無法克服的局限性。化療和放療一直是人們治療腫瘤特別是中晚期腫瘤的主要武器,但是這兩種療法缺乏特異性,因此,雖然具有一定的療效,但患者生活質(zhì)量差,常常因?yàn)闊o法耐受其毒副作用而停止治療。此外,腫瘤細(xì)胞對(duì)化放療敏感性的逐漸降低及隨之而來的高復(fù)發(fā)率也阻礙了這兩種治療手段成為攻克腫瘤的最終方法。腫瘤的分子靶向治療是近年來研究的熱點(diǎn)和亮點(diǎn),是根本意義上的腫瘤特異性治療手段,其中單克隆抗體是這一領(lǐng)域的基石。 選擇與惡性腫瘤抗原特異性結(jié)合的單克隆抗體,利用基因工程手段進(jìn)行商業(yè)化大量生產(chǎn),然后連接上對(duì)腫瘤有殺傷性的負(fù)載如放射性核素~(131)I,這種具有靶向性的放療技術(shù)叫做生物導(dǎo)向治療技術(shù)或放射免疫治療(Radioimmunotherapy,RIA),具有靶向性好、毒性小等優(yōu)點(diǎn),非常適合用于腫瘤的內(nèi)放射治療。多年來,人們嘗試過采用針對(duì)腫瘤細(xì)胞膜抗原的單克隆抗體進(jìn)行放射免疫治療,但是針對(duì)腫瘤細(xì)胞膜抗原的單克隆抗體存在如下各種問題:①目前還沒有發(fā)現(xiàn)所有腫瘤共同的抗原,不管是腫瘤特異性抗原(TSA)還是腫瘤相關(guān)性抗原(TAA),都只表達(dá)于部分腫瘤,因此,對(duì)不同的腫瘤需要不同的單克隆抗體;②由于腫瘤的不均質(zhì)性,只有部分腫瘤細(xì)胞表面表達(dá)抗原,該類單抗注入體內(nèi)后,只有少量單抗聚集在腫瘤部位,導(dǎo)致放射性核素積聚較少;③抗原調(diào)變:腫瘤細(xì)胞表達(dá)某抗原一段時(shí)間后,不再表達(dá)該抗原,而可能表達(dá)另一種抗原。因此抗腫瘤細(xì)胞膜抗原的單抗用于實(shí)體瘤的治療效果不夠理想,尋找新的腫瘤抗原、探索治療實(shí)體瘤的新方法就顯得十分必要。 研究人員發(fā)現(xiàn),盡管癌細(xì)胞和正常細(xì)胞之間有許多相似的特性使得我們的治療手段左右為難,但它們之間仍然存在明顯的差異。在不同階段的細(xì)胞變化周期中,癌組織中變性和死亡細(xì)胞占據(jù)很高的比例,同時(shí)胞膜完整性喪失,細(xì)胞膜表面滲透性異常。而正常組織只有很少細(xì)胞以很慢的速度壞死,且壞死的細(xì)胞會(huì)快速有序地被組織清除,它們?cè)谒劳鲞^程中只有核碎裂,沒有異常的膜滲透改變。以往的治療均著眼于殺死活的癌細(xì)胞,忽略了變性壞死細(xì)胞。經(jīng)過測(cè)算發(fā)現(xiàn),與正常組織不同,50%左右的腫瘤細(xì)胞在分裂后很快出現(xiàn)變性。由于腫瘤中血供不足及巨噬細(xì)胞反應(yīng)異常,使變性細(xì)胞越來越多,在腫瘤中心位置形成大片壞死區(qū),成為惡性實(shí)體腫瘤的典型特征。 利用惡性實(shí)體腫瘤的這一病理特征,研究人員提出了與之相對(duì)應(yīng)的一種新的腫瘤治療模式:腫瘤壞死治療(Tumor Necrosis Therapy,TNT)。TNT屬于腫瘤分子靶向治療的范疇,其基本原理是利用腫瘤組織的微血管內(nèi)皮細(xì)胞的不連續(xù)性,基底膜的不完整性和腫瘤細(xì)胞膜的高通透性特點(diǎn),應(yīng)用能與細(xì)胞內(nèi)核抗原成份結(jié)合的靶向分子攜帶殺瘤物質(zhì),滯留或定位于腫瘤組織內(nèi),造成腫瘤的特異性殺傷。這些靶向分子主要是抗細(xì)胞核內(nèi)某些組份的抗體,殺瘤物質(zhì)為放射性核素、藥物、毒素或某些抗瘤細(xì)胞因子等。瘤細(xì)胞殺傷后釋出的靶向分子又可進(jìn)入相鄰的瘤組織造成進(jìn)一步的殺傷,如此使殺瘤范圍不斷擴(kuò)大,直至正常組織細(xì)胞。由于TNT靶向分子在正常組織中的高清除、低滯留,因而在保證較好殺瘤效果的同時(shí),對(duì)周圍正常組織的損傷卻很小。 腫瘤細(xì)胞核人鼠嵌合單抗(chTNT)是Peregrine開發(fā)成功的一種針對(duì)腫瘤壞死組織的新型抗體,具有明確的抗腫瘤作用,但是,其作為一種鼠/人嵌合單抗必然有其自身的不足。鼠源抗體的人源化是一項(xiàng)比較繁雜的技術(shù),而且仍然存在一定程度的宿主抗體反應(yīng)。隨著生物技術(shù)的發(fā)展,單克隆抗體必然要經(jīng)歷從鼠源單抗、鼠/人嵌合單抗、人源化單抗到全人單抗的發(fā)展階段。因此,作為臨床治療用抗體,相對(duì)于嵌合抗體與人源化抗體而言,全人源抗體無疑是最佳的選擇。目前,以抗體庫技術(shù)和人-人雜交瘤技術(shù)為手段的全人源抗體的制備技術(shù)已經(jīng)較為成熟,因此有必要通過此類技術(shù)制備以腫瘤壞死區(qū)變性壞死細(xì)胞核為靶點(diǎn)的可用于TNT的全人源化單克隆抗體。Fab片段擁有重鏈Fd基因與完整的輕鏈基因,這種小分子抗體保持了抗體活性,大小為完整IgG的1/3。因其不含有Fc段,分子量小,免疫原性低,組織穿透力相對(duì)較強(qiáng),故在腫瘤治療上有其優(yōu)越性,適合用于TNT治療。 本研究擬構(gòu)建抗核抗體Fab片段噬菌體組合文庫,并利用噬菌體表面呈現(xiàn)技術(shù)(Phage Display)篩選(Panning)針對(duì)雙鏈DNA的人源性抗核抗體Fab片段,為進(jìn)一步的放免靶向治療奠定基礎(chǔ)。 研究方法: 1)從4名間接免疫熒光法檢測(cè)抗核抗體(均質(zhì)型)滴度大于1:10000的自身免疫病患者外周血中分離單個(gè)核細(xì)胞(PBMNC),抽提總RNA,反轉(zhuǎn)錄獲取cDNA文庫。 2)以獲取的cDNA文庫為模版,PCR法擴(kuò)增輕鏈κ、λ基因及免疫球蛋白分子重鏈Fd基因。 3)將輕鏈克隆入pComb3Hss載體以構(gòu)建輕鏈文庫。 4)將重鏈基因載入κ/λ-pComb3Hss載體,構(gòu)建完成組合文庫。 5)將重組質(zhì)粒轉(zhuǎn)化大腸桿菌XL1-Blue,用輔助噬菌體M13KO7感染,隨機(jī)的組合文庫表達(dá)于絲狀噬菌體表面,完成噬菌體表面展示。 6)通過4輪淘篩,富集已構(gòu)建的人源性抗核抗體Fab噬菌體抗體庫,間接ELISA法鑒定4輪淘篩后抗核抗體Fab噬菌體抗體,提取陽性克隆的噬菌粒DNA,切除gⅢ基因片段,自連接后轉(zhuǎn)化大腸桿菌XL1-Blue,以IPTG誘導(dǎo)表達(dá)可溶性人源性抗核抗體Fab片段;應(yīng)用間接ELISA法及熒光免疫法對(duì)表達(dá)產(chǎn)物進(jìn)行鑒定。 7)制備用于純化人IgG Fab片段的親和層析柱。 8)利用制備的親和層析柱純化人源性抗核抗體Fab片段。 9)Western blotting及間接免疫熒光法鑒定純化產(chǎn)物。 實(shí)驗(yàn)結(jié)果: 1)從外周血單個(gè)核細(xì)胞中抽提得到總RNA,并成功反轉(zhuǎn)錄獲得cDNA文庫。 2)PCR法擴(kuò)增了大小均為660bp左右的輕鏈κ、λ基因及重鏈Fd基因,并成功構(gòu)建了庫容為2×10~4的輕鏈基因抗體庫(輕鏈庫)和庫容為4×10~4的人Fab抗體庫(組合文庫)。 3)通過噬菌體表面展示技術(shù),在組合文庫的基礎(chǔ)上獲得了噬菌體滴度為2×10~9pfu/ml的富含人源性抗核抗體Fab片段的噬菌體抗體庫。 4)通過固相化抗原吸附篩選法篩選獲得2個(gè)陽性克隆,切除gⅢ基因片段,實(shí)現(xiàn)了可溶性人源性抗核抗體Fab片段的表達(dá)。 5)間接ELISA法檢測(cè)結(jié)果顯示:制備的可溶性人源性抗核抗體Fab片段呈現(xiàn)抗dsDNA陽性,抗核糖核蛋白陰性;間接免疫熒光法結(jié)果顯示:Hep2細(xì)胞和猴肝臟組織細(xì)胞核顯示均質(zhì)型熒光,綠蠅短膜蟲的動(dòng)基體顯示均質(zhì)型熒光。證實(shí)獲得的可溶性人源性抗核抗體Fab片段具有特異性結(jié)合雙鏈DNA的免疫學(xué)活性。 6)制備完成可用于純化人IgG Fab片段的親和層析柱。 7)利用制備的親和層析柱純化獲得一定濃度和數(shù)量的人源性抗核抗體Fab片段,經(jīng)Western blotting證實(shí)為人IgG Fab片段,間接免疫熒光法明確其具有一定的抗dsDNA的免疫學(xué)活性。 結(jié)論: 通過自身免疫性疾病患者的外周血單個(gè)核細(xì)胞獲得了cDNA文庫,并據(jù)此成功構(gòu)建了人源性抗核抗體Fab片段組合文庫,依靠噬菌體表面展示技術(shù)獲得了相應(yīng)的噬菌體抗體庫。通過固相化抗原吸附篩選法篩選獲得了陽性克隆,并實(shí)現(xiàn)了可溶性抗核抗體Fab片段的表達(dá),該抗核抗體Fab片段具有一定的特異性抗dsDNA的免疫學(xué)活性,為進(jìn)一步的腫瘤免疫靶向治療奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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1 錢新宇;人源性抗核抗體Fab片段抗體庫的構(gòu)建篩選及鑒定[D];南方醫(yī)科大學(xué);2008年

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本文編號(hào)：1478480