本文選題：全人源單抗 + 炭疽重組PA疫苗　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2015年博士論文

【摘要】：炭疽芽胞桿菌(Bacillus anthracis)致死率極高,是人畜共患烈性傳染病-炭疽病(Anthrax)的病原體。并且炭疽芽胞桿菌來源廣泛、易于培養(yǎng),被作為一種A類生物戰(zhàn)劑進(jìn)行管理,其防生和反恐一直以來都具有重要的軍事意義和社會意義。炭疽的防控手段包括預(yù)防和治療,二者分別主要依賴于炭疽疫苗和抗菌素,但是這兩種藥物均需在感染前或感染初期立即使用。而炭疽毒素中和抗體在宿主感染后,能快速有效中和體內(nèi)產(chǎn)生的炭疽毒素,同時發(fā)揮補(bǔ)體依賴細(xì)胞毒性作用和抗體依賴細(xì)胞毒性作用,是目前最有前景的炭疽治療藥物。自從1975年雜交瘤技術(shù)出現(xiàn)后,多年來單抗治療主要依賴于鼠源單抗。但是鼠源單抗半衰期短、毒副作用明顯,大大制約了其臨床應(yīng)用。全人源單克隆抗體應(yīng)用于人體時具有無免疫原性、半衰期長,國外大多數(shù)新開發(fā)項(xiàng)目現(xiàn)已轉(zhuǎn)向全人源單抗的研發(fā)。目前全人源單抗制備的方法主要是通過人人雜交瘤技術(shù)、轉(zhuǎn)基因鼠技術(shù)、抗體庫技術(shù)和單細(xì)胞PCR技術(shù),其中通過單細(xì)胞PCR技術(shù)可直接從人單個B細(xì)胞中擴(kuò)增抗體基因,無需細(xì)胞融合培養(yǎng)和抗體多輪篩選的過程,能夠在最短7天內(nèi)篩選獲得有效的全人源單抗,而其他幾種抗體制備方法均需要數(shù)周甚至數(shù)月,建立這種快速高通量抗體篩選平臺,將有助于提升我國新發(fā)突發(fā)傳染病的應(yīng)對能力;并且,單細(xì)胞PCR技術(shù)完整保留了抗體重鏈輕鏈在人體內(nèi)的天然配對方式,因此可在獲得治療抗體的同時更加直觀、深入地研究人體免疫機(jī)制。但是,由于單個細(xì)胞內(nèi)基因拷貝數(shù)非常低,單細(xì)胞PCR技術(shù)的實(shí)現(xiàn)難度大,目前國內(nèi)罕見成功通過該技術(shù)獲得單抗的研究團(tuán)隊(duì)。目前大部分炭疽單抗仍然是鼠源單抗,少數(shù)研究者從大猩猩、轉(zhuǎn)基因鼠和人體中篩選獲得了炭疽單抗,而我國仍未見全人源的抗炭疽毒素單抗報道。鑒于炭疽毒素作用機(jī)制的復(fù)雜性,研發(fā)更多的針對不同中和表位的炭疽全人源單抗,能夠有效避免炭疽毒素被人為突變帶來的生物恐怖威脅,為炭疽的治療提供更多選擇。鑒于此,本文目的在于建立流式分選-單細(xì)胞PCR快速高通量的抗體篩選技術(shù)平臺,并通過該技術(shù)平臺從重組炭疽保護(hù)性抗原(protective antigen,PA)疫苗免疫的志愿者體內(nèi),篩選獲得全人源PA中和抗體,并對單抗的親和力、抗原結(jié)合表位、毒素中和機(jī)制、單抗聯(lián)合用藥效果等進(jìn)行深入研究。首先對一名男性志愿者在第0天和28天兩次免疫了重組PA疫苗,并在第0、28、35天采集志愿者血液樣品。為確保能夠在合適的時間點(diǎn)采集志愿者陽性的血液樣品,從而提高后續(xù)PA抗體篩選的陽性率,本文通過ELISA和TNA的方法,對不同時間點(diǎn)血清中PA結(jié)合和中和抗體滴度進(jìn)行測量。結(jié)果表明,免疫第35天血清中PA結(jié)合抗體和中和抗體滴度均明顯升高。同時,通過ELISpot的方法,對不同時間點(diǎn)血液中抗體分泌細(xì)胞的數(shù)量變化進(jìn)行檢測。結(jié)果顯示抗PA抗體陽性的抗體分泌細(xì)胞占總Ig G陽性的抗體分泌細(xì)胞的比例,從免疫后第28天的0.8%上升至免疫后第35天的23.8%,可見抗PA抗體陽性的抗體分泌細(xì)胞個數(shù)大幅升高。表明重組PA疫苗在志愿者體內(nèi)有效激發(fā)了體液免疫反應(yīng),在免疫后第35天時體液免疫水平顯著升高,為后續(xù)抗體的分選提供保證。為了實(shí)現(xiàn)單個B細(xì)胞的分選和抗體基因的單細(xì)胞PCR擴(kuò)增,我們嘗試了多種細(xì)胞分選和單細(xì)胞PCR的方法,在經(jīng)歷多次失敗后,最終采用流式細(xì)胞分選抗體分泌細(xì)胞,以及多重引物組合的單細(xì)胞反轉(zhuǎn)錄-巢式兩步PCR的方法,成功從人單個B細(xì)胞中擴(kuò)增獲得抗體基因。在單細(xì)胞PCR條件優(yōu)化的過程中,我們發(fā)現(xiàn)引物設(shè)計、酶的種類和模板量對于擴(kuò)增效率的影響較大,而退火溫度對于擴(kuò)增效果影響甚微。在成功實(shí)現(xiàn)研究中抗體分選的關(guān)鍵性技術(shù)-流式分選單個抗體分泌細(xì)胞和單細(xì)胞PCR技術(shù)后,采集了志愿者免疫后第35天的血液樣品,通過密度梯度沉降法從血液樣品中分離外周血單個核細(xì)胞(peripheral blood mononuclear cell,PBMC),在對一組抗體分泌細(xì)胞表面特異的白細(xì)胞分化抗原簇(cluster of differentiation,CD)分子進(jìn)行熒光染色后,通過流式細(xì)胞分選儀從PBMC中分選獲得2112個單個抗體分泌細(xì)胞克隆,通過單細(xì)胞PCR擴(kuò)增單個細(xì)胞中的抗體基因。結(jié)果顯示,529個單細(xì)胞克隆重鏈和輕鏈同時擴(kuò)增成功的比率,即單細(xì)胞PCR的陽性率約為25%。在硬件條件滿足的情況下,僅需1~2天即可完成以上單細(xì)胞的分選和抗體基因擴(kuò)增的過程。如果采用傳統(tǒng)的克隆方法,構(gòu)建529對抗體基因的表達(dá)質(zhì)粒,非常耗時費(fèi)力。為了快速、高通量表達(dá)抗體基因,我們設(shè)計了可直接通過PCR融合啟動子、前導(dǎo)序列、抗體全長基因、多聚A尾(poly(A)tail)的線性表達(dá)框。我們對線性表達(dá)框PCR條件進(jìn)行優(yōu)化,并使用一株對照抗體驗(yàn)證線性表達(dá)框所表達(dá)抗體的表達(dá)量和功能。結(jié)果顯示,經(jīng)線性表達(dá)框方法表達(dá)的抗體濃度可達(dá)0.4μg/m L,且良好保持了抗體中和效果,能夠滿足后續(xù)抗體篩選實(shí)驗(yàn)的要求,通過我們設(shè)計的線性表達(dá)框可在最短3天內(nèi)表達(dá)供后續(xù)篩選用的抗體。在確保方法的可行性后,我們按照優(yōu)化的重疊延伸PCR條件,構(gòu)建了529對抗體重鏈和輕鏈線性表達(dá)框,并將配對的基因共轉(zhuǎn)染至HEK 293T細(xì)胞中進(jìn)行表達(dá)。繼而,通過ELISA和TNA的方法對529株全人源單抗進(jìn)行PA結(jié)合和中和活性的檢測,并鑒定了單抗的特異結(jié)合的PA結(jié)構(gòu)域。結(jié)果顯示,我們成功通過建立的抗體篩選平臺技術(shù)獲得34株P(guān)A結(jié)合單抗,其中4株單抗(2A6、4A3、8H6、22F1)具有PA中和活性。中和單抗22F1與PA不結(jié)合,而是通過與PA的變構(gòu)體PA63結(jié)合發(fā)揮作用。另外,研究發(fā)現(xiàn)PA結(jié)合單抗中有一種能夠加速細(xì)胞死亡的毒素增強(qiáng)抗體(Toxin enhancing m Ab),占PA結(jié)合單抗的比例達(dá)55.9%之多。為了進(jìn)一步探究PA中和抗體和毒素增強(qiáng)抗體的特性,構(gòu)建4株P(guān)A中和抗體和1株毒素增強(qiáng)抗體8A7的表達(dá)質(zhì)粒,轉(zhuǎn)染并純化抗體蛋白;并從以下幾個方面對抗體的功能進(jìn)行研究:1)通過BLI技術(shù)測定了5株重點(diǎn)研究的單抗的親和力,結(jié)果顯示除22F1外單抗親和力均在納摩爾級;2)測定4株中和單抗在體內(nèi)和體外的中和活性,結(jié)果顯示4株中和單抗中22F1單抗中和活性最強(qiáng),其體外保護(hù)效果較上市PA單抗報道結(jié)果高出近一個數(shù)量級,而單抗2A6僅在體外具有保護(hù)活性;3)鑒定中和單抗的中和機(jī)制,結(jié)果表明8H6單抗對PA呈現(xiàn)抗體濃度依賴的酶切抑制作用,4A3單抗通過促進(jìn)PA63的降解而抑制PA七聚體形成,4A3這種類似酶活作用的毒素中和機(jī)制尚未見報道,提示本研究可能發(fā)現(xiàn)了一株新中和機(jī)制的PA中和單抗。毒素增強(qiáng)單抗8A7能促進(jìn)PA七聚體的形成,并與PA七聚體結(jié)合,提示其毒素增強(qiáng)作用的可能機(jī)制;4)分析單抗的聯(lián)合用藥效果,結(jié)果表明2A6和4A3、8H6和22F1表現(xiàn)出較弱的協(xié)同作用;2A6和8H6、2A6和22F1表現(xiàn)出無關(guān)作用;4A3和8H6、4A3和22F1表現(xiàn)出拮抗作用。而單抗聯(lián)合用藥協(xié)同效果最為顯著的,是毒素增強(qiáng)單抗8A7與僅在體外具有中和活性的單抗2A6,二者聯(lián)合用藥能夠在體內(nèi)發(fā)揮高水平的毒素中和效果。結(jié)果提示我們,在人體內(nèi)之所以大量產(chǎn)生毒素增強(qiáng)抗體的可能原因是,這種抗體在血液中能夠輔助其他抗體發(fā)揮良好的機(jī)體保護(hù)效果。并且,8A7單抗針對的PA結(jié)構(gòu)域3是目前普遍認(rèn)為的PA非中和位點(diǎn),根據(jù)本文所得結(jié)果推測針對PA結(jié)構(gòu)域3表位的單抗,在人體內(nèi)能夠通過與其他抗體協(xié)同發(fā)揮重要的生物學(xué)功能,這種輔助性單抗在抗體篩選過程中容易被遺漏,但是其在“雞尾酒”治療中發(fā)揮的重要作用不容忽視。綜上所述,本文建立了基于流式分選和單抗PCR技術(shù)的快速抗體篩選平臺,能夠?yàn)樾掳l(fā)突發(fā)傳染病抗體研究提供技術(shù)儲備;篩選制備了多株不同中和機(jī)制的全人源PA中和單抗,其中包含新中和機(jī)制的單抗和高中和活性的單抗,是具有前景的炭疽治療藥物;同時,對抗體中和機(jī)制、表位、聯(lián)合用藥效果的研究,也為炭疽毒素作用機(jī)理研究、疫苗設(shè)計和“雞尾酒”抗體藥物設(shè)計提供有力依據(jù);
[Abstract]:Bacillus anthracis (Bacillus anthracis) is highly lethal and is the pathogen of human zoonotic strong infectious disease, anthrax (Anthrax). And Bacillus anthracis is widely used as a kind of biological warfare agent to manage, and has important military and social significance. The prevention and anti terrorism of anthrax has been of great importance in the prevention and treatment of anthrax. Control means include prevention and treatment, the two are mainly dependent on anthrax vaccine and antibiotics, but both of these two drugs need to be used immediately before or in the early stage of infection. The anthrax neutralization antibody and antibody can quickly and effectively neutralize the anthrax toxin produced in the body after the infection of the host, and play the complement dependent cytotoxic effect and the antibody. The toxic effects of LYC are the most promising anthrax treatment drugs. Since the emergence of hybridoma technology in 1975, McAbs have been mainly dependent on rat McAbs for many years. But the half life of the mAb is short and the toxic and side effects are obvious, which greatly restrict its clinical application. Most of the new development projects in foreign countries have now turned to the R & D of all human McAbs. At present, the methods of preparing all human monoclonal antibodies are mainly through human hybridoma technology, transgenic mouse technology, antibody library technology and single cell PCR technology, in which single cell PCR technology can directly amplify the antibody genes from individual B cells. The process of cell fusion and antibody multi wheel screening can screen effective total human monoclonal antibodies in the shortest 7 days, and several other antibody preparation methods need several weeks or even months. The establishment of this rapid and high flux antibody screening platform will help to improve the ability to respond to new onset infectious diseases in China; and single cell PCR technology. The natural pairing of antibody heavy chain light chain in human body is preserved, so it can be more intuitionistic and in-depth study on human immune mechanism while obtaining therapeutic antibodies. However, the realization of single cell PCR technology is very difficult because of the very low gene copy number within single cell. The research team. Most of the anthrax monoclonal antibodies are still murine McAbs. A few researchers have screened the anthrax McAbs from gorillas, transgenic mice and human bodies. However, there is no full human anti anthrax monoclonal antibody reported in our country. In view of the complexity of the mechanism of anthrax toxin, more anthrax against different neutralization epitopes has been developed. Source McAbs can effectively avoid the bioterrorism threat of anthrax toxin caused by human mutation, and provide more options for the treatment of anthrax. In view of this, the aim of this paper is to establish a rapid and high flux antibody screening technology platform for flow separation single cell PCR, and the technology platform from the recombinant anthrax protective antigen (protective antigen, PA). All human PA neutralization antibodies were screened in vaccinated volunteers, and the affinity of mAb, antigen binding epitopes, toxin neutralization mechanism, and the effect of McAb combined use were studied. First, a male volunteer was immunized with recombinant PA vaccine at zeroth and 28 days, and the blood samples were collected on day 0,28,35. In order to ensure that the positive blood samples can be collected at the appropriate time point to improve the positive rate of subsequent PA antibody screening, the PA binding and neutralizing antibody titer in serum at different time points were measured by ELISA and TNA. The results showed that the titer of PA binding antibody and neutralizing antibody in the serum of thirty-fifth days were all immunized. At the same time, the number of antibody secreting cells in blood at different time points was detected by ELISpot method. The results showed that the proportion of antibody secreting cells with positive anti PA antibody secreting cells accounted for the total Ig G positive antibody secreting cells, rising from 0.8% days after immunization to thirty-fifth days after immunization, and the anti PA antibody positive was found. The number of antibody secreting cells increased significantly. It showed that the recombinant PA vaccine effectively stimulated the humoral immune response in the volunteers. The level of humoral immunity increased significantly at thirty-fifth days after immunization, which provided a guarantee for the separation of subsequent antibodies. In order to realize the separation of single B cells and the single cell PCR amplification of the antibody base, we tried a variety of details. The method of cell sorting and single cell PCR, after many failures, was finally used to separate the antibody secreting cells by flow cytometry and the single cell reverse transcription two step PCR with multiple primers combination. The antibody gene was successfully amplified from individual B cells. The primer design was found in the optimization of single cell PCR conditions. The type of enzyme and the amount of template have great influence on the amplification efficiency, and the annealing temperature has little effect on the amplification effect. After the successful implementation of the antibody separation of single antibody secreting cells and single cell PCR technology, the blood samples were collected thirty-fifth days after the volunteers' immunization, and the density gradient sedimentation method was used. After separating the peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) from the blood samples, after the fluorescent staining of a group of specific leukocyte differentiation antigen clusters (cluster of differentiation, CD) molecules on a group of antibody secreting cells, 2112 single antibody secreting cells were selected from PBMC through a flow cytometry. Cloning, the antibody genes in single cells were amplified by single cell PCR. The results showed that the ratio of 529 single cell clone heavy chain and light chain amplification successfully, that is, the positive rate of single cell PCR is about 25%. when the hardware conditions are satisfied, only 1~2 days can be used to complete the separation of single cell and the process of amplification of antibody genes. It is very time-consuming and time-consuming to construct 529 pairs of antibody gene expression plasmids with the traditional cloning method. In order to express the antibody genes rapidly and high throughput, we designed the linear expression frame that can directly through the PCR fusion promoter, the preamble sequence, the full length gene of the antibody and the poly A tail (poly (A) tail). We optimize the linear expression frame PCR condition, and we optimize the linear expression frame of the linear expression frame. A control antibody was used to verify the expression and function of the antibody expressed in the linear expression frame. The results show that the antibody concentration in the linear expression frame method can reach 0.4 g/m L, and it keeps the antibody neutralizing effect well, and can meet the requirements of the follow-up antibody screening experiment. The linear expression frame designed by us can be used in the shortest day for 3 days. After ensuring the feasibility of the method, we constructed 529 pairs of antibody heavy chain and light chain linear expression frame according to the optimized overlap extension PCR conditions, and co transfected the paired genes into HEK 293T cells for expression. Then, 529 all human monoclonal antibodies were combined by ELISA and TNA. The specific binding PA domain of the monoclonal antibody was identified and identified. The results showed that we successfully obtained 34 PA binding monoclonal antibodies by establishing the antibody screening platform technique, of which 4 monoclonal antibodies (2A6,4A3,8H6,22F1) had PA neutralization activity. The neutralizing monoclonal antibody 22F1 was not combined with PA, but it played a role by combining with the PA's allosteric PA63. In addition, the study found that there is a toxin enhanced antibody (Toxin enhancing m Ab) that can accelerate cell death in PA binding monoclonal antibody, which accounts for more than 55.9% of the proportion of PA binding monoclonal antibodies. In order to further explore the characteristics of PA neutralization antibody and toxin enhanced antibody, 4 expression plasmids of PA neutralizing antibody and 1 toxin enhanced antibody 8A7 are constructed and transfected and purified. The antibody function was studied in the following aspects: 1) the affinity of 5 key monoclonal antibodies was measured by BLI technology. The results showed that the affinity of McAbs except 22F1 was at nanmore level; 2) neutralization activity in vivo and in vivo was measured in 4 neutralization McAbs in vivo and in vitro, and the results showed that the neutralization activity of 22F1 monoclonal antibodies in 4 neutralization McAbs. In vitro, its protective effect is nearly one order of magnitude higher than that of the PA monoclonal antibody reported in the market, while the monoclonal antibody 2A6 only has protective activity in vitro; 3) the neutralization mechanism of neutralization mAb is identified. The results show that the 8H6 monoclonal antibody has the inhibitory effect on the concentration dependent enzyme of the antibody to PA, and the 4A3 single antibody inhibits the formation of PA seven polymer by promoting the degradation of PA63, 4A 3 the toxin neutralization mechanism similar to the enzyme activity has not yet been reported, suggesting that a new neutralization mechanism of PA neutralization McAb may be discovered in this study. Toxin enhanced monoclonal antibody 8A7 can promote the formation of PA seven polymer and combine with PA seven polymer, suggesting the possible mechanism of the enhancement of the toxin; 4) analysis of the effect of the combination of McAbs and the results of 2A 6 and 4A3,8H6 and 22F1 showed a weak synergistic effect; 2A6 and 8H6,2A6 and 22F1 showed irrelevant effects; 4A3 and 8H6,4A3 and 22F1 showed antagonism. The synergistic effect of mAb combined drugs was the most significant, and the toxin enhanced the monoclonal antibody 8A7 and the neutralization activity of the monoclonal antibody only in vitro, and the combination of the two was able to play high water in the body. The results suggest that the possible cause of a large number of toxin enhanced antibodies in the human body is that the antibody can assist other antibodies in the blood to protect the body's protective effect. And the PA domain 3 of the 8A7 monoclonal antibody is a commonly considered PA non neutralization site, according to the results obtained in this article. It is presumed that the monoclonal antibody against the 3 epitopes of the PA domain can play an important biological function in coordination with other antibodies in the human body. This auxiliary monoclonal antibody is easily omitted during the antibody screening process, but its important role in the "cocktail" treatment can not be ignored. The rapid antibody screening platform for anti PCR technology can provide technical reserve for the study of new onset infectious disease antibodies; screening and preparing all human PA neutralization McAbs with different neutralization mechanisms, including new neutralization mAb, high school and active mAb, is a promising anthrax treatment drug; meanwhile, antibody neutralization mechanism, The study of epitope and combination effect also provides a strong basis for the research of the mechanism of anthrax toxin, the design of vaccine and the design of cocktail antibody drugs.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R517.2

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