【摘要】：目的：帕妥木單抗(Panitumumab)是一種已上市的特異性靶向表皮生長(zhǎng)因子受體(EGFR)的抗體。然而由于其是一種IgG2亞類(lèi)抗體,抗體依賴(lài)的細(xì)胞介導(dǎo)的細(xì)胞毒作用(ADCC)較弱。因此我們將帕妥木單抗的可變區(qū)移植到IgG1亞類(lèi)的骨架區(qū)后得到新型抗體Pan,以提高其ADCC活性。接下來(lái),利用ProbodyTM技術(shù)對(duì)Pan進(jìn)行改造，，構(gòu)建了可被尿激酶型纖溶酶原激活因子(uPA)激活的新型前抗體Pan-P,以期能減輕抗EGFR抗體引起的毒副反應(yīng)。綜上所述,本課題旨在構(gòu)建具有強(qiáng)抑瘤效能且可被選擇性激活的前抗體,以達(dá)到在增強(qiáng)療效的同時(shí)降低其皮膚毒性的目的。方法：首先,利用基因工程技術(shù)將帕妥木單抗的可變區(qū)基因連接到IgG1亞類(lèi)抗體Fc段,轉(zhuǎn)入CHO細(xì)胞中表達(dá)、純化得到新型抗體Pan。利用以下手段對(duì)其進(jìn)行表征分析：SDS-PAGE等分析其分子量和純度；Biacore和ELISA測(cè)定抗體的親和力和抗原結(jié)合力；CCK-8試驗(yàn)測(cè)定抗體對(duì)A431細(xì)胞的生長(zhǎng)抑制效應(yīng)；LC-MS測(cè)定Pan的糖型比例；ADCC報(bào)告基因試驗(yàn)評(píng)估其體外ADCC活性。最后,在荷瘤小鼠體內(nèi)評(píng)估其抑瘤活性。其次,利用ProbodyTM技術(shù)對(duì)Pan抗體進(jìn)行改造。改造方法為在其輕鏈N端添加了封閉肽、連接肽及酶切底物肽等序列而得到了前抗體Pan-P。利用以下方法對(duì)其進(jìn)行表征分析：SDS-PAGE、LC-MS分析其可被uPA特異性酶切的特性；Biacore、ELISA及FACS表征其可被uPA酶切且激活的特性；CCK-8試驗(yàn)測(cè)定其激活后對(duì)A431及DiFi細(xì)胞生長(zhǎng)抑制的活性；冰凍組織原位染色技術(shù)評(píng)估Pan-P在結(jié)直腸癌患者腫瘤組織的酶切激活特性；同時(shí)在荷瘤小鼠體內(nèi)評(píng)估Pan-P的酶切激活后靶向性。最后,在荷瘤小鼠體內(nèi)評(píng)估其抑瘤活性。結(jié)果：我們獲得了Pan抗體,經(jīng)分析其純度大于99%。經(jīng)測(cè)定,Pan的親和力約為7.4×10-10M,與帕妥木單抗相似。同時(shí),CCK-8試驗(yàn)也證實(shí)其保持了帕妥木單抗對(duì)A431細(xì)胞的生長(zhǎng)抑制效能。更重要的是,我們發(fā)現(xiàn)在Pan的糖型比例中,非巖藻糖修飾的糖型比率為33%左右,這可能有助于增強(qiáng)其ADCC活性。在ADCC報(bào)告基因試驗(yàn)中,Pan介導(dǎo)ADCC效應(yīng)的效能顯著高于帕妥木單抗。尤其值得注意的是,Pan顯示的體內(nèi)抑瘤效能較之帕妥木單抗更強(qiáng)。在此基礎(chǔ)上改造得到的前抗體Pan-P可被酶切激活而恢復(fù)完全功能活性。具體結(jié)果如下：SDS-PAGE、LC-MS等試驗(yàn)證實(shí)uPA可對(duì)Pan-P進(jìn)行特異性酶切。Biacore、ELISA及CCK-8等試驗(yàn)證實(shí)Pan-P可被酶切激活而恢復(fù)與Pan相似的功能活性。更重要的是，Pan-P在結(jié)直腸癌患者腫瘤組織中亦可被酶切激活。最后,在荷瘤小鼠體內(nèi)證實(shí)了Pan-P的靶向性及抑瘤活性。結(jié)論：綜上所述,我們最終得到了具有酶激活特性及增強(qiáng)的抗腫瘤效能的前抗體Pan-P。未來(lái)用于結(jié)直腸癌等的治療時(shí),將有望增強(qiáng)治療效果并緩解抗EGFR拮抗劑對(duì)正常組織的毒性,提升治療指數(shù)。
[Abstract]:Objective: Patumumab (Panitumumab) is a specific antibody targeting epidermal growth factor receptor (EGFR). However, because it is a IgG2 subclass antibody, antibody dependent cell mediated cytotoxicity of (ADCC) is weak. So we transplanted the variable region of Patumumab into the skeleton region of IgG1 subclass and obtained a new antibody Pan, to improve its ADCC activity. After that, Pan was modified by ProbodyTM technique to construct a novel prokaryotic antibody (Pan-P,) activated by urokinase type plasminogen activator (uPA) in order to reduce the toxicity caused by anti-EGFR antibody. In conclusion, the aim of this study is to construct proantibodies which have strong tumor inhibition and can be selectively activated in order to enhance the therapeutic effect and reduce the skin toxicity. Methods: firstly, the variable region gene of the patumab antibody was linked to the Fc segment of IgG1 subclass antibody by genetic engineering technique, and expressed in CHO cells. The novel antibody Pan. was purified and purified. The molecular weight and purity of A431 cells were analyzed by SDS-PAGE, Biacore and ELISA were used to determine the affinity and antigen binding ability of A431 cells, and the growth inhibition of A431 cells was determined by CCK-8 assay. LC-MS and ADCC reporter gene assay were used to determine the proportion of Pan and ADCC activity in vitro. Finally, tumor inhibitory activity was evaluated in tumor-bearing mice. Secondly, Pan antibody was modified by ProbodyTM technique. The pre-antibody Pan-P. was obtained by adding the sequence of blocking peptide, ligating peptide and digesting substrate peptide to the N-terminal of its light chain. The characterization analysis was carried out by the following methods: SDS-PAGE,LC-MS, Biacore,ELISA and FACS were used to characterize the activity of the enzyme digested by uPA, and the characteristics of uPA specific enzyme digestion were analyzed by SDS-PAGE,LC-MS, Biacore,ELISA and FACS, respectively, and the results showed that it could be digested and activated by uPA. The growth inhibition activity of A431 and DiFi cells was determined by CCK-8 assay, and the activation characteristics of Pan-P in tumor tissue of colorectal cancer were evaluated by in situ staining technique. At the same time, the target activity of Pan-P was evaluated in tumor-bearing mice. Finally, tumor inhibitory activity was evaluated in tumor-bearing mice. Results: Pan antibody was obtained and its purity was higher than 99%. The affinity of Pan was about 7.4 脳 10 ~ (-10) M, which was similar to that of Patumumab. At the same time, CCK-8 test also confirmed that it maintained the growth inhibitory effect of Patumumab on A 431 cells. More importantly, we found that the ratio of non-fucose-modified sugars to Pan is about 33%, which may help to enhance its ADCC activity. In the ADCC reporter gene test, the efficiency of Pan mediated ADCC effect was significantly higher than that of Patumumab. In particular, Pan showed stronger tumor inhibition than Patumumab. On this basis, the pre-antibody Pan-P can be activated by enzyme digestion to restore its full functional activity. The results were as follows: SDS-PAGE,LC-MS et al confirmed that uPA could specifically endonuclease Pan-P, and Biacore,ELISA and CCK-8 showed that Pan-P could be activated by enzyme digestion to restore the functional activity similar to Pan. More importantly, Pan-P can also be activated by enzyme digestion in tumor tissues of colorectal cancer patients. Finally, Pan-P targeting and tumor-inhibiting activity were confirmed in tumor-bearing mice. Conclusion: to sum up, we finally got pre-antibody Pan-P. with enzyme activation and enhanced anti-tumor effect. In future treatment of colorectal cancer, it is expected to enhance the therapeutic effect, alleviate the toxicity of EGFR antagonist to normal tissue, and improve the therapeutic index.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R730.5

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉彥仿,付勇;重視抗體工程[J];中華病理學(xué)雜志;2003年01期

2 安懷杰,劉志剛,俞煒源;治療性全抗體的表達(dá)及其研究進(jìn)展[J];生物技術(shù)通訊;2004年03期

3 劉彥仿;第七屆國(guó)際抗體工程年會(huì)簡(jiǎn)訊[J];細(xì)胞與分子免疫學(xué)雜志;1997年02期

4 谷仁燁 ,姚月歌;抗體醫(yī)療的最新進(jìn)展[J];日本醫(yī)學(xué)介紹;2004年05期

5 唐文淵;能否用抗體代替抗生素[J];國(guó)外畜牧學(xué)(豬與禽);2005年05期

6 楊磊;張春明;王德芝;;體外展示技術(shù)及其在抗體工程中的應(yīng)用[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2009年13期

7 楊靖;孫衛(wèi)兵;鄺玉;;抗體的系統(tǒng)性檢測(cè)方法[J];四川生理科學(xué)雜志;2013年03期

8 周正任;抗體工程[J];微生物學(xué)雜志;1996年04期

9 陸詩(shī)華;李昌本;趙壽元;;抗體工程[J];科學(xué);1997年01期

10 周正任;抗體工程[J];日本醫(yī)學(xué)介紹;1998年12期

相關(guān)重要報(bào)紙文章 前4條

1 忠良　小莉;江蘇生物抗體工程與藥物創(chuàng)制研究院通過(guò)專(zhuān)家論證[N];常州日?qǐng)?bào);2009年

2 北京華經(jīng)縱橫經(jīng)濟(jì)信息中心 生物醫(yī)藥部;國(guó)際抗體治療藥物技術(shù)“熱氣騰騰”[N];醫(yī)藥經(jīng)濟(jì)報(bào);2005年

3 王博;抗體工程的新紀(jì)元[N];北京科技報(bào);2010年

4 北京華經(jīng)縱橫經(jīng)濟(jì)信息中心生物醫(yī)藥部;國(guó)際抗體治療藥物熱點(diǎn)聚焦[N];醫(yī)藥經(jīng)濟(jì)報(bào);2005年

相關(guān)博士學(xué)位論文 前4條

1 呂明;新型抗CD3小分子抗體的設(shè)計(jì)及其功能研究[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2007年

2 陶俊;人源性抗FGF-2中和性抗體的篩選，表達(dá)及鑒定[D];南方醫(yī)科大學(xué);2010年

3 阮承邁;抗HBsAg抗體在CHO細(xì)胞中表達(dá)量提高研究[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2002年

4 楊峗;靶向表皮生長(zhǎng)因子受體的新型前抗體制備及抗腫瘤功能研究[D];中國(guó)人民解放軍醫(yī)學(xué)院;2015年

相關(guān)碩士學(xué)位論文 前7條

1 李楊;抗β-淀粉樣蛋白鼠源融合抗體在Pichia pastoris中的表達(dá)、純化及性質(zhì)研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2006年

2 張付凱;抗對(duì)硫磷基因工程四價(jià)抗體在大腸桿菌中的高效表達(dá)[D];中國(guó)農(nóng)業(yè)科學(xué)院;2011年

3 相雙雙;基于GFP分子的抗體設(shè)計(jì)與改造[D];福建農(nóng)林大學(xué);2014年

4 楊濱;HAb18G/CD147及其單抗的分子模建對(duì)接和抗體人源化設(shè)計(jì)的研究[D];第四軍醫(yī)大學(xué);2007年

5 安懷杰;全抗體在CHO細(xì)胞的高效表達(dá)[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2004年

6 王曉娜;抗結(jié)核桿菌晶體蛋白人源IgA抗體研制[D];廣西醫(yī)科大學(xué);2012年

7 李存;抗禽流感病毒H5N1分泌型IgA在CHO細(xì)胞中的表達(dá)及免疫學(xué)活性分析[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2011年

本文編號(hào)：2344264