摘要
肺癌是目前世界上死亡率最高的恶性肿瘤之一,与20世纪初相比,现在肺癌已经成为威胁人类健康和生命的最大敌人之一。目前肺癌的治疗主要采取外科手术结合放疗和化疗的综合治疗。但肺癌早期少有症状或症状不明显,因此导致患者出现症状就诊者多属中晚期,疗效不佳。肺癌总的5年生存率在发达国家约为15%,我国低于10%。寻找新的治疗手段成为提高肿瘤治疗效果的突破口。其中,肿瘤的靶向治疗具有良好的发展前景。肿瘤靶向治疗强调治疗的特异性,在杀伤肿瘤细胞的同时尽可能保护正常细胞。其中,基于放射免疫治疗的原理,将放射性核素偶联靶向性特异抗体,可将抗体介导的细胞杀伤效应与放射性核素的治疗作用结合起来,从而提高肿瘤治疗的效果。
靶向治疗成功的关键在于抗体的制备及特异性靶点的选择。作为最具应用潜力的单克隆抗体,经历了鼠源性抗体的人源化改造、小分子抗体及文库展示抗体三个阶段。其中,以噬菌体展示文库抗体为代表的第三代抗体是基因工程抗体技术的新发展。
细胞的氧化还原状态与肿瘤的形成密切相关,参与细胞氧化还原的蛋白可用于研究肿瘤的发生和发展。Peroxiredoxin蛋白是新近发现的一类过氧化物酶,在清除活性氧族中具有重要作用,可以保护细胞免受过氧化反应造成的膜损伤。Peroxiredoxin I(Prx I)是Peroxiredoxin蛋白家族成员之一,在肺腺癌细胞中高表达。Prx I可以减少活性氧自由基的产生,其表达上调增强了肿瘤抗氧化能力,是抗凋亡和化疗耐药的重要原因。因此,Prx I可以作为肺腺癌治疗的特异性靶点。
为克服鼠源性单克隆抗体在人体应用时的异源性及治疗中大分子抗体穿透力较差的弊端,针对肺腺癌细胞高表达的Prx I蛋白,我们尝试利用噬菌体抗体库技术构建肺腺癌相关人源单链抗体库,制备抗Prx I肺腺癌人源单链抗体,评价其抑制肺腺癌细胞生长的能力,并标记放射性核素后行荷人肺腺癌裸鼠放免显像研究。
目的:通过噬菌体展示技术制备抗Prx I肺腺癌人源单链抗体并检测抗体性能,抗体标记放射性核素行放射免疫显像研究,评价肺癌放免靶向治疗的可行性,为肺癌的治疗提供新的辅助手段。
方法与结果:
第一部分噬菌体抗体文库的构建
1.人源单链抗体的制备:以肺腺癌病人癌旁淋巴结作为抗体B细胞来源,提取淋巴结总RNA。经RT–PCR法扩增得到cDNA文库。以cDNA为模板扩增VH和VL基因片段。再以VH和VL基因片段作为延伸连接肽的模板,分别用各自对应引物扩增VH–linker和VL–linker,再通过SOE–PCR反应扩增得到scFv,引入酶切位点Sfi I和Not I,胶回收PCR产物即得到scFv基因片段。结果表明,总RNA的电泳凝胶上可见2条带18 S与28 S;VH基因的大小约为370 bp,VL基因的大小约为350 bp,组装scFv基因大小约为750 bp。
2.重组噬菌体载体pCANTAB–5E的制备:纯化的单链抗体PCR产物分别经Sfi I和Not I双酶切反应后,与噬菌体载体pCANTAB–5E进行拼接。电击穿孔法将纯化的拼接产物转化感受态大肠杆菌TG1,得到2.8×107cfu/μg克隆。行随机质粒鉴定及双酶切鉴定,阳性插入率为88% (44/50)。阳性质粒行基因序列分析检测单链抗体的完整性。
3.噬菌体抗体的表达:用2×YT培养液收集上述阳性质粒,在M13KO7辅助噬菌体感染条件下,无糖环境诱导scFv片段的噬菌体表达。
第二部分噬菌体抗体库的筛选
1.肺腺癌细胞株对抗体库的筛选:先以正常人支气管上皮细胞HBE16负性筛选后,以肺腺癌A549细胞对噬菌体抗体库进行3轮富集,对抗体库富集前后滴度测定结果显示噬菌体收获率逐渐升高。
2.Prx I抗原对抗体库的筛选:将经A549细胞筛选的抗体库进一步用Prx I纯化抗原进行3轮筛选富集。一共进行6轮筛选富集。第1轮噬菌体收获率为1.3×10-6,第6轮噬菌体收获率为2.3×10-4,是第1轮的180倍。
3.可溶性scFv抗体的表达:将ELISA证实的强阳性噬菌体抗体株感染大肠杆菌E.coli HB2151,经IPTG (1 mmol/L)诱导培养后获得可溶性scFv抗体。经HitrapTM Anti–E Tag亲和层析柱纯化后得到纯化的可溶性抗体。
4.可溶性抗体的鉴定:SDS–PAGE检测可溶性抗体:将可溶性抗体行SDS–PAGE检测。考马斯亮蓝R250染色后观察可见在约30 ku处有条带出现,证实scFv抗体片段在大肠杆菌HB2151中实现可溶性表达。细胞ELISA测定可溶性抗体的免疫活性:肺腺癌细胞A549的A405 nm值0.63±0.08,乳腺癌细胞MDA–MB–435的A405 nm值0.36±0.05,正常支气管上皮细胞HBE16的A405 nm值0.37±0.06。
结果显示,可溶性抗体具有较高的特异性,能与肺腺癌细胞A549结合。抗体亲和力测定:通过竞争ELISA评估可溶性抗体与A549细胞的亲和力。结果显示,当抗体1:250稀释时,IgG的结合抑制率为4.5%;当抗体1:1稀释时,结合抑制率增加到66.1%。免疫细胞化学法测定可溶性抗体特异性:免疫细胞化学法检测表明,与MDA-MB-435细胞和HBE16细胞比较,A549细胞明显深染,证实scFv抗体与肺腺癌细胞A549特异性结合。
第三部分噬菌体抗体抑制肺腺癌细胞增殖研究
1.可溶抗体内摄量测定:用氯胺T法将放射性核素标记抗体,纯化后与肺腺癌A549细胞37℃孵育30 min和120 min。以未筛选的scFv及抗iASPP scFv(实验室自备)作为抗体对照,以4℃孵育条件作为温度对照。孵育结束后PBS洗涤细胞,再以2% SDS溶解细胞,收集洗脱液和溶胞产物,分别测定洗脱液和溶胞产物的放射性计数。
结果表明,相对于对照组,37℃条件下Prx I特异性肺腺癌单链抗体能有效结合、进入细胞内。
2.MTT及流式细胞术评价细胞增殖、凋亡情况:单链抗体作用于A549细胞72 h后进行MTT分析,发现抗体对细胞有剂量依赖的抗增殖作用。AnnexinV–FITC/PI法流式细胞仪检测A549细胞凋亡,抗体干预组细胞凋亡率较对照组明显增加。
3.Prx I蛋白表达水平测定:单链抗体干预A549细胞72 h后,提取总蛋白,进行Western blot检测。
结果显示,样品组Prx I蛋白表达水平低于对照组,证实可溶抗体干预后细胞Prx I蛋白表达受到抑制,Prx I表达量约为对照组的60%。
第四部分单链抗体的放射性核素标记及鉴定
氯胺T法将131I标记可溶性单链抗体,经Sephadex G200层析柱纯化后测定抗体的放射性核素标记率、比活度、放射化学纯度以及37℃条件下核素标记抗体与人血清孵育后放射化学纯度变化。
结果显示,抗体的131I标记率为83.2±6.5%,比活度为2.8±0.2 MBq/μg,放射化学纯度为95.6±3.7%。核素标记抗体与新鲜人血清孵育后测放射化学纯度,第48 h测得值与第1 h比较无明显降低,证实稳定性较好。
第五部分核素标记抗体在荷肺腺癌裸鼠模型体内的分布及SPECT显像
通过尾静脉将核素标记的单链抗体注射荷人肺腺癌裸鼠,不同时间点处死裸鼠,测定肿瘤及各组织%ID/g(每克组织百分注射剂量率)。同样裸鼠模型在不同时间点行SPECT显像。
结果显示,注射131I–scFv后,肿瘤组织对抗体的摄取量逐渐增加,荷瘤裸鼠模型的瘤/血、瘤/肌肉放射性比值逐渐升高,并在第48 h达到峰值(瘤/血、瘤/肌肉比值分别为4.19±0.13、4.89±0.56)。通过SPECT显像观察到在肿瘤区域的放射性浓聚,肿瘤组织得以显像,以第48 h时显像最为清晰。
结论:本研究利用噬菌体展示技术制备抗Prx I肺腺癌人源单链抗体,检测抗体性能及抑制肺腺癌细胞增殖能力,经放射性核素标记后行体内分布研究及放免显像。结果表明成功利用噬菌体展示技术制备全人源特异性肺腺癌相关单链抗体,该抗体能有效抑制肺腺癌细胞增殖。经放射性核素标记的单链抗体符合放免显像要求,靶向性较强,得到了较为满意的放免显像结果,为肿瘤的放免诊断及靶向治疗研究提供支持。
Currently, lung cancer is one of the malignant tumors with the highest mortality around the world. Nowadays, the lung cancer has become one of the greatest enemies to human health and life comparing that at the early 20th century. Surgery, chemotherapy and radiation therapy are the main methods of treatments in clinics. However, there is no significant symptom of the inchoate lung cancer, and most lung cancer patients are in advanced stage when diagnosed, leading to an unsatisfactory curative effect. The 5 year–survival rate of lung cancer in developing country is about 15%, and the number in our country is less than 10%. Finding new therapeutic tool is the breakthrough to enhance the curative effect of lung cancer. The targeted therapy of cancers has the good prospects for the development. Specificity therapy is the salient feature of targeted therapy, and the normal cells can be protected when the malignant tumor cells have been killed. Based on the principle of radioimmunotherapy, to couple the radionuclide with the specificity targeted therapy antibodies, we can combine the antibody–mediated cell killing effect and the therapeutic action of radionuclide together, in order to enhance the curative effect.
The key to a successful targeted therapy depends on the choice and construction of antibodies. The monoclonal antibody (McAb) is the most potential therapy antibody. There are three phases of the McAb development: humanization reform of the murine McAb phase, micromolecule antibody phase, and phage display library phase. The phage display antibody is the deputy of the 3rd phase antibody, representing a new development of gene engineering antibody technology.
The oxidoreduction status has close relation with the tumour generation. The oxidoreduction protein can be used for tumor study. Peroxiredoxins are newly discovered peroxidase proteins, playing an important role in active oxygen clearance. Peroxiredoxin I(Prx I)is a member of Peroxiredoxin family, overexpressing in lung adenocarcinoma. Prxs can depress the generation of active oxyradicals in cells, and protect cells from apoptosis. Thus, Prx I is a candidate therapy target of lung adenocarcinoma.
In order to overcome the malpractice of the heterology and low penetrating power of the murine McAb in clinic use, we attempted to prepare the human single–chain variable fragment (scFv) antibody by phage display technology with an target of lung adenocarcinoma cells overexpressing Prx I. The proliferation inhibition ability will be studied, and the antibodies will be used for radioimmunoimaging in bearing lung adenocarcinoma nude mice.
Objective: To prepare the anti–Prx I lung adenocarcinoma human single–chain antibodies, and detect the antibody efficiency. Label the scFv antibody with radionuclide 131I and make radioimmunoimaging study in bearing lung adenocarcinoma nude mice. This study will lay the foundation for lung cancer radioimmunotherapy, and provide useful adjunct treatment to lung cancer patients.
Methods and results:
Part 1 Construction of phage antibody library
1. Preparation of human scFv antibody: The B cells were obtained from the lymph nodes near the lung adenocarcinoma, and the total RNA was extracted. The cDNA gene were prepared and amplified by RT–PCR. The primer pairs for VH and VL gene amplified were determined by graticule bolting. Take the cDNA as a template, the VH and VL gene were amplified by PCR. Then, the VH–linker and VL–linker were amplified from the VH and VL, fragments with their respective primers. The amplified VH–linker and VL–linker fragments were then connected and amplified by SOE–PCR to form scFv, and the Sfi I and Not I restriction sites were inlet in scFv. The scFv fragments were purified by gel electrophoresis. The gel electrophoresis results showed that the total RNA of B cells has 2 bands (28S and 18S). A 370 bp band and 350 bp band were observed on the gel, representing the VH and VL gene respectively. The scFv fragments were detected on gel as a 750 bp band.
2. Ligation of scFv fragments into phage vector: After Sfi I/Not I digestion, the purified scFv fragments were cloned into phage display vector pCANTAB–5E. Ligation mixtures were used to transform Escherichia coli TG1 through electroporation, and 2.8×107cfu/μg clones were obtained. The clones were then identified by Sfi I/Not I digestion analysis, the positive insert rate was 88% (44/50). The positive clones were then analyzed by sequence analysis.
3. Expressing of phage antibody: The positive plasmids were collected by 2×YT culture medium. The scFv fragments were induced for expression with help phage M13KO7 in a sugar–free environment. Part 2 Phage antibody library selection
1. Screening on lung adenocarcinoma cells: Before selection, normal human bronchial epithelial cells (HBE16) were used to deplete the phage library of nonspecific binders. Then, the phage library were screened on lung adenocarcinoma cells A549 3 rounds. The number of eluted phages increased after 3 rounds panning.
2. Screening on Prx I: The phage library after 3 rounds panning on A549 cells were then selected on Prx I for 3 rounds. So, the panning were performed 6 rounds altogether. The harvest rate of first round was 1.3×10-6, and after 6 rounds panning, the number was 2.3×10-4, which increased 180 times.
3. Expressing of soluble scFv antibody: The colonies giving high signals against Prx I were chosen for soluble expression. E. coli HB215l was infected with these chosen colonies with 1 mmol/L IPTG. The scFv fragments were purified over a HitrapTM Anti–E Tag column.
4. The identification of soluble antibodies: SDS–PAGE identification: The soluble antibodies were identified by SDS–PAGE. The protein of antibodies was separated on a polyacrylamide gel and stained with Coomassie Blue R–250. A clear 30 ku band was observed, confirming the scFv fragments had been soluble expressed in E. coli HB215l. The reactivity of soluble scFv analyzed by ELISA: The ELISA result showed the A405 nm value of scFv on A549 cells was 0.63±0.08, and the value on MDA–MB–435 cells and HBE16 cells were 0.36±0.05 and 0.37±0.06, which confirming the specificity of the scFv to lung adenocarcinoma cells. Antibody affinity analysis by competitive ELISA: The results showed that, at high concentrations of cell extract (1:1 diluted), the purified scFv showed a significant inhibition effect to competition antibody IgG (inhibition rate, 66.1%), whereas the inhibition rate of the low concentration group (1:250 diluted) descended to 4.5%. immunocytochemistry analysis: The result showed that the Prx I scFv stained the A549 cells much more significant than that on MDA–MB–435 cells and HBE16 cells, indicating the Prx I–specific purified scFv with high affinity to A549 cells.
Part 3 Proliferation inhibition study on lung adenocarcinoma cells
1. Internalization study: The scFv were labeled with radionuclide 125I using the chloramine T method. The A549 cells were incubated with purified antibodies at 37°C for 30 min and 120 min. As the controls, unselected scFv and irrelevant antibody anti–iASPP scFv (prepared in our laboratory) were labeled with 125I as well. The temperature control for 125I–scFv was held at 4°C for 120 min. The cells were washed, for the removal of surface bound 125I-scFv. Then, the cells were lysed with 2% SDS and cell lysate radioactivity was measured on aγ–counter. The total amount of 125I–scFv bound to cells was estimated from the sum of the acid washes (surface bound 125I–scFv) and cell lysate (internalized 125I–scFv) radioactivity. The result showed that, compared with the control group, the Prx I specificity scFv can combine the A549 cells and be internalized effectly.
2. MTT and flow cytometry analysis: After 72 h incubation with A549 cells, The MTT analysis was operated. The scFv showed a dose-dependent growth inhibition to A549 cells. The apoptosis of A549 cells were analyzed by flow cytometry (AnnexinV–FITC/PI). The scFv caused a significant apoptosis comparing with the control group.
3. Expression of Prx I protein analysis: After 72 h incubation with scFv, the expression of Prx I protein in A549 cells was examined by Western blot. Compared with the control group, scFv caused a decrease in Prx I expression in A549 cells, and the Prx I protein expressed in A549 cells was about 60% of that in control group.
Part 4 Radiolabeling of scFv and identification
The soluble scFv was radiolabeled with 131I using the chloramine T method and purified by gel–filtration on a Sephadex G200 column. The labeling yield, specific activity, radiochemical purity and the stability of 131I-scFv in serum were tested. The results showed that the labeling yield was 83.2±2.8%,specific activity was 2.8±0.2MBq/μg, and radiochemical purity was 95.6±3.7%. After 48 h incubation with fresh human serum, the radiochemical purity of the 131I–scFv showed no significant depression. Part 5 Biodistribution study and SPECT imaging:
The nude mice bearing lung adenocarcinoma were injected via the tail vein with purified 131I–scFv. At various times, the tumor and major organs were removed from groups, and were weighed and counted on aγ-counter to determine the %ID/g. The lung adenocarcinoma bearing nude mice were injected with 131I–scFv and fixed on boards for radioimmunoimaging analysis at designated times. The results showed that the tumor/blood and tumor/muscle values were increased after injection, and reached the peak of 4.19±0.13 and 4.89±0.56 at 48 h. The radioactivity was aggregated in tumor locations and the tumor imaging was clearly observed.
Conclusion: We prepared the anti–Prx I lung adenocarcinoma human scFv antibodies by phage display technology. The specificity and proliferation ability were analyzed. The scFv antibodies were labeled with radionuclide. Biodistribution study and SPECT imaging were performed. The results showed that the lung adenocarcinoma specificity human scFv antibodies have been prepared successfully. The scFv showed strong antiproliferative effects on lung adenocarcinoma cells. The satisfactory radioimmunoimaging effect and specific affinity to target tumor implicates its potential application in lung adenocarcinoma imaging diagnosis and immunotherapy.
引文
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