人源抗鼻咽癌LMP-1胞外区抗体Fab免疫毒素的制备及抗鼻咽癌实验研究

发布时间：2018-05-12 07:20

本文选题：噬菌体展示 + 人源抗体　；参考：《南京医科大学》2010年博士论文

【摘要】：鼻咽癌是来源于鼻咽上皮的高度恶性肿瘤,肿瘤进展极易侵犯颅底等重要结构,并较早发生颈部淋巴结转移和远处转移。鼻咽癌表现为种族和地区分布的特点,是我国十大高发恶性肿瘤之一,其发病率为20/100,000,已引发严重健康问题。鼻咽癌是由Epstein-Barr病毒(EBV)的潜伏感染、环境因素和宿主的基因遗传因素共同参与的多步骤交互作用而逐步形成的复杂性疾病。所有的鼻咽癌细胞都含有EBV基因组,并表达EBNA1,EBER1,EBER2,和LMP1,LMP2A,LMP2B,这使得这些病毒蛋白成为理想的肿瘤标志物,其中LMP1(latent membrane protein1,LMP1)在鼻咽上皮细胞的转化和癌变过程中的作用倍受关注,是目前公认的病毒癌蛋白。它以其独特的蛋白分子结构参与鼻咽癌发生和发展的全过程。以LMP1为靶标,设计并制备各种具有中和作用或靶向功能的抗体将会为鼻咽癌的靶向治疗提供新的方法。本研究拟以噬菌体抗体展示技术筛选人源抗LMP1胞外区抗体Fab(命名为hleaFab),对筛选获得的hleaFab进行表达、纯化以及功能鉴定。进一步通过体外实验显示该抗体与鼻咽癌细胞HNE2-LMP1胞外区特异性结合特性和靶向功能。在此基础上,制备免疫毒素hleaFab-丝裂霉素C(hleaFab-MMC),并通过体内和体外实验研究其靶向抗鼻咽癌作用。方法 1.通过全人源抗体库筛选人源抗LMP1胞外区抗体Fab(hleaFab)。利用细胞与抗原固相筛选相结合的方法进行筛选人源噬菌体抗体库(Fab抗体库)。第1、2、3、6轮通过细胞的差减筛选,第4、5、7轮通过固相筛选。其中,细胞差减筛选以鼻咽癌细胞HNE2为阴性细胞,鼻咽癌细胞HNE2-LMP1为阳性细胞;固相筛选以包被GST-LMP1融合蛋白为筛选抗原。筛选7轮后经噬菌体酶联免疫吸附反应(phage enzyme linked immunosorbent assay,phage ELISA)进行阳性克隆鉴定。 2.原核表达hleaFab抗体, hleaFab的纯化和hleaFab的功能鉴定。HleaFab噬菌体感染大肠杆菌Top 10 F’,培养细菌至对数生长期,IPTG诱导表达;表达蛋白产物使用Protein L亲和层析柱纯化;纯化产物经ELISA、流式细胞技术以及免疫荧光技术进行鉴定。 3.靶向鼻咽癌细胞LMP1胞外区人源抗体Fab-丝裂霉素C免疫毒素的制备及鉴定。HleaFab转换缓冲体系,活化抗hleaFab和丝裂霉素(Mytomycin C),将两者混合反应(hleaFab:Mytomycin C=1:1);脱盐,转换缓冲体系至PBS,纯化得到免疫毒素hleaFab-MMC。以体外培养的鼻咽癌细胞HNE2-LMP1为研究对象,以相同的hleaFab-MMC、MMC、hleaFab摩尔浓度(1600、800、400、200、100、50、25、12.5、6.25、3.125、1.563nmol/L)分别作用于鼻咽癌细胞系HNE2-LMP1 , MTT法检测hleaFab-MMC对人鼻咽癌细胞系HNE2-LMP1生长的抑制作用;流式细胞术检测hleaFab-MMC对鼻咽癌细胞HNE2-LMP1细胞凋亡和细胞周期的影响并结合荧光显微镜观察药物干预前后细胞形态学的变化。 4.观察靶向鼻咽癌细胞LMP1胞外区人源抗体Fab-MMC免疫毒素体内抑瘤效应。首先建立人鼻咽癌细胞HNE2-LMP1裸鼠移植瘤模型。二步法建模:细胞注射移植法,采用人鼻咽癌细胞HNE2-LMP1 2×10~6/ml注射于7只裸鼠一侧肩后腿外侧皮下,成瘤4只;微型瘤块移植法,将瘤块移植于43只裸鼠一侧后腿外侧皮下,成瘤40只。病理学及免疫组化检测移植瘤。挑选肿瘤大小基本一致、生活习性正常的32只。32只鼻咽癌HNE2-LMP1裸鼠动物模型随机分为四组,每组8只:hleaFab- MMC组、hleaFab组、MMC组、NS(生理盐水)组。肿瘤局部注射给药,间隔3天用药一次,每次200μl,连续3次,共9天。分别注射抗体hleaFab (0.8mg/ml,1.6×10~(-5) mol/kg)、hleaFab- MMC(0.8mg/ml ,1.6×10~(-5)mol/kg)、MMC(0.005mg/ml ,1.6×10~(-5)mol/kg)、NS(生理盐水)。每五天测量肿瘤直径一次,观察肿瘤体积大小并进行相关检测。测量5次后处死裸鼠,将移植瘤完整切除,称其重量,计算抑瘤率,在光学显微镜观察移植瘤细胞形态变化,采用免疫组化法检测各组瘤组织中潜伏膜蛋白1 (latent membrane protein 1,LMP1)的表达情况。结果 1.基因测序显示本实验获得Fab抗体基因序列第30号和36号2株轻链为κ链的抗LMP1胞外区抗体Fab(命名为hleaFab)。 2. HleaFab(36号)在原核表达系统内能够表达并正确组装;使用Protein L亲和层析柱纯化获得了纯度较高的抗体hleaFab并保留了抗原结合能力。ELISA、流式细胞技术以及免疫荧光技术检测结果显示,hleaFab能够与GST-LMP1胞外区融合蛋白及鼻咽癌细胞HNE2-LMP1胞外区特异性结合,而不与鼻咽癌细胞HNE2结合。 3. HleaFab-MMC组在1600-200nmol/L时保持在70%以上的细胞生长抑制率,其后的细胞抑制率随着浓度的递减而递减,呈剂量依赖关系,其半数细胞生长抑制率IC50约为3.7μg/ml;hleaFab组中,细胞生长抑制率随着hleaFab抗体浓度的递减而递减,呈剂量依赖关系,其细胞生长半数抑制率IC50约为44μg/ml,MMC在本实验的浓度范围内最大的细胞生长抑制率为37.9%,未能达到细胞的半数抑制。荧光显微镜观察hleaFab-MMC作用于鼻咽癌细胞HNE2-LMP1后可见细胞凋亡特征的形态学改变:细胞悬浮增多,细胞质内含物增多,细胞体积缩小、变圆、核固缩、碎裂,细胞折光性减弱,细胞内出现颗粒样物质,培养液中有较多的碎片。流式细胞仪分析发现, hleaFab-MMC组、MMC组、hleaFab组、PBS组细胞凋亡的比率分别13.88%、3.04%、2.78%、2.10%。HleaFab-MMC组与MMC组、hleaFab组、PBS组相比较,hleaFab-MMC在相同浓度作用下表现出较高的细胞凋亡率,同时hleaFab-MMC组细胞相应的S期细胞比率增加为76.35%,MMC组、hleaFab组、PBS组的S期细胞比率仅为34.94%、41.51%、28.38%。 4.细胞注射移植法7只裸鼠,成瘤4只,成瘤率57%;微型瘤块移植法43只裸鼠,成瘤40只,成瘤率93%。病理学及免疫组化检测移植瘤具备鼻咽癌细胞HNE2-LMP1表型。治疗后第10天、第15天、第20天测量肿瘤体积显示hleaFab-MMC组、MMC组肿瘤较hleaFab组、NS组生长缓慢,肿瘤体积差异有统计学意义(P0.05)。在疗程的第20天hleaFab-MMC组与MMC组相比,肿瘤体积差异有统计学意义(P0.05)。HleaFab组、hleaFab-MMC组、MMC组的抑瘤率分别为12%、36.7%、19.7%,hleaFab-MMC抑瘤率明显增加。hleaFab-MMC组与其它三组相比,平均瘤重明显减轻,差异有统计学意义(P0.05)。MMC组与NS组相比,平均瘤重差异有统计学意义(P=0.0200.05);免疫组化结果显示hleaFab-MMC组与对照组NS组相比,LMP1的表达量明显减少,差异有统计学意义(P=0.0290.05)。结论 1.通过全人源抗体库,采用差减筛选法可以获得抗LMP1胞外区抗体hleaFab。 2. HleaFab可以在原核生物大肠杆菌Top 10 F’中表达,并组装具有生物活性LMP1胞外区特异性抗体hleaFab。 3.在较低的浓度100 nmol/l作用下,免疫毒素hleaFab-MMC对鼻咽癌细胞HNE2-LMP1有明显的细胞抑制作用;免疫毒素hleaFab-MMC对鼻咽癌细胞HNE2-LMP1的生长抑制作用的机制之一是促进细胞凋亡;免疫毒素hleaFab-MMC能够影响鼻咽癌细胞HNE2-LMP1细胞周期变化,增加S期的比率。 4.免疫毒素hleaFab-MMC明显的抑制人鼻咽癌HNE2-LMP1 BALB/C荷瘤裸鼠体内肿瘤生长;免疫毒素hleaFab-MMC下调肿瘤细胞LMP1的表达。总之,本研究制备免疫毒素hleaFab-MMC具有靶向抑制LMP1阳性的裸鼠鼻咽癌移植瘤效应。其抑瘤机制可能为:下调HNE2-LMP1鼻咽癌肿瘤细胞LMP1的表达;诱导细胞凋亡;增加S期HNE2-LMP1鼻咽癌细胞比例,提高鼻咽癌细胞对化疗药物敏感性。因此本研究表明人源抗鼻咽癌细胞LMP1胞外区抗体hleaFab-MMC具有潜在的靶向治疗鼻咽癌临床应用价值。
[Abstract]:Nasopharyngeal carcinoma is a highly malignant tumor derived from the nasopharyngeal epithelium. The progression of the tumor is easily infringed on the important structures such as the base of the skull, and the cervical lymph node metastases and distant metastases occur earlier. Nasopharyngeal carcinoma is characterized by ethnic and regional distribution. It is one of the ten major malignant tumors in China. Its incidence is 20/100000, which has caused serious health problems. Cancer of the pharynx is the latent infection of the Epstein-Barr virus (EBV), the multi step interaction of the environmental factors and the genetic factors of the host. All the nasopharyngeal carcinoma cells contain the EBV genome, and the expression of EBNA1, EBER1, EBER2, and LMP1, LMP2A, LMP2B, which make these viral proteins ideal The role of LMP1 (latent membrane protein1, LMP1) in the transformation of nasopharyngeal epithelial cells and the carcinogenesis of the nasopharyngeal epithelial cells has attracted much attention. It is recognized as a viral oncoprotein. It participates in the whole process of nasopharyngeal carcinoma with its unique protein molecular structure. With LMP1 as the target, it is designed and prepared with various neutralization effects. Targeted antibody will provide a new method for targeted therapy of nasopharyngeal carcinoma.
In this study, a phage antibody display technique was designed to screen the human anti LMP1 extracellular domain antibody Fab (named hleaFab), and to express, purify and functional identification of the selected hleaFab. The specific binding and targeting function of the antibody to the HNE2-LMP1 extracellular region of nasopharyngeal carcinoma cells were further demonstrated by in vitro experiments. On this basis, the preparation of the antibody was prepared. Immunotoxin hleaFab- mitomycin C (hleaFab-MMC) was used to study its targeting anti nasopharyngeal carcinoma function in vivo and in vitro.
Method
1. the human anti LMP1 extracellular domain antibody Fab (hleaFab) was screened by the whole human source antibody library. The human phage antibody library (Fab antibody library) was screened by the combination of cell and antigen solid-phase screening. The 1,2,3,6 wheel was screened by cell differentiation, and the 4,5,7 wheel was screened by solid phase screening. The cell differential screening was used to screen the HNE2 of nasopharyngeal cancer cells to be negative. The HNE2-LMP1 of nasopharyngeal carcinoma cells was positive cells, and the solid phase screening was screened by GST-LMP1 fusion protein as the screening antigen. After screening 7 rounds, the positive clones were identified by the phage phage enzyme linked immunosorbent assay, phage ELISA.
2. prokaryotic expression hleaFab antibody, hleaFab purification and hleaFab function identification,.HleaFab phage infection of Escherichia coli Top 10 F ', cultured bacteria to logarithmic growth period, IPTG induced expression; expression protein products were purified by Protein L affinity chromatography column, and the purified product was identified by ELISA, flow cytometry and immunofluorescence technology.
3. target Fab- mitomycin C immuno toxin of human nasopharyngeal carcinoma cell LMP1 and identification of.HleaFab conversion buffer system, activation of anti hleaFab and Mitomycin (Mytomycin C), mixed reaction (hleaFab:Mytomycin C=1:1), desalination, conversion buffer system to PBS, purification of immunotoxin hleaFab-MMC. in vitro culture In nasopharyngeal carcinoma cell HNE2-LMP1, the same hleaFab-MMC, MMC, and hleaFab molar concentration (1600800400200100,50,25,12.5,6.25,3.125,1.563nmol/L) were used respectively in the nasopharyngeal carcinoma cell line HNE2-LMP1, and the MTT method was used to detect the inhibitory effect of hleaFab-MMC on the HNE2-LMP1 growth of human nasopharyngeal carcinoma cell lines; flow cytometry was used to detect hleaFab-M. Effects of MC on apoptosis and cell cycle of nasopharyngeal carcinoma cell line HNE2-LMP1 were observed, and the morphological changes of cells were observed by fluorescence microscope.
4. to observe the anti tumor effect of human antibody Fab-MMC immuno toxin in the extracellular region of nasopharyngeal carcinoma cell LMP1. First, the model of human nasopharyngeal carcinoma HNE2-LMP1 nude mice transplanted tumor was established. Two step method modeling: cell injection transplantation, HNE2-LMP1 2 x 10~6/ml of human nasopharyngeal carcinoma cells were injected into the lateral posterior leg of the shoulder of the human nasopharyngeal carcinoma cells, and the tumor was 4. The tumor block transplantation method was used to transplant the tumor to the lateral hind leg of 43 nude mice and 40. The tumor was detected by pathology and immunohistochemistry. 32.32 nasopharyngeal carcinoma HNE2-LMP1 nude mice were randomly divided into four groups: hleaFab- MMC group, hleaFab group, MMC group, and NS (physiological salt). Water group. Local injection of the tumor at a interval of 3 days, 200 L each time, 3 times for 9 days. The antibody hleaFab (0.8mg/ml, 1.6 x 10~ (-5) mol/kg), hleaFab- MMC (0.8mg/ml, 1.6 x 10~ (-5) mol/kg) were injected, and the diameter of the tumor was measured every five days to observe the size of the tumor. After 5 times of measurement, the nude mice were killed, the transplanted tumor was completely removed, the weight was measured, the tumor suppressor rate was calculated, the morphological changes of the transplanted tumor cells were observed in the optical microscope, and the expression of the latent membrane protein 1 (latent membrane protein 1, LMP1) in the tumor tissues was detected by immunohistochemistry.
Result
1. gene sequencing showed that the Fab antibody gene sequence thirtieth and 36 were obtained from 2 light chain LMP1 chain Fab antibodies (named hleaFab).
2. HleaFab (No. 36) could be expressed and correctly assembled in the prokaryotic expression system. The purified antibody hleaFab was obtained by using Protein L affinity chromatography column and the antigen binding capacity.ELISA was retained. The results of flow cytometry and immunofluorescence assay showed that hleaFab could be used with the fusion protein of GST-LMP1 extracellular domain and nasopharyngeal carcinoma. The extracellular domain of HNE2-LMP1 cells specifically bind to HNE2 but not with nasopharyngeal carcinoma cell line.
3. HleaFab-MMC group kept the cell growth inhibition rate above 70% at the time of 1600-200nmol/L, and the subsequent cell inhibition rate decreased with the decrease of concentration, which was dose-dependent, and the growth inhibition rate of half cells was about 3.7 mu g/ml, and the growth inhibition rate of cell growth decreased with decreasing of hleaFab antibody concentration in hleaFab group, in a dose dependent manner. The median inhibitory rate of cell growth was about 44 IC50 g/ml, and the maximum inhibitory rate of cell growth was 37.9% within the concentration range of this experiment, which failed to reach half of the inhibition of cell growth. The morphological changes of cell withering characteristics after hleaFab-MMC were observed after HNE2-LMP1 in nasopharyngeal carcinoma cells: cell suspension increased, and thin cell suspension was observed by fluorescence microscopy. The cell volume decreased, the cell volume reduced, the cell became round, the nuclear condensation, the fragmentation, the cell refraction weakened, the cells appeared granular material, and there were more fragments in the culture fluid. Flow cytometry found that the rate of cell apoptosis in group hleaFab-MMC, MMC, hleaFab, and PBS was 13.88%, 3.04%, 2.78%, 2.10%.HleaFab-MMC and MMC, hleaFa In group B, compared with group PBS, hleaFab-MMC showed higher cell apoptosis rate under the same concentration, and the ratio of S cells in group hleaFab-MMC increased to 76.35%, MMC, hleaFab, and PBS groups were only 34.94%, 41.51%, 28.38%..
4. cells were implanted in 7 nude mice, 4 tumor formation and 57% of tumor formation, 43 nude mice and 40 tumor cells with microtumor mass transplantation. The tumor rate 93%. pathology and immunohistochemistry detected the HNE2-LMP1 phenotype of nasopharyngeal carcinoma cells. Tenth days after treatment, fifteenth days and twentieth days, the tumor volume showed hleaFab-MMC group, group MMC tumor was compared to hleaFab group and NS group. The tumor volume difference was statistically significant (P0.05). The tumor volume difference between group hleaFab-MMC and MMC group was statistically significant (P0.05) in group.HleaFab, group hleaFab-MMC and group MMC on twentieth days of treatment. The tumor inhibition rate in group MMC was 12%, 36.7%, 19.7%, and hleaFab-MMC inhibition rate increased significantly compared with the other three groups, the average tumor was compared with the other three groups. The difference was statistically significant (P0.05) and the average tumor weight difference between group.MMC and NS group was statistically significant (P=0.0200.05), and the immunohistochemical results showed that the expression of LMP1 in hleaFab-MMC group was significantly decreased compared with the control group NS group, and the difference was statistically significant (P=0.0290.05).
conclusion
1. through the whole human antibody library, the anti LMP1 extracellular domain antibody hleaFab. can be obtained by the subtractive screening method.
2. HleaFab can be expressed in prokaryotic E.coli Top 10 F "and assembled with bioactive LMP1 extracellular domain specific antibody hleaFab..
3. under the action of low concentration of 100 nmol/l, immuno toxin hleaFab-MMC has obvious inhibitory effect on nasopharyngeal carcinoma cell HNE2-LMP1, and one of the mechanisms of the inhibitory effect of immuno toxin hleaFab-MMC on nasopharyngeal carcinoma cell HNE2-LMP1 is to promote cell apoptosis, and immuno toxin hleaFab-MMC can affect the HNE2-LMP1 cell cycle of nasopharyngeal carcinoma cells. Phase change, increase the ratio of S period.
4. immuno toxin hleaFab-MMC significantly inhibited the growth of human nasopharyngeal carcinoma HNE2-LMP1 BALB/C tumor bearing nude mice, and immuno toxin hleaFab-MMC downregulated the expression of LMP1 in the tumor cells.
In conclusion, the immuno toxin hleaFab-MMC prepared in this study has the effect of targeting LMP1 positive nude mice with nasopharyngeal carcinoma, which may be: down regulation of the expression of LMP1 in HNE2-LMP1 nasopharyngeal carcinoma cells, inducing apoptosis, increasing the proportion of HNE2-LMP1 nasopharyngeal carcinoma cells in S stage, and raising the sensitivity of high nasopharyngeal cancer cells to chemotherapeutic drugs. Studies have shown that human anti NPC LMP1 extracellular domain antibody hleaFab-MMC has potential clinical value in targeting nasopharyngeal carcinoma.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63

【参考文献】