鼠抗人B7-H4单克隆抗体的研制及B7-H4分子在肿瘤细胞中表达特性的分析

发布时间：2018-02-26 23:04

本文关键词： B7-H4 单克隆抗体单核细胞肿瘤基因工程抗体　出处：《苏州大学》2007年硕士论文　论文类型：学位论文

【摘要】： B7-H4分子是新近发现的B7家族又一负性协同刺激分子。尽管其mRNA在淋巴和非淋巴组织中呈广泛性表达,但由于受到转录后水平的严格调控,B7-H4蛋白仅在正常组织中呈现微弱的表达。越来越多的研究发现,在肿瘤组织如卵巢癌、肺癌、乳腺癌、肾癌和腺样囊性癌等的肿瘤细胞以及肿瘤相关巨噬细胞中异常高表达B7-H4分子。在B7-H4信号的作用下,肿瘤周围的CD3+T细胞数量显著减少,活化CTL的增殖受到明显抑制,机体抵抗肿瘤的免疫应答大为削弱。与此同时,B7-H4分子还能抑制肿瘤自身的凋亡,促进上皮细胞恶性转化,为肿瘤免疫逃逸提供了必要的条件。临床研究发现,肿瘤细胞B7-H4的表达与临床和病理学特征息息相关。高表达B7-H4的患者往往肿瘤恶性程度高,预后结果差。此外,在肿瘤患者血清中高水平可溶性B7-H4分子的存在,还为肿瘤患者的临床诊断,尤其是卵巢癌的早期发现,提供了新的检测指标和依据。为此,继负性协同刺激分子B7-H1,B7-H4成为研究肿瘤免疫逃逸和临床诊断治疗的新热点。本研究旨在以高表达B7-H4的基因转染细胞为抗原,应用细胞融合技术,研制鼠抗人B7-H4单克隆抗体,为进一步探求B7-H4分子在肿瘤免疫逃逸中的生物学特性和意义提供有效的研究手段,进而为临床诊断、预后和治疗开拓新的道路。一.鼠抗人B7-H4分子功能性单克隆抗体的研制及生物学特性的鉴定【目的】研制鼠抗人B7-H4单克隆抗体,为探讨人B7-H4分子在免疫细胞上的表达特性及其介导的负性信号对人T淋巴细胞抑制作用的效应提供必备的研究手段。【方法】以高表达人B7-H4分子的基因转染细胞株293T/B7-H4为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,并以该基因转染细胞293T/B7-H4作为阳性筛选细胞,以转空质粒的对照细胞293T/Mock作为阴性对照细胞。经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,获得能特异分泌鼠抗人B7-H4分子单克隆抗体的杂交瘤细胞株;采用细胞核染色体计数、Ig亚型快速定性试纸法、竞争结合抑制以及Western blot等实验对获得的杂交瘤细胞株及单克隆抗体进行生物学特性的鉴定;用间接免疫荧光法初步分析单抗对正常人外周血淋巴细胞表面B7-H4蛋白的识别作用以及单核-树突状细胞诱导过程中B7-H4分子表达的变化。向293T/B7-H4与T细胞的混合反应体系中加入单抗3C8,用3H-TdR掺入法检测单抗对B7-H4信号抑制活化T细胞增殖的阻断效应。【结果】成功获得了1株持续、稳定分泌鼠抗人B7-H4单克隆抗体的杂交瘤细胞株3C8,细胞核型分析显示,杂交瘤细胞株的染色体数目在100条以上,超过小鼠B细胞和SP2/0细胞的染色体数,为两者融合体细胞;快速定性试纸分析显示,这株单抗轻链为κ链,重链为IgG1亚类; Western blot分析结果显示单抗3C8能与B7-H4分子特异性结合,形成阳性条带;单抗识别的抗原表位分析结果表明,单抗3C8与商品化抗体H74识别的抗原表位较为接近,而与科室已有的一株鼠抗人B7-H4单抗1G7的抗原识别表位完全不同。流式细胞仪检测结果表明,单抗3C8能检测到健康人外周血CD14+单核细胞上B7-H4的表达,而在CD4+、CD8+、CD19+和CD56+淋巴细胞上则均未检测到B7-H4分子。此外,在单核-树突状细胞的诱导过程中,IL-4能下调B7-H4的表达,而LPS刺激imDC后则能上调表达B7-H4,GM-CSF对B7-H4表达的影响不明显。3H-TdR结果显示,单抗3C8能有效地阻断B7-H4信号对活化T细胞增殖的抑制效应。【结论】成功获得了1株持续、稳定分泌鼠抗人B7-H4阻断型单克隆抗体的杂交瘤细胞株,其不同的抗原表位为进一步研制可溶性B7-H4分子ELISA试剂盒奠定了良好的基础。同时,健康人外周血CD14+单核细胞上B7-H4表达的变化以及单抗3C8对B7-H4信号地阻断作用,为进一步研究机体免疫自稳和免疫调节开拓了道路。二.B7-H4分子在肿瘤细胞中表达特性的初步研究【目的】探寻肿瘤细胞和肿瘤微环境中B7-H4分子的表达特性,为进一步研究B7-H4与肿瘤免疫逃逸及其相互关系开拓新的途径。【方法】采用间接免疫荧光标记法和流式细胞术分析不同来源人肿瘤细胞株以及肿瘤患者胸、腹水中CD14+单核细胞膜表面B7-H4分子的表达;采用细胞免疫组化分析B7-H4分子在肿瘤(肺癌)细胞株中的分布;Western blot鉴定肿瘤和肿瘤微环境中B7-H4蛋白的表达形式和糖基化修饰水平;同时运用RT-PCR技术检测B7-H4分子在mRNA水平上的不同剪接形式,探求不同表达形式的B7-H4分子与肿瘤逃逸的相关关系。【结果】在多株肿瘤细胞,尤其是上皮性肿瘤细胞株(如:肺癌、胃癌)表面存在B7-H4分子的表达。肿瘤患者胸、腹水中的单核细胞不同于健康人外周血单一的B7-H4+单核细胞,存在B7-H4+和B7-H4-两个群体。细胞免疫组化实验显示,肿瘤细胞株胞浆中也存在大量的B7-H4分子。Western blot结果表明,肿瘤细胞株、肿瘤浸润性单核细胞以及健康人外周血单核细胞中B7-H4分子量大小均在34KD左右,分子量较小,而RT-PCR结果则显示,在这些肿瘤和肿瘤相关的细胞中没有检测到B7-H4分子剪接体的存在。【结论】多株肿瘤细胞以及肿瘤患者胸、腹水中CD14+单核细胞虽然都以正常大小的B7-H4cDNA转录本为主,但其表达的B7-H4蛋白分子量较小,糖基化水平不高。低糖基化水平B7-H4分子在肿瘤和肿瘤微环境中所发挥的调控作用及机制值得关注和进一步深入探讨。此外,肿瘤患者胸、腹水中CD14+B7-H4+和CD14+B7-H4-是否分别代表着具有不同免疫调节功能的单核巨噬细胞亚群仍有待进一步探明。三.抗人B7-H4单克隆抗体3C8可变区基因的克隆及嵌合轻、重链基因的拼接【目的】在已获得的功能型单抗3C8的基础上,克隆和拼接嵌合轻、重链基因,为抗体人源化构建和改造铺平道路。【方法】根据抗体保守的框架区和前导序列设计简并引物,采用RT-PCR技术克隆出单抗3C8的轻重链可变区;应用生物信息学方法对所扩增的序列进行CDR区的分析和二级结构定位、天然信号肽预测、抗体可变区二级拓扑结构分析以及三维模拟。采用TP-PCR法将分析正确的鼠源性抗体可变区基因分别与人IgG1和κ链恒定区进行拼接,形成嵌合轻、重链基因。【结果】成功克隆到了含有信号肽序列的鼠抗人B7-H4单克隆抗体轻、重链可变区基因;经生物信息学分析,抗体轻链可变区由V1-117*01和J1*01两个亚群基因重排后形成,而重链可变区则由V1-14*01和J4*01以及D2-2*01三个亚群基因重排后形成。其轻、重链信号肽切割位点分别位于SSS-DS和VHS-EV,在第19-20个氨基酸之间。二级拓扑结构和三维模拟显示,所获序列除信号肽区为α-螺旋外,其他部分主要由β-折叠构成,抗体CDR区位于反向平行的β-折叠之间,暴露在整个蛋白的表面,可与抗原相互结合。经PCR拼接获得的嵌合轻、重链基因大小分别约为750bp和1500bp,与理论值相一致。【结论】抗体可变区基因的成功克隆及拼接,可变区CDR区域、二级拓扑结构分析以及三维结构的模拟,为进一步展开抗体3C8的人源化以及基于DNA点突变技术的抗体亲和力改变实验打下了坚实的基础。综上所述,本项研究成功获得了1株能稳定分泌特异性鼠抗人B7-H4分子的新型功能型单抗,它能有效阻断B7-H4信号;其抗体可变区基因的克隆及嵌合轻、重链的拼接为继续开展抗体人源化和靶向B7-H4分子的肿瘤免疫治疗提供了必要的物质基础。此外,多株肿瘤细胞以及肿瘤患者胸、腹水中CD14+单核细胞虽然都以正常大小的B7-H4cDNA转录本为主,但其表达的B7-H4蛋白分子量较小,糖基化水平不高。低糖基化B7-H4分子在健康人外周血和肿瘤患者胸、腹水中CD14+单核细胞上表达强度的差异,也将为我们探索机体免疫自稳和肿瘤免疫逃逸提供新的线索。
[Abstract]:B7 - H4 molecule is a newly discovered B7 family and a negative co - stimulatory molecule . Although its mRNA is widely expressed in lymph and non - lymphoid tissues , B7 - H4 protein can be expressed weakly in tumor cells such as ovarian cancer , lung cancer , breast cancer , renal cancer and cystic carcinoma . The purpose of this study was to develop murine anti - human B7 - H4 monoclonal antibody with high expression of B7 - H4 gene - transfected cells . To further explore the biological characteristics and significance of B7 - H4 molecule in tumor immune escape , this study provides a new way for clinical diagnosis , prognosis and treatment . 1 . Preparation and characterization of murine anti - human B7 - H4 functional monoclonal antibody Objective To study the anti - human B7 - H4 monoclonal antibody of murine anti - human B7 - H4 , and provide necessary research methods for the study of the expression of B7 - H4 molecules on immune cells and the effect of negative signals mediated by human B7 - H4 on human T lymphocytes . A hybridoma cell line with monoclonal antibody against B7 - H4 in human peripheral blood was obtained by means of indirect immunofluorescence and flow cytometry . The results showed that monoclonal antibody 3C8 could inhibit the expression of B7 - H4 in human peripheral blood CD14 + monocytes . Conclusion The monoclonal antibody of B7 - H4 monoclonal antibody against human B7 - H4 has been successfully developed . The different epitopes of the hybridoma cell line have established a good foundation for further development of the soluble B7 - H4 molecule ELISA kit . At the same time , the expression of B7 - H4 in the peripheral blood CD14 + monocytes in healthy human peripheral blood and the blocking effect of monoclonal antibody 3C8 on B7 - H4 signal have been successfully developed , which has opened the way for further study on the immune homeostasis and immune regulation of the organism . Preliminary study on the expression of B7 - H4 molecule in tumor cells Objective To explore the expression of B7 - H4 molecules in tumor cells and tumor microenvironment , and explore new ways to further study B7 - H4 and tumor immune escape and their correlation . Methods : The expression of B7 - H4 and B7 - H4 in tumor and ascites were analyzed by indirect immunofluorescence and flow cytometry . The expressions of B7 - H4 in tumor and tumor microenvironment were analyzed by immunohistochemistry . Western blot was used to identify the expression of B7 - H4 protein in tumor and tumor microenvironment . Western blot was used to identify the expression of B7 - H4 protein in tumor and tumor microenvironment . The expression of B7 - H4 and B7 - H4 in peripheral blood mononuclear cells of tumor cells , tumor infiltrating mononuclear cells and healthy human peripheral blood mononuclear cells showed that the molecular weight of B7 - H4 in tumor cell lines , tumor infiltrating mononuclear cells and healthy human peripheral blood mononuclear cells was less than 34KD , and the molecular weight of B7 - H4 was low . III . Cloning of anti - human B7 - H4 monoclonal antibody 3C8 variable region gene and splicing of chimeric light and heavy chain genes Objective : To clone and splice the chimeric light and heavy chain genes on the basis of the obtained functional monoclonal antibody 3C8 , and pave the way for the construction and transformation of humanized antibody humanized antibody . According to the conserved framework region and leader sequence of antibody , the monoclonal antibody 3C8 heavy chain variable region was cloned by RT - PCR technique , and the amplified sequence was analyzed by using bioinformatics method , secondary structure localization , natural signal peptide prediction , secondary topological structure analysis of antibody variable region and three - dimensional simulation . The light weight and heavy chain signal peptide cleavage sites were composed of V1 - 117 * 01 and J4 * 01 and D2 - 2 * 01 three sub - group genes . The light weight and heavy chain signal peptide cleavage sites were located between 19 - 20 amino acids . Conclusion The successful cloning and splicing , CDR region , secondary topological structure analysis and 3D structure simulation of antibody variable region gene can provide a solid foundation for further developing humanized antibody 3C8 and antibody affinity change experiment based on DNA point mutation technology . In conclusion , a novel functional monoclonal antibody capable of stably secreting specific mouse anti - human B7 - H4 molecule is successfully obtained in this study , which can effectively block the B7 - H4 signal , and it can effectively block the B7 - H4 signal , and the splicing of the heavy chain is a necessary material basis for the continuation of antibody humanized and targeted B7 - H4 molecule tumor immunotherapy .

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R730.3

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