【作者】 王华茂；

【导师】 顾健人；

【作者基本信息】 复旦大学 ， 病原生物学， 2009， 博士

【摘要】 抗体治疗是生物治疗的一个重要方面,也是肿瘤生物治疗的一个重要方向。其中抗表皮生长因子受体（EGFR）治疗性抗体药物已经在多种肿瘤治疗中应用。但是EGFR在多种正常细胞和组织中均有表达,使得EGFR抗体具有一定的局限性,而表皮生长因子受体3型突变体（EGFRvⅢ）仅在肿瘤中存在,因此相对于EGFR,EGFRvⅢ是一个更为理想的靶点。本研究旨在开发抗EGFRvⅢ的单克隆抗体以及验证其在肿瘤治疗中的有效性,为肿瘤治疗药物开发提供一个具有潜在价值的治疗性抗体。第一部分抗EGFRvⅢ单克隆抗体的制备及其特征分析【目的】寻求可以与表皮生长因子受体3型突变体（EGFRvⅢ）受体特异性结合的单克隆抗体。【方法】以EGFRvⅢ重组蛋白和NIH-3T3 EGFRvⅢ稳定转染细胞系为抗原免疫BLAB/c小鼠,利用杂交瘤技术进行脾细胞和SP2/0骨髓瘤细胞系融合筛选得到特异性单克隆抗体细胞株。通过western blot、免疫组化和体内荧光成像等方法鉴定抗体的特异性。利用SPR技术测定抗体亲和力常数,ELISA方法测定抗体亚型,通过抗原分段表达并ELISA方法鉴定抗体结合表位。【结果】通过杂交瘤融合技术,经过融合、筛选和克隆化,获得了多株杂交瘤细胞株。其中单克隆抗体12H23能和表达EGFRvⅢ的U87-EGFRvⅢ细胞结合,和过表达EBFR的A431细胞有较弱的结合,但是和野生型的U87细胞没有明显的结合能力。活体荧光成像分析表明12H23可以富集到U87-EGFRvⅢ种植瘤中。对抗体进一步分析显示12H23属于IgG1亚型,识别的序列包含在EGFR 287—302aa（CC16多肽）内,该多肽的C端对抗体的结合更重要。另外该抗体具有较高的亲和力,其与抗原表位结合的解离常数Kd=1.73×10^-10M。【结论】我们利用EGFRvⅢ重组蛋白和NIH-3T3稳转细胞系混合免疫的方法,成功获得了具有与EGFRvⅢ高效结合的单克隆抗体。第二部分EGFRvⅢ在肝癌中的存在与作用【目的】鉴定EGFRvⅢ在肝癌中的存在与作用。【方法】通过western blot及RT-PCR等方法鉴定肝癌细胞系中EGFRvⅢ的表达情况,通过免疫沉淀、westernblot和RT-PCR方法鉴定肝癌组织中EGFRvⅢ的存在。利用稳定转染EGFR或EGFRvⅢ的Huh-7细胞系（分别称为Huh7-EGFR和Huh7-EGFRvⅢ）,通过细胞迁移、MTT等实验测定EGFRvⅢ对肝癌细胞系的生长以及对5氟尿嘧啶（5-FU）耐药性的影响。通过体内的成瘤实验测定EGFRvⅢ对肝癌细胞系成瘤能力进行鉴定。【结果】多种肝癌细胞系和肝癌组织中有检测到EGFRvⅢ的存在。EGFRvⅢ阳性的细胞生长速度较快,并且对5-FU较不敏感。Huh7-EGFRvⅢ细胞表现出较快的体内外生长、较强的迁移及较高的5-FU抵抗能力。【结论】EGFRvⅢ在肝癌细胞和肝癌组织中存在,它能够促进肝癌细胞的成瘤,同时和肝癌的5-FU敏感性有关。第三部分12H23单抗对表达EGFRvⅢ的肝癌细胞系的作用及其与5-FU联合对肝癌作用及初步机理研究【目的】验证抗EGFRvⅢ单克隆抗体12H23单独作用或与5-FU联合对肝癌的治疗作用。【方法】通过体外的MTT细胞毒性实验和体内的Huh7-EGFRvⅢ种植肿瘤模型的治疗作用,来验证12H23抗体对细胞增殖的抑制作用和潜在的治疗价值。通过western blot和荧光定量PCR等方法鉴定治疗前后相关基因的表达情况。【结果】12H23抗体在体外可抑制细胞增殖并促进5-FU的杀伤作用。体内治疗实验的结果显示25 mg/kg的12H23抗体可以显著抑制细胞肿瘤的生长,抑制率到达59.8%。此外相对较低浓度（12.5 mg/kg或5 mg/kg）的12H23抗体和5-FU（25 mg/kg）对Huh7-EGFRvⅢ种植瘤均有联合治疗作用。机理研究表明12H23可以通过降低DPD、TS和E2F-1的表达及提高OPRT表达来增强肝癌对5-FU的敏感性。【结论】12H23单抗具有对EGFRvⅢ表达阳性的肝癌的潜在治疗价值。12H23可望和5-FU联用增加对EGFRvⅢ阳性的肝癌的疗效。第四部分EGFRvⅢ人鼠嵌合单抗的制备及其初步特征分析【目的】重组表达EGFRvⅢ嵌合抗体并对抗体特性进行初步分析。【方法】通过RACE方法获得12H23抗体可变区基因序列,通过基因重组技术构建人鼠嵌合抗体的表达质粒,然后转染CHO细胞,筛选得到稳定表达抗体的细胞株,并通过ELISA免疫组合等方法,验证抗体的亲和力和特异性。【结果】成功获得了稳定高表达嵌合抗体的CHO细胞株,表达纯化的C12抗体经ELISA检测显示C12抗体和12H23识别同样的位点,具有类似的亲和力。免疫组化显示,C12抗体可以和EGFRvⅢ以及表达EGFR的Huh-7细胞结合,而与野生型的Huh-7细胞没有明显结合。【结论】我们成功构建了EGFRvⅢ的特异性嵌合抗体,并实现了在CHO细胞上的稳定高表达。而该嵌合抗体C12很好的保留了鼠源的12H23的亲和力和特异性,为后续进一步的临床前和临床实验提供了一个坚实的基础。更多还原

【Abstract】 EGFRvⅢhave only been observed in cancer but not normal tissues which make it an ideal target for cancer therapy.Antibody therapy has been developed to be potential option for treating cancer.In this study, we developed a monoclonal antibody 12H23 that can specifically recognize EGFRvⅢand also overexpressing EGFR.Additionally,we found EGFRvⅢhave played an important role in HCC tumorigenesis and 5-FU resistance. Hence we tried to use the anti-EGFRvⅢmAb 12H23 for therapy of HCC.The results shown that mAb 12H23 can inhibit EGFRvⅢpositive Huh-7 growth in vivo,and enhance therapeutic effect of 5-FU,which make it a promising agent of HCC therapy.Thereafter,we succefully made the chimeric antibody C12 derived from mAb 12H23.The CHO cell line stably expressing chimeric antibody C12 with a yield of 20 pg/cell/day.Moreover,mAb C12 remains most of the binding affinity and specificity of mAb 12H23.Thus,mAb C12 is promising for the preclinical study.SectionⅠEGFRvⅢtargeting antibody development【Objective】To obtain candidate anti-EGFRvⅢantibodies.【Methods】Recombinant EGFRvⅢand NIH-3T3-EGFRvⅢcell line were used for immunization of BLAB/c mice.Anti-EGFRvⅢantibodies were obtained by fusion of spleen B cell with SP2/0 cell and following hybridoma cloning. Western blot,immuno-staining and FACS were applied to identify antibody specificity.In vivo fluorescent imaging system was used to monitor the in vivo distribution of cyanin 5.5 labeled mAb 12H23.Antibody affinity was measured by SPR analysis while antibody subtype and epitope were identified by Elisa analysis.【Results】An antibody clone 12H23 was obtained by hybridoma screening.In FACS assay,the antibody has strong binding to U87-EGFRvⅢ（stably expressing EGFRvⅢ） cells,a relative week binding to A431 cell（overexpressing EGFR）,while has no obviously binding to Wt U87 cell.Results from the in vivo fluorescent imaging clearly showed the accumulation of mAb 12H23 to U87-EGFRvⅢxenograft but not U87 xenograft.Further study showed mAb 12H23 is an IgGl subtype, and its binding epitope is located in CC16 peptide（287aa-302aa of EGFR）. SPR analysis also showed that 12H23 bind the epitope at a dissociation constant Kd=1.73×10^-10M.【Conclusion】We have successfully obtained a candidate antibody targeting EGFRvⅢ.SectionⅡEpidermal Growth Factor Receptor vⅢenhance the tumorigenicity and 5-FU resistance in Human Hepatocellular Carcinoma【Objective】To determine the biological significance of EGFRvⅢin HCC including tumor progression and chemotherapy resistance.【Methods】To delineate biological characteristic of EGFRvⅢin human HCC cells, different HCC cell lines were evaluated for EGFRvⅢexpression,cell growth and drug resistance.At the same time,EGFRvⅢexpression cassette was introduced by lentivirus vector into representative Huh-7 cell line. The EGFRvⅢ-expressing transfectant was compared with its parent cell for cell proliferation and 5-FU induced apoptosis resistance.【Results】Expression of EGFRvⅢin Huh-7 cells produced constitutively activated EGFRvⅢreceptors.And the EGFRvⅢtransfectants exhibited an about 1.5 fold in cell proliferation in vitro and 2.7 fold in vivo.Huh7-EGFRvⅢcells also showed about 3 fold increase of migration when quantified by transwell migration assay.In addition,Huh7-EGFRvⅢshowed significantly contribution to 5-FU resistance in vitro and in vivo.【Conclusion】EGFRvⅢnot only plays a pivotal role in tumorigenicity of human hepatocellular carcinoma but also contribute to the 5-FU resistance of HCC.SectionⅢAn anti-EGFRvⅢantibody 12H23 as an effective therapeutic agent solely or combining with 5-FU in Human Hepatocellular Carcinoma【Objective】To examine the therapeutic effect of anti-EGFRvⅢantibody 12H23 solely or combining with 5-FU on Human Hepatocellular Carcinoma therapy.【Methods】MTT assay were performed of evaluate effect of mAb 12H23 or combination with 5-FU on HCC cells and HCC tumor xenograft therapy with mAb 12H23 or combination with 5-FU also been performed for in vivo effects. The enzymes correlated with 5-FU（TS,DPD,OPRT） and transcription factor E2F-1 in the cells treating with the indicated drugs also were examined.【Results】mAb 12H23 can strongly inhibit the growth of Huh7-EGFRvⅢxenograft in vivo.Additionally,12H23 and 5-FU have synergetic growth inhibition effect on the Huh7-EGFRvⅢboth in vitro and in vivo.mab 12H23 treatment on Huh7-EGFRvⅢcan downregulate the expression of E2F-1,TS and DPD while upregulate that of OPRT.【Conclusion】Antibody 12H23 have potential to be used as therapeutics solely or combination with 5-FU for the treatment of EGFRvⅢ-positive HCC.mAb 12H23 treatment can downregulate the expression of E2F-1,TS and DPD while upregulate that of OPRT.SectionⅣEngineering and characterization of chimeric monoclonal antibody（C12） for EGFRvⅢtargeting therapy 【Objective】To produce chimeric mAb derived from 12H23.【Methods】Genes encoding variable fragments were amplified from 12H23 hybridoma,and cloned into expression vector carrying constant region of human heavy chain and light chain.After transfection,CHO-DG44 cell line stably expressing the chimeric antibody named as C12 was selected with methotrexate（MTX）.Thereafter,immunostaining and ELISA were applied to analyze the bingding characteristics of the chimeric antibody C12.【Results】The vector encoding the chimeric antibody C12 was successfully constructed and introduced into CHO cells.CHO cells can stably express C12 with a yield of 20 pg/cell/day.Additionally,C12 showed almost the same binding affinity and specificity as its parent antibody 12H23.【Conclusion】The chimeric monoclonal antibody C12 derived from 12H23 was successfully generated.C12 remains most of the binding affinity and specificity of 12H23.更多还原