bFGF全人单抗的研制及其抑瘤效果研究

Anti-bFGF antibody is an potential anti-tumor agent for remarkably suppressing angiogenesis of tumor tissue, tumor growth and metastasis. In Previous research, We abtained one mouse monoclonal antibody against bFGF which significantly inhibited tumor growth in vivo and in vitro. Our research showed there were high level anti-bFGF antibodies in arthritis patients, amyotrophic lateral sclerosis patients and systemic lupus erythematosus patients. In future work, we are going to obain human monoclonal antibodies, binding to the receptor-binding regions of bFGF, from the peripheral blood lymphocytes of the autoimmune disease patients by anti-bFGF Ig+ Immunomagnetic Sorting, CD19+IgG+anti-bFGF IgG+ Flow Cytometry Sorting and single cell RT-PCR . We will evaluate the anti-angiogenesis activity and anti-A549 xenograft cancer growth activity of the anti-bFGF human monoclonal antibodies. And we hope to obtain 1-2 anti-bFGF human monoclonal antibodies. Our methods can reduce the workload obtaining neutralizing antibodies, and gain entirely human antibodies. This research will establish the foundation for a novel antineoplastic antibody drug.

碱性成纤维细胞生长因子（bFGF or FGF2）抗体能显著抑制肿瘤血管新生、肿瘤生长和转移等，有望开发成新的抗肿瘤药物。鉴于肌萎缩侧索硬化症等自身免疫病患者血清中广泛存在高滴度bFGF自身抗体的特点，本研究拟采用免疫磁珠分选法和流式细胞仪从bFGF抗体阳性自身免疫病患者外周血PBMC中分选出表达bFGF抗体（识别bFGF受体结合位点）的记忆性B细胞，进而采用单细胞PCR克隆抗体可变区基因；构建完整bFGF全人单抗真核表达载体，转染HEK293T细胞进行表达；采用CCK-8、Transwell、流式细胞术等方法研究bFGF全人单抗体内外对人肺癌A549肿瘤血管新生、肿瘤生长和转移的影响，以期获得1-2株具有高效抑瘤作用的bFGF全人单抗。本研究采用抗原特异的人单记忆性B细胞PCR直接获得全人单抗，可直接用于临床；并且大大降低筛选中和抗体的工作量，为研制新靶标的肿瘤抗体药物奠定基础。

碱性成纤维细胞生长因子（basic fibroblast growth factor , bFGF or FGF2）抗体能显著抑制肿瘤血管新生、肿瘤生长和转移等，有望开发成新的抗肿瘤药物。实验室前期研究发现自身免疫病患者血清中广泛存在高滴度的bFGF自身抗体。本研究首先采用生物素化bFGF偶联磁珠分选表达bFGF抗体的B细胞；然后利用FGFR1竞争结合bFGF，获得靶向bFGF受体结合位点的B细胞。采用单细胞PCR克隆识别bFGF的全人单抗可变区基因；构建完整全人抗体真核表达载体AbVec-hIgG1-bFGF、AbVec-hIgLambda-bFGF，转染HEK293A细胞进行诱导表达。为了更好模拟bFGF的天然构象，利用酵母表达载体pYD2将bFGF展示于EBY100酵母细胞表面，流式细胞仪检测获得的人源bFGF抗体可特异的与bFGF结合。通过检测不同肺癌细胞株鼠肺癌LL/2、人肺癌A549、H460、95-D等bFGF、FGFR1 mRNA及其蛋白表达水平，确定以LL/2肺癌细胞为靶细胞，研究bFGF人源抗体体内外抑瘤效果。体外细胞增殖、迁移及成管等实验表明，获得的人源bFGF抗体能够抑制血管内皮细胞HUVEC增殖及成管、抑制LL/2肿瘤细胞增殖及迁移；体内抑瘤实验结果表明，bFGF人源抗体能够抑制LL/2肺癌增长，并且能够抑制其向肺脏转移。可能机制是bFGF人源抗体通过下调 AKT(T-308) 、 GSK3-β(Ser-9)的磷酸化和抑制Snail的表达，剂量依赖性的上调LL/2肺癌细胞E-cadherin的表达来增强细胞间连接，进而抑制肿瘤细胞的迁移。