嵌合抗CD20基因工程抗体的构建表达及其功能研究

【作者】 赖增祖；

【导师】 杨纯正； 朱祯平；

【作者基本信息】 中国协和医科大学 ， 基因工程抗体及肿瘤分子药理， 2000， 博士

【摘要】 本研究采用噬菌体表面显示技术克隆抗CD20抗体轻、重链可变区基因，构建嵌合抗CD20抗体及其Fab’片段，在哺乳动物细胞和大肠杆菌中进行表达；并对Fab’抗体片段的体内外抗瘤活性进行研究，以期将抗CD20抗体HI47推向B淋巴细胞瘤临床治疗应用。 一 轻重链可变区基因克隆及单链抗体库的构建 常规提取HI47杂交瘤细胞总RNA，利用RT-PCR法扩增抗体轻重链可变区基因片段，经Overlap PCR，利用连接链（G₄S）₃基因片段将抗体轻重链基因连接构成单链抗体（ScFv）基因，将ScFv基因重组到pCANTAB 5E载体上，构建抗CD20单链抗体噬菌体文库（约3×10⁶克隆），以表达CD20分子的Daudi细胞为靶抗原，经Panning，ELISA法筛选，获得多个具有CD20结合活性的克隆，对克隆6的基因序列进行序列测定分析，结果显示克隆6的基因序列完符合PDB文库中的抗体基本框架序列，是抗体基因序列。 二 嵌合抗体的构建表达及活性鉴定 利用PCR从表达载体pCANTAB 5Ecd20ScFv上扩增抗CD20抗体轻重链可变区基因，将VH、VL基因克隆到表达载体pYZF中，构建抗CD20抗体表达载体pYZFcd20，并在大肠杆菌中进行高效可溶性分泌表达Fab’，表达量可达3mg／ml。 本研究对嵌合抗CD20 Fab’片段的体内外活性进行了进一步的研究，竞争性免疫荧光抑制试验结果显示，Fab’特异结合Daudi细胞表面CD20，MTT法测定结果表明，Fab’表达产物抑制Daudi细胞、Raji细胞的生长，IC₅₀分别为69μg／ml、26μg／ml。³H掺入试验显示，嵌合抗CD20 Fab’不抑制Daudi细胞、Raji细胞DNA合成，但对RNA合成具有抑制作用。以50mg／kg剂量，四天一更多还原

【Abstract】 To make monoclonal antibody HI47 enter the clinic therapy of B cell lymphoma, genes of variable region of HI47 were cloned by phage display technology, chimeric antibody was constructed to low HAMA of HI47 and expressed in mammalian cell and Ecoli.1 Cloning of antibody variable region genes and construction of anti-CD20 ScFv phage display libraryExtract total RNA of HI47 hybridoma cell, amplify the genes of antibody variable region of heave chain（VH） and light chain（VL） by RT-PCR, VH and VL were linked by overlap PCR with （G₄S）₃ linker to construct ScFv, ScFv was then cloned into the vector pCANTAB 5E to construct the anti-CD20 ScFv phage display library, through panning, screening by ELISA with Daudi cell as target cell and getting several clones binding specially to CD20, result from the DNA sequencing of clone 6 indicated that this sequence is an antibody gene sequence.2 Construction, expression and function study of chimeric anti-CD20 antibodyAmplified genes of VH and VL of HI47 from the anti-CD20 ScFv expression vector pCANTAB5Ecd20 by PCR, then cloned VH and VL into the vector pYZF containing human antibody CH1 and CL to construct Fab’ expression vector pYZFcd20, transformed 16C9 with pYZFcd20 and expressed Fab’ in 16C9, The yield of Fab’ in 16C9 is about 3mg/ml.Bioactivities of Fab’ was studied in vivo and vitro. competitive inhibition of immune fluorescence experiment show that the Fab’ inhibits the binding of monoclonal antibody HI47 to CD20 on the surface of Raji cell. Growth inhibition of Daudi and Raji cell by Fab’ were observed by MTT method. Fab’ inhibited the synthesis of RNA but not DNA of Daudi cell and Raji cell. Experimental therapy of Raji cell lymphoma in dose of 50mg/kg, every four days once, 6 times in a nude mouse xenograft model of Raji cell showed that Fab’ has a good anti-tumor effect on Raji cell lymphoma, the tumor growth inhibition rate is about 56%. Induction of tumor cell apoptosis maybe one of anti-tumor mechanisms of chimeric anti-CD20 Fab’ antibody fragment .VH and VL genes were cloned into pDR2 containing human antibody CH1,更多还原