抗CD20嵌合抗体片段Fab′突变体的表达和活性研究

One Amino Acid Mutation in an Anti-CD20 Antibody Fragment That Affects the Yield Bacterial Secretion and the Affinity

利用PCR方法从抗CD2 0单链抗体 (ScFv)表达载体上扩增抗CD2 0抗体轻链可变区基因 (VL)、重链可变区基因 (VH) ,同时在抗体的可变区引入突变 ,然后将VH、VL 基因重组到Fab′表达载体pYZF1中 ,构建抗CD2 0嵌合抗体Fab′片段表达载体 ,并在大肠杆菌 16c9中进行高效可溶性分泌表达。经大量的筛选 ,获得一个产量和活性均有所提高的突变克隆。其突变位点在轻链可变区的CDR1区 ,即G77→A(Ser→Asn)。突变的抗体的表达量为每克干菌 3 8mg ,而未突变抗体的表达量为每克干菌 1.3mg。突变体的亲和力常数Ka为 2 .2× 10 9L mol,约为突变前的 2倍。竞争性免疫荧光抑制实验表明 ,突变的Fab′片段能竞争性抑制鼠源性抗CD2 0抗体HI4 7和CD2 0表达细胞Raji细胞的结合 ,使HI4 7的结合阳性率由 98%下降至 37.5 5 % 。

Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non Hodgkin's B cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti CD20 antibody Fab′ fragment, PCR was used to mutate the anti CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab′ was constructed and expressed in E.coli . The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti CD20 antibody (HI 47 ) was successful mutated as Ser (GAG) Asn (CAG). The soluble expression of mutated anti CD20 Fab′ (CD20 7) was 3.8 mg/g dry cell weight, while the parent (CD20 2) was 1.3 mg/g dry cell weight. The affinity constant K a of CD20 7 was 2 2×10 9 L/mol. The primary results of competitive assays by FACS showed that CD20 7 could partially block the sites through which parent antibody (HI 47 ) bind to Raji cells. There was difference in the Raji cells (CD20+) binding activity between the mutant CD20 7 and parent CD20 2. The site mutation of anti CD20 Fab′ gene make it possible that the anti CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti CD20 Fab′ genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti CD20 engineering antibody (for instance: Fab'Diabody and miniantibody), and make it possible for anti CD20 antibody to be applied to tumor therapy in civil in the future.