二硫键稳定的抗CD3/抗CD19微型双功能抗体的表达及活性测定

Construction and Expression of Disulphide Stabilized AntiCD3/AntiCD19 Diabody

构建了抗CD3/抗CD19(Diabody)微型双功能抗体,并且为增强其稳定性进一步改造为二硫键稳定的抗CD3/抗CD19(ds-Diabody)微型双功能抗体,表达并纯化后测定其生物学活性。构建二硫键稳定的抗CD3/抗CD19表达载体p AYZds CD3CD19,转化感受态大肠杆菌16C9进行原核表达,分离纯化后,经12%SDS-PAGE及Westen-blot鉴定。应用间接免疫荧光和竞争性免疫荧光结合流式细胞分析技术(FACS)检测ds-Diabody与CD19+Raji细胞和CD3+Jurkat细胞的结合活性。改造后的ds-Diabody能够在原核表达系统中进行可溶性表达,ds-Diabody在非还原性SDS-PAGE中,在45 k处可见一条带,Westen-blot在同一位置显示有带,在还原性SDSPAGE中45 k处条带消失,在28 k和26 k各有一条带,Western-blot在28 k显示有带,这些均与理论相符,45 k为二硫键稳定的二聚体,在还原剂巯基乙醇作用下二硫键断开,形成28 k和26 k的2条带。FACS检测ds-Diabody可与CD19+Raji细胞和CD3+Jurkat细胞特异结合,并能竞争性抑制单抗HIT3a和HIT19a与上述细胞的结合。说明改造后的二硫键稳定的抗CD3/抗CD19双功能抗体既能在原核系统中进行可溶性表达,同时又保留了与细胞的结合活性。

A diabody is a promising immunotherapy which is capable of recruiting a polyclonal T cells to specific antibody targetedexpressing tumor cells. However,the heavy chain( VH) and the light chain( VL) domains of diabodies dissociate easily because they are associated non-covalently. So,it is necessary to remodel the diabody to increase its stability in order to enhance the antitumor activity. Cysteine residues were introduced into the V domains of the anti CD3 segment of the anti CD3 / anti CD19 diabody by recombinant gene engineering technology to generate its disulfide stabilized( ds) format. Diabody and ds-diabody were both expressed in E. coli strain 16C9 and purified by 6 × His-tag affinity chromatography. And then,they were analyzed by Coomassie brilliant blue staining and western blot. The binding activity of the two diabodies was analyzed by flow cytometry. The Ser-100 of anti CD3 VLand Gly-44 of anti CD3 VHof diabody were successfully replaced by cysteines to generate the ds diabody. They were both produced and purified from E. coli 16C9 cells with excellent yields ranging from 4 to 5 mg / L of culture medium. Under reducing conditions,the ds diabody resolved and detected as two bands of approximately 28 ku and 26 ku,consistent with those of the diabody. However,under non-reducing conditions,the ds diabody was detected as one band at approximately 40 ku. Western blot analysis using an anti-His tag antibody proved that the two sc Fvs were linked by the disulfide bond. The ds diabody bound to both CD3-positive Jurkat cells and CD19-positive Raji cells as efficiently as the parental diabody. It was successful to construct the expression vector of ds-anti CD3 /anti CD19 diabody,and the soluble ds-diabody was purified with high yield and high affinity to target cells.