Wang D, Berven E, Li Q, Uckun F, Kersey J H
University of Minnesota Cancer Center, Department of Laboratory Medicine/Pathology, University of Minnesota, Minneapolis 55455, USA.
Bioconjug Chem. 1997 Jan-Feb;8(1):64-70. doi: 10.1021/bc9600776.
In this report, we present the production of a dimeric form of anti-CD19 scFv, the FVS191cys (scFv')2. Anti-CD19 scFv FVS191cys was constructed by engineering a cysteine residue at the C terminus of the V1, domain of scFv FVS191. FVS191cys (scFv')2 was formed through a disulfide bond between two FVS191cys molecules. To optimize the yield of FVS191cys (scFv')2, the effects of oxidation time, buffer pH, and temperature on the formation of dimeric scFv were analyzed. Our study indicates that the formation of FVS191cys (scFv')2 is oxidation time- and buffer pH-dependent; a high pH buffer facilitates the formation of disulfide-linked (scFv')2. The maximum yield of FVS191cys (scFv')2 can be achieved when FVS191cys is air-oxidized at 4 degrees C, in buffer with a pH of 8.5-9. The biological activity of FVS191cys (scFv')2 was analyzed by ELISA and an internalization assay. FVS191cys (scFv')2 has a CD19 binding ability similar to that of its parental mAb B43 and is internalized by CD19 positive Nalm 6 cells. This study indicates that FVS191cys (scFv')2 is a potential candidate for tumor diagnosis or therapy.
在本报告中，我们展示了抗CD19单链抗体片段（scFv）的二聚体形式FVS191cys（scFv'）2的制备。抗CD19 scFv FVS191cys是通过在scFv FVS191的V1结构域C末端工程化一个半胱氨酸残基构建而成的。FVS191cys（scFv'）2是通过两个FVS191cys分子之间的二硫键形成的。为了优化FVS191cys（scFv'）2的产量，分析了氧化时间、缓冲液pH值和温度对二聚体scFv形成的影响。我们的研究表明，FVS191cys（scFv'）2的形成依赖于氧化时间和缓冲液pH值；高pH值缓冲液有利于二硫键连接的（scFv'）2的形成。当FVS191cys在4℃、pH值为8.5 - 9的缓冲液中进行空气氧化时，可实现FVS191cys（scFv'）2的最大产量。通过酶联免疫吸附测定（ELISA）和内化试验分析了FVS191cys（scFv'）2的生物学活性。FVS191cys（scFv'）2具有与其亲本单克隆抗体B43相似的CD19结合能力，并被CD19阳性的Nalm 6细胞内化。本研究表明，FVS191cys（scFv'）2是肿瘤诊断或治疗的潜在候选物。
