Humphreys D P, Vetterlein O M, Chapman A P, King D J, Antoniw P, Suitters A J, Reeks D G, Parton T A, King L M, Smith B J, Lang V, Stephens P E
Celltech Therapeutics, Slough, Berkshire, UK.
J Immunol Methods. 1998 Aug 1;217(1-2):1-10. doi: 10.1016/s0022-1759(98)00061-1.
Fab's with hinges based on the human gamma1 sequence containing 1, 2, or 4 cysteines have been produced by high level Escherichia coli periplasmic secretion, and coupled in vitro by reduction/oxidation to form F(ab')2. We find that the F(ab')2 made with hinges containing 2 or 4 cysteines have a high level (approximately 70%) of multiple disulphide bonds. These F(ab')2 molecules have an increased pharmacokinetic stability as measured by area under the curve compared to those made by direct coupling through a single disulphide bond. One particular molecule containing 4 hinge cysteines has a greater pharmacokinetic stability than a F(ab')2 formed by chemical cross-linking. F(ab')2 made from the Fab' with 4 hinge cysteines is also relatively resistant to chemical reduction in vitro allowing partial reduction to expose reactive hinge thiols. These hinge sequences provide a simple method for producing robust F(ab')2 in vitro, obviating the need to use chemical cross-linkers, and provide a route to hinge specific chemical modification with thiol-reactive conjugates.
基于含1个、2个或4个半胱氨酸的人γ1序列的带有铰链的Fab片段已通过高水平的大肠杆菌周质分泌产生，并在体外通过还原/氧化偶联形成F(ab')2。我们发现，由含2个或4个半胱氨酸的铰链制成的F(ab')2具有高水平（约70%）的多个二硫键。与通过单个二硫键直接偶联制成的F(ab')2相比，这些F(ab')2分子通过曲线下面积测量显示出更高的药代动力学稳定性。一种含有4个铰链半胱氨酸的特定分子比通过化学交联形成的F(ab')2具有更高的药代动力学稳定性。由具有4个铰链半胱氨酸的Fab'制成的F(ab')2在体外也相对耐化学还原，允许部分还原以暴露反应性铰链硫醇。这些铰链序列提供了一种在体外生产稳定F(ab')2的简单方法，无需使用化学交联剂，并提供了一条用硫醇反应性偶联物进行铰链特异性化学修饰的途径。
