Desplancq D, King D J, Lawson A D, Mountain A
Oncology Department, Celltech Research, Slough, UK.
Protein Eng. 1994 Aug;7(8):1027-33. doi: 10.1093/protein/7.8.1027.
A systematic study has been performed on the relationship between linker length, relative orientation of variable domains, multimerization behaviour and antigen binding activity for single chain Fvs (scFvs) of the tumour-binding antibody B72.3. Thirteen scFv variants with linkers comprising up to six repeats of the motif Gly-Gly-Gly-Gly-Ser were studied. All these scFvs showed a tendency to form dimers or higher molecular weight species, and this tendency decreased with increasing linker length. The dimers and higher molecular weight forms may arise from head to tail intermolecular association of VH and VL domains. For each linker length, scFvs with the organization VL-linker-VH showed greater binding activity than those with the organization VH-linker-VL. In fact, for the latter organization only the variant with a 30 amino acid linker showed good binding activity, suggesting that (i) for B72.3 the C-terminus of VH or the N-terminus of VL makes a structural contribution to antigen binding, and (ii) shorter linkers interfere with this contribution. Antigen binding studies on scFvs should be interpreted with caution because of their tendency to multimerize. Such multimerization can be minimized by using linkers longer than those in common use.
针对肿瘤结合抗体B72.3的单链Fv片段（scFv），对连接子长度、可变结构域的相对方向、多聚化行为和抗原结合活性之间的关系进行了系统研究。研究了13种连接子包含多达六个Gly-Gly-Gly-Gly-Ser基序重复序列的scFv变体。所有这些scFv都显示出形成二聚体或更高分子量物种的倾向，并且这种倾向随着连接子长度的增加而降低。二聚体和更高分子量形式可能源于VH和VL结构域的头对头分子间缔合。对于每种连接子长度，具有VL-连接子-VH结构的scFv比具有VH-连接子-VL结构的scFv表现出更高的结合活性。事实上，对于后一种结构，只有具有30个氨基酸连接子的变体显示出良好的结合活性，这表明（i）对于B72.3，VH的C末端或VL的N末端对抗原结合有结构贡献，以及（ii）较短的连接子会干扰这种贡献。由于scFv有形成多聚体的倾向，因此对其进行抗原结合研究时应谨慎解释。通过使用比常用连接子更长的连接子，可以将这种多聚化降至最低。
