Vaughan T J, Williams A J, Pritchard K, Osbourn J K, Pope A R, Earnshaw J C, McCafferty J, Hodits R A, Wilton J, Johnson K S
Cambridge Antibody Technology Ltd., Unit B3, Melbourn, Cambridgeshire, U.K.
Nat Biotechnol. 1996 Mar;14(3):309-14. doi: 10.1038/nbt0396-309.
To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.
为了生成一个稳定的资源库，以便能够快速分离出针对任何给定抗原的高亲和力人源抗体，我们使用了来自43名未免疫的人类供体的功能性V基因片段，构建了一个展示在噬菌体表面的1.4×10¹⁰个单链Fv（scFv）片段的文库。片段被克隆到噬菌粒载体中，无需亚克隆即可产生噬菌体展示的scFv和可溶性scFv。引入了一个六组氨酸标签，以便通过镍螯合层析快速纯化scFv。这种文库形式将分离单克隆抗体片段所需的时间缩短至两周以内。所有测得的结合亲和力均显示Kd＜10 nM，解离速率为10⁻³至10⁻⁴ s⁻¹，这些特性通常与二次免疫应答产生的抗体相关。其中最好的scFv，一种抗荧光素抗体（0.3 nM）和一种针对半抗原DTPA的抗体（0.8 nM），是首批从天然文库中分离出的具有亚纳摩尔结合亲和力的抗体。已经分离出了针对具有免疫抑制和毒性的阿霉素的抗体，以及针对类固醇激素雌二醇的高亲和力和高特异性抗体。这项工作表明，传统的杂交瘤技术可能会被大型噬菌体文库所取代，事实证明这些文库是特异性、高亲和力人源单克隆抗体的稳定可靠来源。
