In vitro selection of bispecific diabody fragments using covalent bicistronic DNA display.
In vitro selection of bispecific diabody fragments using covalent bicistronic DNA display.
复制标题
使用共价双顺反子 DNA 展示体外选择双特异性双抗体片段。
DOI：
10.1016/j.bbrc.2016.07.113
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
N.
中科院分区：
文献类型：
--
作者：
Nakayama;M.;Komiya;S.;Fujiwara;K.;Horisawa;K.;Doi;N.
具有两个不同抗原结合位点的双特异性抗体已广泛用于多种医学应用。体外进化可以提高抗体片段的活性和稳定性。虽然小的双特异性抗体片段如双抗体的亲和力和稳定性可以通过体外展示技术进一步优化，但双特异性抗体片段的无细胞展示还没有报道。在本研究中，我们应用共价双顺反子DNA展示技术进行异源二聚体双抗体的体外筛选。首先，我们证实了体外转录翻译系统合成的双抗体的抗原结合活性。然而，当我们在原理验证实验中进行模型双抗体文库的DNA展示选择时，没有观察到双抗体基因的富集，这可能是由于双抗体异二聚体的低产率。为了克服这个问题，我们在双抗体异源二聚体的VH-VL界面处引入半胱氨酸残基。使用二硫键稳定的双抗体，我们成功地从模型文库中富集了双抗体基因。我们的研究结果表明，共价双顺反子DNA展示技术可以用于提高双特异性双抗体片段的稳定性和亲和力。
Bispecific antibodies with two different antigen-binding sites have been widely used for a variety of medical applications. The activity and stability of antibody fragments can be improved byin vitroevolution. Although the affinity and stability of small bispecific antibody fragments such as diabodies can be further optimized byin vitrodisplay technologies, cell-free display of bispecific antibody fragments has not been reported. In this study, we applied a covalent bicistronic DNA display for thein vitroselection of heterodimeric diabodies. First, we confirmed the antigen-binding activities of a diabody synthesized by anin vitrotranscription and translation system. However, when we performed DNA-display selection of a model diabody library in a proof-of-principle experiment, no enrichment of the diabody gene was observed, likely due to a low yield of the diabody heterodimer. To overcome this issue, we introduced cysteine residues at the VH-VLinterface of the diabody heterodimer. Using the disulfide-stabilized diabodies, we successfully enriched the diabody gene from a model library. Our results indicate that the covalent bicistronic DNA display technique could be useful for improving the stability and affinity of bispecific diabody fragments.