Bivalent V_HHs have been shown to display better functional affinity compared with their monovalent counterparts. Bivalency can be achieved either by inserting a hinge region between both V_HHs units or by using modules that lead to dimerization. In this report, a small self-associating peptide originating from the tetramerization domain of p53 was developed as a tool for devicing nanobody dimerization. This E3 peptide was evaluated for the dimerization of an anti-eGFP nanobody (nano-eGFP-E3) whose activity was compared to a bivalent anti-eGFP constructed in tandem using GS rich linker. The benefit of bivalency in terms of avidity and specificity was assessed in different in vitro and in cellulo assays. In ELISA and SPR, the dimeric and tandem formats were nearly equivalent in terms of gain of avidity compared to the monovalent counterpart. However, in cellulo, the nano-eGFP-E3 construct showed its superiority over the tandem format in terms of specificity with a highest and better ratio signal-to-noise. All together, the E3 peptide provides a universal suitable tool for the construction of dimeric biomolecules, in particular antibody fragments with improved functional affinity.

与单价对应物相比，二价 V _H H 已显示出更好的功能亲和力。双价可以通过在两个 V _H Hs 单元之间插入铰链区或使用导致二聚化的模块来实现。在本报告中，开发了一种源自 p53 的四聚化结构域的小型自缔合肽，作为设计纳米抗体二聚化的工具。评估该 E3 肽的抗 eGFP 纳米抗体 (nano-eGFP-E3) 的二聚化，将其活性与使用富含 GS 的接头串联构建的二价抗 eGFP 进行比较。在不同的体外和纤维素中评估了双价在亲和力和特异性方面的益处化验。在 ELISA 和 SPR 中，与单价对应物相比，二聚体和串联形式在亲和力增加方面几乎相同。然而，在纤维素中，nano-eGFP-E3 构建体在具有最高和更好的信噪比的特异性方面表现出优于串联格式。总之，E3 肽为构建二聚体生物分子，特别是具有改进功能亲和力的抗体片段提供了一种通用的合适工具。