Target‐binding small proteins are promising alternatives to conventional monoclonal antibodies (mAbs), offering advantages in terms of tissue penetration and manufacturing costs. Recently, a design strategy to create small proteins called fluctuation‐regulated affinity proteins (FLAPs) consisting of a structurally immobilized peptide from the complementarity‐determining region (CDR) loops of mAbs (CDR‐derived peptide) and a protein scaffold was developed. Because mAb paratopes are usually composed of multiple CDRs, FLAPs with multiple binding peptides may have an enhanced target‐binding capability. Here, a strategy to create FLAPs bearing dual CDR‐derived peptides (D‐FLAPs) using the anti‐human epithelial growth factor receptor type 2 (HER2) mAb trastuzumab as a basis is developed. Computationally selected CDR‐derived peptides are first grafted onto two adjacent loops of the fibronectin type III domain (FN3) scaffold, yielding 80 D‐FLAP candidates. After computational screening based on their similarity to the parental mAb with regard to the conformation of paratope residues, two candidates are selected. After further evaluation with ELISA, one D‐FLAP with HYTTPP and GDGFYA peptides from CDR‐L3 and CDR‐H3 of the parental mAb, respectively, is found to bind HER2 with a dissociation constant of 58 nm. This method applies to various mAb drugs and allows the rational design of small protein alternatives.

中文翻译：

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通过将双CDR衍生肽接枝到小蛋白支架上来重建抗HER2抗体对位。

具有靶标结合作用的小蛋白是常规单克隆抗体（mAb）的有前途的替代品，在组织渗透和制造成本方面具有优势。最近，开发了一种设计策略来创建称为波动调节亲和蛋白（FLAP）的小蛋白，该蛋白由mAb（CDR衍生肽）的互补决定区（CDR）环和蛋白支架组成的结构固定肽组成。由于mAb对位通常由多个CDR组成，因此具有多个结合肽的FLAP可能具有增强的靶结合能力。在此，制定了一种策略，以抗人上皮生长因子受体2型（HER2）mAb曲妥珠单抗为基础，构建带有双CDR衍生肽（D-FLAP）的FLAP。通过计算选择的CDR衍生肽首先被移植到纤连蛋白III型结构域（FN3）支架的两个相邻环上，产生80个D-FLAP候选物。在基于其与亲本单克隆抗体的互补位残基构象的相似性进行计算筛选后，选择了两个候选物。经过ELISA进一步评估后，发现一种D-FLAP与亲本mAb的CDR-L3和CDR-H3中的HYTTPP和GDGFYA肽分别结合，其解离常数为58 n米。此方法适用于各种mAb药物，并允许合理设计小蛋白替代品。