趋化因子受体CXCR4参与干细胞和免疫细胞的发育和迁移,但也涉及多种癌症的肿瘤进展和转移。拮抗配体(CXCL12)诱导的CXCR4信号是,因此,治疗兴趣。目前,市场上有两种小分子CXCR4拮抗剂用于动员造血干细胞。其他具有改善效力和安全性的分子正在开发用于不同适应症,包括癌症.此外,与CXCR4靶向分子AMD3100(Plerixafor)相比,靶向CXCR4的多个拮抗纳米抗体显示出相似或更好的效力,通过二价衍生物的积极结合进一步增强。在这项研究中,我们旨在比较各种多价纳米抗体形式的亲和力,这些形式可能受到亲和力的不同影响。通过融合到柔性GS-接头,人IgG1的Fc区,不同的C4bp/CLR多聚结构域,或通过与三价接头支架的定点缀合,我们产生了不同类型的多价纳米体,其价数从二价到十价不等。其中,C端融合,尤其是人类Fc,在抑制CXCL12介导的Gαi-或β-抑制素募集中,效力增加2-log倍和3-log倍最有利,分别。总的来说,我们描述了产生能够诱导受体聚集的多价和高效CXCR4拮抗纳米抗体的策略,并得出结论,与Fc尾融合导致最高的亲合力效应,而与铰链接头无关。
The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or β-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.