Potent and Efficacious Inhibition of CXCR2 Signaling by Biparatopic Nanobodies Combining Two Distinct Modes of Action
Potent and Efficacious Inhibition of CXCR2 Signaling by Biparatopic Nanobodies Combining Two Distinct Modes of Action
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DOI：
10.1124/mol.114.094821
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发表时间：
2015-02-01
影响因子：
3.6
通讯作者：
Cromie, K. D.
中科院分区：
文献类型：
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作者：
Bradley, M. E.;Dombrecht, B.;Cromie, K. D.
趋化因子和趋化因子受体是炎症性疾病和恶性肿瘤的关键调节因子。在这里，我们描述了选择性阻断CXCR2的纳米体的鉴定和药理学特性，CXCR2是所有趋化因子受体中最混杂的。鉴定了两类选择性单价纳米体，详细的表位定位显示它们与CXCR2受体上不同的、不重叠的表位结合。n端结合或1类单价纳米体具有个位数纳摩尔范围的效力，但在高激动剂浓度下缺乏完全的效力。相比之下，细胞外环结合或2类单价纳米体的效力较低，但在重组和中性粒细胞的体外实验中更有效和竞争性地抑制cxcr2介导的功能反应。除了阻断CXCL1（生长相关癌基因α）和CXCL8（白细胞介素-8）介导的CXCR2信号传导外，这两类纳米体都表现出相反的激动剂行为。二价纳米体和双异位纳米体通过甘氨酸/丝氨酸连接剂分别由相同或不同类别的纳米体组合而成。有趣的是，受体突变和竞争研究表明，双异位纳米体能够在一个或两个CXCR2受体分子内贪婪地结合表位。最重要的是，双异位纳米体在效力和功效方面优于单价和二价纳米体。
Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene alpha) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy.