An interferon α2 mutant optimized by phage display for IFNAR1 binding confers specifically enhanced antitumor activities
An interferon α2 mutant optimized by phage display for IFNAR1 binding confers specifically enhanced antitumor activities
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DOI：
10.1074/jbc.m610115200
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发表时间：
2007-04-13
影响因子：
4.8
通讯作者：
Schreiber, Gideon
中科院分区：
文献类型：
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作者：
Kalie, Eyal;Jaitin, Diego A.;Schreiber, Gideon
所有α干扰素（IFN α）都以低亲和力结合IFNAR1受体亚基。增加结合亲和力被证明可以特异性地增加IFN α 2的抗增殖能力。在这里，我们通过随机选择IFN α 2上三个先前被证明与IFNAR1弱结合的位置，构建了一个噬菌体展示库。所选择的结合最紧密的变异，由突变H57Y、E58N和Q61S （YNS）组成，与野生型IFN α 2相比，与IFNAR1的结合紧密度为60倍，与IFN β相比紧密度为3倍。YNS与IFNAR2的结合与野生型IFN α 2相当。与野生型IFN α 2相比，YNS突变体在WISH细胞中的抗增殖能力提高了150倍，而其抗病毒活性仅提高了3.5倍。高抗增殖活性与诱导凋亡有关，正如膜联蛋白V结合试验所证明的那样，并与特定基因诱导有关，特别是TRAIL。为了确定YNS突变体在异种移植癌症模型中的效力，我们每周两次将其注射到携带移植的MDA231人乳腺癌细胞的裸鼠身上。5周后，用YNS治疗的小鼠没有肿瘤残留，而用野生型IFN α 2治疗的大多数小鼠都有可见的肿瘤。与野生型IFN α 2相比，这些肿瘤的组织学分析显示YNS具有显著的抗血管生成作用。这项工作证明了详细的生物物理理解在蛋白质工程过程中的应用，产生了具有高度提高的生物效力的干扰素变体。
All alpha-interferons ( IFN alpha) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFN alpha 2. Here, we constructed a phage display library by randomizing three positions on IFN alpha 2 previously shown to confer weak binding to IFNAR1. The tightest binding variant selected, comprised of mutations H57Y, E58N, and Q61S ( YNS), was shown to bind IFNAR1 60-fold tighter compared with wild-type IFN alpha 2, and 3-fold tighter compared with IFN beta. Binding of YNS to IFNAR2 was comparable with wild-type IFN alpha 2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFN alpha 2, whereas its antiviral activity was increased by only 3.5-fold. The high antiproliferative activity was related to an induction of apoptosis, as demonstrated by annexin V binding assays, and to specific gene induction, particularly TRAIL. To determine the potency of the YNS mutant in a xenograft cancer model, we injected it twice a week to nude mice carrying transplanted MDA231 human breast cancer cells. After 5 weeks, no tumors remained in mice treated with YNS, whereas most mice treated with wild-type IFN alpha 2 showed visible tumors. Histological analysis of these tumors showed a significant anti-angiogenic effect of YNS, compared with wild-type IFN alpha 2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an interferon variant with highly increased biological potency.