Parallel in vivo and in vitro selection using phage display identifies protease-dependent tumor-targeting peptides.
Parallel in vivo and in vitro selection using phage display identifies protease-dependent tumor-targeting peptides.
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DOI：
10.1074/jbc.m110.138297
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发表时间：
2010-07-16
期刊：
影响因子：
--
通讯作者：
Tsien RY
中科院分区：
文献类型：
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作者：
Whitney M;Crisp JL;Olson ES;Aguilera TA;Gross LA;Ellies LG;Tsien RY
我们最近开发了可激活的细胞穿透肽（ACPP），其将造影剂靶向到基质金属蛋白酶活性的体内部位，例如肿瘤。在这里，我们使用平行的体内和体外选择与噬菌体展示，以确定新的肿瘤归巢ACPP的一级序列或靶蛋白酶没有偏见。具体地，将展示ACPP文库的噬菌体注射到荷瘤小鼠中，然后从解剖的肿瘤分离裂解的噬菌体，或者基于肿瘤提取物对正常组织提取物的选择性裂解进行分离。选择的序列被合成为荧光标记的肽，并通过用组织提取物消化确认肿瘤特异性切割。最有效切割的肽含有底物序列RLQLKL，并以高达5倍的对比度标记来自几种癌症模型的肿瘤和转移。这种独特鉴定的ACPP不被基质金属蛋白酶或各种凝血因子切割，但被纤溶酶和弹性蛋白酶有效切割，这两种酶在肿瘤中异常过表达。在没有合理设计的情况下鉴定靶向肿瘤表达的蛋白酶的ACPP突出了无偏选择方案对于开发潜在治疗剂的价值。
We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.