In vivo phage display screening for tumor vascular targets in glioblastoma identifies a llama nanobody against dynactin-1-p150Glued
In vivo phage display screening for tumor vascular targets in glioblastoma identifies a llama nanobody against dynactin-1-p150Glued
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胶质母细胞瘤中肿瘤血管靶标的体内噬菌体展示筛选鉴定出针对 dynactin-1-p150Glued 的美洲驼纳米抗体
作者：
S. V. van Lith;Ilse Roodink;J. Verhoeff;Petri I. Mäkinen;J. P. Lappalainen;S. Ylä;J. Raats;E. van Wijk;R. Roepman;S. Letteboer;K. Verrijp;W. Leenders
弥漫性神经胶质瘤是以浸润性生长为特征的原发性脑癌。而高级别胶质瘤的特征性表现为坏死周围新生血管形成，大的肿瘤区域也在预先存在的血管上茁壮成长。临床研究表明，药物抑制血管生成过程并不能提高胶质母细胞瘤患者的生存率。然而，直接靶向肿瘤血管可能仍然是一种令人感兴趣的治疗方法，因为它允许切断肿瘤细胞的血液供应。这种肿瘤血管靶向需要鉴定肿瘤特异性血管靶向剂（TVTA）。在这里，我们描述了一种新的TVTA，C-C7，我们通过在弥漫性胶质瘤的原位小鼠模型中对美洲驼纳米抗体噬菌体展示文库进行体内生物淘选来鉴定。我们发现，C-C7识别胶质瘤异种移植物和临床胶质瘤样本中的肿瘤血管亚群。此外，C-C7识别动脉粥样硬化病变中的巨噬细胞和活化的内皮细胞。通过使用C-C7作为诱饵在酵母-2-杂交（Y2H）筛选中，我们鉴定了dynactin-1-p150Glued作为其结合配偶体。通过用C-C7和商业抗dynactin-1-p150Glued抗体的共免疫染色以及通过共免疫沉淀/蛋白质印迹研究证实了相互作用。正常脑血管不表达dynactin-1-p150Glued，并且其表达在抗VEGF治疗下降低，表明dynactin-1-p150Glued是活化的内皮细胞的标志物。总之，我们表明，体内噬菌体展示结合Y2H筛选提供了一种强有力的方法来识别肿瘤靶向纳米抗体及其结合伴侣。使用这种方法的组合，我们确定dynactin-1-p150Glued作为一种新的靶向蛋白激活的内皮细胞和巨噬细胞。
Diffuse gliomas are primary brain cancers that are characterised by infiltrative growth. Whereas high-grade glioma characteristically presents with perinecrotic neovascularisation, large tumor areas thrive on pre-existent vasculature as well. Clinical studies have revealed that pharmacological inhibition of the angiogenic process does not improve survival of glioblastoma patients. Direct targeting of tumor vessels may however still be an interesting therapeutic approach as it allows pinching off the blood supply to tumor cells. Such tumor vessel targeting requires the identification of tumor-specific vascular targeting agents (TVTAs). Here we describe a novel TVTA, C-C7, which we identified via in vivo biopanning of a llama nanobody phage display library in an orthotopic mouse model of diffuse glioma. We show that C-C7 recognizes a subpopulation of tumor blood vessels in glioma xenografts and clinical glioma samples. Additionally, C-C7 recognizes macrophages and activated endothelial cells in atherosclerotic lesions. By using C-C7 as bait in yeast-2-hybrid (Y2H) screens we identified dynactin-1-p150Glued as its binding partner. The interaction was confirmed by co-immunostainings with C-C7 and a commercial anti-dynactin-1-p150Glued antibody, and via co-immunoprecipitation/western blot studies. Normal brain vessels do not express dynactin-1-p150Glued and its expression is reduced under anti-VEGF therapy, suggesting that dynactin-1-p150Glued is a marker for activated endothelial cells. In conclusion, we show that in vivo phage display combined with Y2H screenings provides a powerful approach to identify tumor-targeting nanobodies and their binding partners. Using this combination of methods we identify dynactin-1-p150Glued as a novel targetable protein on activated endothelial cells and macrophages.
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影响因子：
158.5
作者：
Hurwitz, H;Fehrenbacher, L;Kabbinavar, F
通讯作者：
Kabbinavar, F
DOI：
10.1056/nejmoa1308573
发表时间：
2014-02-20
期刊：
The New England journal of medicine
影响因子：
--
作者：
Gilbert MR;Dignam JJ;Armstrong TS;Wefel JS;Blumenthal DT;Vogelbaum MA;Colman H;Chakravarti A;Pugh S;Won M;Jeraj R;Brown PD;Jaeckle KA;Schiff D;Stieber VW;Brachman DG;Werner-Wasik M;Tremont-Lukats IW;Sulman EP;Aldape KD;Curran WJ Jr;Mehta MP
通讯作者：
Mehta MP
通过酵母中的相互作用陷阱筛选，分离与色素性视网膜炎 GTP 酶调节剂相互作用的视网膜蛋白。
影响因子：
--
作者：
Roepman,R;Schick,D;Ferreira,PA
通讯作者：
Ferreira,PA
影响因子：
3.3
作者：
Tokito, MK;Howland, DS;Holzbaur, ELF
通讯作者：
Holzbaur, ELF
影响因子：
15.9
作者：
Rajotte, D;Arap, W;Ruoslahti, E
通讯作者：
Ruoslahti, E