Construction of human naive Fab library and characterization of anti-Met Fab fragment generated from the library
Construction of human naive Fab library and characterization of anti-Met Fab fragment generated from the library
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DOI：
10.1385/mb:31:1:041
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发表时间：
2005-09-01
影响因子：
2.6
通讯作者：
Cao, B
中科院分区：
文献类型：
--
作者：
Liao, YJ;Zhao, P;Cao, B
受体酪氨酸激酶Met及其配体肝细胞生长因子(HGF)/散射因子(SF)的不适当表达通常与侵袭性实体瘤表型(血管生成、侵袭和转移)和临床预后不良有关。我们设计和构建了一个大的人类天然抗原结合片段(Fab)噬菌体展示文库，其多样性为2.0x101，该文库可以快速分离抗原特异性的人类抗体片段。通过对转Met基因的S 114细胞进行生物扫描，成功地从该文库中筛选出一段针对Met的Fab片段(命名为hFab-Met-1)。免疫沉淀、Western blotting和流式细胞术鉴定hFab-Met-1的特异性。结果表明，hFab-Met-1以天然构象与Met胞外区发生反应。此外，通过Madine-Darby犬肾细胞散射和尿激酶型纤溶酶原激活物试验进行的功能分析表明，hFab-Met-1与小鼠完整的单抗Met5相比，不是HGF/Met信号的激动剂。为了证实hFab-Met-1在体内与表达Met的肿瘤的相互作用，在表达Met和HGF/SF的小鼠异种移植瘤中进行了I-125标记的hFab-Met-1的核成像。注射后I~48it(PI)进行全身核素扫描。肿瘤相关活动早在I it Pi时就被成像，并在某些动物中直到24 it Pi时仍可见。正如预期的那样，在早期图像中，肾脏的活动最高，而在后来的图像中，甲状腺活动变得主要。综上所述，hFab-Met-1在体外和体内都能与Met相互作用，在临床诊断和治疗中具有广阔的应用前景。
Inappropriate expression of the receptor tyrosine kinase Met and its ligand hepatocyte growth factor (HGF)/scatter factor (SF) is usually associated with an aggressive solid tumor phenotype (angiogenesis, invasiveness, and metastasis) and poor clinical prognosis. We report here the design and construction of a large, human naive antigen-binding fragment (Fab) phage-display library with a diversity of 2.0 x 101, which allows rapid isolation of antigen-specific human antibody fragments. A Fab fragment specifically against Met (designated hFab-Met-1) was successively selected from this library by using biopanning on Met-transfected cell line S 114. The specificity of hFab-Met-1 was characterized by immunoprecipitation, Western blotting, and flow cytometry. The results demonstrate that hFab-Met-1 reacts with the extracellular domain of Met in its native conformation. Moreover, functional analysis by Madine-Darby canine kidney cell scattering and urokinase-type plasminogen activator assays demonstrated that hFab-Met-1 is not an agonist to HGF/Met signaling compared with a murine intact monoclonal antibody (MAb) Met5. To confirm that hFab-Met-1 interacts with Met-expressing tumors in vivo, I-125-labeled hFab-Met-1 was nuclear-imaged in a mouse xenograft of Met- and HGF/SF-expressing human leiomyosarcoma. Total body scintigrams were obtained between I and 48 It postinjection (PI). Tumor-associated activity was imaged as early as I It PI, and remained visible in some animals as late as 24 It PI. As expected, activity was highest in the kidneys in early images, whereas thyroid activity became predominant in later images. In conclusion, hFab-Met-1 interacts with Met both in vitro and in vivo, and is a promising candidate for clinical diagnosis and therapeutics.