人源抗滋养层细胞表面抗原-2基因工程抗体Fab的制备及特性分析

Preparation and Characterization of Human Anti-Trop-2 Engineering Antibody Fab

应用噬菌体抗体库技术制备全人源抗滋养层细胞表面抗原-2(Trop-2)特异性Fab抗体片段.抗体库经细胞筛选和固相抗原筛选,获得特异性的阳性克隆.阳性载体经核酸序列分析后,构建工程菌,经IPTG诱导表达,SDS-PAGE和Western blot分析,呈现28ku和32ku大小的两条蛋白质条带.Fab分子经流式细胞术、细胞免疫荧光检测,结果表明,Fab能够与BxPc3细胞膜蛋白特异性结合,而与NIH3T3细胞不结合.免疫共沉淀与质谱分析结果表明,该Fab分子能够与Trop-2蛋白特异性结合.免疫组化显示,该抗体可结合胰腺癌细胞膜蛋白,在细胞培养液中加入Fab,能够抑制BxPc3细胞的生长.以上研究结果提示,该抗体有望成为胰腺癌临床影像诊断或治疗的候选分子.

Fully human antibody fragment Fab that specifically binding to Trop-2 （trophoblast cell-surface antigens 2, Trop-2）, was selected from phage display antibody library. Positive phage-displayed antibody clones were selected on live cell lines and immobilized protein. The purified Fab was verified by SDS-PAGE and Western blot, which showed two bands at about 28 ku and 32 ku at the expected sizes. To analyze the immunological characters of Fab for Trop-2 binding, flow cytometry, immunoprecipitation assays and mass spectrometry were set up and carried out with BxPc3 and NIH3T3 cell lines. The results demonstrated Fab could bind native Trop-2 specifically on the BxPc3 cell surface. Peptide mapping fingerprint showed that the protein which bound is Trop-2 protein. Immunohistochemistry detection illuminated Fab could bind the membrance protein of pancreatic cancer tissue. In vitro cell growth inhibition assay showed that anti-Trop-2 Fab could inhibit the Trop-2 positive cell BxPc3 growth, it illuminated that anti-Trop-2 Fab bound the Trop-2 on Trop-2 positive cell surface. For Trop-2 negative cell line NIH3T3, no significant inhibition among the different dosages of Fab. The results showed that anti-Trop-2 Fab antibody fragments could recognize Trop-2 extracellular domain in native conformation, making them as potential powerful reagents for clinical therapeutic application.