DataSheet_1_Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2.doc

Angiogenesis is considered one of the hallmarks of cancer and plays a critical role in the development of tumor. Fibroblast growth factor 2 (FGF-2) is a member of the FGF family and participates in excessive cancer cell proliferation and tumor angiogenesis. Thus, targeting FGF-2 was considered to be a promising anti-tumor strategy. A disulfide-stabilized diabody (ds-Diabody) against FGF-2 was produced in Pichia pastoris (GS115) by fermentation and the anti-tumor activity was analyzed. The novel 10-L fed batch fermentation with newly designed media was established, and the maximum production of the ds-Diabody against FGF-2 reached 210.4 mg/L. The ds-Diabody against FGF-2 was purified by Ni2+ affinity chromatography and DEAE anion exchange chromatography. The recombinant ds-Diabody against FGF-2 could effectively inhibit proliferation, migration, and invasion of melanoma and glioma tumor cells stimulated by FGF-2. Furthermore, xenograft tumor model assays showed that the ds-Diabody against FGF-2 had potent antitumor activity in nude mice by inhibiting tumor growth and angiogenesis. The tumor growth inhibition rate of melanoma and glioma was about 70 and 45%, respectively. The tumor angiogenesis inhibition rate of melanoma and glioma was about 64 and 51%, respectively. The results revealed that the recombinant ds-Diabody against FGF-2 may be a promising anti-tumor drug for cancer therapy.

血管生成（Angiogenesis）被视作癌症的核心标志性特征之一，在肿瘤发生发展进程中发挥关键调控作用。成纤维细胞生长因子2（Fibroblast growth factor 2, FGF-2）属于FGF家族成员，参与肿瘤细胞异常增殖与肿瘤血管生成过程。因此，靶向FGF-2被认为是极具潜力的抗肿瘤治疗策略。本研究通过毕赤酵母（Pichia pastoris, GS115）发酵制备了针对FGF-2的二硫键稳定双抗（disulfide-stabilized diabody, ds-Diabody），并对其抗肿瘤活性开展了系统分析。研究建立了采用新型定制培养基的10L补料分批发酵工艺，靶向FGF-2的ds-Diabody最高表达产量可达210.4 mg/L。采用Ni²⁺亲和层析与DEAE阴离子交换层析两步法对靶向FGF-2的ds-Diabody进行纯化。体外实验结果显示，该重组靶向FGF-2的ds-Diabody可有效抑制FGF-2刺激诱导的黑色素瘤与神经胶质瘤细胞的增殖、迁移与侵袭能力。此外，异种移植瘤模型（xenograft tumor model）体内实验表明，靶向FGF-2的ds-Diabody可通过抑制肿瘤生长与血管生成，在裸鼠体内展现出强效抗肿瘤活性。其中，黑色素瘤与神经胶质瘤的肿瘤生长抑制率分别约为70%与45%，肿瘤血管生成抑制率分别约为64%与51%。本研究结果证实，该重组靶向FGF-2的ds-Diabody有望成为一种极具临床转化价值的抗肿瘤治疗候选药物。