Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.
Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.
复制标题
靶向血管内皮生长因子 165 和程序性死亡 1 的新型全人源双特异性单链双抗体在毕赤酵母中的优化表达和表征以及体内抗肿瘤活性评价
DOI：
10.3390/ijms19102900
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发表时间：
2018-09-25
影响因子：
5.6
通讯作者：
Liu Y
中科院分区：
文献类型：
--
作者：
Xiong C;Mao Y;Wu T;Kang N;Zhao M;Di R;Li X;Ji X;Liu Y
双特异性抗体，可以同时结合同一或不同抗原上的两个不同的表位，最近成为各种疾病研究的有吸引力的候选者。本研究成功构建并表达了一种能与毕赤酵母中血管内皮生长因子165 （VEGF165）和程序性死亡-1 （PD-1）结合的全人双特异性单链糖体（BsDb）。在最佳表达条件（甲醇浓度为1%，pH为4.0，接种量OD600 = 4，诱导时间为96 h）下，该BsDb的最大产量约为20 mg/L。重组BsDb采用镍-硝基三乙酸（Ni-NTA）柱层析一步纯化，纯度大于95%。间接酶联免疫吸附试验（ELISA）和夹心ELISA分析表明，纯化后的BsDb可同时特异性结合VEGF165和PD-1，亲和度分别为124.78 nM和25.07 nM。此外，BsDb不仅能有效抑制vegf165刺激的人脐静脉内皮细胞（HUVECs）的增殖、迁移和成管，还能通过阻断PD-1/PD-L1的共刺激，显著提高活化T细胞的增殖和INF-γ的产生。此外，BsDb通过抑制肿瘤血管生成和激活肿瘤微环境中的免疫反应，在HT29异种移植肿瘤小鼠中显示出强大的抗肿瘤活性。基于这些结果，我们首次制备了一种潜在的双特异性抗体药物，可以同时靶向VEGF165和PD-1。这项工作为开发血管生成抑制和免疫检查点阻断联合治疗癌症的新策略提供了稳定的基础。
Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.