Takei Junko, Ohishi Tomokazu, Kaneko Mika K, Harada Hiroyuki, Kawada Manabu, Kato Yukinari
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan.
Biochem Biophys Rep. 2020 Aug 30;24:100801. doi: 10.1016/j.bbrep.2020.100801. eCollection 2020 Dec.
Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. A strong correlation has been reported between PD-L1 expression in tumor cells and negative prognosis in cancer patients. Previously, we established an anti-PD-L1 monoclonal antibody (mAb), LMab-13 (IgG, kappa), by immunizing mice with PD-L1-overexpressing CHO-K1 cells. LMab-13 specifically reacts with endogenous PD-L1 in lung cancer cell lines in flow cytometry and Western blot applications, and stains a plasma membrane-like pattern in lung cancer tissues via immunohistochemical analysis. In this study, we investigated whether LMab-13 reacts with oral cancer cell lines and exerts antitumor activities. Because LMab-13 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), we first converted the subclass of LMab-13 from IgG into IgG (13-mG), and further produced a defucosylated version (13-mG-f) using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 13-mG-f was confirmed using fucose-binding lectins, such as and lectins. The dissociation constants ( ) for 13-mG-f in SAS and HSC-2 oral cancer cells were determined via flow cytometry to be 2.8 × 10 M and 4.8 × 10 M, respectively, indicating that 13-mG-f possesses extremely high binding affinity. analysis demonstrated that 13-mG-f showed moderate ADCC and CDC activities against SAS and HSC-2 oral cancer cells. analysis revealed that 13-mG-f significantly reduced tumor development in SAS and HSC-2 xenografts in comparison to control mouse IgG, even after injection seven days post-tumor inoculation. Taken together, these data demonstrate that treatment with 13-mG-f may represent a useful therapy for patients with PD-L1-expressing oral cancers.
程序性细胞死亡配体1（PD-L1）是一种I型跨膜糖蛋白，表达于抗原呈递细胞和包括黑色素瘤及肺癌细胞在内的多种肿瘤细胞上。据报道，肿瘤细胞中PD-L1的表达与癌症患者的不良预后之间存在强相关性。此前，我们通过用过量表达PD-L1的CHO-K1细胞免疫小鼠，制备了一种抗PD-L1单克隆抗体（mAb）LMab-13（IgG，κ）。在流式细胞术和蛋白质印迹应用中，LMab-13能与肺癌细胞系中的内源性PD-L1特异性反应，并且通过免疫组织化学分析在肺癌组织中染出类似质膜的图案。在本研究中，我们调查了LMab-13是否与口腔癌细胞系反应并发挥抗肿瘤活性。由于LMab-13缺乏抗体依赖性细胞毒性（ADCC）和补体依赖性细胞毒性（CDC），我们首先将LMab-13的亚类从IgG转化为IgG（13-mG），并进一步使用缺乏岩藻糖基转移酶8（FUT8）的ExpiCHO-S（BINDS-09）细胞制备了去岩藻糖基化版本（13-mG-f）。使用岩藻糖结合凝集素（如 和 凝集素）确认了13-mG-f的去岩藻糖基化。通过流式细胞术测定13-mG-f在SAS和HSC-2口腔癌细胞中的解离常数（ ）分别为2.8×10 M和4.8×10 M，表明13-mG-f具有极高的结合亲和力。 分析表明，13-mG-f对SAS和HSC-2口腔癌细胞表现出中等程度的ADCC和CDC活性。 分析显示，即使在肿瘤接种后7天注射，与对照小鼠IgG相比，13-mG-f也显著减少了SAS和HSC-2异种移植瘤的生长。综上所述，这些数据表明用13-mG-f治疗可能是对表达PD-L1的口腔癌患者的一种有效治疗方法。
