Kaneko Mika K, Ohishi Tomokazu, 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.
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, 18-24 Miyamoto, Numazu-shi, Shizuoka, 410-0301, Japan.
Biochem Biophys Rep. 2020 Oct 10;24:100826. doi: 10.1016/j.bbrep.2020.100826. eCollection 2020 Dec.
Overexpression of podocalyxin (PODXL) is associated with progression, metastasis, and poor outcomes in several cancers. PODXL also plays an important role in the development of normal tissues. For antibody-based therapy to target PODXL-expressing cancers using monoclonal antibodies (mAbs), cancer-specificity is necessary to reduce the risk of adverse effects to normal tissues. In this study, we developed an anti-PODXL cancer-specific mAb (CasMab), named as PcMab-60 (IgM, kappa) by immunizing mice with soluble PODXL, which is overexpressed in LN229 glioblastoma cells. The PcMab-60 reacted with the PODXL-overexpressing LN229 (LN229/PODXL) cells and MIA PaCa-2 pancreatic cancer cells in flow cytometry but did not react with normal vascular endothelial cells (VECs), whereas one of non-CasMabs, PcMab-47 showed high reactivity for not only LN229/PODXL and MIA PaCa-2 cells but also VECs, indicating that PcMab-60 is a CasMab. Next, we engineered PcMab-60 into a mouse IgG-type mAb, named as 60-mG, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed a core fucose-deficient type of 60-mG, named as 60-mG-f, to augment its ADCC activity. analysis revealed that 60-mG-f exerted antitumor activity in MIA PaCa-2 xenograft models at a dose of 100 μg/mouse/week administered three times. These results suggested that 60-mG-f could be useful for antibody-based therapy against PODXL-expressing pancreatic cancers.
足突细胞粘附分子（PODXL）的过表达与多种癌症的进展、转移及不良预后相关。PODXL在正常组织发育中也发挥重要作用。对于使用单克隆抗体（mAb）靶向表达PODXL的癌症进行基于抗体的治疗而言，癌症特异性对于降低对正常组织产生不良反应的风险是必要的。在本研究中，我们通过用在LN229胶质母细胞瘤细胞中过表达的可溶性PODXL免疫小鼠，开发了一种抗PODXL癌症特异性mAb（CasMab），命名为PcMab-60（IgM，κ链）。在流式细胞术中，PcMab-60与过表达PODXL的LN229（LN229/PODXL）细胞和MIA PaCa-2胰腺癌细胞反应，但不与正常血管内皮细胞（VEC）反应，而非CasMab之一的PcMab-47不仅对LN229/PODXL和MIA PaCa-2细胞，而且对VEC都显示出高反应性，这表明PcMab-60是一种CasMab。接下来，我们将PcMab-60改造为小鼠IgG型mAb，命名为60-mG，以增加抗体依赖性细胞毒性（ADCC）。我们进一步开发了一种核心岩藻糖缺陷型的60-mG，命名为60-mG-f，以增强其ADCC活性。分析显示，60-mG-f以100μg/小鼠/周的剂量给药三次，在MIA PaCa-2异种移植模型中发挥抗肿瘤活性。这些结果表明，60-mG-f可能对基于抗体的靶向表达PODXL的胰腺癌治疗有用。
