7B7: a novel antibody directed against the Ku70/Ku80 heterodimer blocks invasion in pancreatic and lung cancer cells
7B7: a novel antibody directed against the Ku70/Ku80 heterodimer blocks invasion in pancreatic and lung cancer cells
复制标题
DOI：
10.1007/s13277-014-1857-5
复制
发表时间：
2014-07-01
期刊：
影响因子：
--
通讯作者：
Larkin, Annemarie
中科院分区：
文献类型：
--
作者：
O'Sullivan, Dermot;Henry, Michael;Larkin, Annemarie
针对高度未满足需求的癌症开发更有效的治疗策略需要不断发现用于治疗性抗体靶向的疾病特异性蛋白靶点。为了鉴定与癌细胞侵袭/转移相关的新型蛋白质，我们在此提出了一种替代抗体靶向细胞表面蛋白的替代方案，该蛋白质在侵袭中具有既定的作用；我们的功能性抗体筛选方法涉及单克隆抗体的分离和选择，主要筛选其抑制肿瘤侵袭的能力。 Mia PaCa-2（一种胰腺导管腺癌 (PDAC) 细胞系，具有高度侵袭性表型）的克隆群被用来生成单克隆抗体，目的是识别直接参与癌症侵袭的膜靶点。选定的 MAb 7B7 可以以剂量响应的方式显着减少 Mia PaCa-2 克隆 3 和 DLKP-M 鳞状肺癌细胞的侵袭。通过免疫沉淀和液相色谱-串联质谱（LC-MS-MS）分析，抗侵袭抗体7B7的靶抗原被确定为异二聚体Ku抗原Ku70/80，这是一种由Ku70和Ku80亚基组成的核心蛋白，参与非同源末端连接（NHEJ）DNA修复。 RNA干扰介导的Ku70和Ku80敲低导致Mia PaCa-2克隆3和DLKP-M细胞的侵袭能力显着降低，表明Ku70/Ku80在功能上参与胰腺癌和肺癌侵袭。免疫组织化学分析证明了 37 个 PDAC 肿瘤中的 Ku70/Ku80 免疫反应性，表明这种异二聚体在这种侵袭性癌症类型中高度表达。这项研究表明，功能性单克隆抗体筛选方法与免疫沉淀/蛋白质组学分析相结合，可以成功地应用于鉴定功能性抗侵入性单克隆抗体和治疗性抗体靶向的潜在新靶点。
Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.