Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Int J Cancer. 2010 Aug 1;127(3):598-611. doi: 10.1002/ijc.25062.
Multidrug resistance protein 3 (MRP3), a multidrug resistance protein identified by serial analysis of gene expression as a glioblastoma multiforme (GBM)-associated molecule, is highly expressed in GBM, but not in normal brain cells. Thus, MRP3 is a candidate for GBM immunotargeting, but to date, no monoclonal antibody has been isolated that can target an extracellular MRP3 epitope. By phage display, we have isolated 3 recombinant, fully human, single-chain Fv (scFv) antibodies, M25, M58 and M89, which specifically react with the extracellular N-terminus of human MRP3. In ELISA, these scFvs reacted only with the peptide used for screening and not with other MRP3-derived peptides. Flow cytometric analysis revealed that these scFv fragments bind specifically to viable human GBM cells displaying different MRP3 expression levels, but not to MRP3-null cells. Furthermore, these scFv antibodies failed to react with tumor cells overexpressing other MRP proteins, including MRP1, MRP2, MRP4 and MRP5. M25 and M58 also bound to viable neurospheres. Iodogen-labeled scFvs demonstrated a yield of 56-76%. The immunoreactive fractions of the radiolabeled M25, M58 and M89 scFvs were 32, 52 and 69%, respectively. M25 exhibited 20% internalization into D2159MG neurospheres, M58, 33% into D54MG cells and M89, 26% into D247MG. Immunohistochemical evaluation of human gliomas to determine the localization of MRP3 antigen using scFvs M25 and M58 showed a dense cytoplasmic and membranous staining pattern. These Fv-based recombinant antibodies, which possess superior tumor penetration capabilities and selectively target tumor cells that express MRP3, may potentially be used in immunotherapy and diagnosis for brain tumors and other cancers.
多药耐药蛋白 3（MRP3）是通过基因表达序列分析鉴定的一种多药耐药蛋白，被认为是胶质母细胞瘤（GBM）相关分子，在 GBM 中高度表达，但在正常脑细胞中不表达。因此，MRP3 是 GBM 免疫靶向的候选物，但迄今为止，尚未分离出能够靶向细胞外 MRP3 表位的单克隆抗体。通过噬菌体展示，我们分离出 3 种重组、完全人源的单链 Fv（scFv）抗体，M25、M58 和 M89，它们特异性地与人类 MRP3 的细胞外 N 端反应。在 ELISA 中，这些 scFv 仅与用于筛选的肽反应，而不与其他 MRP3 衍生肽反应。流式细胞术分析显示，这些 scFv 片段特异性地与表达不同 MRP3 水平的活人类 GBM 细胞结合，但与 MRP3 缺失细胞不结合。此外，这些 scFv 抗体不能与过度表达其他 MRP 蛋白的肿瘤细胞反应，包括 MRP1、MRP2、MRP4 和 MRP5。M25 和 M58 也与活的神经球结合。碘代标记的 scFv 的产量为 56-76%。放射性标记的 M25、M58 和 M89 scFv 的免疫反应性部分分别为 32%、52%和 69%。M25 有 20%内化到 D2159MG 神经球中，M58 有 33%内化到 D54MG 细胞中，M89 有 26%内化到 D247MG 中。使用 scFv M25 和 M58 对人类脑肿瘤进行免疫组织化学评估，以确定 MRP3 抗原的定位，结果显示细胞质和膜状染色模式密集。这些基于 Fv 的重组抗体具有优越的肿瘤穿透能力，并且选择性地靶向表达 MRP3 的肿瘤细胞，可能潜在地用于脑肿瘤和其他癌症的免疫治疗和诊断。
