Kiesgen Stefan, Liebers Nora, Cremer Martin, Arnold Ulrich, Weber Tobias, Keller Armin, Herold-Mende Christel, Dyckhoff Gerhard, Jäger Dirk, Kontermann Roland E, Arndt Michaela A E, Krauss Jürgen
Department of Medical Oncology, National Center for Tumor Diseases, Heidelberg University Hospital, Im Neuenheimer Feld 460, 69120 Heidelberg, Germany.
Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
Protein Eng Des Sel. 2014 Oct;27(10):331-7. doi: 10.1093/protein/gzu040.
Due to its frequent overexpression in a variety of solid tumors the epidermal growth factor receptor (EGFR) is a well-established target for therapeutic interventions in epithelial cancers. In order to target EGFR in head and neck cancer, we have generated a ribonuclease (RNase) fusion protein comprising a humanized anti-EGFR antibody single-chain Fv fragment (scFv) and Ranpirnase, an RNase from Rana pipiens. Fusion of Ranpirnase to the N-terminus of the scFv via a flexible glycine-serine linker (G4S)3 resulted in very poor cytotoxicity of the fusion protein. As endosomal accumulation and lysosomal degradation have been reported to diminish the antitumor efficacy of ribonuclease or toxin-based immunoagents, we explored a fusion peptide from dengue virus that has been reported to be involved in the endosomal escape of the virus. This peptide was introduced as a linker between Ranpirnase and the scFv moiety. The modified immunoRNase exhibited exceptionally high cytotoxicity toward EGFR-expressing head and neck cell lines without affecting specificity. These results indicate that endosomal entrapment needs to be considered for Ranpirnase-based immunoagents and might be overcome by the use of tailored transduction domains from viral proteins.
由于表皮生长因子受体（EGFR）在多种实体瘤中频繁过表达，它是上皮癌治疗干预的一个成熟靶点。为了在头颈癌中靶向EGFR，我们构建了一种核糖核酸酶（RNase）融合蛋白，其包含人源化抗EGFR抗体单链Fv片段（scFv）和来自豹蛙的核糖核酸酶Ranpirnase。通过柔性甘氨酸 - 丝氨酸接头（G4S）3将Ranpirnase融合到scFv的N端，导致融合蛋白的细胞毒性非常低。由于据报道内体积累和溶酶体降解会降低基于核糖核酸酶或毒素的免疫剂的抗肿瘤功效，我们探索了一种来自登革病毒的融合肽，据报道该肽参与病毒的内体逃逸。该肽被引入作为Ranpirnase和scFv部分之间的接头。修饰后的免疫核糖核酸酶对表达EGFR的头颈细胞系表现出极高的细胞毒性，且不影响特异性。这些结果表明，基于Ranpirnase的免疫剂需要考虑内体截留问题，并且可以通过使用来自病毒蛋白的定制转导结构域来克服。
