Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Cytokine. 2011 Sep;55(3):402-8. doi: 10.1016/j.cyto.2011.05.024. Epub 2011 Jun 22.
Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling.
血小板生成素是一种有效的细胞因子，通过其同源受体 c-Mpl 促进造血干细胞（HSCs）的增殖。因此，通过识别人工配体的受体模拟 c-Mpl 信号对于实现基因修饰 HSCs 的特异性扩增是有吸引力的。在这里，我们提出了一种使用能够被特定抗原激活的抗体/受体嵌合体来选择性扩增基因修饰细胞的系统。我们构建了一种抗体/c-Mpl 嵌合体，其中荧光素抗体的单链 Fv（ScFv）与促红细胞生成素受体的胞外 D2 结构域以及 c-Mpl 的跨膜/胞质结构域相连。当该嵌合体在依赖白细胞介素（IL）-3 的前 B 细胞系 Ba/F3 中表达时，在荧光素缀合的 BSA（BSA-FL）作为特定抗原存在的情况下，基因修饰细胞被选择性地扩增。此外，用携带抗体/c-Mpl 嵌合体基因的逆转录病毒转导的高度纯化的小鼠 HSCs 在 BSA-FL 的刺激下在体外增殖，并且细胞在体内保留了长期的重编程能力。这些结果表明，抗体/c-Mpl 嵌合体能够模拟野生型 c-Mpl 信号转导。
