Bioprocessing Technology Institute, Agency for Science Technology and Research (A*STAR), 20 Biopolis Way, #06-01, Centros S138668, Singapore.
J Biotechnol. 2011 May 20;153(3-4):77-85. doi: 10.1016/j.jbiotec.2011.03.017. Epub 2011 Mar 30.
Human embryonic stem cells (hESC) possess great potential for applications in regenerative medicine due to their ability to differentiate into any cell type in the body. However, it is crucial to remove residual undifferentiated hESC from the differentiated population to avoid teratoma formation in vivo. The monoclonal antibody, mAb 84, has been shown to bind and kill undifferentiated hESC and is very useful for the elimination of contaminating undifferentiated hESC prior to transplantation. As mAb 84 is an IgM, its large size may impede penetration into embryoid bodies (EB) or cell clumps. To improve penetration, four antibody fragment formats of mAb 84 were engineered and expressed in Escherichia coli: Fab 84, scFv 84, scFv 84-diabody and scFv 84-HTH. All 4 fragments bound specifically to hESC, but only scFv 84-HTH, a single chain variable fragment with a dimerizing helix-turn-helix motif, could recapitulate the cytotoxicity of mAb 84 on multiple hESC lines. The results suggest that multivalency and flexibility between the antigen-binding sites may be essential features required for killing of hESC by mAb 84 and its derivatives. Imaging of EB treated with scFv 84-HTH or mAb 84 showed an even distribution of scFv 84-HTH throughout the EB whereas mAb 84 was localized more to the periphery.
人类胚胎干细胞（hESC）由于其能够分化为体内任何细胞类型的能力，在再生医学应用方面具有巨大的潜力。然而，必须从分化群体中去除残留的未分化 hESC，以避免体内形成畸胎瘤。单克隆抗体 mAb 84 已被证明可以结合并杀死未分化的 hESC，并且在移植前消除污染的未分化 hESC 非常有用。由于 mAb 84 是 IgM，其较大的尺寸可能会阻碍其进入胚状体（EB）或细胞团。为了提高穿透力，设计并在大肠杆菌中表达了 mAb 84 的四种抗体片段形式：Fab 84、scFv 84、scFv 84-二抗体和 scFv 84-HTH。所有 4 个片段都特异性地与 hESC 结合，但只有 scFv 84-HTH（具有二聚化螺旋-转角-螺旋模体的单链可变片段）能够重现 mAb 84 对多种 hESC 系的细胞毒性。结果表明，抗原结合位点之间的多价性和灵活性可能是 mAb 84 及其衍生物杀死 hESC 所必需的关键特征。用 scFv 84-HTH 或 mAb 84 处理的 EB 的成像显示，scFv 84-HTH 在整个 EB 中均匀分布，而 mAb 84 则更多地定位于外围。
