Cytotoxic antibody fragments for eliminating undifferentiated human embryonic stem cells
Cytotoxic antibody fragments for eliminating undifferentiated human embryonic stem cells
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DOI：
10.1016/j.jbiotec.2011.03.017
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发表时间：
2011-05-20
影响因子：
4.1
通讯作者：
Wong, Victor V. T.
中科院分区：
文献类型：
--
作者：
Lim, Denis Y. X.;Ng, Yi-Han;Wong, Victor V. T.
人类胚胎干细胞(HESC)具有分化为体内任何细胞类型的能力，在再生医学中具有巨大的应用潜力。然而，从分化的群体中去除残留的未分化的hESC对于避免体内形成畸胎瘤至关重要。单抗mAb84可结合和杀伤未分化的hESC，对移植前清除污染的未分化的hESC非常有用。由于mAb84是一种IgM，其大小可能会阻碍其对类胚体(EB)或细胞团的渗透。为了提高通透性，设计了四种抗体片段形式，并在大肠杆菌中表达：Fab 84、ScFv 84、ScFv 84-diabody和ScFv 84-HTH。4个片段均与hESC特异结合，但只有单链可变片段scFv 84-HTH具有二聚螺旋-转角螺旋基序，可以概括mAb84在多个hESC株上的细胞毒作用。结果提示，抗原结合位点之间的多价性和灵活性可能是mAb84及其衍生物杀伤hESC所必需的基本特征。经ScFv84-HTH或mAb84处理的EB成像显示，scFv84-HTH在EB中分布均匀，而mAb84则更多地分布在边缘。(C)2011年，由爱思唯尔出版。
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. (C) 2011 Published by Elsevier B.V.