Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1
Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1
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DOI：
10.1038/sj.bjc.6602847
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发表时间：
2005-11-28
影响因子：
8.8
通讯作者：
Deonarain, MP
中科院分区：
文献类型：
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作者：
Pericleous, LM;Richards, J;Deonarain, MP
人源化HMFG-1免疫球蛋白已被广泛开发为MUCI表达肿瘤的临床免疫抑制剂。我们已经从该抗体构建了单链Fv（scFv）和Fab片段，并且表明这两种物种都保留了它们对MUCI的特异性。scFv比Fab更不稳定且更不可溶。完整IgG和Fab片段的结合动力学的详细分析显示，对MUCI合成肽的亲和力低（IgG约100 nM，Fab约10 μ M），具有特别低但相似的解离速率常数（0.031 - 0.095 s（-1））。与细胞表面上的天然抗原的结合好两个数量级以上。共聚焦免疫荧光显微镜显示IgG和Fab都迅速内化（IgG在15分钟内内化）并共定位于早期内体。这项工作提供了一个升值的结合，内化和运输动力学，重要的是未来的治疗方法的基础上，这种抗体的发展。
The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUCI expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUCI. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUCI synthetic peptides is low ( approximately 100 nM for the IgG and 10 mu M for the Fab), with particularly low but similar dissociation rate constants (0.031 - 0.095 s(-1)). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly ( the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody.