Monoclonal antibody 667 recognizes the variable region a motif of the ecotropic retrovirus CasBrE envelope glycoprotein and inhibits Env binding to the viral receptor
Monoclonal antibody 667 recognizes the variable region a motif of the ecotropic retrovirus CasBrE envelope glycoprotein and inhibits Env binding to the viral receptor
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DOI：
10.1128/jvi.77.20.10984-10993.2003
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发表时间：
2003-10-01
影响因子：
5.4
通讯作者：
Pelegrin, M
中科院分区：
文献类型：
--
作者：
Dreja, H;Gros, L;Pelegrin, M
单克隆抗体（MAb）667是一种中和小鼠单克隆抗体，可识别亲嗜性嗜神经性小鼠逆转录病毒CasBrE的包膜糖蛋白（Env），但不识别其他小鼠逆转录病毒的包膜糖蛋白。由于667可用于抗病毒基因治疗的临床前研究以及研究逆转录病毒感染的早期事件，我们已经克隆了它的cDNA，并对其进行了详细的分子表征。基于斑点技术的实验表明，667识别位于受体结合结构域的可变区A中的12个氨基酸的线性表位。丙氨酸扫描实验表明，表位内的六个氨基酸对于MAb结合是关键的。其中之一，D-57，是不存在于任何其他鼠逆转录病毒Env，这表明该残基在667的选择性的关键作用。MAb 667重链和轻链cDNA通过瞬时转染Cos-7细胞进行功能表征。酶联免疫吸附试验和Biacore研究表明，Cos-7细胞产生的重组667单克隆抗体（r667）和亲本杂交瘤产生的单克隆抗体（h667）的特异性以及抗原结合的热力学和动力学性质相似。然而，h667显示含有污染性逆转录病毒和/或逆转录病毒样颗粒，其干扰病毒结合和中和实验。这些污染物可以通过严格的纯化方案成功地去除。重要的是，这种纯化的667可以完全阻止逆转录病毒结合靶细胞，并在中和试验中与转染的Cos-7细胞产生的r667 MAb一样有效。总之，本研究表明，MAb 667中和病毒的主要机制是阻断CasBrE Env的逆转录病毒受体结合结构域。此外，本研究的结果构成了对直接使用杂交瘤细胞培养上清液研究逆转录病毒细胞感染的初始事件以及进行体内中和实验的警告，并表明重组抗体或高度纯化的抗体对于这些目的是优选的。
Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D-57, is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.