Ager S, Nilson B H, Morling F J, Peng K W, Cosset F L, Russell S J
Cambridge Centre for Protein Engineering, England.
Hum Gene Ther. 1996 Nov 10;7(17):2157-64. doi: 10.1089/hum.1996.7.17-2157.
Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.
通过将五种针对人细胞表面抗原的不同单链抗体片段（scFv）与莫洛尼氏鼠白血病病毒（Moloney）的表面糖蛋白（SU）融合，展示在鼠嗜亲性逆转录病毒载体上。通过将scFv与莫洛尼氏鼠白血病病毒SU的+7位残基或+1位残基融合，以及在结构域之间插入不同长度的接头序列，改变scFv与SU糖蛋白之间的间距。所有嵌合包膜都能有效地整合到载体颗粒中，并能通过其展示的抗体片段与人细胞结合，但不能感染细胞。scFv与SU糖蛋白之间的间距对包膜表达效率或病毒整合效率没有显著影响，不影响嵌合包膜的结合特性，也不影响展示scFv的载体将靶向基因递送至人细胞的效率。然而，对于鼠成纤维细胞，嵌合包膜载体的感染性受结构域间间隔长度的强烈影响。当单链抗体通过三肽接头与SU残基+7融合时，滴度非常低，而当它们通过七肽接头与SU残基+1融合时，滴度大大提高（高达10^5倍）。这些结果表明嵌合包膜糖蛋白结构域之间空间相互作用的重要性，可能对人类基因治疗的逆转录病毒载体设计有影响。
