T-CELL-TARGETED IMMUNOFUSION PROTEINS FROM ESCHERICHIA-COLI
T-CELL-TARGETED IMMUNOFUSION PROTEINS FROM ESCHERICHIA-COLI
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DOI：
10.1074/jbc.270.25.14951
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发表时间：
1995-06-23
影响因子：
4.8
通讯作者：
FISHWILD, DM
中科院分区：
文献类型：
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作者：
BETTER, M;BERNHARD, SL;FISHWILD, DM
细胞靶向结构域和细胞毒性蛋白之间的融合蛋白应该是特别有效的治疗试剂。我们构建了一个免疫融合蛋白家族，将人源化Fab、F（ab '）（2）或H65抗体的单链抗体形式（其识别人T细胞表面上的CD 5抗原）与植物核糖体失活蛋白白树蛋白连接。我们推断，这种免疫融合将与之前描述的H65和白树蛋白的化学缀合物一样有效地杀死人类靶细胞（Better M.，Bernhard，S. L.，Fishwild，D. M.，诺兰，私人助理，鲍尔河，巴西-地J.，Kung，黄毛菊A. H. C.的方法，和卡罗尔，S. F.（1994）J.Biol.Chem.269，9644-9650），如果识别结构域和催化结构域都保持活性，并且可以发现结构域之间的适当连接。所有的免疫融合蛋白通过一个共同的过程进行纯化，并测试对抗原阳性的人细胞的细胞毒性。在融合家族成员中观察到20-60倍范围的细胞毒活性，并且鉴定了几种融合蛋白，其活性与有效的化学缀合物大致相同。基于这些构建体，可以通过适当选择抗体结构域和核糖体失活蛋白来控制免疫融合亲合力和效力。
Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking humanized Fab, F(ab')(2), or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A. H. C., and Carroll, S. F. (1994) J. Biol. Chem. 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found. Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form. All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells. A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates. Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome-inactivating protein.