Liu S, Netzel-Arnett S, Birkedal-Hansen H, Leppla S H
Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland 20892-4350, USA.
Cancer Res. 2000 Nov 1;60(21):6061-7.
Matrix metalloproteinases (MMPs) are overexpressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. To exploit these characteristics in the design of tumor cell-selective cytotoxins, we constructed two mutated anthrax toxin protective antigen (PA) proteins in which the furin protease cleavage site is replaced by sequences selectively cleaved by MMPs. These MMP-targeted PA proteins were activated rapidly and selectively on the surface of MMP-overexpressing tumor cells. The activated PA proteins caused internalization of a recombinant cytotoxin, FP59, consisting of anthrax toxin lethal factor residues 1-254 fused to the ADP-ribosylation domain of Pseudomonas exotoxin A. The toxicity of the mutated PA proteins for MMP-overexpressing cells was blocked by hydroxamate inhibitors of MMPs, including BB94, and by a tissue inhibitor of matrix metalloproteinases (TIMP-2). The mutated PA proteins killed MMP-overexpressing tumor cells while sparing nontumorigenic normal cells when these were grown together in a coculture model, indicating that PA activation occurred on the tumor cell surface and not in the supernatant. This method of achieving cell-type specificity is conceptually distinct from, and potentially synergistic with, the more common strategy of retargeting a protein toxin by fusion to a growth factor, cytokine, or antibody.
基质金属蛋白酶（MMPs）在多种肿瘤组织和细胞系中过度表达，其表达与肿瘤侵袭和转移高度相关。为了在肿瘤细胞选择性细胞毒素的设计中利用这些特性，我们构建了两种突变的炭疽毒素保护性抗原（PA）蛋白，其中弗林蛋白酶切割位点被MMPs选择性切割的序列所取代。这些靶向MMP的PA蛋白在MMP过度表达的肿瘤细胞表面迅速且选择性地被激活。激活的PA蛋白导致一种重组细胞毒素FP59内化，FP59由与铜绿假单胞菌外毒素A的ADP-核糖基化结构域融合的炭疽毒素致死因子1 - 254位残基组成。MMP的羟基肟酸抑制剂（包括BB94）和基质金属蛋白酶组织抑制剂（TIMP - 2）可阻断突变的PA蛋白对MMP过度表达细胞的毒性。在共培养模型中，当非致瘤性正常细胞与MMP过度表达的肿瘤细胞一起生长时，突变的PA蛋白可杀死肿瘤细胞而不损伤正常细胞，这表明PA的激活发生在肿瘤细胞表面而非上清液中。这种实现细胞类型特异性的方法在概念上与通过与生长因子、细胞因子或抗体融合来重新靶向蛋白毒素的更常见策略不同，并且可能具有协同作用。
