Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates
Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates
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DOI：
10.1074/jbc.m510026200
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发表时间：
2006-04-14
影响因子：
4.8
通讯作者：
Wahl, AF
中科院分区：
文献类型：
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作者：
Sutherland, MSK;Sanderson, RJ;Wahl, AF
嵌合抗CD 30单克隆抗体cAC 10与抗有丝分裂剂单甲基澳瑞他汀E（MMAE）或F（MMAF）连接，在体外和体内产生强效和高度CD 30选择性抗肿瘤活性。这些药物通过缬氨酸瓜氨酸（vc）二肽键连接，设计用于在血清中的高稳定性和溶酶体组织蛋白酶对完全活性药物的条件切割和推定释放。为了表征导致有效药物递送的生化过程，我们检查了在CD 30表面抗原与靶细胞相互作用后，母体抗体和两种抗体-药物缀合物cAC 10 vc-MMAE和cAC 10 vc-MMAF的细胞内运输、内化和代谢。cAC 10及其偶联物均与靶细胞结合，并以相似的方式内化。亚细胞分级分离和免疫荧光研究表明，进入靶细胞的抗体和抗体-药物缀合物迁移到溶酶体。这两个物种的贩运被网格蛋白介导的内吞作用的抑制剂阻断，表明药物偶联不会改变抗体-抗原复合物的命运。将cAC 10 vc-MMAE或cAC 10 vc-MMAF与纯化的组织蛋白酶B或通过亚细胞分级分离制备的富集溶酶体组分孵育，导致活性游离药物释放。半胱氨酸蛋白酶抑制剂，但不是天冬氨酸或丝氨酸蛋白酶抑制剂，阻断抗体-药物缀合物代谢和随之而来的靶细胞的细胞毒性，并产生增强的细胞内水平的完整的缀合物。这些发现表明，除了运输到溶酶体之外，组织蛋白酶B和可能的其他溶酶体半胱氨酸蛋白酶是药物释放所必需的，并且为开发可被细胞内蛋白酶裂解的抗体-药物缀合物以靶向递送抗癌治疗剂提供了机理基础。
The chimeric anti-CD30 monoclonal antibody cAC10, linked to the antimitotic agents monomethyl auristatin E (MMAE) or F (MMAF), produces potent and highly CD30-selective anti-tumor activity in vitro and in vivo. These drugs are appended via a valinecitrulline (vc) dipeptide linkage designed for high stability in serum and conditional cleavage and putative release of fully active drugs by lysosomal cathepsins. To characterize the biochemical processes leading to effective drug delivery, we examined the intracellular trafficking, internalization, and metabolism of the parent antibody and two antibody-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, following CD30 surface antigen interaction with target cells. Both cAC10 and its conjugates bound to target cells and internalized in a similar manner. Subcellular fractionation and immunofluorescence studies demonstrated that the antibody and antibody-drug conjugates entering target cells migrated to the lysosomes. Trafficking of both species was blocked by inhibitors of clathrin-mediated endocytosis, suggesting that drug conjugation does not alter the fate of antibody-antigen complexes. Incubation of cAC10vc-MMAE or cAC10vc-MMAF with purified cathepsin B or with enriched lysosomal fractions prepared by subcellular fractionation resulted in the release of active, free drug. Cysteine protease inhibitors, but not aspartic or serine protease inhibitors, blocked antibody-drug conjugate metabolism and the ensuing cytotoxicity of target cells and yielded enhanced intracellular levels of the intact conjugates. These findings suggest that in addition to trafficking to the lysosomes, cathepsin B and perhaps other lysosomal cysteine proteases are requisite for drug release and provide a mechanistic basis for developing antibody-drug conjugates cleavable by intracellular proteases for the targeted delivery of anti-cancer therapeutics.