Oxidizing potential of endosomes and lysosomes limits intracellular cleavage of disulfide-based antibody-drug conjugates
Oxidizing potential of endosomes and lysosomes limits intracellular cleavage of disulfide-based antibody-drug conjugates
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DOI：
10.1073/pnas.0509035102
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发表时间：
2005-12-13
影响因子：
11.1
通讯作者：
Scales, SJ
中科院分区：
文献类型：
--
作者：
Austin, CD;Wen, XH;Scales, SJ
抗体-药物结合疗法需要用与肿瘤特异性抗体共价连接的细胞毒性化合物靶向杀伤癌细胞，并在治疗几种人类癌症方面显示出希望。目前在抗体和细胞毒性药物之间加入二硫键连接物的抗体-药物结合物设计的灵感来自间接证据，表明内体系统内的氧化还原电位正在降低。据推测，抗原依赖的内吞作用会导致二硫键连接物的减少和细胞内游离药物的释放，但缺乏对这种机制的直接证明。为了确定在抗HER2抗体曲妥珠单抗(Herceptin，Genentech)的胞内循环过程中，二硫代N-琥珀酰亚胺4-(2-吡二硫基)戊酸酯(SPP)连接体是否会减少，我们合成了曲妥珠单抗-SPP-罗丹明红结合物，并利用该荧光团的自猝灭特性建立了连接体切割试验。在乳腺癌SKBr3细胞中，没有观察到SPP连接子的裂解，这是通过内化荧光去猝灭检测到的。相反，当被格尔达那霉素转移到溶酶体途径时，结合物确实显示出荧光猝灭，这部分是由于蛋白质降解而不是二硫键还原所致。为了了解连接子还原效率低下的原因，我们通过表达融合到各种细胞内蛋白的氧化还原敏感的GFP变体来测量内细胞室的氧化还原电位。出乎意料的是，我们发现回收的内小体、晚期的内小体和溶酶体并没有减少，而是氧化，并与内质网中的条件相当。这些结果表明，细胞内还原不太可能解释二硫键连接的抗菌药物偶联物的效力。
Antibody-drug conjugate therapy entails targeted killing of cancer cells with cytotoxic compounds covalently linked to tumor-specific antibodies and shows promise in the treatment of several human cancers. Current antibody-drug conjugate designs that incorporate a disulfide linker between the antibody and cytotoxic drug are inspired by indirect evidence suggesting that the redox potential within the endosomal system is reducing. It is presumed that antigen-dependent endocytosis leads to disulfide linker reduction and intracellular release of free drug, but direct demonstration of such a mechanism is lacking. To determine whether the disulfide N-succinimidyl 4-(2-pyridyidithio)pentanoate (SPP) linker would be reduced during endocytic recycling of the anti-HER2 antibody trastuzumab (Herceptin, Genentech), we synthesized a trastuzumab-SPP-Rhodamine red conjugate and developed a linker cleavage assay by using the self-quenching property of this fluorophore. In breast carcinoma SKBr3 cells, no SPP linker cleavage was observed, as detected by fluorescence dequenching upon internalization. By contrast, the conjugate did display fluorescence dequenching when diverted to the lysosomal pathway by geldanamycin, an effect partly due to proteolytic degradation rather than disulfide reduction. To understand why linker reduction was inefficient, we measured redox potentials of endocytic compartments by expressing a redox-sensitive variant of GFP fused to various endocytic proteins. Unexpectedly, we found that recycling endosomes, late endosomes, and lysosomes are not reducing, but oxidizing and comparable with conditions in the endoplasmic reticulum. These results suggest that intracellular reduction is unlikely to account for the potency of disulfide-linked antibod-drug conjugates.