Knewtson Kelsey E, Perera Chamani, Hymel David, Gao Zhe, Lee Molly M, Peterson Blake R
Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66045, United States.
ACS Omega. 2019 Jul 31;4(7):12955-12968. doi: 10.1021/acsomega.9b01585.
Antibody-drug conjugates are an important class of cancer therapeutics. These agents generally bind a specific cell surface receptor, undergo receptor-mediated endocytosis, and enter the endosomal-lysosomal system, where the environment in these organelles facilitates the release of a membrane-permeable cytotoxin. By using a membrane-impermeable cytotoxin, we describe here a method that allows the cytotoxicity of an antibody conjugate to be triggered by co-administration with an endosome-disruptive peptide that exhibits low toxicity. This approach was validated by conjugation of an anionic derivative of the tubulin-binding cytotoxin colchinol methyl ether to lysine residues of the HER2-targeting antibody trastuzumab (Herceptin) via a disulfide. When this antibody binds HER2 on SKBR3 breast cancer cells and undergoes endocytosis, the membrane-impermeable cytotoxin is released, but it becomes trapped in endosomes, resulting in relatively low cytotoxicity (IC > 1 μM). However, co-administration with an essentially nontoxic (IC > 10 μM) cholesterol-linked endosome-disruptive peptide promotes the release of this small molecule into the cytoplasm, conferring subnanomolar cytotoxic potency (IC = 0.11 ± 0.07 nM). Studies of a structurally related fluorophore conjugate revealed that the endosome-disruptive peptide does not substantially enhance cleavage of the disulfide ( = 8 ± 2 h) within endosomes, suggesting that the mechanism of endosomal escape involves the efflux of some small molecules without facilitating substantial influx of reduced glutathione.
抗体药物偶联物是一类重要的癌症治疗药物。这些药物通常结合特定的细胞表面受体，经历受体介导的内吞作用，并进入内体-溶酶体系统，在这些细胞器中的环境有利于释放一种可透过膜的细胞毒素。通过使用一种不能透过膜的细胞毒素，我们在此描述一种方法，该方法允许通过与一种毒性低的内体破坏肽共同给药来触发抗体偶联物的细胞毒性。这种方法通过将微管蛋白结合细胞毒素秋水仙醇甲醚的阴离子衍生物通过二硫键与靶向HER2的抗体曲妥珠单抗（赫赛汀）的赖氨酸残基偶联来验证。当这种抗体结合SKBR3乳腺癌细胞上的HER2并经历内吞作用时，不能透过膜的细胞毒素被释放，但它被困在内体中，导致相对较低的细胞毒性（IC>1μM）。然而，与一种基本无毒（IC>10μM）的胆固醇连接的内体破坏肽共同给药可促进这种小分子释放到细胞质中，赋予亚纳摩尔级的细胞毒性效力（IC=0.11±0.07 nM）。对一种结构相关的荧光团偶联物的研究表明，内体破坏肽不会显著增强内体内二硫键的裂解（半衰期=8±2小时），这表明内体逃逸的机制涉及一些小分子的流出，而不会促进大量还原型谷胱甘肽的流入。
