Antibody–Drug Conjugate that Exhibits Synergistic Cytotoxicity with an Endosome–Disruptive Peptide

[Image: see text] 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(50) > 1 μM). However, co-administration with an essentially nontoxic (IC(50) > 10 μM) cholesterol-linked endosome-disruptive peptide promotes the release of this small molecule into the cytoplasm, conferring subnanomolar cytotoxic potency (IC(50) = 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 (t(1/2) = 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.