Lysosomal trafficking and cysteine protease metabolism confer target-specific cytotoxicity by peptide-linked anti-CD30-auristatin conjugates.
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 valine-citrulline (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.
Department of Molecular Oncology & Immunology, Seattle Genetics Inc., 21823-30th Drive SE, Bothell, WA 98021, USA.
May S Kung
MS
Sutherland
Russell J
RJ
Sanderson
Kristine A
KA
Gordon
Jamie
J
Andreyka
Charles G
CG
Cerveny
Changpu
C
Yu
Timothy S
TS
Lewis
Damon L
DL
Meyer
Roger F
RF
Zabinski
Svetlana O
SO
Doronina
Peter D
PD
Senter
Che-Leung
CL
Law
Alan F
AF
Wahl
D000906:Antibodies / Q000737:chemistry; D018951:Antigens, CD20 / Q000737:chemistry; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002401:Cathepsin B / Q000737:chemistry; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D010450:Endopeptidases / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D020128:Inhibitory Concentration 50; D017730:Ki-1 Antigen / Q000737:chemistry*; D008247:Lysosomes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009842:Oligopeptides / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D000906:Antibodies; D018951:Antigens, CD20; D000970:Antineoplastic Agents; D015853:Cysteine Proteinase Inhibitors; D017730:Ki-1 Antigen; D009842:Oligopeptides; D010455:Peptides; C105791:soblidotin; D001616:beta-Galactosidase; D010450:Endopeptidases; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B