HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 15, 10540-10547, April 14, 2006

This Article Full Text Full Text (PDF) All Versions of this Article: 281/15/10540    most recent M510026200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sutherland, M. S. K. Articles by Wahl, A. F. Search for Related Content PubMed PubMed Citation Articles by Sutherland, M. S. K. Articles by Wahl, A. F. Social Bookmarking                      What's this?

Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates^*

From the Departments of Molecular Oncology & Immunology, and Chemistry, Seattle Genetics, Inc., Bothell, Washington 98021

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.

Received for publication, September 12, 2005 , and in revised form, February 14, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular Oncology & Immunology, Seattle Genetics, Inc. 21823-30th Dr. SE, Bothell, WA 98021. Tel.: 425-527-4610; Fax: 425-527-4609; E-mail: awahl{at}seagen.com.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				H.-P. Gerber, M. Kung-Sutherland, I. Stone, C. Morris-Tilden, J. Miyamoto, R. McCormick, S. C. Alley, N. Okeley, B. Hayes, F. J. Hernandez-Ilizaliturri, et al. Potent antitumor activity of the anti-CD19 auristatin antibody drug conjugate hBU12-vcMMAE against rituximab-sensitive and -resistant lymphomas Blood, April 30, 2009; 113(18): 4352 - 4361. [Abstract] [Full Text] [PDF]

				A. G. Polson, J. Calemine-Fenaux, P. Chan, W. Chang, E. Christensen, S. Clark, F. J. de Sauvage, D. Eaton, K. Elkins, J. M. Elliott, et al. Antibody-Drug Conjugates for the Treatment of Non-Hodgkin's Lymphoma: Target and Linker-Drug Selection Cancer Res., March 15, 2009; 69(6): 2358 - 2364. [Abstract] [Full Text] [PDF]

				C. W. Wright and C. S. Duckett The Aryl Hydrocarbon Nuclear Translocator Alters CD30-Mediated NF-{kappa}B-Dependent Transcription Science, January 9, 2009; 323(5911): 251 - 255. [Abstract] [Full Text] [PDF]

				Y. Chen, S. Clark, T. Wong, Y. Chen, Y. Chen, M. S. Dennis, E. Luis, F. Zhong, S. Bheddah, H. Koeppen, et al. Armed Antibodies Targeting the Mucin Repeats of the Ovarian Cancer Antigen, MUC16, Are Highly Efficacious in Animal Tumor Models Cancer Res., May 15, 2007; 67(10): 4924 - 4932. [Abstract] [Full Text] [PDF]