Lysosomal Trafficking and Cysteine Protease Metabolism Confer Target-specific Cytotoxicity by Peptide-linked Anti-CD30-Auristatin Conjugates
2006; Elsevier BV; Volume: 281; Issue: 15 Linguagem: Inglês
10.1074/jbc.m510026200
ISSN1083-351X
AutoresMay S.K. Sutherland, Russell J. Sanderson, Kristine A. Gordon, Jamie B. Andreyka, Charles G. Cerveny, Changpu Yu, Timothy S. Lewis, Damon L. Meyer, Roger F. Zabinski, Svetlana O. Doronina, Peter D. Senter, Che‐Leung Law, Alan F. Wahl,
Tópico(s)Cellular transport and secretion
ResumoThe 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. 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. Cancer is the second leading cause of mortality in the United States, resulting in >500,000 American deaths annually. 2American Cancer Society Cancer Facts and Figures, available online. The unmet medical need for more effective anticancer therapeutics, especially for strategies that can focus toxicity to tumor cells and away from normal tissues, has lead to the development of monoclonal antibodies (mAbs) 3The abbreviations used are: mAb, monoclonal antibody; MMAE, monomethyl auristatin E; MMAF, monomethyl auristatin F; vc, valine-citrulline; ADC, mAb-drug conjugate; PBS, phosphate-buffered saline. linked to immunotoxins, radionuclides, or cytotoxic drugs, to provide selective elimination of antigen-positive target cells. The first such clinically approved agent, Gemtuzumab ozogamicin (Mylotarg), an anti-CD33 mAb linked to the potent DNA damaging agent calicheamicin, is used for the treatment of patients with relapsed acute myeloid leukemia (1Sievers E.L. Larson R.A. Stadtmauer E.A. Estey E. Lowenberg B. Dombret H. Karanes C. Theobald M. Bennett J.M. Sherman M.L. Berger M.S. Eten C.B. Loken M.R. van Dongen J.J. Bernstein I.D. Appelbaum F.R. J. Clin. Oncol. 2001; 19: 3244-3254Crossref PubMed Scopus (743) Google Scholar). Mylotarg and multiple other mAb-drug conjugates (ADCs) and mAbtoxin conjugates currently in development use primarily disulfide or hydrazone linkers sensitive to the reductive or acidic environment of the tumor cell (2Omelyanenko V. Gentry C. Kopeckova P. Kopecek J. Int. J. Cancer. 1998; 75: 600-608Crossref PubMed Scopus (93) Google Scholar, 3Chan S.Y. Gordon A.N. Coleman R.E. Hall J.B. Berger M.S. Sherman M.L. Eten C.B. Finkler N.J. Cancer Immunol. Immunother. 2003; 52: 243-248Crossref PubMed Scopus (54) Google Scholar, 4Di Paolo C. Willuda J. Kubetzko S. Lauffer I. Tschudi D. Waibel R. Pluckthun A. Stahel R.A. Zangemeister-Wittke U. Clin. Cancer Res. 2003; 9: 2837-2848PubMed Google Scholar, 5Tolcher A.W. Ochoa L. Hammond L.A. Patnaik A. Edwards T. Takimoto C. Smith L. de Bono J. Schwartz G. Mays T. Jonak Z.L. Johnson R. DeWitte M. Martino H. Audette C. Maes K. Chari R.V. Lambert J.M. Rowinsky E.K. J. Clin. Oncol. 2003; 21: 211-222Crossref PubMed Scopus (174) Google Scholar, 6Hassan R. Bera T. Pastan I. Clin. Cancer Res. 2004; 10: 3937-3942Crossref PubMed Scopus (355) Google Scholar). These linkers readily deliver and liberate free drug or toxin within the target cell and yet are relatively unstable in circulation compared with the circulating half-life of the mAb (7Sanderson R.J. Hering M.A. James S.F. Sun M.M. Doronina S.O. Siadak A.W. Senter P.D. Wahl A.F. Clin. Cancer Res. 2005; 11: 843-852PubMed Google Scholar), resulting in premature drug release. An alternative approach is to employ ADC linkers of protease-cleavable dipeptides. These combine qualities of high stability in serum or plasma with efficient drug release potentially by lysosomal proteases (8Dubowchik G.M. Firestone R.A. Padilla L. Willner D. Hofstead S.J. Mosure K. Knipe J.O. Lasch S.J. Trail P.A. Bioconjug. Chem. 2002; 13: 855-869Crossref PubMed Scopus (388) Google Scholar). Using this strategy, we recently described a new ADC of the anti-CD30 mAb cAC10 (9Wahl A.F. Klussman K. Thompson J.D. Chen J.H. Francisco L.V. Risdon G. Chace D.F. Siegall C.B. Francisco J.A. Cancer Res. 2002; 62: 3736-3742PubMed Google Scholar), appended to the anti-tubulin agent, monomethyl auristatin E (MMAE), via a cathepsin-cleavable valine-citrulline (vc) linker (10Doronina S.O. Toki B.E. Torgov M.Y. Mendelsohn B.A. Cerveny C.G. Chace D.F. DeBlanc R.L. Gearing R.P. Bovee T.D. Siegall C.B. Francisco J.A. Wahl A.F. Meyer D.L. Senter P.D. Nat. Biotechnol. 2003; 21: 778-784Crossref PubMed Scopus (892) Google Scholar, 11Francisco J.A. Cerveny C.G. Meyer D.L. Mixan B.J. Klussman K. Chace D.F. Rejniak S.X. Gordon K.A. DeBlanc R. Toki B.E. Law C.L. Doronina S.O. Siegall C.B. Senter P.D. Wahl A.F. Blood. 2003; 102: 1458-1465Crossref PubMed Scopus (674) Google Scholar). This drug linker system was shown to be highly stable in vitro and in vivo (7Sanderson R.J. Hering M.A. James S.F. Sun M.M. Doronina S.O. Siadak A.W. Senter P.D. Wahl A.F. Clin. Cancer Res. 2005; 11: 843-852PubMed Google Scholar, 12Hamblett K.J. Senter P.D. Chace D.F. Sun M.M. Lenox J. Cerveny C.G. Kissler K.M. Bernhardt S.X. Kopcha A.K. Zabinski R.F. Meyer D.L. Francisco J.A. Clin. Cancer Res. 2004; 10: 7063-7070Crossref PubMed Scopus (891) Google Scholar), and when applied to multiple mAbs, the resulting ADCs were selectively potent and effective against cognate antigen-positive tumor cells and tumor xenografts (LeY (8Dubowchik G.M. Firestone R.A. Padilla L. Willner D. Hofstead S.J. Mosure K. Knipe J.O. Lasch S.J. Trail P.A. Bioconjug. Chem. 2002; 13: 855-869Crossref PubMed Scopus (388) Google Scholar), CD30 (11Francisco J.A. Cerveny C.G. Meyer D.L. Mixan B.J. Klussman K. Chace D.F. Rejniak S.X. Gordon K.A. DeBlanc R. Toki B.E. Law C.L. Doronina S.O. Siegall C.B. Senter P.D. Wahl A.F. Blood. 2003; 102: 1458-1465Crossref PubMed Scopus (674) Google Scholar), TMEFF2 (13Afar D.E. Bhaskar V. Ibsen E. Breinberg D. Henshall S.M. Kench J.G. Drobnjak M. Powers R. Wong M. Evangelista F. O'Hara C. Powers D. DuBridge R.B. Caras I. Winter R. Anderson T. Solvason N. Stricker P.D. Cordon-Cardo C. Scher H.I. Grygiel J.J. Sutherland R.L. Murray R. Ramakrishnan V. Law D.A. Mol. Cancer Ther. 2004; 3: 921-932PubMed Google Scholar), CD20 (14Law C.L. Cerveny C.G. Gordon K.A. Klussman K. Mixan B.J. Chace D.F. Meyer D.L. Doronina S.O. Siegall C.B. Francisco J.A. Senter P.D. Wahl A.F. Clin. Cancer Res. 2004; 10: 7842-7851Crossref PubMed Scopus (102) Google Scholar), and EphB2 (15Mao W. Luis E. Ross S. Silva J. Tan C. Crowley C. Chui C. Franz G. Senter P. Koeppen H. Polakis P. Cancer Res. 2004; 64: 781-788Crossref PubMed Scopus (111) Google Scholar)). One premise of this drug delivery technology is that the mAb-antigen complex on the cell surface will internalize, traffic to the lysosomes, and be metabolized by lysosomal proteases to release free drug. ADC efficacy therefore depends in part on mAb-antigen interaction at the cell surface triggering the internalization, trafficking, and subsequent release of the active cytotoxic payload. Thus, conjugates comprised of different drug linkers or with different mAbs against the same target can vary significantly in their utility. For example, anti-CD20 ADC incorporating doxorubicin (16Braslawsky G.R. Kadow K. Knipe J. McGoff K. Edson M. Kaneko T. Greenfield R.S. Cancer Immunol. Immunother. 1991; 33: 367-374Crossref PubMed Scopus (82) Google Scholar, 17Sapra P. Allen T.M. Cancer Res. 2002; 62: 7190-7194PubMed Google Scholar) or ricin-A (18Goulet A.C. Goldmacher V.S. Lambert J.M. Baron C. Roy D.C. Kouassi E. Blood. 1997; 90: 2364-2375Crossref PubMed Google Scholar) were ineffective as anti-tumor agents, whereas anti-CD20 conjugates of vcMMAE were highly effective against CD20+ tumors (14Law C.L. Cerveny C.G. Gordon K.A. Klussman K. Mixan B.J. Chace D.F. Meyer D.L. Doronina S.O. Siegall C.B. Francisco J.A. Senter P.D. Wahl A.F. Clin. Cancer Res. 2004; 10: 7842-7851Crossref PubMed Scopus (102) Google Scholar). Interestingly, the anti-CD20 mAb remained on the cell surface, whereas the anti-CD20vc-MMAE conjugate was readily internalized, resulting in rapid cell cycle arrest and apoptosis (14Law C.L. Cerveny C.G. Gordon K.A. Klussman K. Mixan B.J. Chace D.F. Meyer D.L. Doronina S.O. Siegall C.B. Francisco J.A. Senter P.D. Wahl A.F. Clin. Cancer Res. 2004; 10: 7842-7851Crossref PubMed Scopus (102) Google Scholar). Alternatively, in targeting CD30, both mAb and anti-CD30 ADC demonstrated comparable binding and internalization rates in CD30+ tumor cells (11Francisco J.A. Cerveny C.G. Meyer D.L. Mixan B.J. Klussman K. Chace D.F. Rejniak S.X. Gordon K.A. DeBlanc R. Toki B.E. Law C.L. Doronina S.O. Siegall C.B. Senter P.D. Wahl A.F. Blood. 2003; 102: 1458-1465Crossref PubMed Scopus (674) Google Scholar), with the ADC inducing rapid mitotic arrest cell and apoptosis (11Francisco J.A. Cerveny C.G. Meyer D.L. Mixan B.J. Klussman K. Chace D.F. Rejniak S.X. Gordon K.A. DeBlanc R. Toki B.E. Law C.L. Doronina S.O. Siegall C.B. Senter P.D. Wahl A.F. Blood. 2003; 102: 1458-1465Crossref PubMed Scopus (674) Google Scholar). Just what governs optimal ADC internalization and trafficking critical for effective drug release is not known. Here we examine the trafficking and intracellular fate of anti-CD30 mAb cAC10 and two cAC10-drug conjugates, cAC10vc-MMAE and cAC10vc-MMAF, in CD30+ T-cell lymphoma cell lines. Using flow cytometry, immunofluorescence, subcellular fractionation, and chemical inhibitors of trafficking and processing, we demonstrate that the cytotoxicity of peptide-linked auristatin ADCs is contingent upon their delivery to the lysosome and the activity of lysosomal cysteine proteases. These studies demonstrate the functional basis of drug delivery by ADCs with protease-cleavable linkers for anti-cancer therapeutics. Flow Cytometry for Antibody and Antibody-Drug Conjugate Internalization and Trafficking—The chimeric anti-CD30 antibody cAC10 was produced as described previously (9Wahl A.F. Klussman K. Thompson J.D. Chen J.H. Francisco L.V. Risdon G. Chace D.F. Siegall C.B. Francisco J.A. Cancer Res. 2002; 62: 3736-3742PubMed Google Scholar) and conjugated to MMAE (11Francisco J.A. Cerveny C.G. Meyer D.L. Mixan B.J. Klussman K. Chace D.F. Rejniak S.X. Gordon K.A. DeBlanc R. Toki B.E. Law C.L. Doronina S.O. Siegall C.B. Senter P.D. Wahl A.F. Blood. 2003; 102: 1458-1465Crossref PubMed Scopus (674) Google Scholar) and monomethyl auristatin F (MMAF) (19Doronina S.O. Mendelsohn B.A. Bovee T.D. Cerveny C.G. Alley S.C. Meyer D.L. Oflazoglu E. Toki B.E. Sanderson R.J. Zabinski R.F. Wahl A.F. Senter P.D. Bioconjug. Chem. 2006; 17: 114-124Crossref PubMed Scopus (409) Google Scholar) to yield cAC10vc-MMAE and cAC10vc-MMAF, respectively. The CD30+ Hodgkin disease L540cy cell line, a derivative of the L540 Hodgkin disease cell line adapted for xenograft growth, was provided by Dr. Harald Stein (Institut fur Pathologie, University of Veinikum Benjamin Franklin, Berlin, Germany). L540cy cells were grown in RPMI 1640 supplemented with 20% heat-inactivated fetal calf sera and antibiotics. L540cy cells (1 × 106 cells/ml) were incubated with 2 μg/ml cAC10, cAC10vc-MMAE, or cAC10vc-MMAF for 30 min on ice, rinsed with ice-cold PBS, and then incubated for 30 min in the presence (cross-linking) or absence (no cross-linking) of 2 μg/ml goat anti-human IgG (Jackson Immunoresearch, West Grove, PA) (20Coffey G.P. Stefanich E. Palmieri S. Eckert R. Padilla-Eagar J. Fielder P.J. Pippig S. J. Pharmacol. Exp. Ther. 2004; 310: 896-904Crossref PubMed Scopus (50) Google Scholar). The cells were rinsed with cold PBS, resuspended in growth media, and incubated at 37 °C. The samples were harvested at various times and processed for flow cytometry. To detect surface-bound ADC, the cells were incubated with 10 μg/ml mouse anti-id antibody to cAC10 for 30 min at 4 °C, washed, and then incubated with 10 μg/ml goat anti-mouse IgG-fluorescein isothiocyanate with minimal cross-reactivity to human IgG (Fcγ-specific, F(ab′)2 fragment; Jackson Immunoresearch). To detect internalized antibody or ADC, the cells were washed with cold PBS, incubated with proteinase K (5 μg/ml for 10 min at 37 °C), washed to remove cell surface-bound antibody, and treated with Cytofix/Cytoperm (BD Biosciences, San Jose, CA) prior to incubation with the anti-id antibody as described above. The cells were assessed by flow cytometry using a Becton Dickinson FACScan. In other experiments, L540cy cells were preincubated with subcellular trafficking inhibitors (21Harrison P.T. Davis W. Norman J.C. Hockaday A.R. Allen J.M. J. Biol. Chem. 1994; 269: 24396-24402Abstract Full Text PDF PubMed Google Scholar, 22Durrbach A. Louvard D. Coudrier E. J. Cell Sci. 1996; 109: 457-465Crossref PubMed Google Scholar, 23Brown B.K. Song W. Traffic. 2001; 2: 414-427Crossref PubMed Scopus (76) Google Scholar, 24Szpaderska A.M. Frankfater A. Cancer Res. 2001; 61: 3493-3500PubMed Google Scholar, 25Booth J.W. Kim M.K. Jankowski A. Schreiber A.D. Grinstein S. EMBO J. 2002; 21: 251-258Crossref PubMed Scopus (76) Google Scholar, 26Mueller A. Kelly E. Strange P.G. Blood. 2002; 99: 785-791Crossref PubMed Scopus (134) Google Scholar, 27Sieczkarski S.B. Whittaker G.R. J. Gen. Virol. 2002; 83: 1535-1545Crossref PubMed Scopus (401) Google Scholar) (10 μm colchicine, 0.5 μm amantadine, 0.1 μm phenylarsine oxide, 20 μm clasto-lactacystin-β-lactone, 40 μm cytochalasin D, 3 mm ammonium chloride, 14 μg/ml chlorpromazine, 10 μg/ml nystatin; Sigma) or cysteine protease inhibitors (3 μm CA074-OME and 20 μm E64d; Calbiochem, San Diego, CA) for 30 min at 4 °C prior to a 3- or 5-h incubation with cAC10 or cAC10-drug conjugates at 37 °C. The cells were processed as described above for internalized antibody except that detection was done by staining with a goat anti-human IgG-fluorescein isothiocyanate-labeled secondary antibody (Jackson Immunoresearch). Quantitative evaluation of the surface expression of CD30 on the Hodgkin disease cell line L540cy and the anaplastic large cell lymphoma cell line (ALCL) Karpas-299 (Deutsche Sammlung von Mikroorganism und Zellkulturen, GmbH) was performed using the QiFiKiT bead assay (DAKO, Carpinteria, CA). Immunofluorescence for Antibody and Antibody-Drug Conjugate Trafficking—L540cy cells (5 × 105 cells/ml in regular medium) were incubated with 1 μg/ml cAC10, cAC10vc-MMAE, or cAC10vc-MMAF for 30 min on ice or for 16 h at 37 °C. After the incubation, the cells were washed with cold PBS to remove unbound antibody and drug conjugates. The cells were fixed and permeabilized with Cytofix/Cytoperm and stained as described previously (14Law C.L. Cerveny C.G. Gordon K.A. Klussman K. Mixan B.J. Chace D.F. Meyer D.L. Doronina S.O. Siegall C.B. Francisco J.A. Senter P.D. Wahl A.F. Clin. Cancer Res. 2004; 10: 7842-7851Crossref PubMed Scopus (102) Google Scholar). cAC10 and its conjugates were detected following incubation with Alexa Fluor 488-labeled goat anti-human IgG (H+L) with minimal cross-reactivity to mouse IgG (Molecular Probes, Eugene, OR). Lysosomal compartments were visualized by staining with Lamp-1 (mouse CD107a antibody, BD Biosciences) and a secondary antibody, Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) with minimal cross-reactivity to human IgG (Molecular Probes). Nuclear compartments were stained with 4′,6′-diamidino-2-phenylindole (Roche Applied Science). Fluorescence images were acquired with a Leitz Orthoplan epifluorescence microscope. In other experiments, the cAC10 antibody was linked to Alexa Fluor 488 reactive dye (Molecular Probes) and incubated with L540cy cells (2 × 105 cells/ml) at 200 ng/ml for 3 h at 37 °C in the presence or absence of trafficking inhibitors (40 μm cytochalasin D or 3 mm ammonium chloride). Lysosomal and nuclear compartments were visualized by staining with LysoTracker and Hoescht DNA dyes, respectively (Molecular Probes). Fluorescence images were taken on fixed cells with a Carl Zeiss Axiovert 200M microscope. Subcellular Fractionation—L540cy cells (5 × 105 cells) were incubated in growth medium with Alexa Fluor 488-labeled cAC10vc-MMAE (8 μg/ml) for 18 h at 37 °C. The cells were harvested, washed with ice-cold PBS, and pelleted at 500 × g for 5 min. Lysosome-enriched fractions were prepared as described previously (Axis-Shield, Oslo, Norway and Ref. 28Castle J.D. Coligan J.E. Dunn B.M. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. 1. John Wiley & Sons, Inc., New York2004: 4.2.1-4.2.57Google Scholar). Briefly, the cell pellets (5 × 107 cells) were incubated on ice for 30 min in cold homogenization buffer (0.25 m sucrose, 1 mm EDTA, 10 mm HEPES, pH 7.4) and gently Dounce homogenized to break open the cells. The homogenate was spun at 3000 × g for 10 min at 4 C. The supernatant was collected and centrifuged at 17,000 × g for 15 min at 4 °C. The resulting pellet was resuspended in homogenization buffer and mixed with 60% OptiPrep (Axis-Shield; final concentration, 20%) before centrifugation at 208,000 × g for 18 h at 4 °C. The samples were then fractionated (5 drops/fraction), and the 25 fractions were assayed for the lysosomal marker, β-galactosidase activity (metabolism of 4-methylumbelliferyl-β-d-galactopyranoside; Sigma), the presence of Alexa Fluor-labeled ADC by fluorimetry (Fusion-HT; Packard Instruments, Meridien, CT), and the loss of drug associated with heavy or light chains on the antibody frame by Western blot analyses with the anti-MMAE SG2.15 antibody (14Law C.L. Cerveny C.G. Gordon K.A. Klussman K. Mixan B.J. Chace D.F. Meyer D.L. Doronina S.O. Siegall C.B. Francisco J.A. Senter P.D. Wahl A.F. Clin. Cancer Res. 2004; 10: 7842-7851Crossref PubMed Scopus (102) Google Scholar). Western Blot Analyses—To evaluate the metabolism of cAC10-drug conjugates, purified cathepsin B (2 units/ml; Calbiochem) was incubated for 3 h at 37°C with 5 μg/ml cAC10vc-MMAE or cAC10vc-MMAF in buffer (2 mm dithiothreitol, 50 mm sodium acetate, pH 5.0) in the presence or absence of 10 μm E64d cysteine protease inhibitor. In other experiments, Karpas and L540cy pooled lysosome-enriched fractions (numbers 10-19) were incubated with 5 μg/ml cAC10vc-MMAE for 18 h in the presence or absence of 20 μm E64d or 3 μm CA074-OME. The reactions were stopped by quick freezing in a dry ice bath. The digests were mixed with Novex sample buffer (Invitrogen), run on 4-20% Tris-Gly or 4-12% Bis-Tris gradient gels (Invitrogen) under reducing conditions, and transferred onto polyvinylidene difluoride membranes (Invitrogen). The membranes were blocked with 2% nonfat dry milk in PBST (PBS+ 0.1% Tween 20) prior to incubation with the mouse SG2.15 antibody to detect drug. This antibody recognizes both MMAE and MMAF (data not shown). Detection was then performed using a horseradish peroxidase-goat anti-mouse IgG (Fcγ specific, Jackson Immunoresearch) followed by ECL (SuperSignal West Pico; Pierce). To detect the heavy and light chains of the antibody, detection was performed using horseradish peroxidase-goat anti-human IgG (Fcγ specific; Jackson Immunoresearch) or horseradish peroxidase-goat anti-human IgG (κ-specific; Southern Biotech, Birmingham, AL), respectively, followed by ECL. Cytotoxicity Assay—Cell viability was measured by Alamar Blue (BIOSOURCE International, Camarillo, CA) dye reduction (10Doronina S.O. Toki B.E. Torgov M.Y. Mendelsohn B.A. Cerveny C.G. Chace D.F. DeBlanc R.L. Gearing R.P. Bovee T.D. Siegall C.B. Francisco J.A. Wahl A.F. Meyer D.L. Senter P.D. Nat. Biotechnol. 2003; 21: 778-784Crossref PubMed Scopus (892) Google Scholar, 14Law C.L. Cerveny C.G. Gordon K.A. Klussman K. Mixan B.J. Chace D.F. Meyer D.L. Doronina S.O. Siegall C.B. Francisco J.A. Senter P.D. Wahl A.F. Clin. Cancer Res. 2004; 10: 7842-7851Crossref PubMed Scopus (102) Google Scholar) or with Celltiter-Glo (Promega, Madison, WI). The results were reported as the IC50 values, the concentration of compound needed to yield a 50% reduction in viability compared with untreated cells (control = 100%). To inhibit ADC metabolism, L540cy cells (8 × 103 cells/well) were pretreated with 20 μm E64d, 0.5 μm CA074-OME, 10 μm calpeptin (Calbiochem), 3 μm N-acetyl-Leu-Leu-Nle-CHO (calpain inhibitor I; Calbiochem), 20 μg/ml pepstatin A (Calbiochem), 20 μm p-aminoethylbenzenesulfonyl fluoride (Calbiochem), 20 μg/ml aprotinin (Sigma), or 25 μm cystamine dihydrochloride (Sigma) for 1 h prior to the addition of cAC10vc-MMAE. The cultures were maintained for 96 h prior to the addition of Celltiter-Glo. The cells were incubated with Celltiter-Glo reagent for 25 min, and the dishes were processed for luminescent readout. In other experiments, Karpas cells (5 × 105 cells) were incubated in growth medium for 4 h at 37 °C prior to the harvesting of cells to generate lysosomal fractions. The fractions were washed with 0.25 m sucrose buffer containing 10 mm HEPES, pH 7.2. Each fraction was incubated with cAC10vc-MMAE (5 μg/ml) for 24 h in reaction buffer (2 mm dithiothreitol, 50 mm sodium acetate, pH 5.0). Afterward, the fractions were diluted 300-fold in RPMI growth medium and added to cultures of CD30-negative Ramos cells (1 × 104 cells/well; American Type Culture Collection, Manassas, VA) in the presence or absence of 2.5 μg/ml mouse anti-MMAE antibody, SG3.190. Cell growth was assessed 96 h later with Alamar Blue. The cells were incubated with the dye for 4 h prior to fluorescence measurement on a Fusion HT plate reader. MMAE and MMAF belong to the dolastatin 10 family of highly potent anti-mitotic agents that inhibit tubulin polymerization (Fig. 1) (10Doronina S.O. Toki B.E. Torgov M.Y. Mendelsohn B.A. Cerveny C.G. Chace D.F. DeBlanc R.L. Gearing R.P. Bovee T.D. Siegall C.B. Francisco J.A. Wahl A.F. Meyer D.L. Senter P.D. Nat. Biotechnol. 2003; 21: 778-784Crossref PubMed Scopus (892) Google Scholar, 19Doronina S.O. Mendelsohn B.A. Bovee T.D. Cerveny C.G. Alley S.C. Meyer D.L. Oflazoglu E. Toki B.E. Sanderson R.J. Zabinski R.F. Wahl A.F. Senter P.D. Bioconjug. Chem. 2006; 17: 114-124Crossref PubMed Scopus (409) Google Scholar). In contrast to MMAE, the charged, carboxylic acid terminus of free MMAF can potentially limit passive transit through cell membranes. The cAC10 antibody-drug conjugates of MMAE and MMAF containing a protease-cleavable vc linker were prepared as described previously (10Doronina S.O. Toki B.E. Torgov M.Y. Mendelsohn B.A. Cerveny C.G. Chace D.F. DeBlanc R.L. Gearing R.P. Bovee T.D. Siegall C.B. Francisco J.A. Wahl A.F. Meyer D.L. Senter P.D. Nat. Biotechnol. 2003; 21: 778-784Crossref PubMed Scopus (892) Google Scholar, 19Doronina S.O. Mendelsohn B.A. Bovee T.D. Cerveny C.G. Alley S.C. Meyer D.L. Oflazoglu E. Toki B.E. Sanderson R.J. Zabinski R.F. Wahl A.F. Senter P.D. Bioconjug. Chem. 2006; 17: 114-124Crossref PubMed Scopus (409) Google Scholar). We have reported that cAC10 and cAC10-vcMMAE were comparable in binding to CD30+ cells and lacked interaction with CD30-negative cells (11Francisco J.A. Cerveny C.G. Meyer D.L. Mixan B.J. Klussman K. Chace D.F. Rejniak S.X. Gordon K.A. DeBlanc R. Toki B.E. Law C.L. Doronina S.O. Siegall C.B. Senter P.D. Wahl A.F. Blood. 2003; 102: 1458-1465Crossref PubMed Scopus (674) Google Scholar). The activities of the free drugs and the conjugates cAC10vc-MMAE and cAC10vc-MMAF were compared on CD30+ lymphoma cell lines including Karpas-299 and L540cy (Table 1). Comparison of free drugs showed that the cell-permeable MMAE was 50-200-fold more effective than MMAF. As an ADC however, cAC10vc-MMAF was significantly more potent than cAC10vc-MMAE (IC50 < than 0.11 nm, p < 0.001). Neither drug conjugate exhibited appreciable activity against multiple antigen-negative cell lines, demonstrating that the potency of these ADCs is antigen-dependent. For example, the IC50 value against the CD30-negative cell line Ramos following 96 h continuous exposure was >75 nm (Table 1).TABLE 1Cytotoxic activities of cAC10vc-MMAE and cAC10vc-MMAF on CD30+ tumor cell linesCell lineTypeCD30aCD30 levels expressed as the number of antibody molecules bound per cell.MMAEMMAFcAC10vc-EcAC10vc-FKarpasALCL (T-cell)290,6760.52 ± 0.29bp < 0.005 to MMAF.101 ± 120.11 ± 0.016bp < 0.005 to MMAF.0.05 ± 0.005L540cyHD (T-cell)587,5111.25 ± 0.81cp < 0.001 to MMAF.65.6 ± 14.30.32 ± 0.032dp < 0.001 to cAC10vc-F.0.11 ± 0.035RamosBurkitt lymphoma00.039 ± 0.02bp < 0.005 to MMAF.58 ± 10>75>75a CD30 levels expressed as the number of antibody molecules bound per cell.b p < 0.005 to MMAF.c p < 0.001 to MMAF.d p < 0.001 to cAC10vc-F. Open table in a new tab To evaluate the trafficking and internalization of cAC10 and its ADC, CD30+ L540cy cells were preincubated with mAb or ADCs and treated with vehicle or anti-human IgG for cross-linking prior to culture at 37 °C. Cross-linked antibodies exhibit increased internalization and cellular clearance (10Doronina S.O. Toki B.E. Torgov M.Y. Mendelsohn B.A. Cerveny C.G. Chace D.F. DeBlanc R.L. Gearing R.P. Bovee T.D. Siegall C.B. Francisco J.A. Wahl A.F. Meyer D.L. Senter P.D. Nat. Biotechnol. 2003; 21: 778-784Crossref PubMed Scopus (892) Google Scholar). At appointed times, the cells were harvested and assessed for surface-bound and intracellular levels of mAb and ADCs by flow cytometry (Fig. 2). Cell surface levels of both mAb and ADCs decreased sharply with time, coincident with increased intracellular levels (Fig. 2), suggesting that they internalized with similar kinetics. By 20 h, the surface levels of mAb and ADCs were ∼60% of the initial levels (Fig. 2A). Intracellular levels rose quickly within the first hour, peaked between 2 and 5 h, and maintained lower but steady levels up to 48 h (Fig. 2B). Cross-linking of the cAC10 antibody and ADCs increased intracellular levels of both the mAb and the conjugates by ∼3-fold over levels observed in the absence of cross-linking (Fig. 2B). Immunofluorescence microscopy was used to localize the internalized mAb and ADCs within L540cy cells (Fig. 3). The cells were incubated with the mAb or ADCs either on ice or at 37 °C for the stated times and imaged immediately or fixed, permeabilized, and then processed for immunofluorescence. The cells incubated on ice and stained for mAb or ADCs (Fig. 3, top panels, green signal) showed diffuse, cell surface-associated staining and no evidence of internalization. Lysosomes, visualized using an antibody to lysosome-associated membrane protein 1 (Lamp-1; Fig. 3, top panels, red signal) or with LysoTracker (bottom panels, red signal) were distinct and punctate. At 37 °C, there was capping and punctate staining for both mAb and ADCs within the L540cy cells and reduced staining on the cell surface. The intracellular mAb and ADC signals co-localized with those for Lamp-1 (Fig. 3, arrows, yellow signal, 16 h incubation) or with LysoTracker (Fig. 3, 3-h incubation, yellow signal, Merged, and data not shown), suggesting that both mAb and ADCs internalized and were transported to the l
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