Research Article ADAM10 Inhibition of Human CD30 Shedding Increases Specificity of Targeted Immunotherapy In vitro Dennis A. Eichenauer,1 Vijaya Lakshmi Simhadri,1 Elke Pogge von Strandmann,1 Andreas Ludwig,3 Vance Matthews,4 Katrin S. Reiners,1 Bastian von Tresckow,1,2 Paul Saftig,3 Stefan Rose-John,2 Andreas Engert,1 and Hinrich P. Hansen1 1Department of Internal Medicine I and 2Center for Molecular Medicine, University Hospital Cologne, Cologne, Germany; 3Department of Biochemistry, University of Kiel, Kiel, Germany; and 4Ludwig Institute for Cancer Research, PO Royal Melbourne Hospital, Victoria, Melbourne, Australia Abstract Due to its selective expression, CD30 is generally well suited for CD30 is a transmembrane protein selectively overexpressed on targeted immunotherapy of CD30-positive lymphomas. Although many human lymphoma cells and therefore an interesting antibody-based reagents against CD30 showed impressive anti- target for antibody-based immunotherapy. However, binding tumor activity in experimental Hodgkin’s lymphoma models, early of therapeutic antibodies stimulates a juxtamembrane cleav- clinical trials suffered from dose-limiting toxicities (4). A problem age of CD30 leading to a loss of target antigen and an that appeared was the formation of complexes containing the enhanced release of the soluble ectodomain of CD30 (sCD30). therapeutic antibody and the soluble ectodomain of CD30 (sCD30). Here, we show that sCD30 binds to CD30 ligand (CD153)– sCD30 is generated by proteolytic cleavage of CD30 (5). The release expressing non-target cells. Because antibodies bind to sCD30, of extracellular domains, also referred to as ectodomain shedding, this results in unwanted antibody binding to these cells via is a general process regulating the function of membrane proteins sCD30 bridging. To overcome shedding-dependent damage of (6). In most cases, shedding is catalyzed by metalloproteinases. The normal cells in CD30-specific immunotherapy, we analyzed tumor necrosis factor-a (TNF-a)–converting enzyme, also known the mechanism involved in the release. Shedding of CD30 can as a disintegrin and metalloproteinase 17 (ADAM17), is the be enhanced by protein kinase C (PKC) activation, implicating metalloproteinase responsible for the ectodomain release of many the disintegrin metalloproteinase ADAM17 but not free membrane proteins, including CD30 (7, 8). A structurally related cytoplasmic calcium. However, antibody-induced CD30 shed- proteinase (ADAM10) is also implicated in certain shedding ding is calcium dependent and PKC independent. This processes, suchas thenon-amyloidogenic processing of Alz- shedding involved the related metalloproteinase ADAM10 as heimer’s amyloid precursor protein (APP; ref. 9) or the cleavage of shown by the use of the preferential ADAM10 inhibitor N-cadherin and E-cadherin (10, 11). GI254023X and by an ADAM10-deficient cell line generated Many studies distinguish between the mechanisms underlying À À from embryonically lethal ADAM10 / mouse. In coculture the constitutive shedding of unstimulated cells and the release from experiments, the antibody-induced transfer of sCD30 from stimulated cells. Classic shedding stimulation is initiated by the the human Hodgkin’s lymphoma cell line L540 to the CD30- protein kinase C (PKC) activator phorbol 12-myristate-13-acetate negative but CD153-expressing human mast cell line HMC-1 (PMA), and there is evidence for an implication of ADAM17 (12). In was inhibited by GI254023X. These findings suggest that some cases, including CD30, membrane protein shedding is also selective metalloproteinase inhibitors blocking antibody- stimulated by antibody binding (6, 13). As the release of soluble induced shedding of target antigens could be of therapeutic antigen from tumor cells has far-reaching implications for targeted value to increase the specificity and reduce side effects of immunotherapy, our group has investigated the use of metal- immunotherapy with monoclonal antibodies. [Cancer Res loproteinase inhibitors to block the release of the target antigen 2007;67(1):332–8] (14). In a mouse model, we showed that an anti-CD30 immunotoxin was only effective against human Hodgkin’s lymphoma tumors Introduction when concomitantly used with the broad-spectrum hydroxamic A common feature of Hodgkin’s lymphoma and large cell acid–based metalloproteinase inhibitor BB-3644 (15). Such nonse- anaplastic lymphoma is the strong surface expression of CD30, lective inhibitors block many different metalloproteinases, includ- a type I transmembrane receptor (1). Healthy donors express ing essential matrix and disintegrin metalloproteinases. They have CD30 exclusively on very few cells, predominantly on activated been used in clinical studies, and the broad specificity resulted in lymphocytes. In contrast, the cognate ligand (CD153), a membrane- more unspecific toxicity than antitumor effects (16). Thus, more anchored type II glycoprotein, is expressed on the cell surface of selective inhibitors are desirable for clinical use. many different cell types, including resting B cells, activated T cells, Here, we analyze the mechanism of antibody-stimulated release mast cells, monocytes, and granulocytes (2, 3). of sCD30. We found that antibody-induced CD30 shedding was not catalyzed by the previously described ADAM17 but instead by ADAM10. Moreover, we show that immunocompetitive sCD30 not only resided in a soluble form in the pericellular environment but Requests for reprints: HinrichHansen, Department of Internal Medicine I, also specifically bound to membrane-anchored CD30 ligand University Hospital Cologne, LFI, Ebene 4, Room 703, Kerpener Str. 62, 50924 Cologne, (CD153) on non-target cells where it generated novel targeting Germany. Phone: 49-221-478-5808; Fax: 49-221-478-3778; E-mail: [email protected]. I2007 American Association for Cancer Research. sites. Application of an ADAM10-selective metalloproteinase doi:10.1158/0008-5472.CAN-06-2470 inhibitor significantly reduced this mistargeting. Cancer Res 2007; 67: (1). January 1, 2007 332 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. ADAM10 Inhibition and Specificity of CD30 Targeting Materials and Methods Construction and expression of CD153-glutathione S-transferase fusion + À protein. The ectodomain of CD153 was amplified by PCR using cDNA from Cells and reagents. We used the CD30 /CD153 cell lines L540 DG75 cells and the primers 5¶-CGACAGGATCCCCGTTCAGAGGACG- (Hodgkin’s lymphoma) and Karpas 299 (large cell anaplastic lymphoma) ¶ ¶ À + À GACTCCATTCCCAAC-3 and 5 -CAAGAGAATTCGTTCAGTCTGAAT- and the CD30 /CD153 cell line DG75 (Burkitt lymphoma) and the CD30 / ¶ À TACTGTATAAGAAGATGGAC-3 .AfterBamHI and EcoRI restriction CD153 cell line Reh (acute lymphoblastic leukemia). In addition, we used the digest, the PCR product was cloned into the glutathione S-transferase mast cell line HMC-1, which was kindly provided by Dr. J. Butterfield (Mayo (GST) expression vector (pGEX-3X, Amersham Pharmacia Biotech, Clinic, Rochester, MN); the ADAM17-targeted mouse fibroblast cells, which Freiburg, Germany). BL21 cells (DE3) were transformed and induced for were received from Dr. R. Black (Amgen Incorporated, Seattle, WA); and 5 hby isopropyl- L-thio-h-D-galactopyranoside (0.5 mmol/L). The bacterial À/À the ADAM10 ( ) cells, which were a gift of Dr. D. Hartmann (Center for pellet was suspended in 20 mmol/L Tris-HCl (pH 8) containing 150 Human Genetics, Leuven, Belgium). The hydroxamate inhibitor BB-3644 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L h-mercaptoethanol, 1% Triton 1 [2S-(2,2-dimethyl-propyl)-N -[2,2-dimethyl-1-(pyridin-2S-ylcarbamoyl)- X-100, and complete protease inhibitor cocktail (Roche Diagnostics, 4 propyl]-N -hydroxy-3R-methoxy-succinamide] was from British Biotech Mannheim, Germany). Lysozyme (0.5 mg/mL) was added to the Pharmaceuticals Ltd. (Oxford, United Kingdom). GW280264 [(2R,3S)-3- suspension before sonication on ice (3Â, 15 s). After centrifugation for (formylhydroxyamino)-2-(2-methyl-1-propyl) hexanoic acid (1S)-5-(benzy- 40 min at 35,000 Â g and 4jC, the recombinant protein was extracted loxycarbamoyl-amino-1-(1,3-thiazol-2-ylcarbamoyl)-1-pentyl amide)] and from the cell lysate by incubation with glutathione-conjugated agarose GI254023X [(2R,3S)-3-( formyl-hydroxyamino)-2-(3-phenyl-1-propyl) buta- beads (GSH-agarose; Amersham Pharmacia Biotech). The beads were noic acid [(1S)-2,2-dimetyl-1-methylcarbamoyl-1-propyl] amide] were from washed with lysis buffer without Triton X-100 and stored in 50 mmol/L GlaxoSmithKline (Stevenage, United Kingdom). Recombinant ADAM10 was Tris-HCl (pH 8). from Merck Biosciences (Schwalbach, Germany), and ADAM17 was from Transfection. Cells were transfected as described previously (17). R&D Systems (Wiesbaden, Germany). Transfection with cDNA for enhanced green fluorescence protein (Clontech, Plasmids. Construction of recombinant CD30Fc. The CD30Fc fusion Palo Alto, CA) was used to monitor the transfection efficiency. protein containing the Fc portion of human IgG1 and the truncated Production and purification of sCD30. 293T cells were transfected ectodomain of human CD30 (deletion between 89Arg and 156Arg) was withCD30Fc or wild-type CD30 cDNA. After 48 hof incubation, CD30Fc or constructed by PCR using the CD30 cDNA clone (17), the HindIII forward sCD30 was purified from the supernatants using Protein A Sepharose CL-4B primer 5¶-GCGAGAAGCTTATGCGCGTCCTCCTCGCCGCG-3¶,andthe beads (Amersham Pharmacia Biotech)