Abstract
Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.
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References
Hale C, Bartholomew M, Taylor V, Stables J, Topley P, Tite J . Recognition of CD52 allelic gene products by CAMPATH-1H antibodies. Immunology 1996; 88: 183–190.
Hale G . The CD52 antigen and development of the CAMPATH antibodies. Cytotherapy 2001; 3 (3): 137–143.
Hale GXM, Tighe HP, Dyer MJ, Waldmann H . The CAMPATH-1 antigen (CDw52). Tissue Antigens 1990; 35: 118–127.
Ratzinger G, Reagan JL, Heller G, Busam KJ, Young JW . Differential CD52 expression by distinct myeloid dendritic cell subsets: implications for alemtuzumab activity at the level of antigen presentation in allogeneic graft-host interactions in transplantation. Blood 2003; 101: 1422–1429.
Buggins AG, Mufti GJ, Salisbury J, Codd J, Westwood N, Arno M et al. Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab. Blood 2002; 100: 1715–1720.
Hale G, Rye PD, Warford A, Lauder I, Brito-Babapulle A . The glycosylphosphatidylinositol-anchored lymphocyte antigen CDw52 is associated with the epididymal maturation of human spermatozoa. J Reprod Immunol 1993; 23: 189–205.
Ginaldi L, De Martinis M, Matutes E, Farahat N, Morilla R, Dyer MJ et al. Levels of expression of CD52 in normal and leukemic B and T cells: correlation with in vivo therapeutic responses to Campath-1H. Leuk Res 1998; 22: 185–191.
Hale G, Dyer MJ, Clark MR, Phillips JM, Marcus R, Riechmann L et al. Remission induction in non-Hodgkin lymphoma with reshaped human monoclonal antibody CAMPATH-1H. Lancet 1988; 2 (8625): 1394–1399.
Pawson R, Dyer MJ, Barge R, Matutes E, Thornton PD, Emmett E et al. Treatment of T-cell prolymphocytic leukemia with human CD52 antibody. J Clin Oncol 1997; 15: 2667–2672.
Osterborg A, Dyer MJ, Bunjes D, Pangalis GA, Bastion Y, Catovsky D et al. Phase II multicenter study of human CD52 antibody in previously treated chronic lymphocytic leukemia* European Study Group of CAMPATH-1H Treatment in Chronic Lymphocytic Leukemia. J Clin Oncol 1997; 15: 1567–1574.
Keating MJ, Flinn I, Jain V, Binet JL, Hillmen P, Byrd J et al. Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood 2002; 99: 3554–3561.
Crowe JS, Hall VS, Smith MA, Cooper HJ, Tite JP . Humanized monoclonal antibody CAMPATH-1H: myeloma cell expression of genomic constructs, nucleotide sequence of cDNA constructs and comparison of effector mechanisms of myeloma and Chinese hamster ovary cell-derived material. Clin Exp Immunol 1992; 87: 105–110.
Hale G, Bright S, Chumbley G, Hoang T, Metcalf D, Munro AJ et al. Removal of T cells from bone marrow for transplantation: a monoclonal antilymphocyte antibody that fixes human complement. Blood 1983; 62: 873–882.
Xia MQ, Hale G, Waldmann H . Efficient complement-mediated lysis of cells containing the CAMPATH-1 (CDw52) antigen. Mol Immunol 1993; 30: 1089–1096.
Hale G, Clark M, Waldmann H . Therapeutic potential of rat monoclonal antibodies: isotype specificity of antibody-dependent cell-mediated cytotoxicity with human lymphocytes. J Immunol 1985; 134: 3056–3061.
Rowan WC, Hale G, Tite JP, Brett SJ . Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes. Int Immunol 1995; 7: 69–77.
Rowan W, Tite J, Topley P, Brett SJ . Cross-linking of the CAMPATH-1 antigen (CD52) mediates growth inhibition in human B- and T-lymphoma cell lines, and subsequent emergence of CD52-deficient cells. Immunology 1998; 95: 427–436.
Zent CS, Chen JB, Kurten RC, Kaushal GP, Marie Lacy H, Schichman SA . Alemtuzumab (CAMPATH 1H) does not kill chronic lymphocytic leukemia cells in serum free medium. Leuk Res 2004; 28: 495–507.
Grdisa M . Sensitivity of B-Cell Chronic Lymphocytic Leukemia to Rituximab and Campath-1H and Correlation with the Expression of Cell Cycle Regulatory Proteins. Croat Med J 2004; 45: 136–141.
Shan D, Ledbetter JA, Press OW . Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 1998; 91: 1644–1652.
Hofmeister JK, Cooney D, Coggeshall KM . Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis. Blood Cells Mol Dis 2000; 26: 133–143.
Pedersen IM, Buhl AM, Klausen P, Geisler CH, Jurlander J . The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism. Blood 2002; 99: 1314–1319.
Byrd JC, Kitada S, Flinn IW, Aron JL, Pearson M, Lucas D et al. The mechanism of tumor cell clearance by rituximab in vivo in patients with B-cell chronic lymphocytic leukemia: evidence of caspase activation and apoptosis induction. Blood 2002; 99: 1038–1043.
Mateo V, Lagneaux L, Bron D, Biron G, Armant M, Delespesse G et al. CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia. Nat Med 1999; 5: 1277–1284.
Nagy ZA, Hubner B, Lohning C, Rauchenberger R, Reiffert S, Thomassen-Wolf E, Zahn S et al. Fully human, HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells. Nat Med 2002; 8: 801–807.
Mone AP, Huang P, Pelicano H, Cheney CM, Green JM, Tso JY . Hu1D10 induces apoptosis concurrent with activation of the AKT survival pathway in human chronic lymphocytic leukemia cells. Blood 2004; 103: 1846–1854.
Suzuki T, Kiyokawa N, Taguchi T, Sekino T, Katagiri YU, Fujimoto J . CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system. J Immunol 2001; 166: 5567–5577.
Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O'Brien S et al. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 1996; 87: 4990–4997.
Weitkamp JH, Crowe Jr JE . Blood donor leukocyte reduction filters as a source of human B lymphocytes. Biotechniques 2001; 31: 464–466.
Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH . Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84: 1415–1420.
Byrd JC, Lucas DM, Mone A, Kitner JB, Drabick JJ, Grever MR . KRN5500: a novel therapeutic agent with in vitro activity against human B-cell chronic lymphocytic leukemia cells mediates cytotoxicity via the intrinsic pathway of apoptosis. Blood 2003; 101: 4547–4550.
Byrd JC, Shinn C, Waselenko JK, Fuchs EJ, Lehman TA, Nguyen PL et al. Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functional p53. Blood 1998; 92: 3804–3816.
Goebel J, Forrest K, Flynn D, Rao R, Roszman TL . Lipid rafts, major histocompatibility complex molecules, and immune regulation. Hum Immunol 2002; 63: 813–820.
Cinek T, Horejsi V . The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases. J Immunol 1992; 149: 2262–2270.
Harder T, Simons K . Clusters of glycolipid and glycosylphosphatidylinositol-anchored proteins in lymphoid cells: accumulation of actin regulated by local tyrosine phosphorylation. Eur J Immunol 1999; 29: 556–562.
Fra AM, Williamson E, Simons K, Parton RG . Detergent-insoluble glycolipid microdomains in lymphocytes in the absence of caveolae. J Biol Chem 1994; 269: 30745–30748.
Harder T, Scheiffele P, Verkade P, Simons K . Lipid domain structure of the plasma membrane revealed by patching of membrane components. J Cell Biol 1998; 141: 929–942.
Fishman PH . Role of membrane gangliosides in the binding and action of bacterial toxins. J Membr Biol 1982; 69: 85–97.
Cheng PC, Dykstra ML, Mitchell RN, Pierce SK . A role for lipid rafts in B cell antigen receptor signaling and antigen targeting. J Exp Med 1999; 190: 1549–1560.
Janes PW, Ley SC, Magee AI . Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J Cell Biol 1999; 147: 447–461.
Edmonds SD, Ostergaard HL . Dynamic association of CD45 with detergent-insoluble microdomains in T lymphocytes. J Immunol 2002; 169: 5036–5042.
Aman MJ, Tosello-Trampont AC, Ravichandran K . Fc gamma RIIB1/SHIP-mediated inhibitory signaling in B cells involves lipid rafts. J Biol Chem 2001; 276: 46371–46378.
Edidin M . The state of lipid rafts: from model membranes to cells. Annu Rev Biophys Biomol Struct 2003; 32: 257–283.
Munro S . Lipid rafts: elusive or illusive? Cell 2003; 115: 377–388.
Simons K, Ikonen E . Functional rafts in cell membranes. Nature 1997; 387 (6633): 569–572.
Brown DA, London E . Functions of lipid Rafts in Biological Membranes. Annual Review of Cell Developemental Biology 1998; 14: 111–136.
Spiegel S, Kassis S, Wilchek M, Fishman PH . Direct visualization of redistribution and capping of fluorescent gangliosides on lymphocytes. J Cell Biol 1984; 99: 1575–1581.
Kellie S, Patel B, Pierce EJ, Critchley DR . Capping of cholera toxin-ganglioside GM1 complexes on mouse lymphocytes is accompanied by co-capping of alpha-actinin. J Cell Biol 1983; 97: 447–454.
Godal T, Henriksen A, Iversen JG, Landaas TO, Lindmo T . Altered membrane-associated functions in chronic lymphocytic leukemia cells. Int J Cancer 1978; 21: 561–569.
Keller P, Simons K . Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol 1998; 140: 1357–1367.
Kilsdonk EP, Yancey PG, Stoudt GW, Bangerter FW, Johnson WJ, Phillips MC et al. Cellular cholesterol efflux mediated by cyclodextrins. J Biol Chem 1995; 270: 17250–17256.
Truman JP, Ericson ML, Choqueux-Seebold CJ, Charron DJ, Mooney NA . Lymphocyte programmed cell death is mediated via HLA class II DR. Int Immunol 1994; 6: 887–896.
Mateo V, Brown EJ, Biron G, Rubio M, Fischer A, Deist FL et al. Mechanisms of CD47-induced caspase-independent cell death in normal and leukemic cells: link between phosphatidylserine exposure and cytoskeleton organization. Blood 2002; 100: 2882–2890.
Deans JP, Li H, Polyak MJ . CD20-mediated apoptosis: signalling through lipid rafts. Immunology 2002; 107: 176–182.
Garcia A, Cayla X, Fleischer A, Guergnon J, Alvarez-Franco Canas F, Rebollo MP et al. Rafts: a simple way to control apoptosis by subcellular redistribution. Biochimie 2003; 85: 727–731.
Semac I, Palomba C, Kulangara K, Klages N, van Echten-Deckert G, Borisch B et al. Anti-CD20 therapeutic antibody rituximab modifies the functional organization of rafts/microdomains of B lymphoma cells. Cancer Res 2003; 63: 534–540.
Li H, Ayer LM, Lytton J, Deans JP . Store-operated cation entry mediated by CD20 in membrane rafts. J Biol Chem 2003; 278: 42427–42434.
Craig MS, Morgan SM, Chan HT, Morgan BP, Filatov AV, Johnson PW et al. Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood 2003; 101: 1045–1052.
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This work was supported by the National Cancer Institute (P01 CA95426), The Leukemia and Lymphoma Society and The D Warren Brown Foundation.
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Mone, A., Cheney, C., Banks, A. et al. Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism. Leukemia 20, 272–279 (2006). https://doi.org/10.1038/sj.leu.2404014
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DOI: https://doi.org/10.1038/sj.leu.2404014
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