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Rituximab and alemtuzumab induce a nonclassic, caspase-independent apoptotic pathway in B-lymphoid cell lines and in chronic lymphocytic leukemia cells

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Abstract

The monoclonal antibodies (MoAbs) alemtuzumab (anti-CD52) and rituximab (anti-CD20) produce objective clinical responses in patients with chronic lymphocytic leukemia (CLL). However, their mechanisms of action are not fully understood. Therefore, we investigated the mechanisms of lymphoma and CLL cell killing by two anti-CD20 antibodies (rituximab, B1) and by alemtuzumab. All antibodies induced complement-independent cell death in B-lymphoid cell lines Raji, Ramos, and Mec-1. The efficiency of cell killing was increased by the addition of human complement in Raji but not Ramos cells. Both alemtuzumab and rituximab also killed freshly isolated CLL cells, with a much stronger response for alemtuzumab (from eight of eight patients) compared to rituximab (from two of six patients). Cell morphology and Western blot analyses revealed that the antibody-induced cell death lacked some typical features of apoptosis such as chromatin condensation or poly-ADP-ribose polymerase (PARP) cleavage. Taken together, the results suggest that the tumor killing activity of these MoAbs is not only mediated by complement-mediated cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC), but also by a nonclassic, caspase-independent apoptotic pathway.

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References

  1. Byrd JC, Murphy T, Howard RS, Lucas MS, Goodrich A, Park K, Pearson M, Waselenko JK, Ling G, Grever MR, Grillo-Lopez AJ, Rosenberg J, Kunkel L, Flinn IW (2001) Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity. J Clin Oncol 19:2153–2164

    CAS  PubMed  Google Scholar 

  2. Huhn D, von Schilling C, Wilhelm M, Ho AD, Hallek M, Kuse R, Knauf W, Riedel U, Hinke A, Srock S, Serke S, Peschel C, Emmerich B (2001) Rituximab therapy of patients with B-cell chronic lymphocytic leukemia. Blood 98:1326–1331

    Article  CAS  PubMed  Google Scholar 

  3. O’Brien SM, Kantarjian H, Thomas DA, Giles FJ, Freireich EJ, Cortes J, Lerner S, Keating MJ (2001) Rituximab dose-escalation trial in chronic lymphocytic leukemia. J Clin Oncol 19:2165–2170

    CAS  PubMed  Google Scholar 

  4. Byrd JC, Kitada S, Flinn IW, Aron JL, Pearson M, Lucas D, Reed JC (2002) 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 99:1038–1043

    Article  CAS  PubMed  Google Scholar 

  5. Schulz H, Klein SK, Rehwald U, Reiser M, Hinke A, Knauf WU, Aulitzky WE, Hensel M, Herold M, Huhn D, Hallek M, Diehl V, Engert A (2002) Phase 2 study of a combined immunochemotherapy using rituximab and fludarabine in patients with chronic lymphocytic leukemia. Blood 100:3115–3120

    Article  CAS  PubMed  Google Scholar 

  6. Keating M, Manshouri T, O’Brien S, Wierda W, Kantarjian H, Washington L, Lerner S, Albitar M (2002) A high proportion of molecular remission can be obtained with a fludarabine, cyclophosphamide, rituximab combination (FCR) in chronic lymphocytic leukemia (CLL). Blood 100:205a

    Google Scholar 

  7. Keating MJ, Flinn I, Jain V, Binet JL, Hillmen P, Byrd J, Albitar M, Brettman L, Santabarbara P, Wacker B, Rai KR (2002) Therapeutic role of alemtuzumab (Campath-1H) in patients who have failed fludarabine: results of a large international study. Blood 99:3554–3561

    Article  CAS  PubMed  Google Scholar 

  8. Rai KR, Freter CE, Mercier RJ, Cooper MR, Mitchell BS, Stadtmauer EA, Santabarbara P, Wacker B, Brettman L (2002) Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine. J Clin Oncol 20:3891–3897

    Article  CAS  PubMed  Google Scholar 

  9. Schulz H, Winkler U, Staak JO, Engert A (2000) The monoclonal antibodies Campath-1h and rituximab in the therapy of chronic lymphocytic leukemia. Onkologie 23:526–532

    Article  PubMed  Google Scholar 

  10. Lundin J, Kimby E, Bjorkholm M, Broliden PA, Celsing F, Hjalmar V, Mollgard L, Rebello P, Hale G, Waldmann H, Mellstedt H, Osterborg A (2002) Phase II trial of subcutaneous anti-CD52 monoclonal antibody alemtuzumab (Campath-1H) as first-line treatment for patients with B-cell chronic lymphocytic leukemia (B-CLL). Blood 100:768–773

    Article  CAS  PubMed  Google Scholar 

  11. Stashenko P, Nadler LM, Hardy R, Schlossman SF (1980) Characterization of a human B lymphocyte-specific antigen. J Immunol 125:1678–1685

    CAS  PubMed  Google Scholar 

  12. Riley JK, Sliwkowski MX (2000) CD20: a gene in search of a function. Semin Oncol 27:17–24

    CAS  Google Scholar 

  13. Tedder TF, Engel P (1994) CD20: a regulator of cell-cycle progression of B lymphocytes. Immunol Today 15:450–454

    Article  CAS  PubMed  Google Scholar 

  14. O’Keefe TL, Williams GT, Davies SL, Neuberger MS (1998) Mice carrying a CD20 gene disruption. Immunogenetics 48:125–132

    Article  CAS  PubMed  Google Scholar 

  15. Xia MQ, Hale G, Lifely MR, Ferguson MA, Campbell D, Packman L, Waldmann H (1993) Structure of the CAMPATH-1 antigen, a glycosylphosphatidylinositol-anchored glycoprotein which is an exceptionally good target for complement lysis. Biochem J 293:633–640

    CAS  PubMed  Google Scholar 

  16. Hale G, Xia MQ, Tighe HP, Dyer MJ, Waldmann H (1990) The CAMPATH-1 antigen (CDw52). Tissue Antigens 35:118–127

    CAS  PubMed  Google Scholar 

  17. Maloney DG, Smith B, Rose A (2002) Rituximab: mechanism of action and resistance. Semin Oncol 29:2–9

    Article  CAS  Google Scholar 

  18. Hofmeister JK, Cooney D, Coggeshall KM (2000) Clustered CD20 induced apoptosis: src-family kinase, the proximal regulator of tyrosine phosphorylation, calcium influx, and caspase 3-dependent apoptosis. Blood Cells Mol Dis 26:133–143

    Article  CAS  PubMed  Google Scholar 

  19. Shan D, Ledbetter JA, Press OW (2000) Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells. Cancer Immunol Immunother 48:673–683

    Article  CAS  PubMed  Google Scholar 

  20. Golay J, Zaffaroni L, Vaccari T, Lazzari M, Borleri GM, Bernasconi S, Tedesco F, Rambaldi A, Introna M (2000) Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 95:3900–3908

    CAS  PubMed  Google Scholar 

  21. Clynes RA, Towers TL, Presta LG, Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 6:443–446

    Article  CAS  PubMed  Google Scholar 

  22. Crowe JS, Hall VS, Smith MA, Cooper HJ, Tite JP (1992) 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 87:105–110

    CAS  PubMed  Google Scholar 

  23. Rowan W, Tite J, Topley P, Brett SJ (1998) 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 95:427–436

    Article  CAS  PubMed  Google Scholar 

  24. Cioca DP, Kitano K (2002) Apoptosis induction by hypercross-linking of surface antigen CD5 with anti-CD5 monoclonal antibodies in B cell chronic lymphocytic leukemia. Leukemia 16:335–343

    Article  CAS  PubMed  Google Scholar 

  25. Nuessler V, Stotzer O, Gullis E, Pelka-Fleischer R, Pogrebniak A, Gieseler F, Wilmanns W (1999) Bcl-2, bax and bcl-xL expression in human sensitive and resistant leukemia cell lines. Leukemia 13:1864–1872

    Article  CAS  PubMed  Google Scholar 

  26. Shan D, Ledbetter JA, Press OW (1998) Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 91:1644–1652

    CAS  PubMed  Google Scholar 

  27. Kaufmann SH (1998) Cell death induced by topoisomerase-targeted drugs: more questions than answers. Biochim Biophys Acta 1400:195–211

    Article  CAS  PubMed  Google Scholar 

  28. Mathas S, Rickers A, Bommert K, Dorken B, Mapara MY (2000) Anti-CD20- and B-cell receptor-mediated apoptosis: evidence for shared intracellular signaling pathways. Cancer Res 60:7170–7176

    CAS  PubMed  Google Scholar 

  29. Cross TG, Scheel-Toellner D, Henriquez NV, Deacon E, Salmon M, Lord JM (2000) Serine/threonine protein kinases and apoptosis. Exp Cell Res 256:34–41

    Article  CAS  PubMed  Google Scholar 

  30. Mateo V, Lagneaux L, Bron D, Biron G, Armant M, Delespesse G, Sarfati M (1999) CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia. Nat Med 5:1277–1284

    Article  CAS  PubMed  Google Scholar 

  31. Drenou B, Blancheteau V, Burgess DH, Fauchet R, Charron DJ, Mooney NA (1999) A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes. J Immunol 163:4115–4124

    CAS  PubMed  Google Scholar 

  32. Rossmann ED, Lundin J, Lenkei R, Mellstedt H, Osterborg A (2001) Variability in B-cell antigen expression: implications for the treatment of B-cell lymphomas and leukemias with monoclonal antibodies. Hematol J 2:300–306

    Article  CAS  PubMed  Google Scholar 

  33. Cardarelli PM, Quinn M, Buckman D, Fang Y, Colcher D, King DJ, Bebbington C, Yarranton G (2002) Binding to CD20 by anti-B1 antibody or F(ab′)(2) is sufficient for induction of apoptosis in B-cell lines. Cancer Immunol Immunother 51:15–24

    Article  CAS  PubMed  Google Scholar 

  34. Chan HT, Hughes D, French RR, Tutt AL, Walshe CA, Teeling JL, Glennie MJ, Cragg MS (2003) CD20-induced lymphoma cell death is independent of both caspases and its redistribution into triton X-100 insoluble membrane rafts. Cancer Res 63:5480–5489

    CAS  PubMed  Google Scholar 

  35. Treon SP, Mitsiades C, Mitsiades N, Young G, Doss D, Schlossman R, Anderson KC (2001) Tumor cell expression of CD59 is associated with resistance to CD20 serotherapy in patients with B-cell malignancies. J Immunother 24:263–271

    Article  CAS  Google Scholar 

  36. Golay J, Lazzari M, Facchinetti V, Bernasconi S, Borleri G, Barbui T, Rambaldi A, Introna M (2001) CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59. Blood 98:3383–3389

    Article  CAS  PubMed  Google Scholar 

  37. Weng WK, Levy R (2001) Expression of complement inhibitors CD46, CD55, and CD59 on tumor cells does not predict clinical outcome after rituximab treatment in follicular non-Hodgkin lymphoma. Blood 98:1352–1357

    Article  CAS  PubMed  Google Scholar 

  38. Bannerji R, Kitada S, Flinn IW, Pearson M, Young G, Reed JC, Byrd JC (2003) Apoptotic-regulatory and complement-protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo Rituximab resistance. J Clin Oncol 21:1466–1471

    Article  PubMed  Google Scholar 

  39. Anderson DR, Grillo-Lopez A, Vams C, Chambers KS, Hanna N (1997) Targeted anti-cancer therapy using rituximab, a chimeric anti-CD20 antibody (IDEC-C2B8) in the treatment of non-Hodgkin’s B-cell lymphoma. Biochem Soc Trans 25:705–708

    CAS  PubMed  Google Scholar 

  40. van der Kolk LE, Grillo-Lopez AJ, Baars JW, Hack CE, van Oers MH (2001) Complement activation plays a key role in the side-effects of rituximab treatment. Br J Haematol 115:807–811

    Article  PubMed  Google Scholar 

  41. Cragg MS, Glennie MJ (2004) Antibody specificity controls in vivo effector mechanisms of anti-CD20. Blood 103:2738–2743

    Article  CAS  PubMed  Google Scholar 

  42. van der Kolk LE, Evers LM, Omene C, Lens SM, Lederman S, van Lier RA, van Oers MH, Eldering E (2002) CD20-induced B cell death can bypass mitochondria and caspase activation. Leukemia 16:1735–1744

    Article  PubMed  Google Scholar 

  43. Lesage S, Steff AM, Philippoussis F, Page M, Trop S, Mateo V, Hugo P (1997) CD4+CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway. J Immunol 159:4762–4771

    CAS  PubMed  Google Scholar 

  44. Bellosillo B, Villamor N, Lopez-Guillermo A, Marce S, Esteve J, Campo E, Colomer D, Montserrat E (2001) Complement-mediated cell death induced by rituximab in B-cell lymphoproliferative disorders is mediated in vitro by a caspase-independent mechanism involving the generation of reactive oxygen species. Blood 98:2771–2777

    Article  CAS  PubMed  Google Scholar 

  45. Stanglmaier M, Reis S, Hallek M (2000) Stimulation of programmed cell death (PCD) and tyrosine kinases by monoclonal antibodies against CD20 and CD52 in B-CLL cells and B-cell lines in vitro. Blood 96:160a

    Google Scholar 

  46. Pedersen IM, Buhl AM, Klausen P, Geisler CH, Jurlander J (2002) 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 99:1314–1319

    Article  CAS  PubMed  Google Scholar 

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Author notes

  1. Michael Stanglmaier

    Present address: Trion Research, Am Klopferspitz 19, 82152, Planegg-Martinsried, Germany

  2. Simone Reis

    Present address: Institut für Pathologie und Rechtsmedizin, Universitätsklinikum Ulm, Prittwitzstrasse. 6, 89075, Ulm, Germany

Authors and Affiliations

  1. Clinical Cooperation Group for Gene Therapy, GSF—National Institute of Health and Environment, Marchioninistr. 25, 81377, Munich, Germany

    Michael Stanglmaier, Simone Reis & Michael Hallek

  2. Klinikum Großhadern, Medical Clinic III, Marchioninistr. 15, 81377, Munich, Germany

    Michael Hallek

  3. Genzentrum, Universität München, Feodor-Lynen-Strasse 25, 81377, Munich, Germany

    Michael Hallek

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  1. Michael Stanglmaier
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Correspondence to Michael Stanglmaier.

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Stanglmaier, M., Reis, S. & Hallek, M. Rituximab and alemtuzumab induce a nonclassic, caspase-independent apoptotic pathway in B-lymphoid cell lines and in chronic lymphocytic leukemia cells. Ann Hematol 83, 634–645 (2004). https://doi.org/10.1007/s00277-004-0917-0

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  • DOI: https://doi.org/10.1007/s00277-004-0917-0

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