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  • Molecular Targets For Therapy (MTT)
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Molecular Targets for Therapy (MTT)

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine

Leukemia volume 18, pages 1215–1222 (2004)Cite this article

Abstract

Immunotoxins are chimeric proteins consisting of a toxin coupled to an antibody. To date, several clinical trials have been conducted, and some are still ongoing, to evaluate their anti-tumor efficacy. In this view, we chemically constructed an anti-CD20 immunotoxin with the mAb Rituximab and the type 1 ribosome-inactivating protein (RIP) saporin-S6, designed for B cells non-Hodgkin's lymphoma (NHL) therapy. This immunotoxin showed a specific cytotoxicity for the CD20+ cell lines Raji and D430B, evidenced by inhibition of protein synthesis, evaluation of apoptosis and clonogenic assay. Upon conjugation, saporin-S6 increased its toxicity on target cells by at least 2 logs, with IC50 values of 0.1–0.3 nM. The percentage of AnnexinV+ cells was over 95% in both cell lines treated with 10 nM immunotoxin. A complete elimination of Raji clones was reached with the 10 nM immunotoxin, whereas a mixture of free RIP and mAb gave about 90% of clonogenic growth. Rituximab/saporin-S6, at 10 nM concentration, also induced apoptosis in 80% of lymphoma cells from NHL patients. Moreover, sensitivity of Raji to Rituximab/saporin-S6 was augmented when cells were coincubated with Fludarabine. The synergistic toxic effect of the two drugs led to a total elimination of the neoplastic population.

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References

  1. Anderson KC, Bates MP, Slaughenhoupt BL, Pinkus GS, Schlossman SF, Nadler LM . Expression of human B cell-associated antigens on leukemias and lymphomas: a model of human B cell differentiation. Blood 1984; 63: 1424–1433.

    CAS  PubMed  Google Scholar 

  2. Maloney DG . Mechanism of action of rituximab. Anticancer Drugs 2001; 12 (Suppl. 2): S1–S4.

    CAS  PubMed  Google Scholar 

  3. McLaughlin P, Grillo-Lopez AJ, Link BK, Levy R, Czuczman MS, Williams ME et al. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J Clin Oncol 1998; 16: 2825–2833.

    Article  CAS  PubMed  Google Scholar 

  4. Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002; 346: 235–242.

    Article  CAS  PubMed  Google Scholar 

  5. Ma D, McDevitt MR, Barendswaard E, Lai L, Curcio MJ, Pellegrini V et al. Radioimmunotherapy for model B cell malignancies using 90Y-labeled anti-CD19 and anti-CD20 monoclonal antibodies. Leukemia 2002; 16: 60–66.

    Article  CAS  PubMed  Google Scholar 

  6. Goldenberg DM . The role of radiolabeled antibodies in the treatment of non-Hodgkin's lymphoma: the coming of age of radioimmunotherapy. Crit Rev Oncol Hematol 2001; 39: 195–201.

    Article  CAS  PubMed  Google Scholar 

  7. Kaminski MS, Estes J, Zasadny KR, Francis IR, Ross CW, Tuck M et al. Radioimmunotherapy with Iodine 131 Itositumomab for relapsed or refractory B-cell non-Hodgkin's lymphoma: updated results and long-term follow-up of the University of Michigan experience. Blood 2000; 96: 1259–1266.

    CAS  PubMed  Google Scholar 

  8. Witzig TE, Gordon LI, Cabanillas F, Czuczman MS, Emmanouilides C, Joyce R et al. Randomized controlled trial of yttrium-90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin's lymphoma. J Clin Oncol 2002; 20: 2453–2462.

    Article  CAS  PubMed  Google Scholar 

  9. Cheson BD . Radioimmunotherapy of non-Hodgkin lymphomas. Blood 2003; 101: 391–398.

    Article  CAS  PubMed  Google Scholar 

  10. Weiden PL, Breitz HB, Press O, Appelbaum JW, Bryan JK, Gaffigan S et al. Pretargeted radioimmunotherapy (PRIT) for treatment of non-Hodgkin's lymphoma (NHL): initial phase I/II study results. Cancer Biother Radiopharm 2000; 15: 15–29.

    Article  CAS  PubMed  Google Scholar 

  11. Pastan I, Kreitman RJ . Immunotoxins for targeted cancer therapy. Adv Drug Deliv Rev 1998; 31: 53–88.

    Article  CAS  PubMed  Google Scholar 

  12. Barbieri L, Battelli MG, Stirpe F . Ribosome-inactivating proteins from plants. Biochim Biophys Acta 1993; 1154: 237–282.

    Article  CAS  PubMed  Google Scholar 

  13. Peumans WJ, Hao Q, Van Damme EJ . Ribosome-inactivating proteins from plants: more than RNA N-glycosidases? FASEB J 2001; 15: 1493–1506.

    Article  CAS  PubMed  Google Scholar 

  14. Barbieri L, Valbonesi P, Bonora E, Gorini P, Bolognesi A, Stirpe F . Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A). Nucleic Acids Res 1997; 25: 518–522.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bolognesi A, Polito L, Lubelli C, Barbieri L, Parente A, Stirpe F . Ribosome-inactivating and adenine polynucleotide glycosylase activity in Mirabilis jalapa L. tissues. J Biol Chem 2002; 277: 13709–13716.

    Article  CAS  PubMed  Google Scholar 

  16. Frankel AE, Kreitman RJ, Sausville EA . Targeted toxins. Clin Cancer Res 2000; 6: 326–334.

    CAS  PubMed  Google Scholar 

  17. Thrush GR, Lark LR, Clinchy BC, Vitetta ES . Immunotoxins: an update. Annu Rev Immunol 1996; 14: 49–71.

    Article  CAS  PubMed  Google Scholar 

  18. Barbieri L, Stoppa C, Bolognesi A . Large-scale chromatographic purification of ribosome-inactivating proteins. J Chromatogr 1987; 408: 235–243.

    Article  CAS  Google Scholar 

  19. Bolognesi A, Polito L, Tazzari PL, Lemoli RM, Lubelli C, Fogli M et al. In vitro anti-tumor activity of anti-CD80 and anti-CD86 immunotoxins containing type 1 ribosome-inactivating proteins. Br J Haematol 2000; 110: 351–361.

    Article  CAS  PubMed  Google Scholar 

  20. Tazzari PL, de Totero D, Bolognesi A, Testoni N, Pileri S, Roncella S et al. An Epstein–Barr virus-infected lymphoblastoid cell line (D430B) that grows in SCID-mice with the morphologic features of a CD30+ anaplastic large cell lymphoma, and is sensitive to anti-CD30 immunotoxins. Haematologica 1999; 84: 988–995.

    CAS  PubMed  Google Scholar 

  21. Allen EH, Schweet RS . Synthesis of hemoglobin in a cell free system. Properties of the complete system. J Biol Chem 1962; 237: 760–767.

    CAS  PubMed  Google Scholar 

  22. Johnson SA . Nucleoside analogues in the treatment of haematological malignancies. Expert Opin Pharmacother 2001; 2: 929–943.

    Article  CAS  PubMed  Google Scholar 

  23. Tazzari PL, Polito L, Bolognesi A, Pistillo MP, Capanni P, Palmisano GL et al. Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model. J Immunol 2001; 167: 4222–4229.

    Article  CAS  PubMed  Google Scholar 

  24. Bolognesi A, Tazzari PL, Olivieri F, Polito L, Falini B, Stirpe F . Induction of apoptosis by ribosome-inactivating proteins and related immuntoxins. Int J Cancer 1996; 68: 349–355.

    Article  CAS  PubMed  Google Scholar 

  25. Carter P . Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001; 1: 118–129.

    Article  CAS  PubMed  Google Scholar 

  26. Boye J, Elter T, Engert A . An overview of the current clinical use of the anti-CD20 monoclonal antibody rituximab. Ann Oncol 2003; 14: 520–535.

    Article  CAS  PubMed  Google Scholar 

  27. Wu M . Are immunoconjugates useful for therapy with autoimmune diseases? Int J Immunopharmacol 1997; 19: 83–93.

    Article  CAS  PubMed  Google Scholar 

  28. Pastan I I, Kreitman RJ . Immunotoxins for targeted cancer therapy. Adv Drug Deliv Rev 1998; 31: 53–88.

    Article  PubMed  Google Scholar 

  29. Frankel AE, Kreitman RJ, Sausville EA . Targeted toxins. Clin Cancer Res 2000; 6: 326–334.

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  32. Bellosillo B, Villamor N, Lopez-Guillermo A, Marce S, Esteve J, Campo E et al. 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 2001; 98: 2771–2777.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  34. Countouriotis A, Moore TB, Sakamoto KM . Cell surface antigen and molecular targeting in the treatment of hematologic malignancies. Stem Cells 2002; 20: 215–229.

    Article  CAS  PubMed  Google Scholar 

  35. Tazzari PL, Barbieri L, Gobbi M, Dinota A, Rizzi S, Bontadini A et al. An immunotoxin containing a rat IgM monoclonal antibody (Campath 1) and saporin 6: effect on T lymphocytes and hemopoietic cells. Cancer Immunol Immunother 1988; 26: 231–236.

    Article  CAS  PubMed  Google Scholar 

  36. Bolognesi A, Tazzari PL, Tassi C, Gromo G, Gobbi M, Stirpe F . A comparison of anti-lymphocyte immunotoxins containing different ribosome-inactivating proteins and antibodies. Clin Exp Immunol 1992; 89: 341–346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dinota A, Tazzari PL, Michieli M, Visani G, Gobbi M, Bontadini A et al. In vitro bone marrow purging of multidrug-resistant cells with a mouse monoclonal antibody directed against Mr 170,000 glycoprotein and a saporin-conjugated anti-mouse antibody. Cancer Res 1990; 50: 4291–4294.

    CAS  PubMed  Google Scholar 

  38. Lazzarino M, Orlandi E, Montillo M, Tedeschi A, Pagnucco G, Astori C et al. Fludarabine, cyclophosphamide, and dexamethasone (FluCyD) combination is effective in pretreated low-grade non-Hodgkin's lymphoma. Ann Oncol 1999; 10: 59–64.

    Article  CAS  PubMed  Google Scholar 

  39. Zinzani PL, Magagnoli M, Moretti L, De Renzo A, Battista R, Zaccaria A et al. Randomized trial of fludarabine versus fludarabine and idarubicin as frontline treatment in patients with indolent or mantle-cell lymphoma. J Clin Oncol 2000; 18: 773–779.

    Article  CAS  PubMed  Google Scholar 

  40. Zinzani PL, Buzzi M, Farabegoli P, Tosi P, Fortuna A, Visani G et al. Induction of ‘in vitro’ apoptosis by Fludarabine in freshly isolated B-chronic lymphocytic leukemia cells. Leuk Lymphoma 1994; 13: 95–97.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was supported by the University of Bologna, funds for selected research topics, from the Ministero della Salute, by the Pallotti's Legacy for Cancer Research, by the Fondazione Cassa di Risparmio in Bologna and by Progetto Strategico Oncologia n.74 (DD 19Ric, 09/01/02) from Ministero Istruzione Università e Ricerca.

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Authors and Affiliations

  1. Dipartimento di Patologia Sperimentale, Università di Bologna, Bologna, Italy

    L Polito, A Bolognesi, V Farini, C Lubelli & F Stirpe

  2. Servizio di Immunoematologia e Trasfusionale, Policlinico ‘S Orsola’, Bologna, Italy

    P L Tazzari & F Ricci

  3. Istituto di Ematologia e Oncologia medica ‘L & A Seràgnoli’, Università di Bologna, Bologna, Italy

    P L Zinzani

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  1. L Polito
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  2. A Bolognesi
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Correspondence to A Bolognesi.

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Polito, L., Bolognesi, A., Tazzari, P. et al. The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine. Leukemia 18, 1215–1222 (2004). https://doi.org/10.1038/sj.leu.2403378

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