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Immunology

Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice

Leukemia volume 29, pages 406–414 (2015)Cite this article

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Abstract

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4–25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3–4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.

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References

  1. Baker M . Upping the ante on antibodies. Nat Biotechnol 2005; 23: 1065–1072.

    Article  CAS  PubMed  Google Scholar 

  2. Cohen J, Wilson A . New challenges to medicare beneficiary access to mAbs. MAbs 2009; 1: 56–66.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Ledford H . 'Biosimilar' drugs poised to penetrate market. Nature 2010; 468: 18–19.

    Article  CAS  PubMed  Google Scholar 

  4. Adams GP, Weiner LM . Monoclonal antibody therapy of cancer. Nat Biotechnol 2005; 23: 1147–1157.

    Article  CAS  PubMed  Google Scholar 

  5. Hong CW, Zeng Q . Awaiting a new era of cancer immunotherapy. Cancer Res 2012; 72: 3715–3719.

    Article  CAS  PubMed  Google Scholar 

  6. 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.

    Article  CAS  PubMed  Google Scholar 

  7. Bannerji R, Kitada S, Flinn IW, Pearson M, Young D, Reed JC et al. Apoptotic-regulatory and complement-protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo rituximab resistance. J Clin Oncol 2003; 21: 1466–1471.

    Article  CAS  PubMed  Google Scholar 

  8. Hubert P, Amigorena S . Antibody-dependent cell cytotoxicity in monoclonal antibody-mediated tumor immunotherapy. Oncoimmunology 2012; 1: 103–105.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Nimmerjahn F, Ravetch JV . Fcgamma receptors: old friends and new family members. Immunity 2006; 24: 19–28.

    Article  CAS  PubMed  Google Scholar 

  10. Weiner LM, Surana R, Wang S . Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 2010; 10: 317–327.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Winiarska M, Glodkowska-Mrowka E, Bil J, Golab J . Molecular mechanisms of the antitumor effects of anti-CD20 antibodies. Front Biosci (Landmark Ed) 2011; 16: 277–306.

    Article  CAS  Google Scholar 

  12. Dunkelberger JR, Song WC . Complement and its role in innate and adaptive immune responses. Cell Res 2010; 20: 34–50.

    Article  CAS  PubMed  Google Scholar 

  13. Zipfel PF, Skerka C . Complement regulators and inhibitory proteins. Nat Rev Immunol 2009; 9: 729–740.

    Article  CAS  PubMed  Google Scholar 

  14. Macor P, Tedesco F . Complement as effector system in cancer immunotherapy. Immunol Lett 2007; 111: 6–13.

    Article  CAS  PubMed  Google Scholar 

  15. Maher J, Adami AA . Antitumor immunity: easy as 1, 2, 3 with monoclonal bispecific trifunctional antibodies? Cancer Res 2013; 73: 5613–5617.

    Article  CAS  PubMed  Google Scholar 

  16. Baeuerle PA, Reinhardt C . Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res 2009; 69: 4941–4944.

    Article  CAS  PubMed  Google Scholar 

  17. Brischwein K, Parr L, Pflanz S, Volkland J, Lumsden J, Klinger M et al. Strictly target cell-dependent activation of T cells by bispecific single-chain antibody constructs of the BiTE class. J Immunother 2007; 30: 798–807.

    Article  CAS  PubMed  Google Scholar 

  18. Haas C, Krinner E, Brischwein K, Hoffmann P, Lutterbuse R, Schlereth B et al. Mode of cytotoxic action of T cell-engaging BiTE antibody MT110. Immunobiology 2009; 214: 441–453.

    Article  CAS  PubMed  Google Scholar 

  19. Baeuerle PA, Kufer P, Bargou R . BiTE: Teaching antibodies to engage T-cells for cancer therapy. Curr Opin Mol Ther 2009; 11: 22–30.

    CAS  PubMed  Google Scholar 

  20. Klinger M, Brandl C, Zugmaier G, Hijazi Y, Bargou RC, Topp MS et al. Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell-engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood 2012; 119: 6226–6233.

    Article  CAS  PubMed  Google Scholar 

  21. Maetzel D, Denzel S, Mack B, Canis M, Went P, Benk M et al. Nuclear signalling by tumour-associated antigen EpCAM. Nat Cell Biol 2009; 11: 162–171.

    Article  CAS  PubMed  Google Scholar 

  22. Blok VT, Daha MR, Tijsma O, Harris CL, Morgan BP, Fleuren GJ et al. A bispecific monoclonal antibody directed against both the membrane-bound complement regulator CD55 and the renal tumor-associated antigen G250 enhances C3 deposition and tumor cell lysis by complement. J Immunol 1998; 160: 3437–3443.

    CAS  PubMed  Google Scholar 

  23. Gelderman KA, Blok VT, Fleuren GJ, Gorter A . The inhibitory effect of CD46, CD55, and CD59 on complement activation after immunotherapeutic treatment of cervical carcinoma cells with monoclonal antibodies or bispecific monoclonal antibodies. Lab Invest 2002; 82: 483–493.

    Article  CAS  PubMed  Google Scholar 

  24. Klepfish A, Gilles L, Ioannis K, Rachmilewitz EA, Schattner A . Enhancing the action of rituximab in chronic lymphocytic leukemia by adding fresh frozen plasma: complement/rituximab interactions & clinical results in refractory CLL. Ann NY Acad Sci 2009; 1173: 865–873.

    Article  CAS  PubMed  Google Scholar 

  25. Klepfish A, Rachmilewitz EA, Kotsianidis I, Patchenko P, Schattner A . Adding fresh frozen plasma to rituximab for the treatment of patients with refractory advanced CLL. QJM 2008; 101: 737–740.

    Article  CAS  PubMed  Google Scholar 

  26. Tedesco F, Bulla B, Fischetti F . Terminal complement complex: regulation of formation and pathophysiological function. In: Szebeni J ed The Complement System: Novel Rules in Health and Disease, pp 97–127 2004.

  27. Garred P, Hetland G, Mollnes TE, Stoervold G . Synthesis of C3, C5, C6, C7, C8, and C9 by human fibroblasts. Scand J Immunol 1990; 32: 555–560.

    Article  CAS  PubMed  Google Scholar 

  28. Langeggen H, Pausa M, Johnson E, Casarsa C, Tedesco F . The endothelium is an extrahepatic site of synthesis of the seventh component of the complement system. Clin Exp Immunol 2000; 121: 69–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Langeggen H, Berge KE, Macor P, Fischetti F, Tedesco F, Hetland G et al. Detection of mRNA for the terminal complement components C5, C6, C8 and C9 in human umbilical vein endothelial cells in vitro. APMIS 2001; 109: 73–78.

    Article  CAS  PubMed  Google Scholar 

  30. Gelderman KA, Tomlinson S, Ross GD, Gorter A . Complement function in mAb-mediated cancer immunotherapy. Trends Immunol 2004; 25: 158–164.

    Article  CAS  PubMed  Google Scholar 

  31. Fishelson Z, Donin N, Zell S, Schultz S, Kirschfink M . Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors. Mol Immunol 2003; 40: 109–123.

    Article  CAS  PubMed  Google Scholar 

  32. Ziller F, Macor P, Bulla R, Sblattero D, Marzari R, Tedesco F . Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59. Eur J Immunol 2005; 35: 2175–2183.

    Article  CAS  PubMed  Google Scholar 

  33. Macor P, Tripodo C, Zorzet S, Piovan E, Bossi F, Marzari R et al. In vivo targeting of human neutralizing antibodies against CD55 and CD59 to lymphoma cells increases the antitumor activity of rituximab. Cancer Res 2007; 67: 10556–10563.

    Article  CAS  PubMed  Google Scholar 

  34. Golay J, Manganini M, Facchinetti V, Gramigna R, Broady R, Borleri G et al. Rituximab-mediated antibody-dependent cellular cytotoxicity against neoplastic B cells is stimulated strongly by interleukin-2. Haematologica 2003; 88: 1002–1012.

    CAS  PubMed  Google Scholar 

  35. Golay J, Zaffaroni L, Vaccari T, Lazzari M, Borleri GM, Bernasconi S et al. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood 2000; 95: 3900–3908.

    CAS  PubMed  Google Scholar 

  36. Di Gaetano N, Cittera E, Nota R, Vecchi A, Grieco V, Scanziani E et al. Complement activation determines the therapeutic activity of rituximab in vivo. J Immunol 2003; 171: 1581–1587.

    Article  CAS  PubMed  Google Scholar 

  37. Di Niro R, Ziller F, Florian F, Crovella S, Stebel M, Bestagno M et al. Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models. BMC Biotechnol 2007; 7: 46.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Fang J, Qian JJ, Yi S, Harding TC, Tu GH, VanRoey M et al. Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol 2005; 23: 584–590.

    Article  CAS  PubMed  Google Scholar 

  39. Hanke T, Szawlowski P, Randall RE . Construction of solid matrix-antibody-antigen complexes containing simian immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen. J Gen Virol 1992; 73 (Pt 3): 653–660.

    Article  CAS  PubMed  Google Scholar 

  40. Boscolo S, Mion F, Licciulli M, Macor P, De Maso L, Brce M et al. Simple scale-up of recombinant antibody production using an UCOE containing vector. N Biotechnol 2012; 29: 477–484.

    Article  CAS  PubMed  Google Scholar 

  41. Macor P, Mezzanzanica D, Cossetti C, Alberti P, Figini M, Canevari S et al. Complement activated by chimeric anti-folate receptor antibodies is an efficient effector system to control ovarian carcinoma. Cancer Res 2006; 66: 3876–3883.

    Article  CAS  PubMed  Google Scholar 

  42. Mezzaroba N, Zorzet S, Secco E, Biffi S, Tripodo C, Calvaruso M et al. New potential therapeutic approach for the treatment of B-Cell malignancies using chlorambucil/hydroxychloroquine-loaded anti-CD20 nanoparticles. PLoS One 2013; 8: e74216.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Biffi S, Garrovo C, Macor P, Tripodo C, Zorzet S, Secco E et al. In vivo biodistribution and lifetime analysis of cy5.5-conjugated rituximab in mice bearing lymphoid tumor xenograft using time-domain near-infrared optical imaging. Mol Imaging 2008; 7: 272–282.

    Article  CAS  PubMed  Google Scholar 

  44. Biffi S, Dal Monego S, Dullin C, Garrovo C, Bosnjak B, Licha K et al. Dendritic polyglycerolsulfate near infrared fluorescent (NIRF) dye conjugate for non-invasively monitoring of inflammation in an allergic asthma mouse model. PLoS One 2013; 8: e57150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Ridgway JB, Presta LG, Carter P . 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 1996; 9: 617–621.

    Article  CAS  PubMed  Google Scholar 

  46. Reed CD, Rast H, Hu WG, Mah D, Nagata L, Masri SA . Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector. Protein Expr Purif 2007; 54: 261–266.

    Article  CAS  PubMed  Google Scholar 

  47. Funakoshi S, Longo DL, Murphy WJ . Differential in vitro and in vivo antitumor effects mediated by anti-CD40 and anti-CD20 monoclonal antibodies against human B-cell lymphomas. J Immunother Emphasis Tumor Immunol 1996; 19: 93–101.

    Article  CAS  PubMed  Google Scholar 

  48. Cragg 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.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  50. Gunn L, Ding C, Liu M, Ma Y, Qi C, Cai Y et al. Opposing roles for complement component C5a in tumor progression and the tumor microenvironment. J Immunol 2012; 189: 2985–2994.

    Article  CAS  PubMed  Google Scholar 

  51. Dobrina A, Pausa M, Fischetti F, Bulla R, Vecile E, Ferrero E et al. Cytolytically inactive terminal complement complex causes transendothelial migration of polymorphonuclear leukocytes in vitro and in vivo. Blood 2002; 99: 185–192.

    Article  CAS  PubMed  Google Scholar 

  52. Markiewski MM, DeAngelis RA, Benencia F, Ricklin-Lichtsteiner SK, Koutoulaki A, Gerard C et al. Modulation of the antitumor immune response by complement. Nat Immunol 2008; 9: 1225–1235.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was financially supported by Regione Friuli Venezia Giulia (Linfonet and AITT—to FT), the Italian Association for Cancer Research (AIRC Project no. 12965/2012—to PM), Fondazione Kathleen Foreman Casali—Trieste (to PM) and Fondazione Sanpaolo (to DS).

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Authors and Affiliations

  1. Department of Life Sciences, University of Trieste, Trieste, Italy

    P Macor, E Secco, N Mezzaroba, S Zorzet, P Durigutto, L De Maso, S Capolla & R Marzari

  2. National Institute for Biological Standards and Control (NIBSC), Hertfordshire, UK

    T Gaiotto

  3. Institute for Maternal and Child Health–IRCCS ‘Burlo Garofolo’, Trieste, Italy

    S Biffi & C Garrovo

  4. Department of Human Pathology, University of Palermo, Palermo, Italy

    C Tripodo

  5. Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano, Italy

    V Gattei

  6. IRCCS Istituto Auxologico Italiano, Milan, Italy

    F Tedesco

  7. Department of Health Sciences and IRCAD, University of Eastern Piedmont, Novara, Italy

    D Sblattero

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Correspondence to P Macor or D Sblattero.

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Macor, P., Secco, E., Mezzaroba, N. et al. Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice. Leukemia 29, 406–414 (2015). https://doi.org/10.1038/leu.2014.185

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