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Thromb Haemost 2017; 117(01): 33-43
DOI: 10.1160/TH16-05-0404
Coagulation and Fibrinolysis
Schattauer GmbH

Combination therapy for inhibitor reversal in haemophilia A using monoclonal anti-CD20 and rapamycin

Moanaro Biswas   *
1   University of Florida, Department of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida, USA
,
Geoffrey L. Rogers   *
1   University of Florida, Department of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida, USA
,
Alexandra Sherman
1   University of Florida, Department of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida, USA
,
Barry J. Byrne
1   University of Florida, Department of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida, USA
,
David M. Markusic
1   University of Florida, Department of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida, USA
,
Haiyan Jiang
2   Biogen, Cambridge, Massachusetts, USA
,
Roland W. Herzog
1   University of Florida, Department of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida, USA
› Author Affiliations Financial support: This work was supported by a sponsored research agreement with Biogen and by NIH grant R01 AI51390 (to RWH).
Further Information

Publication History

Received: 25 May 2016

Accepted after major revision: 12 September 2016

Publication Date:
01 December 2017 (online)

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Summary

Development of antibodies (inhibitors) against coagulation factor VIII (FVIII) is a major complication of intravenous replacement therapy in haemophilia A (HA). Current immune tolerance induction (ITI) regimens are not universally effective. Rituximab, a B cell-depleting antibody against CD20, has shown mixed results for inhibitor reversal in patients. This study aims to develop a combinatorial therapy for inhibitor reversal in HA, using anti-murine CD20 (anti-mCD20) antibody and rapamycin, which targets both B and T cell responses. Additionally, it extensively characterises the role of the IgG backbone in B cell depletion by anti-CD20 antibodies. For this, inhibitors were generated in BALB/c-HA mice by weekly IV injection of FVIII. Subsequently, anti-mCD20 (18B12) with IgG2a or IgG1 backbone was injected IV in two doses three weeks apart and B cell depletion and recovery was characterised. Rapamycin was administered orally 3x/week (for 1 month) while continuing FVIII injections. Altering the IgG backbone of anti-mCD20 from IgG2a to IgG1 reduced overall depletion of B cells (including memory B cells), and marginal zone, B-10, and B-1b cells were specifically unaffected. While neither antibody was effective alone, in combination with rapamycin, anti-mCD20 IgG2a but not IgG1 was able to reverse inhibitors in HA mice. This regimen was particularly effective for starting titres of ∼10 BU. Although IgG1 anti-mCD20 spared potentially tolerogenic B cell subsets, IgG2a directed sustained hyporesponsiveness when administered in conjunction with rapamycin. This regimen represents a promising treatment for inhibitor reversal in HA, as both of these compounds have been extensively used in human patients.

Supplementary Material to this article is available at www.thrombosis-online.com.

Keywords

Anti-mCD20 - factor VIII - haemophilia - inhibitor - rapamycin

* These authors contributed equally to this work.


 

  • References

  • 1 Manco-Johnson MJ, Abshire TC, Shapiro AD. et al. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N Engl J Med 2007; 357: 535-544.
    CrossrefPubMedGoogle Scholar
  • 2 Peyvandi F, Mannucci PM, Garagiola I. et al. A Randomized Trial of Factor VIII and Neutralizing Antibodies in Hemophilia A. N Engl J Med 2016; 374: 2054-2064.
    CrossrefPubMedGoogle Scholar
  • 3 Halimeh S, Bidlingmaier C, Heller C. et al. Risk factors for high-titre inhibitor development in children with hemophilia A: results of a cohort study. BioMed Res Int 2013; 2013: 901975.
    PubMedGoogle Scholar
  • 4 Gouw SC, van den Berg HM, Oldenburg J. et al. F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review and meta-analysis. Blood 2012; 119: 2922-2934.
    CrossrefPubMedGoogle Scholar
  • 5 Santagostino E, Mancuso ME, Rocino A. et al. Environmental risk factors for inhibitor development in children with haemophilia A: a case-control study. Br J Haematol 2005; 130: 422-427.
    CrossrefPubMedGoogle Scholar
  • 6 Hay CR, DiMichele DM. The principal results of the International Immune Tolerance Study: a randomized dose comparison. Blood 2012; 119: 1335-1344.
    CrossrefPubMedGoogle Scholar
  • 7 Mariani G, Siragusa S, Kroner BL. Immune tolerance induction in hemophilia A: a review. Semin Thromb Hemost 2003; 29: 69-76.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 8 Franchini M, Mannucci PM. Inhibitor eradication with rituximab in haemophilia: where do we stand?. Br J Haematol 2014; 165: 600-608.
    CrossrefPubMedGoogle Scholar
  • 9 Carcao M, St Louis J, Poon MC. et al. Rituximab for congenital haemophiliacs with inhibitors: a Canadian experience. Haemophilia 2006; 12: 7-18.
    PubMedGoogle Scholar
  • 10 Leissinger C, Josephson CD, Granger S. et al. Rituximab for treatment of inhibitors in haemophilia A. A Phase II study. Thromb Haemost 2014; 112: 445-458.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 11 Sack BK, Merchant S, Markusic DM. et al. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy. PloS one 2012; 07: e37671.
    CrossrefPubMedGoogle Scholar
  • 12 Liu CL, Ye P, Lin J. et al. Anti-CD20 as the B-Cell Targeting Agent in a Combined Therapy to Modulate Anti-Factor VIII Immune Responses in Hemophilia a Inhibitor Mice. Frontiers Immunol 2014; 04: 502.
    PubMedGoogle Scholar
  • 13 Zhang AH, Skupsky J, Scott DW. Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice. Blood 2011; 117: 2223-2226.
    CrossrefPubMedGoogle Scholar
  • 14 Irani V, Guy AJ, Andrew D. et al. Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. Mol Immunol 2015; 67: 171-182.
    CrossrefPubMedGoogle Scholar
  • 15 Cerutti A, Cols M, Puga I. Marginal zone B cells: virtues of innate-like antibody-producing lymphocytes. Nat Rev Immunol 2013; 13: 118-132.
    CrossrefPubMedGoogle Scholar
  • 16 Baumgarth N. The double life of a B-1 cell: self-reactivity selects for protective effector functions. Nat Rev Immunol 2011; 11: 34-46.
    CrossrefPubMedGoogle Scholar
  • 17 Gong Q, Ou Q, Ye S. et al. Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy. J Immunol 2005; 174: 817-826.
    CrossrefPubMedGoogle Scholar
  • 18 Lykken JM, Candando KM, Tedder TF. Regulatory B10 cell development and function. Int Immunol 2015; 27: 471-477.
    CrossrefPubMedGoogle Scholar
  • 19 Bray GL, Kroner BL, Arkin S. et al. Loss of high-responder inhibitors in patients with severe hemophilia A and human immunodeficiency virus type 1 infection: a report from the Multi-Center Hemophilia Cohort Study. Am J Hematol 1993; 42: 375-379.
    CrossrefPubMedGoogle Scholar
  • 20 Qian J, Collins M, Sharpe AH. et al. Prevention and treatment of factor VIII inhibitors in murine hemophilia A. Blood 2000; 95: 1324-1329.
    PubMedGoogle Scholar
  • 21 Moghimi B, Sack BK, Nayak S. et al. Induction of tolerance to factor VIII by transient co-administration with rapamycin. J Thromb Haemost 2011; 09: 1524-1533.
    CrossrefPubMedGoogle Scholar
  • 22 Nayak S, Cao O, Hoffman BE. et al. Prophylactic immune tolerance induced by changing the ratio of antigen-specific effector to regulatory T cells. J Thromb Haemost 2009; 07: 1523-1532.
    CrossrefPubMedGoogle Scholar
  • 23 Nayak S, Sarkar D, Perrin GQ. et al. Prevention and Reversal of Antibody Responses Against Factor IX in Gene Therapy for Hemophilia B. Frontiers Microbiol 2011; 02: 244.
    PubMedGoogle Scholar
  • 24 Biswas M, Sarkar D, Kumar SR. et al. Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell-dependent induction of CD4+CD25+FoxP3+ Treg. Blood 2015; 125: 2937-2947.
    CrossrefPubMedGoogle Scholar
  • 25 Cao O, Hoffman BE, Moghimi B. et al. Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B. Mol Ther 2009; 17: 1733-1742.
    CrossrefPubMedGoogle Scholar
  • 26 Sarkar D, Biswas M, Liao G. et al. Expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia. Mol Therapy Methods Clin Develop 2014; 01: 14030.
    PubMedGoogle Scholar
  • 27 Lykken JM, DiLillo DJ, Weimer ET. et al. Acute and chronic B cell depletion disrupts CD4+ and CD8+ T cell homeostasis and expansion during acute viral infection in mice. J Immunol 2014; 193: 746-756.
    CrossrefPubMedGoogle Scholar
  • 28 Qadura M, Waters B, Burnett E. et al. Immunoglobulin isotypes and functional anti-FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice. Haemophilia 2011; 17: 288-295.
    CrossrefPubMedGoogle Scholar
  • 29 Elder ME, Nayak S, Collins SW. et al. B-Cell depletion and immunomodulation before initiation of enzyme replacement therapy blocks the immune response to acid alpha-glucosidase in infantile-onset Pompe disease. J Pediatr 2013; 163: 847-854. e1
    CrossrefPubMedGoogle Scholar
  • 30 Antun A, Monahan PE, Manco-Johnson MJ. et al. Inhibitor recurrence after immune tolerance induction: a multicenter retrospective cohort study. J Thromb Haemost 2015; 13: 1980-1988.
    CrossrefPubMedGoogle Scholar
  • 31 Laros-van Gorkom BA, Falaise C, Astermark J. Immunosuppressive agents in the treatment of inhibitors in congenital haemophilia A and B--a systematic literature review. Eur J Haematol Suppl 2014; 76: 26-38.
    PubMedGoogle Scholar
  • 32 Cao O, Loduca PA, Herzog RW. Role of regulatory T cells in tolerance to coagulation factors. J Thromb Haemost 2009; 07 (Suppl. 01) 88-91.
    CrossrefPubMedGoogle Scholar
  • 33 Collins PW, Mathias M, Hanley J. et al. Rituximab and immune tolerance in severe hemophilia A: a consecutive national cohort. J Thromb Haemost 2009; 07: 787-794.
    CrossrefPubMedGoogle Scholar
  • 34 Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nat Rev Immunol 2009; 09: 324-337.
    CrossrefPubMedGoogle Scholar
  • 35 Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood 2005; 105: 4743-4748.
    CrossrefPubMedGoogle Scholar
  • 36 Hackstein H, Taner T, Zahorchak AF. et al. Rapamycin inhibits IL-4--induced dendritic cell maturation in vitro and dendritic cell mobilization and function in vivo. Blood 2003; 101: 4457-4463.
    CrossrefPubMedGoogle Scholar
  • 37 Zeiser R, Leveson-Gower DB, Zambricki EA. et al. Differential impact of mammalian target of rapamycin inhibition on CD4+CD25+Foxp3+ regulatory T cells compared with conventional CD4+ T cells. Blood 2008; 111: 453-462.
    CrossrefPubMedGoogle Scholar
  • 38 Delgoffe GM, Kole TP, Zheng Y. et al. The mTOR kinase differentially regulates effector and regulatory T cell lineage commitment. Immunity 2009; 30: 832-844.
    CrossrefPubMedGoogle Scholar
  • 39 Battaglia M, Stabilini A, Migliavacca B. et al. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol 2006; 177: 8338-8347.
    CrossrefPubMedGoogle Scholar
  • 40 Matsue H, Yang C, Matsue K. et al. Contrasting impacts of immunosuppressive agents (rapamycin, FK506, cyclosporin A, and dexamethasone) on bidirectional dendritic cell-T cell interaction during antigen presentation. J Immunol 2002; 169: 3555-3564.
    CrossrefPubMedGoogle Scholar
  • 41 Lui SL, Yung S, Tsang R. et al. Rapamycin prevents the development of nephritis in lupus-prone NZB/W F1 mice. Lupus 2008; 17: 305-313.
    CrossrefPubMedGoogle Scholar
  • 42 Esposito M, Ruffini F, Bellone M. et al. Rapamycin inhibits relapsing experimental autoimmune encephalomyelitis by both effector and regulatory T cells modulation. J Neuroimmunol 2010; 220: 52-63.
    CrossrefPubMedGoogle Scholar
  • 43 Zhang AH, Rossi RJ, Yoon J. et al. Tolerogenic nanoparticles to induce immunologic tolerance: Prevention and reversal of FVIII inhibitor formation. Cell Immunol 2016; 301: 74-81.
    CrossrefPubMedGoogle Scholar
  • 44 Streif W, Escuriola Ettingshausen C, Linde R. et al. Inhibitor treatment by rituximab in congenital haemophilia A - Two case reports. Hamostaseologie 2009; 29: 151-154.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 45 Yanaba K, Bouaziz JD, Haas KM. et al. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. Immunity 2008; 28: 639-650.
    CrossrefPubMedGoogle Scholar
  • 46 Huang H, Benoist C, Mathis D. Rituximab specifically depletes short-lived auto-reactive plasma cells in a mouse model of inflammatory arthritis. Proc Natl Acad Sci USA 2010; 107: 4658-4663.
    CrossrefPubMedGoogle Scholar
  • 47 Markusic DM, Hoffman BE, Perrin GQ. et al. Effective gene therapy for haemophilic mice with pathogenic factor IX antibodies. EMBO Mol Med 2013; 05: 1698-1709.
    CrossrefPubMedGoogle Scholar
  • 48 Khodadadi L, Cheng Q, Alexander T. et al. Bortezomib Plus Continuous B Cell Depletion Results in Sustained Plasma Cell Depletion and Amelioration of Lupus Nephritis in NZB/W F1 Mice. PloS one 2015; 10: e0135081.
    CrossrefPubMedGoogle Scholar
  • 49 Doerfler PA, Nayak S, Corti M. et al. Targeted approaches to induce immune tolerance for Pompe disease therapy. Mol Ther Methods Clin Dev 2016; 03: 15053.
    CrossrefPubMedGoogle Scholar
  • 50 Cousens LP, Mingozzi F, van der Marel S. et al. Teaching tolerance: New approaches to enzyme replacement therapy for Pompe disease. Hum Vaccin Immunother 2012; 08: 1459-1464.
    CrossrefPubMedGoogle Scholar