High-Dose IV Administration of Rasburicase Suppresses Anti-rasburicase Antibodies, Depletes Rasburicase-Specific Lymphocytes, and Upregulates Treg Cells


Therapeutic proteins can be potent agents for treating serious diseases, but in many patients these proteins provoke antibody responses that blunt therapeutic efficacy. Intravenous administration of high doses of some proteins induces immune tolerance, but the mechanisms underlying this effect are poorly understood. As a model to study tolerance induction in mice, we used rasburicase, a commercial recombinant uricase used for the treatment of hyperuricemia. Intraperitoneal (i.p.) injection of rasburicase without or with alum adjuvants induced a clear anti-rasburicase antibody response, but intravenous (i.v.) injection did not. The lack of response to i.v. rasburicase was apparently due to active immune suppression since i.v.-treated mice showed blunted antibody and reduced T cell responses to subsequent i.p. injections of rasburicase. This blunted response was associated with a decrease in rasburicase-specific B cell and T cell responses and an increase in proportion of CD4+ FoxP3+ regulatory T cells (Treg) in the spleen. We examined the number of lymphocytes in peripheral blood after rasburicase i.v. injection. Rasburicase caused a transient reduction in B and T cells, but a robust and sustained depletion of rasburicase-specific B cells. Further experiments showed that rasburicase i.v. injection decreased the number of lymphocytes and was associated with apoptosis of both B cells and activated T cells and that the enhanced percentage of Treg cells was likely mediated by a macrophage-dependent pathway. Thus, our data suggest that apoptosis and depletion of antigen-specific B lymphocytes and upregulation of Treg cells may play important roles in the immune suppression induced by intravenous administration of a therapeutic protein.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


  1. 1.

    Baldo BA. Enzymes approved for human therapy: indications, mechanisms and adverse effects. BioDrugs. 2015;29(1):31–55.

    CAS  PubMed  Google Scholar 

  2. 2.

    Carter PJ. Introduction to current and future protein therapeutics: a protein engineering perspective. Exp Cell Res. 2011;317(9):1261–9.

    CAS  PubMed  Google Scholar 

  3. 3.

    Baker MP, et al. Immunogenicity of protein therapeutics: the key causes, consequences and challenges. Self Nonself. 2010;1(4):314–22.

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Blumberg RS, Lillicrap D, G.F.I.T.G. Ig. Tolerogenic properties of the Fc portion of IgG and its relevance to the treatment and management of hemophilia. Blood. 2018;131(20):2205–14.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Kalden JR, Schulze-Koops H. Immunogenicity and loss of response to TNF inhibitors: implications for rheumatoid arthritis treatment. Nat Rev Rheumatol. 2017;13(12):707–18.

    CAS  PubMed  Google Scholar 

  6. 6.

    Endres RO, Grey HM. Antigen recognition by T cells. II. Intravenous administration of native or denatured ovalbumin results in tolerance to both forms of the antigen. J Immunol. 1980;125(4):1521–5.

    CAS  PubMed  Google Scholar 

  7. 7.

    Ettingshausen CE, Kreuz W. The immune tolerance induction (ITI) dose debate: does the International ITI Study provide a clearer picture? Haemophilia. 2013;19(Suppl 1):12–7.

    CAS  PubMed  Google Scholar 

  8. 8.

    Kubisz P, Plamenová I, Hollý P, Stasko J. Successful immune tolerance induction with high-dose coagulation factor VIII and intravenous immunoglobulins in a patient with congenital hemophilia and high-titer inhibitor of coagulation factor VIII despite unfavorable prognosis for the therapy. Med Sci Monit. 2009;15(6):CS105–11.

    PubMed  Google Scholar 

  9. 9.

    Dargaud Y, Pavlova A, Lacroix-Desmazes S, Fischer K, Soucie M, Claeyssens S, et al. Achievements, challenges and unmet needs for haemophilia patients with inhibitors: report from a symposium in Paris, France on 20 November 2014. Haemophilia. 2016;22(Suppl 1):1–24.

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    McFarland HI, et al. Amelioration of autoimmune reactions by antigen-induced apoptosis of T cells. Adv Exp Med Biol. 1995;383:157–66.

    CAS  PubMed  Google Scholar 

  11. 11.

    Chao H, Walsh CE. Induction of tolerance to human factor VIII in mice. Blood. 2001;97(10):3311–2.

    CAS  PubMed  Google Scholar 

  12. 12.

    Astermark J. Immune tolerance induction in patients with hemophilia A. Thromb Res. 2011;127(Suppl 1):S6–9.

    CAS  PubMed  Google Scholar 

  13. 13.

    Jacobs MJ, van den Hoek A, van de Putte L, van den Berg W. Anergy of antigen-specific T lymphocytes is a potent mechanism of intravenously induced tolerance. Immunology. 1994;82(2):294–300.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Liblau RS, Tisch R, Shokat K, Yang X, Dumont N, Goodnow CC, et al. Intravenous injection of soluble antigen induces thymic and peripheral T-cells apoptosis. Proc Natl Acad Sci U S A. 1996;93(7):3031–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Li H, Zhang GX, Chen Y, Xu H, Fitzgerald DC, Zhao Z, et al. CD11c+CD11b+ dendritic cells play an important role in intravenous tolerance and the suppression of experimental autoimmune encephalomyelitis. J Immunol. 2008;181(4):2483–93.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Faust KB, Finke D, Klempt-Giessing K, Randers K, Zachrau B, Schlenke P, et al. Antigen-induced B cell apoptosis is independent of complement C4. Clin Exp Immunol. 2007;150(1):132–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Yoshida T, Higuchi T, Hagiyama H, Strasser A, Nishioka K, Tsubata T. Rapid B cell apoptosis induced by antigen receptor ligation does not require Fas (CD95/APO-1), the adaptor protein FADD/MORT1 or CrmA-sensitive caspases but is defective in both MRL-+/+ and MRL-lpr/lpr mice. Int Immunol. 2000;12(4):517–26.

    CAS  PubMed  Google Scholar 

  18. 18.

    Garay RP, el-Gewely MR, Labaune JP, Richette P. Therapeutic perspectives on uricases for gout. Joint Bone Spine. 2012;79(3):237–42.

    CAS  PubMed  Google Scholar 

  19. 19.

    Allen KC, Champlain AH, Cotliar JA, Belknap SM, West DP, Mehta J, et al. Risk of anaphylaxis with repeated courses of rasburicase: a Research on Adverse Drug Events and Reports (RADAR) project. Drug Saf. 2015;38(2):183–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Fitzgerald DC, Zhang GX, Yu S, Cullimore ML, Zhao Z, Rostami A. Intravenous tolerance effectively overcomes enhanced pro-inflammatory responses and experimental autoimmune encephalomyelitis severity in the absence of IL-12 receptor signaling. J Neuroimmunol. 2012;247(1–2):32–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Zhang GX, Yu S, Li Y, Ventura ES, Gran B, Rostami A. A paradoxical role of APCs in the induction of intravenous tolerance in experimental autoimmune encephalomyelitis. J Neuroimmunol. 2005;161(1–2):101–12.

    CAS  PubMed  Google Scholar 

  22. 22.

    Yan Y, Zhang GX, Gran B, Fallarino F, Yu S, Li H, et al. IDO upregulates regulatory T cells via tryptophan catabolite and suppresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis. J Immunol. 2010;185(10):5953–61.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Wakabayashi H, Fukushima H, Yamada T, Kawase M, Shirataki Y, Satoh K, et al. Inhibition of LPS-stimulated NO production in mouse macrophage-like cells by Barbados cherry, a fruit of Malpighia emarginata DC. Anticancer Res. 2003;23(4):3237–41.

    CAS  PubMed  Google Scholar 

  24. 24.

    Guan Y, Yu S, Zhao Z, Ciric B, Zhang GX, Rostami A. Antigen presenting cells treated in vitro by macrophage colony-stimulating factor and autoantigen protect mice from autoimmunity. J Neuroimmunol. 2007;192(1–2):68–78.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Corradin G, Engers HD. Inhibition of antigen-induced T-cell clone proliferation by antigen-specific antibodies. Nature. 1984;308(5959):547–8.

    CAS  PubMed  Google Scholar 

  26. 26.

    Xu H, Oriss TB, Fei M, Henry AC, Melgert BN, Chen L, et al. Indoleamine 2,3-dioxygenase in lung dendritic cells promotes Th2 responses and allergic inflammation. Proc Natl Acad Sci U S A. 2008;105(18):6690–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Yan Y, Li Z, Zhang GX, Williams MS, Carey GB, Zhang J, et al. Anti-MS4a4B treatment abrogates MS4a4B-mediated protection in T cells and ameliorates experimental autoimmune encephalomyelitis. Apoptosis. 2013;18(9):1106–19.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Green DR, Droin N, Pinkoski M. Activation-induced cell death in T cells. Immunol Rev. 2003;193:70–81.

    CAS  PubMed  Google Scholar 

  29. 29.

    Roberts AI, Devadas S, Zhang X, Zhang L, Keegan A, Greeneltch K, et al. The role of activation-induced cell death in the differentiation of T-helper-cell subsets. Immunol Res. 2003;28(3):285–93.

    PubMed  Google Scholar 

  30. 30.

    Blander JM. The many ways tissue phagocytes respond to dying cells. Immunol Rev. 2017;277(1):158–73.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Gordan S, Biburger M, Nimmerjahn F. bIgG time for large eaters: monocytes and macrophages as effector and target cells of antibody-mediated immune activation and repression. Immunol Rev. 2015;268(1):52–65.

    CAS  PubMed  Google Scholar 

  32. 32.

    Zent CS, Elliott MR. Maxed out macs: physiologic cell clearance as a function of macrophage phagocytic capacity. FEBS J. 2017;284(7):1021–39.

    CAS  PubMed  Google Scholar 

  33. 33.

    Gorczyca W, et al. Immunophenotypic pattern of myeloid populations by flow cytometry analysis. Methods Cell Biol. 2011;103:221–66.

    CAS  PubMed  Google Scholar 

  34. 34.

    Ateshkadi A, Johnson CA, Oxton LL, Hammond TG, Bohenek WS, Zimmerman SW. Pharmacokinetics of intraperitoneal, intravenous, and subcutaneous recombinant human erythropoietin in patients on continuous ambulatory peritoneal dialysis. Am J Kidney Dis. 1993;21(6):635–42.

    CAS  PubMed  Google Scholar 

  35. 35.

    Bettelli E, Oukka M, Kuchroo VK. TH-17 cells in the circle of immunity and autoimmunity. Nat Immunol. 2007;8(4):345–50.

    CAS  PubMed  Google Scholar 

  36. 36.

    Shevach EM. Mechanisms of Foxp3+ T regulatory cell-mediated suppression. Immunity. 2009;30(5):636–45.

    CAS  Google Scholar 

  37. 37.

    von Boehmer H. Mechanisms of suppression by suppressor T cells. Nat Immunol. 2005;6(4):338–44.

    Google Scholar 

  38. 38.

    Hoyer LW. Future approaches to factor VIII inhibitor therapy. Am J Med. 1991;91(5A):40S–4S.

    CAS  PubMed  Google Scholar 

  39. 39.

    Goodnow CC, Vinuesa CG, Randall KL, Mackay F, Brink R. Control systems and decision making for antibody production. Nat Immunol. 2010;11(8):681–8.

    CAS  PubMed  Google Scholar 

  40. 40.

    Packard, T.A., et al., B cell receptor affinity for insulin dictates autoantigen acquisition and B cell functionality in autoimmune diabetes. J Clin Med, 2016;5(11).

  41. 41.

    Caulfield MJ, Shaffer D. Immunoregulation by antigen/antibody complexes. I. Specific immunosuppression induced in vivo with immune complexes formed in antibody excess. J Immunol. 1987;138(11):3680–3.

    CAS  PubMed  Google Scholar 

  42. 42.

    Halstead SB, Mahalingam S, Marovich MA, Ubol S, Mosser DM. Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. Lancet Infect Dis. 2010;10(10):712–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Tada M, Suzuki T, Ishii-Watabe A. Development and characterization of an anti-rituximab monoclonal antibody panel. MAbs. 2018;10(3):370–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Wang XY, Wang B, Wen YM. From therapeutic antibodies to immune complex vaccines. NPJ Vaccines. 2019;4:2.

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Gallo P, Goncalves R, Mosser DM. The influence of IgG density and macrophage Fc (gamma) receptor cross-linking on phagocytosis and IL-10 production. Immunol Lett. 2010;133(2):70–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Cashman KS, Jenks SA, Woodruff MC, Tomar D, Tipton CM, Scharer CD, et al. Understanding and measuring human B-cell tolerance and its breakdown in autoimmune disease. Immunol Rev. 2019;292(1):76–89.

    CAS  PubMed  Google Scholar 

  47. 47.

    Cyster JG, Allen CDC. B cell responses: cell interaction dynamics and decisions. Cell. 2019;177(3):524–40.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Gonzalez SF, Degn SE, Pitcher LA, Woodruff M, Heesters BA, Carroll MC. Trafficking of B cell antigen in lymph nodes. Annu Rev Immunol. 2011;29:215–33.

    CAS  PubMed  Google Scholar 

  49. 49.

    Tsubata T. B-cell tolerance and autoimmunity. F1000Res. 2017;6:391.

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Gotot J, Dhana E, Yagita H, Kaiser R, Ludwig-Portugall I, Kurts C. Antigen-specific Helios, Neuropilin-1 Tregs induce apoptosis of autoreactive B cells via PD-L1. Immunol Cell Biol. 2018;96(8):852–62.

    CAS  PubMed  Google Scholar 

  51. 51.

    Zhao DM, Thornton AM, DiPaolo RJ, Shevach EM. Activated CD4+CD25+ T cells selectively kill B lymphocytes. Blood. 2006;107(10):3925–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Kavai M, Szegedi G. Immune complex clearance by monocytes and macrophages in systemic lupus erythematosus. Autoimmun Rev. 2007;6(7):497–502.

    CAS  PubMed  Google Scholar 

  53. 53.

    Leslie RG. Macrophage handling of soluble immune complexes. Immunol Today. 1980;1(4):78–84.

    CAS  PubMed  Google Scholar 

  54. 54.

    Leslie RG. Macrophage interactions with antibodies and soluble immune complexes. Immunobiology. 1982;161(3–4):322–33.

    CAS  PubMed  Google Scholar 

  55. 55.

    Leslie RG. Macrophage handling of soluble immune complexes: evaluation of mechanisms involved in the selective clearance of complexes from the circulation. Mol Immunol. 1985;22(5):513–9.

    CAS  PubMed  Google Scholar 

  56. 56.

    Leslie RG. Complex aggregation: a critical event in macrophage handling of soluble immune complexes. Immunol Today. 1985;6(6):183–7.

    CAS  PubMed  Google Scholar 

  57. 57.

    Ronnelid J, et al. Immune complex-mediated cytokine production is regulated by classical complement activation both in vivo and in vitro. Adv Exp Med Biol. 2008;632:187–201.

    PubMed  Google Scholar 

  58. 58.

    Esparza I, Green R, Schreiber RD. Inhibition of macrophage tumoricidal activity by immune complexes and altered erythrocytes. J Immunol. 1983;131(5):2117–21.

    CAS  PubMed  Google Scholar 

  59. 59.

    Virgin HW 4th, et al. Immune complex effects on murine macrophages. II. Immune complex effects on activated macrophages cytotoxicity, membrane IL 1, and antigen presentation. J Immunol. 1985;135(6):3744–9.

    CAS  PubMed  Google Scholar 

  60. 60.

    Virgin HW 4th, et al. Suppression of immune response to Listeria monocytogenes: mechanism(s) of immune complex suppression. Infect Immun. 1985;50(2):343–53.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Virgin HW 4th, Wittenberg GF, Unanue ER. Immune complex effects on murine macrophages. I. Immune complexes suppress interferon-gamma induction of Ia expression. J Immunol. 1985;135(6):3735–43.

    CAS  PubMed  Google Scholar 

  62. 62.

    Feldman GM, Chuang EJ, Finbloom DS. IgG immune complexes inhibit IFN-gamma-induced transcription of the Fc gamma RI gene in human monocytes by preventing the tyrosine phosphorylation of the p91 (Stat1) transcription factor. J Immunol. 1995;154(1):318–25.

    CAS  PubMed  Google Scholar 

  63. 63.

    Boekhoudt GH, Frazier-Jessen MR, Feldman GM. Immune complexes suppress IFN-gamma signaling by activation of the FcgammaRI pathway. J Leukoc Biol. 2007;81(4):1086–92.

    CAS  PubMed  Google Scholar 

  64. 64.

    Boekhoudt GH, McGrath AG, Swisher JFA, Feldman GM. Immune complexes suppress IFN-gamma-induced responses in monocytes by activating discrete members of the SRC kinase family. J Immunol. 2015;194(3):983–9.

    CAS  PubMed  Google Scholar 

  65. 65.

    Swisher JF, Feldman GM. The many faces of FcgammaRI: implications for therapeutic antibody function. Immunol Rev. 2015;268(1):160–74.

    CAS  PubMed  Google Scholar 

  66. 66.

    Issara-Amphorn J, Surawut S, Worasilchai N, Thim-uam A, Finkelman M, Chindamporn A, et al. The synergy of endotoxin and (1→3)-β-D-Glucan, from gut translocation, worsens sepsis severity in a lupus model of Fc gamma receptor IIb-deficient mice. J Innate Immun. 2018;10(3):189–201.

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Wu Z, Zhou J, Prsoon P, Wei X, Liu X, Peng B. Low expression of FCGRIIB in macrophages of immune thrombocytopenia-affected individuals. Int J Hematol. 2012;96(5):588–93.

    CAS  PubMed  Google Scholar 

  68. 68.

    Heyman B. Antibodies as natural adjuvants. Curr Top Microbiol Immunol. 2014;382:201–19.

    CAS  PubMed  Google Scholar 

  69. 69.

    Lambour J, Naranjo-Gomez M, Piechaczyk M, Pelegrin M. Converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play. Emerg Microbes Infect. 2016;5(8):e92.

    CAS  PubMed  Google Scholar 

  70. 70.

    Beutier H, Gillis CM, Iannascoli B, Godon O, England P, Sibilano R, et al. IgG subclasses determine pathways of anaphylaxis in mice. J Allergy Clin Immunol. 2017;139(1):269–80 e7.

    CAS  PubMed  Google Scholar 

  71. 71.

    Chen B, Vousden KA, Naiman B, Turman S, Sun H, Wang S, et al. Humanised effector-null FcgammaRIIA antibody inhibits immune complex-mediated proinflammatory responses. Ann Rheum Dis. 2019;78(2):228–37.

    CAS  PubMed  Google Scholar 

  72. 72.

    Gao CH, Dong HL, Tai L, Gao XM. Lactoferrin-containing immunocomplexes drive the conversion of human macrophages from M2- into M1-like phenotype. Front Immunol. 2018;9:37.

    PubMed  PubMed Central  Google Scholar 

  73. 73.

    Kang S, Rogers JL, Monteith AJ, Jiang C, Schmitz J, Clarke SH, et al. Apoptotic debris accumulates on hematopoietic cells and promotes disease in murine and human systemic lupus erythematosus. J Immunol. 2016;196(10):4030–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  74. 74.

    Weingartner E, Golding A. Direct control of B cells by Tregs: an opportunity for long-term modulation of the humoral response. Cell Immunol. 2017;318:8–16.

    CAS  PubMed  Google Scholar 

  75. 75.

    Baptista AP, et al. The chemoattractant receptor Ebi2 drives intranodal naive CD4+ T cell peripheralization to promote effective adaptive immunity. Immunity. 2019;50(5):1188–201 e6.

    CAS  PubMed  Google Scholar 

  76. 76.

    Germain, R.N. EMBL keynote lecture—Imaging the immune system. [From the EMBO | EMBL Symposium: Seeing is believing—imaging the processes of life]. 2017 April 11, 2018. https://www.youtube.com/watch?v=bman-ttEfno

  77. 77.

    Hamuro L, Kijanka G, Kinderman F, Kropshofer H, Bu DX, Zepeda M, et al. Perspectives on subcutaneous route of administration as an immunogenicity risk factor for therapeutic proteins. J Pharm Sci. 2017;106(10):2946–54.

    CAS  PubMed  Google Scholar 

Download references


We thank Mark Kukuruga and Adovi Akue in the FDA CBER Flow Cytometric Core Facility for their helpful technical assistance.

Author information



Corresponding author

Correspondence to Gerald M. Feldman.

Ethics declarations

Competing Interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Xu, H., Feldman, G.M. & Max, E.E. High-Dose IV Administration of Rasburicase Suppresses Anti-rasburicase Antibodies, Depletes Rasburicase-Specific Lymphocytes, and Upregulates Treg Cells. AAPS J 22, 80 (2020). https://doi.org/10.1208/s12248-020-00461-0

Download citation


  • apoptosis
  • immune suppression
  • lymphocyte
  • macrophage
  • therapeutic protein