Acta Neuropathologica

, Volume 125, Issue 6, pp 829–840 | Cite as

C1q-targeted monoclonal antibody prevents complement-dependent cytotoxicity and neuropathology in in vitro and mouse models of neuromyelitis optica

  • Puay-Wah Phuan
  • Hua Zhang
  • Nithi Asavapanumas
  • Michael Leviten
  • Arnon Rosenthal
  • Lukmanee Tradtrantip
  • A. S. Verkman
Original Paper


Neuromyelitis optica (NMO) is an autoimmune disorder with inflammatory demyelinating lesions in the central nervous system, particularly in the spinal cord and optic nerve. NMO pathogenesis is thought to involve binding of anti-aquaporin-4 (AQP4) autoantibodies to astrocytes, which causes complement-dependent cytotoxicity (CDC) and downstream inflammation leading to oligodendrocyte and neuronal injury. Vasculocentric deposition of activated complement is a prominent feature of NMO pathology. Here, we show that a neutralizing monoclonal antibody against the C1q protein in the classical complement pathway prevents AQP4 autoantibody-dependent CDC in cell cultures and NMO lesions in ex vivo spinal cord slice cultures and in mice. A monoclonal antibody against human C1q with 11 nM binding affinity prevented CDC caused by NMO patient serum in AQP4-transfected cells and primary astrocyte cultures, and prevented complement-dependent cell-mediated cytotoxicity (CDCC) produced by natural killer cells. The anti-C1q antibody prevented astrocyte damage and demyelination in mouse spinal cord slice cultures exposed to AQP4 autoantibody and human complement. In a mouse model of NMO produced by intracerebral injection of AQP4 autoantibody and human complement, the inflammatory demyelinating lesions were greatly reduced by intracerebral administration of the anti-C1q antibody. These results provide proof-of-concept for C1q-targeted monoclonal antibody therapy in NMO. Targeting of C1q inhibits the classical complement pathway directly and causes secondary inhibition of CDCC and the alternative complement pathway. As C1q-targeted therapy leaves the lectin complement activation pathway largely intact, its side-effect profile is predicted to differ from that of therapies targeting downstream complement proteins.


NMO Aquaporin-4 Complement Neuroinflammation Autoimmunity 



This work was supported by grants EY13574, EB00415, DK35124, HL73856, DK86125 and DK72517 from the National Institutes of Health, and a grant from the Guthy-Jackson Charitable Foundation. This work was also supported by a sponsored research agreement from Annexon Inc. (Palo Alto, CA, USA). We thank Dr. Jeffrey Bennett (Univ. Colorado Denver, Aurora, CO, USA) for providing recombinant monoclonal NMO antibody and for Accelerated Cure (Waltham, MA, USA) for providing human NMO sera.

Conflict of interest

Drs. Leviten and Rosenthal are employed by Annexon, Inc.


  1. 1.
    Aberer W (2012) Hereditary angioedema treatment options: the availability of new therapies. Ann Med 44(6):523–529PubMedCrossRefGoogle Scholar
  2. 2.
    Asgari N, Khorooshi R, Lillevang ST, Owens T (2013) Complement-dependent pathogenicity of brain-specific antibodies in cerebrospinal fluid. J Neuroimmunol 254(1–2):76–82PubMedCrossRefGoogle Scholar
  3. 3.
    Ayzenberg I, Kleiter I, Schröder A, Hellwig K, Chan A, Yamamura T, Gold R (2013) Interleukin 6 receptor blockade in patients with neuromyelitis optica nonresponsive to anti-CD20 therapy. JAMA Neurol 70(3):394–397PubMedCrossRefGoogle Scholar
  4. 4.
    Bennett JL, Lam C, Kalluri SR, Saikali P, Bautista K, Dupree C, Glogowska M, Case D, Antel JP, Owens GP, Gilden D, Nessler S, Stadelmann C, Hemmer B (2009) Intrathecal pathogenic anti-aquaporin-4 antibodies in early neuromyelitis optica. Ann Neurol 66(5):617–629PubMedCrossRefGoogle Scholar
  5. 5.
    Botto M, Agnola CD, Bygrave AE, Thompson EM, Cook HT, Petry F, Loos M, Pandolfi PP, Walport MJ (1998) Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 19:56–59PubMedCrossRefGoogle Scholar
  6. 6.
    Botto M, Walport MJ (2002) C1q, autoimmunity and apoptosis. Immunobiol 205:395–406CrossRefGoogle Scholar
  7. 7.
    Crane JM, Lam C, Rossi A, Gupta T, Bennett JL, Verkman AS (2011) Binding affinity and specificity of neuromyelitis optica autoantibodies to aquaporin-4 M1/M23 isoforms and orthogonal arrays. J Biol Chem 286(18):16516–16524PubMedCrossRefGoogle Scholar
  8. 8.
    Gadjeva MG, Rouseva MM, Zlatarova AS, Reid KBM, Kishore U, Kojouharova MS (2008) Interaction of human C1q with IgG and IgM: revisited. Biochemistry 47(49):13093–13102PubMedCrossRefGoogle Scholar
  9. 9.
    Georgy MS, Pongracic JA (2012) Chapter 22: hereditary and acquired angioedema. Allergy Asthma Proc 33(Suppl 1):S73–S76PubMedCrossRefGoogle Scholar
  10. 10.
    Ghai R, Waters P, Roumenina LT, Gadjeva M, Kojouharova MS, Reid KB, Sim RB, Kishore U (2007) C1q and its growing family. Immunobiol 212(4–5):253–266CrossRefGoogle Scholar
  11. 11.
    Gómez-Almaguer D (2012) Monoclonal antibodies in the treatment of immune thrombocytopenic purpura (ITP). Hematology 17(Suppl 1):S25–S27PubMedGoogle Scholar
  12. 12.
    Gomez AM, Van Den Broeck J, Vrolix K, Janssen SP, Lemmens MA, Van Der Esch E, Duimel H, Frederik P, Molenaar PC, Martínez-Martínez P, De Baets MH, Losen M (2010) Antibody effector mechanisms in myasthenia gravis pathogenesis at the neuromuscular junction. Autoimmunity 43:353–370PubMedCrossRefGoogle Scholar
  13. 13.
    Greenberg BM, Graves D, Remington G, Hardeman P, Mann M, Karandikar N, Stuve O, Monson N, Frohman E (2012) Rituximab dosing and monitoring strategies in neuromyelitis optica patients: creating strategies for therapeutic success. Mult Scler 18(7):1022–1026PubMedCrossRefGoogle Scholar
  14. 14.
    Hengstman GJ, Wesseling P, Frenken CW, Jongen PJ (2007) Neuromyelitis optica with clinical and histopathological involvement of the brain. Mult Scler 13(5):679–682PubMedCrossRefGoogle Scholar
  15. 15.
    Hinson SR, McKeon A, Fryer JP, Apiwattanakul M, Lennon VA, Pittock SJ (2009) Prediction of neuromyelitis optica attack severity by quantitation of complement-mediated injury to aquaporin-4-expressing cells. Arch Neurol 66:1164–1167PubMedCrossRefGoogle Scholar
  16. 16.
    Hinson SR, Pittock SJ, Lucchinetti CF, Roemer SF, Fryer JP, Kryzer TJ, Lennon VA (2007) Pathogenic potential of IgG binding to water channel extracellular domain in neuromyelitis optica. Neurology 69(24):2221–2231PubMedCrossRefGoogle Scholar
  17. 17.
    Jacob A, McKeon A, Nakashima I, Sato DK, Elsone L, Fujihara K, de Seze J (2013) Current concept of neuromyelitis optica (NMO) and NMO spectrum disorders. J Neurol Neurosurg Psych 19:475–479Google Scholar
  18. 18.
    Jarius S, Paul F, Franciotta D, Waters P, Zipp F, Hohlfeld R, Vincent A, Wildemann B (2008) Mechanisms of disease: aquaporin-4 antibodies in neuromyelitis optica. Nat Clin Pract Neurol 4(4):202–214PubMedGoogle Scholar
  19. 19.
    Jarius S, Wildemann B (2010) AQP4 antibodies in neuromyelitis optica: diagnostic and pathogenetic relevance. Nature Rev Neurol 6(7):383–392CrossRefGoogle Scholar
  20. 20.
    Kieseier BC, Stüve O, Dehmel T, Goebels N, Leussink VI, Mausberg AK, Ringelstein M, Turowski B, Aktas O, Antoch G, Hartung HP (2012) Disease amelioration with Tocilizumab in a treatment-resistant patient with neuromyelitis optica: implication for cellular immune responses. Arch Neurol 24:1–4Google Scholar
  21. 21.
    Kim SH, Kim W, Huh SY, Lee KY, Jung IJ, Kim HJ (2013) Clinical efficacy of plasmapheresis in patients with neuromyelitis optica spectrum disorder and effects on circulating anti-aquaporin-4 antibody levels. J Clin Neurol 9(1):36–42PubMedCrossRefGoogle Scholar
  22. 22.
    Kira J (2011) Autoimmunity in neuromyelitis optica and opticospinal multiple sclerosis: astrocytopathy as a common denominator in demyelinating disorders. J Neurol Sci 311(1–2):69–77PubMedCrossRefGoogle Scholar
  23. 23.
    Kishore U, Reid KBM (2000) C1q: structure, function, and receptors. Immunopharmacol 49:159–170CrossRefGoogle Scholar
  24. 24.
    Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Köhl J (2009) The role of the anaphylatoxins in health and disease. Mol Immunol 46(14):2753–2766PubMedCrossRefGoogle Scholar
  25. 25.
    Kuroda H, Fujihara K, Takano R, Takai Y, Takahashi T, Misu T, Nakashima I, Sato S, Itoyama Y, Aoki M (2013) Increase of complement fragment C5a in cerebrospinal fluid during exacerbation of neuromyelitis optica. J Neuroimmunol 254(1–2):178–182PubMedCrossRefGoogle Scholar
  26. 26.
    Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR (2005) IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med 202(4):473–477PubMedCrossRefGoogle Scholar
  27. 27.
    Lucchinetti CF, Mandler RN, McGavern D, Bruck W, Gleich G, Ransohoff RM, Trebst C, Weinshenker B, Wingerchuk D, Parisi JE, Lassmann H (2002) A role for humoral mechanisms in the pathogenesis of Devic’s neuromyelitis optica. Brain 125(Pt 7):1450–1461PubMedCrossRefGoogle Scholar
  28. 28.
    Mader S, Gredler V, Schanda K, Rostasy K, Dujmovic I, Pfaller K, Lutterotti A, Jarius S, Di Pauli F, Kuenz B, Ehling R, Hegen H, Deisenhammer F, Aboul-Enein F, Storch MK, Koson P, Drulovic J, Kristoferitsch W, Berger T, Reindl M (2011) Complement activating antibodies to myelin oligodendrocyte glycoprotein in neuromyelitis optica and related disorders. J Neuroinflammation 8:184PubMedCrossRefGoogle Scholar
  29. 29.
    Manderson AP, Botto M, Walport MJ (2004) The role of complement in the development of systemic lupus erythematosus. Annu Rev Immunol 22:431–456PubMedCrossRefGoogle Scholar
  30. 30.
    Misu T, Fujihara K, Kakita A, Konno H, Nakamura M, Watanabe S, Takahashi T, Nakashima I, Takahashi H, Itoyama Y (2007) Loss of aquaporin 4 in lesions of neuromyelitis optica: distinction from multiple sclerosis. Brain 130(Pt 5):1224–1234PubMedCrossRefGoogle Scholar
  31. 31.
    Nayak A, Pedneka L, Reid KB, Kishore U (2012) Complement and non-complement activating functions of C1q: a prototypical innate immune molecule. Innate Immun 18(2):350–363PubMedCrossRefGoogle Scholar
  32. 32.
    Papadopoulos MC, Verkman AS (2012) Aquaporin 4 and neuromyelitis optica. Lancet Neurol 11(6):535–544PubMedCrossRefGoogle Scholar
  33. 33.
    Parker C (2009) Eculizumab for paroxysmal nocturnal haemoglobinuria. Lancet 373(9665):759–767PubMedCrossRefGoogle Scholar
  34. 34.
    Phuan PW, Ratelade J, Rossi A, Tradtrantip L, Verkman AS (2012) Complement-dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein assembly in orthogonal arrays. J Biol Chem 287(17):13829–13839PubMedCrossRefGoogle Scholar
  35. 35.
    Pittock SJ, Lennon VA, McKeon A, Mandrekar J, Weinshenker BG, Lucchinetti CF, O’Toole O, Wingerchuk DM (2013) Eculizumab in AQP4-IgG-positive relapsing neuromyelitis optica spectrum disorders: an open-label pilot study. Lancet Neurol (in press)Google Scholar
  36. 36.
    Ratelade J, Zhang H, Saadoun S, Bennett JL, Papadopoulos MC, Verkman AS (2012) Neuromyelitis optica IgG and natural killer cells produce NMO lesions in mice without myelin loss. Acta Neuropathol 123(6):861–872PubMedCrossRefGoogle Scholar
  37. 37.
    Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R, Newman RA, Hanna N, Anderson DR (1994) Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 83(2):435–445PubMedGoogle Scholar
  38. 38.
    Ricklin D, Hajishengallis G, Lambris JD (2010) Complement: a key system for immune surveillance and homeostatis. Nat Immunol 11(9):785–797PubMedCrossRefGoogle Scholar
  39. 39.
    Ricklin D, Lambris JD (2013) Progress and trends in complement therapeutics. Adv Exp Med Biol 735:1–22PubMedGoogle Scholar
  40. 40.
    Risitano AM (2013) Paroxysmal nocturnal hemoglobinuria and the complement system: recent insights and novel anticomplement strategies. Adv Exp Med Biol 735:155–172PubMedGoogle Scholar
  41. 41.
    Roemer SF, Parisi JE, Lennon VA, Benarroch EE, Lassmann H, Bruck W, Mandler R, Weinshenker BG, Pittsock SJ, Wingerchuk DM, Lucchinetti CF (2007) Pattern-specific loss of aquaporin-4 immunoreactivity distinguishes neuromyelitis optica from multiple sclerosis. Brain 130(Pt 5):1194–1205PubMedCrossRefGoogle Scholar
  42. 42.
    Saadoun S, Waters P, Bell BA, Vincent A, Verkman AS, Papadopoulos MC (2010) Intra-cerebral injection of neuromyelitis optica immunoglobulin G and human complement produces neuromyelitis optica lesions in mice. Brain 133(Pt 2):349–361PubMedCrossRefGoogle Scholar
  43. 43.
    Saadoun S, Waters P, MacDonald C, Bell BA, Vincent A, Verkman AS, Papadopoulos MC (2012) Neutrophil protease inhibition reduces neuromyelitis optica-immunoglobulin G-induced damage in mouse brain. Ann Neurol 71(3):323–333PubMedCrossRefGoogle Scholar
  44. 44.
    Sabater L, Giralt A, Boronat A, Hankiewicz K, Blanco Y, Llufriu S, Alberch J, Graus F, Saiz A (2009) Cytotoxic effect of neuromyelitis optica antibody (NMO-IgG) to astrocytes: an in vitro study. J Neuroimmunol 215:31–35PubMedCrossRefGoogle Scholar
  45. 45.
    Sato D, Callegaro D, Lana-Peixoto MA, Fujihara K (2012) Treatment of neuromyelitis optica: an evidence based review. Arq Neuropsiquiatr 70(1):59–66PubMedCrossRefGoogle Scholar
  46. 46.
    Schäfer MK, Schwaeble WJ, Post C, Salvati P, Calabresi M, Sim RB, Petry F, Loos M, Weihe E (2000) Complement C1q is dramatically up-regulated in brain microglia in response to transient global cerebral ischemia. J Immunol 164:5446–5452PubMedGoogle Scholar
  47. 47.
    Tam SH, Sassoli PM, Jordan RE, Nakada MT (1998) Abciximab (ReoPro, chimeric 7E3 Fab) demonstrates equivalent affinity and functional blockade of glycoprotein IIb/IIIa and ανβ3 integrins. Circulation 98(11):1085–1091PubMedCrossRefGoogle Scholar
  48. 48.
    Tradtrantip L, Asavapanumas N, Verkman AS (2013) Therapeutic cleavage of anti-aquaporin-4 autoantibody in neuromyelitis optica by an IgG-selective proteinase. Mol Pharmacol (in press)Google Scholar
  49. 49.
    Tradtrantip L, Ratelade J, Zhang H, Verkman AS (2013) Enzymatic deglycosylation converts pathogenic neuromyelitis optica anti-aquaporin-4 immunoglobulin G into therapeutic antibody. Ann Neurol 73(1):77–85PubMedCrossRefGoogle Scholar
  50. 50.
    Tradtrantip L, Zhang H, Anderson MO, Saadoun S, Phuan PW, Papadopoulos MC, Bennett JL, Verkman AS (2012) Small molecule inhibitors of NMO-IgG binding to aquaporin-4 reduce astrocyte cytotoxicity in neuromyelitis optica. FASEB J 26:2197–2208PubMedCrossRefGoogle Scholar
  51. 51.
    Tradtrantip L, Zhang H, Saadoun S, Phuan PW, Lam C, Papadopoulos MC, Bennett JL, Verkman AS (2012) Anti-aquaporin-4 monoclonal antibody blocker therapy for neuromyelitis optica. Ann Neurol 71(3):314–322PubMedCrossRefGoogle Scholar
  52. 52.
    Tüzün E, Li J, Saini SS, Yang H, Christadoss P (2008) Targeting classical complement pathway to treat complement mediated autoimmune diseases. Adv Exp Med Biol 632:265–272PubMedGoogle Scholar
  53. 53.
    Van Herle K, Behne JM, Van Herle A, Blaschke TF, Smith TJ, Yeaman MR (2012) Integrative continuum: accelerating therapeutic advances in rare autoimmune diseases. Annu Rev Pharmacol Toxicol 52:523–547PubMedCrossRefGoogle Scholar
  54. 54.
    Vedove CD, Del Giglio M, Schena D, Girolomoni G (2009) Drug-induced lupus erythematosus. Arch Dermatol Res 301(1):99–105PubMedCrossRefGoogle Scholar
  55. 55.
    Wallis R, Mitchell DA, Schmid R, Schwaeble WJ, Keeble AH (2010) Paths reunited: initiation of the classical and lectin pathways of complement activation. Immunobiol 215:1–11CrossRefGoogle Scholar
  56. 56.
    Wilde B, van Paassen P, Witzke O, Tervaert JW (2011) New pathophysiological insights and treatment of ANCA-associated vasculitis. Kidney Int 79:599–612PubMedCrossRefGoogle Scholar
  57. 57.
    Wingerchuk DM (2013) Neuromyelitis optica: potential roles for intravenous immunoglobulin. J Clin Immunol 33(Suppl 1):S33–S37PubMedCrossRefGoogle Scholar
  58. 58.
    Wingerchuk DM, Lennon VA, Lucchinetti CF, Pittock SJ, Weinshenker BG (2007) The spectrum of neuromyelitis optica. Lancet Neurol 6(9):805–815PubMedCrossRefGoogle Scholar
  59. 59.
    Wingerchuk DM, Lennon VA, Pittock SJ, Lucchinetti CF, Weinshenker BG (2006) Revised diagnostic criteria for neuromyelitis optica. Neurology 66(10):1485–1489PubMedCrossRefGoogle Scholar
  60. 60.
    Zipfel PF, Mihlan M, Skerka C (2007) The alternative pathway of complement: a pattern recognition system. Adv Exp Med Biol 598:80–92PubMedCrossRefGoogle Scholar
  61. 61.
    Zipfel PF, Skerka C (2009) Complement regulators and inhibitory proteins. Nature Rev Immunol 9:729–740Google Scholar
  62. 62.
    Zhang H, Bennett JL, Verkman AS (2011) Ex vivo spinal cord slice model of neuromyelitis optica reveals novel immunopathogenic mechanisms. Ann Neurol 70(6):943–954PubMedCrossRefGoogle Scholar
  63. 63.
    Zhang H, Verkman AS (2013) Eosinophil pathogenicity mechanisms and therapeutics in neuromyelitis optica. J Clin Invest 123(5):2306–2316PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Puay-Wah Phuan
    • 1
  • Hua Zhang
    • 1
  • Nithi Asavapanumas
    • 1
  • Michael Leviten
    • 2
  • Arnon Rosenthal
    • 2
  • Lukmanee Tradtrantip
    • 1
  • A. S. Verkman
    • 1
  1. 1.Departments of Medicine and PhysiologyUniversity of CaliforniaSan FranciscoUSA
  2. 2.Annexon, IncPalo AltoUSA

Personalised recommendations