Complement Targeted Therapy for Myasthenia Gravis Erdem Tüzün, M.D. Department of Neurology Istanbul Faculty of Medicine, Istanbul University Istanbul, Turkey Premkumar Christadoss, M.D. Department of Microbiology and Immunology University of Texas Medical Branch 301 University Blvd. Galveston, Texas, USA

Complement system is involved in Myasthenia Gravis AChR-Ab + MuSK-Ab + ? Seronegative ? Experimental Autoimmune Myasthenia Gravis (EAMG) Active Immunization Passive Transfer Ocular EAMG model

Experimental Autoimmune Myasthenia Gravis AChR Source 1° immunization: 20mg TAChR/CFA per mouse s.c. 28 days 2° immunization: 20mg TAChR/CFA per mouse s.c. 28 days EAMG

Ocular muscles are rarely involved in this model Grade 0: Normal Grade 1: Muscle weakness following exercise Grade 2: Grade 1 symptoms at rest Grade 3: Moribund Ocular muscles are rarely involved in this model

Immunopathogenesis of EAMG NK? Class II Peptide (a146-162) CD4 TCR AChR-Ab producing cells Complement pathway activation IL-2 IFN-g IL-18 B7 CD 28 APC T helper Proliferation and Differentiation IL-10, IFN-g, TNF-a, IL-6, IL-12, IL-2, IL-18 AChR CD40L/CD40 IL-1, IL-12 C’ C’ AChR-specific memory B cells B cell AChR-specific memory T cell Damage to the neuromuscular junction

Complement on NMJ TAChR in CFA-BTx a TAChR in CFA-C3 CFA-BTx CFA-C3

Immunization with human-AChR -subunit induces ocular symptoms normal partial complete

Complement-antibody-clinical severity correlations C3 levels correlate with clinical severity of AChR-Ab-positive generalized MG patients with no treatment (Liu A, Muscle Nerve, 2009) MG patients with high AChR-Ab levels have lower C3 and C4 concentrations suggesting increased complement consumption (Romi F, J Neuroimmunol, 2005) Clinical grades Serum immune complex (OD) Grip strength (gr) Serum immune complex (OD) TAChR-immunized B6 mice TAChR-immunized RIIIS/J mice

C1 C4b C2a MASP1 MASP2 MBL C3 C5 MAC (C5bC9) C3b Bb C3bBb D COMPLEMENT PATHWAYS CLASSICAL PATHWAY C1 C4b C2a MASP1 MASP2 MBL C3 C5 MAC (C5bC9) MBL PATHWAY COMMON PATHWAY C3b Bb C3bBb D ALTERNATIVE PATHWAY Which pathway is involved in EAMG?

Complement KO mice are resistant to EAMG B6 imm. B6 imm. B6 imm. Lewis rats Passive B6 imm. B6 imm.

C1qrs C4b C2a C5a MASP1 MASP2 MBL C3 C5 MAC (C5bC6 C7C8C9) C3b Bb COMPLEMENT PATHWAYS CLASSICAL PATHWAY C1qrs C4b C2a C5a MASP1 MASP2 MBL C3 C5 MAC (C5bC6 C7C8C9) MBL PATHWAY C3b Bb C3bBb COMMON PATHWAY D ? ALTERNATIVE PATHWAY

Complement targeted therapy for EAMG Lewis rats Passive Lewis rats Passive Lewis rats Passive Lewis rats Passive Wistar rats Passive

Anti-C5 complement monoclonal antibodies Eculizumab  paroxysmal nocturnal hemoglobinuria Pexelizumab  coronary heart disease ! Risk for infections !

Can EAMG be treated by classical pathway inhibition? Tuzun E. et al, 2003 C4+/+ C4+/- C4-/- EAMG incidence Anti-AChR IgG C4+/+ C4-/- C4 KO mice display normal immune functions Normal cytokine production Normal lymphocyte proliferation Normal germinal center in spleen Normal lymph node cell counts Intact alternative pathway for host defense against microorganisms C3 IgG RED  -BTx binding GREEN  C3, or IgG deposits

Anti-C1q Prevents EAMG B6 mice 200 μg Day -7 200 μg Day -4 100 μg 2 times a week – 5 weeks AChR imm. AChR imm. Clinical incidence % IL-6 NMJ deposits pg/ml Anti-C1q Prevents EAMG Tuzun E. et al., 2006

EAMG mice treated with 10μg anti-C1q 2 times a week-4 weeks Anti-C1q enhances serum immune complex levels Clinical grades Anti-C1q induces glomerular C3 and IgG deposits Grip strengths Anti-C1q Treatment Prevents EAMG (Tuzun E. et al., 2007)

Complement targeted therapy for EAMG B6 imm. Lewis rats Passive Lewis rats Passive Lewis rats Passive Lewis rats Passive Wistar rats Passive

Other targets, double hit method? Prevention – 0.01 mg IL-1ra Prevention – 0.5 mg anti-IL-6 0.01 mg IL-1ra placebo 0.5 mg anti-IL-6 PBS Reduced serum C3 levels Reduced serum C1q levels

EAMG-resistant KO strains Reduced serum complement levels CD59 KO FcRIII KO Reduced NMJ complement deposits IFN- KO IL-5 KO ICOS KO

Non-AChR Ab mediated MG, alternative pathway Leite et al., 2008  complement activating IgG1 in anti-MuSK+ and seronegative MG Shiraishi et al., 2005  2 of 8 anti-MuSK+ patients display NMJ C3 deposits FBb *;p=0.045 * *

THANKS! UTMB Istanbul University Premkumar Christadoss Vuslat Yılmaz Saini Shamsher Huan Yang Benjamin Scott Elzbieta Goluszko Jing Li Istanbul University Vuslat Yılmaz Güher Saruhan-Direskeneli Feza Deymeer Piraye Oflazer Yeşim Parman