Developing Immunotherapy for Autoimmune Diseases Premkumar Christadoss, M.B.B.S. Department of Microbiology and Immunology University of Texas Medical Branch 301 University Blvd. Galveston, Texas 77555-1070 pchrista@utmb.edu

Generalized Myasthenia Gravis

MG

Neuromuscular Junction (NMJ) Conti-Fine, B.M. et al. J. Clin. Invest. (116) 2843-22854, 2006

NMJ in MG Normal MG

Electronmicroscopy Study of NMJ of an MG Patient Engel et al. Mayo clinic proc. 52:267, 1977

AChR is a transmembrane glycoprotein formed by five homologous subunits in the stoichiometry a2bgd or a2bed. The molecular weights of the subunits range between 45 and 55 kDa. The a subunit is Considered to be the highly immunogenic region.

Primary immunization: 20 microgram AChR/CFA EAMG induction AChR Source Primary immunization: 20 microgram AChR/CFA 28-30 days Boost: 20 microgram AChR/CFA 28-30 days Monitor for clinical EAMG Immunopathological evaluation

MG in Mice Normal MG

Molecular Mechanisms of EAMG Damage to the neuromuscular junction Complement activation AChR APC IL-1, IL-12 CD4 Class II Peptide (a146-162) CD4 TCR B7 CD 28 CD40L/CD40 AChR-specific memory T cell Proliferation and Differentiation IL-10, TNF-a, IL-6, IL-12, IL-18 AChR-specific memory B cells AChR-Ab Plasma cells NK IFN-g IL-18 B

COMPLEMENT PATHWAYS C1 C4b C2a MASP1 MASP2 MBL C3 C5 MAC (C5bC9) C3b CLASSICAL PATHWAY C1 C4b C2a MASP1 MASP2 MBL C3 C5 MAC (C5bC9) MBL PATHWAY C3b Bb C3bBb ALTERNATIVE PATHWAY

AChR-immunized C3-/- and C4-/- mice are resistant to clinical EAMG Tuzun -Christadoss. J. Immunol. 171:3847, 2003

Serum anti-AChR antibody levels of AChR-immunized mice Tuzun- Christadoss. J. Immunol. 171:3847, 2003

RED  -bungarotoxin binding (NMJ) GREEN  C3, MAC or IgG deposits IF Studies Reveal IgG Deposits But Not C3 or MAC Deposits at the NMJs of Mice with C3 or C4 Deficiency C4+/+ C4-/- C3-/- C3 MAC IgG RED  -bungarotoxin binding (NMJ) GREEN  C3, MAC or IgG deposits Tuzun –Christadoss J. Immunol. 171:3847, 2003

Immune Intervention Disease specific Antigen/organ specific I. AChR T cell epitope tolerance II. AChR B cell epitope tolerance Disease specific I. Anti-Proinflammatory Cytokine a. Soluble TNFR (etanercept) b. IL1-Ra c. Anti-IL-6 II. Blocking classical complement pathway a. Anti C1q/C2/C4

Targeting Classical Complement Pathway

Anti-C1q Administration Suppresses EAMG Tuzun-Christadoss, J.Neuroimmunol.174:157-167, 2006

Dual Effect of Anti-C1q Tuzun-Christadoss, J.Neuroimmunol.174:157-167, 2006

Anti-C1q Ab Treats EAMG B6 RIII Tuzun-Christadoss, J.Neuroimmunol, 182: 167-176, 2007

Anti-C1q Ab Treatment Suppresses AChR and Dominant Peptide Specific IL-6 Production

Effect of Cytokine Deficiencies in Clinical EAMG Normal IL-4 IL-10 Gene Depletion IFN-g Anti-AChR Ab Disease IL-12 IL-18 IL-6 TNF-a p55p75 20 40 60 80 100 120 % clinical disease and anti-AChR antibodies

IL-6 and TNF in EAMG Th Th B B TNF TNF AChR specific IL6 IL-6 GC formation Activation of B cells and generation of effector B cells. Potentiates production of IgG anti-AChR antibodies (pathogenic) B B Activates C3 Promotes EAMG pathology

Targeting ProinflammatorCytokines A. Soluble Recombinant HumanTNFR (Etanercept)

Soluble TNFR (Etanercept) Treats EAMG Christadoss and Goluszko J. Neuroimmunol, 122:186, 2002

Etanercept Treatment Fails to Suppress Serum Anti-AChR Ab Christadoss and Goluszko, J. Neuro. Immunol. 122:186, 2002.

A pilot Trial of Etanercept in the Treatment of Steroid-Dependent MG *+ Mean change in QMG score from basline at 6 months was - 2.9 (p=0.041). Mean change in MMT at 6 months was - 8.4 (p=0.020). Mean decrease in prednisone dose from baseline to end of study was 17.5 mg/48 hr dose (p=0.0084). Etanercept was well tolerated, and no severe adverse reaction observed + 11 patients enrolled; 8 completed, and 2 patients withdrawn due to disease worsening.

Immunological Effect of Etanercept No reduction in plasma anti-AChR antibody. Peripheral blood CD4 and B cell (CD19+) counts rose steadily during the 24 week study. Patients who had higher increases in their cytokine levels had a worse outcome.

Targeting ProinflammatorCytokines b. Recombinant Human IL1-Ra

Activation of the Adaptive Immune System with IL-1

IL-1Ra Treatment Prevents Clinical EAMG stopped Yang –Christadoss, J. Immunol. 175:2018, 2005

IL-1Ra Treats EAMG Yang –Christadoss, J. Immunol. 175:2018, 2005 P<0.05 P<0.05 P<0.05

Possible Consequence of Down Regulating IL-1 by IL-1Ra in Mice with Clinical EAMG CD40L, OX40 Expression on T cells Inflammatory cytokines IFN-g, IL-2, IL-1, IL-6, TNF-a Anti-AChR IgG , IgG1 and C3 Anti-AChR antibodies and complement mediated NMJ pathology

IL-6 in MG: Multiple Hit AChR Specific NMJ CD4 IL-6 B IgG2b -C1q C4-C3- C5-9 C3

IL-6 – A Danger Molecule in EAMG ! 1. IL-6 deficient mice are resistant to EAMG and produce less C3 2. C3 and FCγRIII deficient mice are resistant to EAMG and produce less IL-6 3. Amelioration of EAMG following anti-C1q treatment is associated with reduced IL-6

Anti-IL-6 Ab Treatment Reduces the Incidence of EAMG Days after second immunization

Anti-IL-6 Ab Treatment Suppresses Serum Anti-AChR IgG and IgG2b Ab

Anti-IL-6 Ab Treatment Suppresses AChR Specific Cytokine Production

Classical Complement Pathway and IL-6 in EAMG Pathogenesis anti-AChR antibody production T helper B cell Antigen presenting cell AChR IL-6 C3 Immune complex formation AChR C1q C1r C1s C1q FcγRIII activation Classical complement pathway activation Stimulation of IL-6, C1q and C3 production Membrane attack complex formation

Balancing the Immune System to Treat Autoimmune Disease (MG) IL-6, TNF C3-C5b-C9 Anti-AChR IgG IL-6, TNF-normal level Healthy Suppress anti-AChR and C5b-C9

Targeting IL-6 and Classical Complement Pathway Suppressed anti-AChR antibody production T helper B cell Antigen presenting cell AChR IL-6 C3 Immune complex formation AChR C1q C1r C1s C1q Classical complement pathway activation FcγRIII activation Stimulation of IL-6, C1q and C3 production Membrane attack complex formation

MG lab in Galveston

MG Lab - Galveston Collaborators Huan Yang Bo Wu Stephen Higgs Erdem Tuzun1,2 Shamsher Saini Andrey Bednov Ben Scott 3 Jing Li1 Iris Wingrow3 Huibin Qi Xiarong Wu Collaborators Huan Yang Bo Wu Stephen Higgs Tian Lin Xio Galen Kaufmann Mat Merigioli Juli Rowin 1MG foundation Osserman/Sosin/McClure Post doctoral Fellows 1,2MDA Research Career Award Recipients 3 MG Foundation Henry Viets Fellow Supported by NIH,MDA, AFM,and MG Foundation