IMMUNOTHERAPIE DU DIABETE AUTO-IMMUN INSERM U1013 INSTITUT UNIVERSITAIRE DE FRANCE
Natural history of type 1 diabetes b cell function (%) Time
AN ALARMING EPIDEMIOLOGICAL TREND EURODIAB registered 29 311 new cases of T1D in children before 15 years of age between 1989-2003. The overall annual increase was 3.9%, New cases in Europe in 2005 : 15 000 (24%: 0-4 years, 35%: 5-9 years, and 41%: 10-14 years). In 2020, the predicted number of new cases is 24 400, with a doubling in numbers in children younger than 5 years. Prevalence under age 15 years is predicted to rise from 94 000 in 2005, to 160 000 in 2020. Doubling of new cases of type 1 diabetes in children younger than 5 years and prevalent cases younger than 15 years will rise by 70%. Patterson et al. Lancet 2009, 373, 1999
40 PLACEBO-CONTROLLED TRIAL OF CYCLOSPORIN A IN RECENTLY DIAGNOSED IDDM (CDF study) EFFECT ON REMISSION RATE Remission (%) 30 20 10 CsA (n = 155) 0 3 6 9 12 15 18 24 Placebo (n = 127) Months G. Feutren et al. Lancet 1986; 2: 119-124
1 year p=0,0195 RITUXIMAB IN T1D Over time p< 0,038 One course 4 doses (375 mg/m 2 ) Over time p< 0,004 M. Pescovitz et al. New Engl J Med 2009, 361: 2143-2152
TWO STRATEGIES TO INDUCE IMMUNE TOLERANCE 1. Administration of b-cell autoantigens 2. Use of monoclonal antibodies mediating T cell signaling
AUTOANTIGEN TRIALS Antigen Trial Reference Oral Insulin New onset T1D Chaillous L et al. The Lancet 356, 545, 2000 Oral Insulin New onset T1D Pozilli P et al. Diabetologia 43, 1000, 2000 APL insulin B chain (NBI-6,024) New onset T1D Walter M et al. Diabetes Care 32, 2036, 2009 Hsp60 p277 New onset T1D Raz I et al. The Lancet 358, 1749, 2001 i.v. + s.c. insulin DPT-1 Oral insulin DPT1 Intranasal insulin Late preclinical T1D Insulin-autoantibody positive relatives Insulin-autoantibody positive relatives Skyler J et al. New Engl J Med 346, 1685, 2002 Skyler J et al. New Engl J Med 346, 1685, 2002 Näntö-Salonen K et al. The Lancet 372, 1746, 2008 Alum formulated GAD Recent onset T1D Ludvigsson J et al. New Engl J Med 359, 1909, 2008
CD3 antibody therapy - Expresses the unique ability to restore selftolerance in established autoimmunity - The therapeutic effect initially demonstrated in the experimental setting was successfully transferred to the clinic - The mode of action involves ' resetting ' of active tolerance
ACTIVE TOLERANCE Tolerance requiring active contribution of antigen-specific activated T cells Related concepts dominant tolerance infectious tolerance immunoregulation T-cell mediated suppression
HETEROGENEITY OF CD4 + REGULATORY T CELLS CD8 + T cell CD4 + CD8 + CD4 + CD25 +high Foxp3 + CD4 + CD25 +high Foxp3 + CD4 + T cell Tr1 IL-10/ TGF-b CD4 + CD25 - Foxp3 - Systemic Ag Th2 IL-4/ IL-10 Th3 TGF-b Oral Ag THYMUS IN AUTOIMMUNE PRONE INDIVIDUALS Ag Stimulation tolerogenic context CD4 + CD25 +low Foxp3 + CD4 + CD25 - Foxp3 - Cell contact TGF-b, GITR, CTLA-4 Ag Stimulation Pro-inflammatory context - - - CD4 + Th1 Effector CD25 - Foxp3 - - -
CD25 % Diabetes Both CD4 + CD25 high and CD4+CD25 low T cells are suppressive in vivo in NOD mice 100 CD4 + CD25 high CD4 + CD25 low CD4 + CD25-80 60 40 20 Diab Diab/CD25 low Diab/CD25 high CD4 0 2 4 6 8 10 Weeks after transfer
REMISSION OF ESTABLISHED DIABETES IN CD3 ANTIBODY-TREATED NOD MICE 1 0 0 % r e m i s s i o n 8 0 6 0 4 0 A n t i - C D 3 C o n t r o l s ( n = 20 ) p < 0. 001 ( n = 16 ) p < 0. 005 2 0 ( n = 1 7 ) 0 ( n = 1 6 ) 2 4 6 8 1 0 1 2 W e e k s T r e a t m e n t ( d 0 - d 5 ) L. Chatenoud et al. Proc.Natl.Acad.Sci. USA 1994; 91: 123-127
-Belghith M et al. Nat Med 9:1202-1208, 2003 -You S et al. Immunol Rev 212:185-202, 2006 - You S et al. (2007) Proc Natl Acad Sci (U S A) 104: 6335-6340, 2007 -Chatenoud L & Bluestone JA. Nat Rev Immunol 7:622-632, 2007 -You S et al. Adv Immunol 100: 13-37, 2008
Mean Clinical Score Mean Clinical Score Mean Clinical Score THERAPY OF ONGOING R-EAE USING CD3 ANTIBODIES 2.5 2 1.5 1 0.5 0 3.5 3 2.5 2 1.5 1 0.5 0 Day 0-4 Treatment Control acd3 F(ab')2 Rx 3.2 2.6 0 2 4 6 8 10 11 13 14 15 17 Days Post Disease Induction Day 6-10 Treatment Day 13-17 Treatment Control acd3 F(ab')2 Rx 2.2 0.4 0 2 4 6 8 10 11 13 14 15 17 Days Post Disease Induction 3.5 3 2.5 2 1.5 1 0.5 0 Control acd3 F(ab')2 1.2 2.8 2.6 Rx 1.6 0.4 2.6 0.6 0 4 8 11 14 17 21 25 29 33 Days Post Disease Induction
Humanized YTH 12.5 (CAMPATH 3) Reshape d Rat YTH 12.5 higg1 Mutation Asn to Ala position 297 (prevents glycosylation) S. Bolt et al European J Immunol, 1993, 23, 403
Treatment of established Type 1 diabetes with a non-mitogenic CD3 antibody Phase II trial Multicenter Placebo Controlled Between 07/06/2000 and 07/03/2003 210 consecutive recent onset diabetic patients were screened 80 patients included 12-39 years old. positive for ICA and/or GAD Abs insulin-treatment <4 weeks 8mg/day of ChAglyCD3 for 6 consecutive days EBV IgG positive. Keymeulen B. N. Engl. J. Med 2005, 352:2598-2608
% IU/kg/day Insulin dose 0.8 0.7 n=27 n=29 n=31 n=31 ChAglyCD3 0.6 n=28 Placebo 0.5 0.4 0.3 0.2 n=31 n=33 n=31 n=33 n=33 n=32 n=31 n=32 n=33 0.1 p=0.004 p<0.001 p=0.001 p=0.001 0.0 0 6 12 18 24 36 48 Months 9 HbA1c 8 7 6 p=0.761 p=0.984 p=0.990 p=0.428 5 0 6 12 18 24 36 48 Months Keymeulen B. Diabetologia 2009, 53: 614-623
Insulin dose U/Kg/day 12 MONTHS AFTER TREATMENT HbA1C % 6 6.5 7 7.5 NS NS NS* * 0.25 NS NS 0.01 0.01 NS NS NS 0.5 NS NS NS 0.75 NS NS: non significant difference between placebo and CD3 antibody-treated patients (Chi Square) Data from European Phase II trial using otelixizumab (Keymeulen B. N. Engl. J. Med 2005, 352:2598-2608 )
Phase III Study : teplizumab Lancet. 2011; 378 :487-497
GENETIC PREDISPOSITION LOSS OF BETA CELL FUNCTION GENETIC PREDISPOSITION GENETIC PREDISPOSITION GENETIC PREDISPOSITION AUTOIMMUNE DIABETES MULTIPLE SCLEROSIS BETA CELL AUTOANTIBODIES BENIGN CD3 mab Therapeutic Window BETA CELL AUTOANTIBODIES RELAPSING REMITTING DISABILITY BETA CELL AUTOANTIBODIES SECONDARY CHRONIC PROGRESSIVE BETA CELL AUTOANTIBODIES PRIMARY CHRONIC PROGRESSIVE OVERT HYPERGLYCEMIA
counts counts mcd8 mcd3 Transgenic Human CD3e-NOD mice thymus spleen NOD NOD-huCD3e NOD NOD-huCD3e 17 <1 2 14 44 1 4 43 hucd3 82 <1 82 2 54 <1 53 <1 2 85 2 87 13 <1 13 <1 mcd4 3 10 3 8 55 32 55 31 mcd3 M1 NOD 38% MFI=142 NOD-huCD3e 40% MFI=72 hucd3 M1 human PBMCs 75% MFI=244 NOD-huCD3e 36% MFI=119
% remission % diabetes % infiltrated islets 100 80 60 40 20 NOD NOD-huCD3e (n = 40) (n = 32) 0 0 5 10 15 20 25 30 35 40 weeks of age 100 80 60 40 20 0 6-wk 12-wk diabetic Otelixizumab islets with invasive infiltration islets with peripheral infiltration islets w/o infiltration 100 80 (n = 26) 60 (n = 23) 40 20 0 anti-cd3 NOD + F(ab ) 2-2C11 NOD-huCD3e + Otelixizumab 5 10 15 20 weeks after treatment
6-wk 12-wk diabetic Otelixizumab merge CD3 FoxP3 Insulin merge
SPU / 10 6 cells SPU / 10 6 cells % diabetes 100 80 60 40 20 CD25 - CD62L - diabetic CD25 - CD62L - Otelixizumab 0 0 2 4 6 8 weeks after transfer IGRP 206-214 PI B:15-23 800 SPLEEN 30 SPLEEN 600 400 200 20 10 0 6wk 16wk diab 2wk >8wk remission 0 6wk 16wk diab 2wk >8wk remission 600 BLOOD 300 BLOOD 400 200 200 100 0 6wk 16wk diab 2wk >8wk remission 0 6wk 16wk diab 2wk >8wk remission
BEFORE TREATMENT CD3Ab TREATMENT AFTER TREATMENT
CONCLUSIONS CD3 antibodies are effective at preserving b cell function and decreasing insulin needs in newly diagnosed Type 1 diabetic patients The effect is long lasting Patients are responsive to unrelated antigens CD3 antibodies induce/ expand TGF-b dependent adaptive Tregs These results are a proof of concept and pave the way for the use of CD3 antibodies as tolerance-promoting agents in various T cell-mediated autoimmune diseases, in transplantation and cell therapies
Incidence of prototype infectious diseases and immune disorders % 100 Hepatitis A % 300 Asthma IDDM 50 Rheumatic fever Tuberculosis 200 Crohn's disease Measles 0 100 Multiple sclerosis 55 65 75 85 95 55 65 75 85 95 Infectious diseases Immune disorders JF Bach, N Engl J Med, 2002
Incidence of IDDM (per 100,000) Incidence of multiple sclerosis (per 100,000) 9.4 1.9 (56-74) 35.8 (87-99) 3.3 19.7 24 (77-85) 3.78 5.3 19.9 3.94 13.7 7.7 1.69 24 7.3 (66-89) 8.05 (54-86) 0.96 (mortality or incidence) 2.2 11.5 (67-90) 1.1 10.9 (85-88) 2.24 5 1.72
Type 1 Diabetes (incidence in children 0 14 years) Incidence per 100,000 children (0 14years) High (> 16) Intermediate (8 16) Low (0 8) No estimate Adapted from : www.eatlas.idf.org
Multiple Sclerosis Prevalence per 100,000 (2007) High (> 60) Intermediate (20-60) Low (0 20) No estimate Adapted from : www.atlasofms.org
Childhood Asthma Proportion of clinical asthma * High (> 10%) Intermediate (5.1% 10%) Low (0% 5%) No estimate Adapted from : Masoli et al. Allergy. 2004:59
Hepatitis A virus antibodies prevalence Risk level High Intermediate Low Adapted from : wwwn.cdc.gov
IDDM incidence in children of migrants from Pakistan to Yorkshire 12 10 Incidence of 8 diabetes /105 6 4 2 0 Pakistan Migrant's children Yorkshire Staines A. (1997) and Bodansky H.J. (1992)
CAUSAL LINK
PREVENTION OF IDDM IN NOD MICE BY INFECTIOUS AGENTS Bacteria Viruses Parasites streptococci salmonella mycobacteria (CFA, BCG, ) LCMV MHV LDHV schistosoma pimworms
% diabetes TGF-b antibody treatment abrogates diabetes protection by OM-85 60 40 20 0,,,,,, 3 7 1 15 19 23 27 Age (weeks) OM-85 anti-tgf-b NOD controls,,,,,,,,,,,,,, NOD OM-85 + anti-tgf-b NOD OM-85
Immune response of dendritic cell to TLR agonists Endotoxins or LPS Mycobacterial soluble factor Proteine MMLV Mannan Fibrinogen HSP70, HSP60 Hyaluronan Beta-defensin Surfactant Protein A Lipoproteins Zymosan P. Gingivalis LPS Lipoteichoic acid Lipoarabinomannan HCMV Trypanosoma cruzi GPI KOmpA HSP70 TLR4 Flagellin TLR2 TLR5 v v lipopeptide TLR6 v MyD-88 upec TLR11 v v NF-kB AP-1 v TRIF TLR3 IRF3 v v ssarn Poly(I:C) v v TLR7 TLR8 v v IFN TLR9 Imidazoquinoline dsarn Imidazoquinoline Bacterial DNA (CpG) Herpes simplex virus 2 Lipopeptide TLR1
Diabetes incidence (%) TLR2, TLR3,TLR4 and TLR7 agonists delay autoimmune diabetes onset TLR2 TLR3 TLR4 TLR7 80 70 60 50 40 30 20 10 0 Non treated P40 p=0.0064 0 5 10 15 20 25 Treatment 100 90 80 70 60 50 40 30 20 10 0 Non treated Poly(I:C) 5 15 25 p=0.00045 80 70 60 50 40 30 20 10 p=0.0085 0 5 10 15 20 25 Age (weeks) Non treated LPS 90 80 70 60 50 40 30 20 10 0 Non treated R848 p=0.0143 5 10 15 20 25 Similar protective results were obtained with TLR agonists : synthetic lipopetide Pam3CSK4 (TLR2 agonist) or dsrna Poly(I)Poly(C)
PROTECTION BY TLR AGONISTS AND IMMUNE REGULATION p <0.05 p <0.05 p <0.05 Thieblemont N et al. PLoS ONE 2010, 5(7): e11484
ALLERGIC ASTHMA IN NOD MICE D0 Immunization 1, 10, 100 µg OVA + Alum i.p./ s.c. D7, D8, D9 OVA challenge D10 Measurement of airway hyperreactivity to metacholine Sacrifice and collection of Sera (cytokines, IgE) BALFs (cells, cytokines) Lungs (cytokines)
Eosinophils in BAL 10 5 /ml p< 0.005 p< 0.005 OVA Araujo LM et al. Eur.J.Immunol. 2004, 34: 327
BALF cell count (10 5 ) Lung IL-4 level (pg/ml) Penh Lung eotaxin level (pg/ml) Prevention of allergic asthma by a TLR2 agonist 9.0 7.5 6.0 4.5 3.0 1.5 Saline/Vehicle OVA/Vehicle Saline/P40 OVA/P40-2 -1 0 1 2 3 4 5 6 7 8 9 28 21 14 7 0 Eosinophils Macrophages Saline OVA P40-treated Saline Neutrophils Lymphocytes OVA Vehicle 6000 p=0,0357 4500 3000 1500 0 OVA Saline OVA Saline P40-treated Vehicle-injected 200 p=0,0736 150 100 50 0 OVA Saline OVA Saline P40-treated Vehicle