1 © DeveloGen AG Prevention Of Type 1 Diabetes Mellitus: Current Status And Future Directions ITAMAR RAZ Hadassah University Hospital Jerusalem Israel

2 © DeveloGen AG Foreward on Type 1A Diabetes Mellitus …  A chronic progressive autoimmune disease  Inflammatory response targeted specifically at beta cells in the islets of Langerhans

3 © DeveloGen AG Preventing the Progression of Diabetes in Its Early Stages = Interfering with the autoimmune disease

4 © DeveloGen AG LOSS OF FIRST PHASE INSULIN RESPONSE TIME Stages in Development of Type 1 Diabetes BETA CELL MASS DIABETES “PRE”- DIABETES GENETIC PREDISPOSITION INSULITIS BETA CELL INJURY NEWLY DIAGNOSED DIABETES MULTIPLE ANTIBODY POSITIVE GENETICALLY AT RISK Modified from G. Eisenbarth, NEJM, 1986

5 © DeveloGen AG Initial Trials …  Prednisone  Prednisone with Azathioprine  Anti Thymocyte Globulin  Cyclosporine A Elliot et al Schernthaner et al Bougneres et al FAILED

6 © DeveloGen AG Treatment of Autoimmune Diabetes: Rationale  Autoimmune diabetes results from destruction of the insulin-producing pancreatic beta-cells.  Beta-cell destruction is mediated by cellular immunity.  Autoimmune diabetes can be stopped by immunological intervention (NOD mice, BB rats, Cys-a clinical studies)  The immunological intervention should be:  Effective  Safe  Disease-specific

7 © DeveloGen AG GAD b Insulin APC NKT T TCR TLR-2 T TCR TLR-2 T CELL MODULATORY APPROACH CyclosporinA Prednisone Azathioprine Cytokines (IL-4, IL-10) Anti CD3Ab Peptide MHC dimers Anti-CD3Ab MHC dimers AUTOANTIGEN VACCINATION Insulin GAD Diapep277 INNATE SYSTEM MODULATION a Galactosylceramide Peptide277(Diapep277) GAD vaccine GAD Insulin B9-23 Al p277 p p a galactosy l-ceramide Diapep 277 Pro-inflammatory effect Anti-inflammatory effect Immune modulatoryagent

8 © DeveloGen AG Immune Modulation in Type 1 Diabetes More questions than answers…

9 © DeveloGen AG Proliferative T-cell Responses Age of mice (weeks) Stimulation index (log)

10 © DeveloGen AG  Gad?  Heat shock protein 60 (HSP60)?  Insulin B9-23 peptide ?

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13 © DeveloGen AG DPT-1 - Kaplan-Meier Curves according to Treatment-Group Assignment NEJM 346:1685, 2002

14 © DeveloGen AG Diabetes Prevention Trial-type 1 (CONT)  CONCLUSION : IN PERSONS AT HIGH RISK, INSULIN AT THE DOSES AND DELIVERY USED IN THIS TRIAL DOES NOT DELAY OR PREVENT TYPE1 DIABETES.

15 © DeveloGen AG Insulin B9-23  Diabetes Prevention Trial-1 1  Sub cutaneous Insulin B chain peptide B: (in NOD mice)  vaccination with DNA encoding peptide B: (in NOD mice) 1. Pozzilli et al 2. Liu et al; Ramiya et al 3. Urbanek-Ruiz et al

16 © DeveloGen AG Glutamic Acid decarboxylase ١ reduction of severity of insulitis and prevention of onset of diabetes in NOD mice was achieved with: ٢ Injection of GAD65 1 ٢ Injection of GAD 67 (isoform) 2 ٢ Anti GAD monoclonal antibodies 3 ٢ GAD-plasmid DNA/DNA vaccine/GAD antisense 4 ٢ A Vaccinia virus expressing GAD 5 ٢ GAD derived peptides 6 1. Ramiya et al; Pleau et al; Petersen et al; Tisch et al 2. Elliott et al 3. Menard et al 4. Yoon et al;Balasa et al; Li et al 5. Jun et al 6. Tisch et al;Sai et al

17 © DeveloGen AG Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. “ a GAD-expressing transgenic plant, given as a dietary supplement, inhibits the development of diabetes in the NOD mouse …” Ma et al. Nat Med. 1997

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20 © DeveloGen AG Low dose linomide in Type I juvenile diabetes of recent onset: a randomised placebo-controlled double blind trial.  42 patients with recent onset diabetes received low dose Linomide for a year.  After 1 year  HbA1C was significantly lower  Insulin requierments where significantly lower  A higher C peptide value after 6 months (in patients with residual C peptide at trial entry) Coutant R Saint-Vincent-de-Paul Hospital, Paris,France. Diabetologia. 1998

21 © DeveloGen AG An immunomodulatory effect? TH-2 cell TH-1 cell IL-4 IL-10 IL-4 IL-10 GAD65 hOKT31 linomide P 277

22 © DeveloGen AG T cell Proliferative Responses to hsp60 P<0.001 P<0.01 Proliferation Index

23 © DeveloGen AG Stressed  -cell Stressed  -cell Hsp60 Promotes Inflammation in  -cell Autoimmunity GAD Th1 IL-12 IL-12 CTL hsp60 Macrophage Destruction of  -cells by autoimmune responses Autoimmune disease TNF  NO IFN  ; TNF  Cell lysis hsp60 insulin

24 © DeveloGen AG And to complicate things…  T cells reactive to HSP60 have been shown to be of the immunomodulatory type TH2  T cells that recognize HSP confer immunity in rat models of arthritis and mouse models of diabetes 1 1. Anderton et al; van Haltren et al; van Eden et al

25 © DeveloGen AG 60 kDa Heat Shock Protein (HSP60) HSP60 p277 Peptide p277: HSP60 positions VLGGGVALLRVIPALDSLTPANED Targeted by T-cells from diabetic NOD mice and Type I IDDM patients. Vaccination with p277 arrests NOD diabetes

26 © DeveloGen AG HSP60 effects macrophages and T cells: Pro- vs Anti-inflammation HSP60 macrophages endothelial cells T cells Activates pro-inflammatory cytokines and matrix metalloproteinase Up-regulates expression of adhesion molecules TLR-4 dependent signaling Activates adhesion to FN and intracellular signaling Down-regulates chemokine receptors and chemotaxis Inhibits IFN  TLR-2 dependent signaling p277

27 © DeveloGen AG LigandCellTLREffect on Inflammation HSP60 MTMT 4242 Up-regulation Down-regulation p277T2Down-regulation Innate Regulation of Inflammation by HSP60 and p277

28 © DeveloGen AG P277-specific clone Th2 IL4 IL10 IL13 Long term effect Activated cells are more sensitive T-cell receptor TLR2 P277-specific clone p277 Effects of p277 on p277-specific T-cells Promotion of cell adhesion Inhibition of migration Modulation of cytokine secretion upon activation

29 © DeveloGen AG DiaPep277 Immunomodulation  - cell IL-1, NO IFN  Th1 T-cell MM IL-4IL-10 Th2 T-cell MM P277

30 © DeveloGen AG DiaPep277 treatment prevents the progression of diabetes in NOD mice Treatment (arrow) of NOD mice with DiaPep277 or Control

31 © DeveloGen AG Insulin Secreting Cells Preserved by Treatment with DiaPep277 The pancreata of 20 weeks old NOD mice were stained with anti-insulin antibodies (8 weeks after administration of treatment). ControlTreatment

32 © DeveloGen AG DiaPep277 reduces IFN  secretion by islet infiltrating T-lymphocytes None p278 DiaPep277 Vehicle 

33 © DeveloGen AG Dosing Scheme for Studies 420 and 431

34 © DeveloGen AG Full Preservation of Endogenous Insulin Secretion in DiaPep277 Treated Patients (study 420) * * * P<0.05  Endogenous insulin secretion tested by I.V. 1mg glucagon stimulation.  AUC calculated for time points 0-20min.  Two-tailed unpaired t-test.

35 © DeveloGen AG Change in Daily Insulin Dose

36 © DeveloGen AG Percentage of patients with %HbA 1c < 7.5

37 © DeveloGen AG Partial Preservation in Extension Study: Need for Continuous Treatment  After a 6-18 no treatment period, patients were re-randomized  Patients on continued DiaPep277 treatment showed the least drop in beta-cell function.

38 © DeveloGen AG Change in %HbA 1c

39 © DeveloGen AG Change in Daily Insulin Dose

40 © DeveloGen AG Study 441/451  Same inclusion/exclusion criteria as in study 420  A total of 64 patients randomized  Female patients included  Dose range: Placebo, 0.04, 0.2, 1 mg DiaPep277.  Low doses were found to be non-effective  The Placebo and 1mg DiaPep277 groups were analyzed in combination with study 420.

41 © DeveloGen AG Combined Analysis of Israeli Trials: Basal Fasting C-peptide is Significantly Preserved N=52

42 © DeveloGen AG Combined Analysis of Israeli Trials: Stimulated Endogenous Insulin Secretion is Preserved  DiaPep277-treated patients fully preserve glucagon-stimulated C-peptide secretion (AUC)

43 © DeveloGen AG Significant Difference in Beta-Cell Function between DiaPep277-Treated and Placebo  The change from baseline in stimulated C-peptide AUC was significant.

44 © DeveloGen AG Combined Analysis of Adult Phase II Studies: Beta-cell Preservation in upper Thertile p=0.04 p=0.000 p=0.21 p=0.06 p=0.56 p=0.50 High responders: Patienrs with high beta-cell reserve at start of treatment

45 © DeveloGen AG Opposite Trends in Glycemic Control of DiaPep277 and Placebo Treated Patients N=54  Overall glycemic control in all patients was good, average Hba1c=7.5%  The trend in DiaPep277-treated patients was for reduced HbA1c, in Placebo-treated for increased HbA1c.

46 © DeveloGen AG Kinetics of Th2 induced shift in DiaPep277-treated patients

47 © DeveloGen AG Change in Cytokine Profile in DiaPep277 Treated Newly Diagnosed T1D Adults

48 © DeveloGen AG Specific Intervention with DiaPep277 TM Re-establishes Balance in the Immune System HealthDiabetes T h1 T h2 T reg Th 1 T h2 T reg Rx DiaPep277 +-

49 © DeveloGen AG Conclusions  Good safety, not differences between Treatment and Placebo in clinical laboratory parameters, adverse events.  DiaPep277 was well tolerated, most common adverse event was an injection site pain & edema that resolved within 1-2 days.  DiaPep277 treatment preserved both basal and stimulated C- peptide secretion.  DiaPep277 treated patients also showed trend for improved glycemia at similar insulin requirement.