Immune Modulation for Prevention of Type 1 Diabetes Peter A. Gottlieb, MD Barbara Davis Center University of Colorado Health Sciences Center Denver, CO

Main Points Type 1 diabetes is an autoimmune disease It is a predictable disease with different phases Approaches to prevention and cure are possible. New insulins, medications and devices will improve therapy in the coming decade.

       NK B Th1 MO Tc1 Target Effector Cells CD4CD25 Th2 NKT Th3 Tr1 Regulatory Cells Cellular Mechanics of Autoimmune Type 1 Diabetes

Progression to Diabetes vs Number of Autoantibodies (GAD, ICA512, Insulin) Percent not Diabetic Years of Follow-up 3 Ab n = Abs n = Abs n = Verge et al, Diabetes 45: , 1996 BDC

Type 1a Diabetes: An Autoimmune Disorder Autoantibodies to islet proteins: insulin, GAD 65, IA-2 (ICA512)Autoantibodies to islet proteins: insulin, GAD 65, IA-2 (ICA512) T cell responses to islet proteinsT cell responses to islet proteins HLA associationHLA association Immunosuppressive drugs can ameliorate the disorder – ex. CyclosporineImmunosuppressive drugs can ameliorate the disorder – ex. Cyclosporine Recurrence of autoimmunity in pancreas transplants between monozygotic twinsRecurrence of autoimmunity in pancreas transplants between monozygotic twins

Prevention of Type 1 diabetes Primary: 1autoimmunity 1b-cell loss 1clinical diabetes Prevention of Type 1 diabetes Primary: 1autoimmunity 1b-cell loss 1clinical diabetes Genetic susceptibility Autoimmunity No autoimmunity Clinical diabetes Complications Clinical remission 1c Secondary 1a 1b  -cell loss Tertiary ?

Secondary Prevention XGoal - induction of diabetes remission and preservation of C-peptide Xnon-antigen-specific interventions Xantigen specific interventions

EDIC: Long Term Benefit of Intensive Treatment -The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group. N Engl J Med 2000;342:381-9.

EDIC: Long Term Benefit of Intensive Treatment -The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group. N Engl J Med 2000;342:381-9.

 -Cell Function and Complications in the Diabetes Control and Complications Trial - Steffes MW, et al. Diabetes Care 26:832–836, 2003

 -Cell Function and Hypoglycemia in the Diabetes Control and Complications Trial - Steffes MW, et al. Diabetes Care 26:832–836, 2003

Past Trials in New Onset Type 1 DM Cyclosporine A - no lasting effect Imuran- no lasting effect Corticosteroids- no lasting effect Plasmapheresis- no lasting effect BCG (Denver) - no effect Nicotinamide (DENIS)- no effect ( At risk ) Gluten-free diet (Italy)- no effect Q fever vaccine s.c. - no effect Nicotinamide and Vitamin E- no effect

Metabolic Effects of AZA and Prednisone at 1 year in New Onset T1DM - Silverstein, et al. NEJM 1988, 319:

Accelerated Loss of C-Peptide with BCG Vaccination at Onset < 12 >=12 Age Fasting C-Peptide Stimulated C-Peptide Adapted from Allen, et al, Diabetes Care 1999, 22:

Ongoing and Proposed Non-antigen Specific Immunotherapy Trials in New Onset Type 1 DM MMF and DZB - Peter Gottlieb, TrialNet Multidose anti-CD3 hOKT3 - Kevan Herold, NY; Lucienne Chatenoud, France HSP 65 p277 s.c. - (Peptor) – Jerry Palmer, Seattle Multi-dose DZB - Henry Rodriguez, Indiana Exanitide and Rapamycin – David Harlan, NIH Oral hIFN-alpha - Staley Brod, Texas Anti-CD20 – Mark Peskovitz, Indiana, TrialNet ATG (Sandostat) – Steve Gitelman, UCSF, ITN, TrialNet Rapamycin and IL-2, Alex Rabinovitch, Canada Fish oil - A-G Ziegler, Germany Diazoxide - E Bjork+A Karlsson, Sweden Lisofylline i.v. - S Kirk, Virginia Vitamin E+nicotinamide - P Pozzilli, Italy

Preservation of Pancreatic Production of Insulin (POPPI) Participating Centers The Barbara Davis Center Indiana University Stanford University University of Florida University of Minnesota Virginia Mason (Washington) Joslin Diabetes Center Columbia University UCSF Children’s Hospital of Los Angeles Toronto, Canada Milan, Italy and Munich, Germany New Centers Existing Centers

Preservation of Pancreatic Production of Insulin (POPPI) study (Mycophenolate Mofetil and Zenapax) MMF protects BB rats from developing DM and is effective in islet allograft transplantation in mice MMF effective in a number of autoimmune conditions and in transplantation DZB effective as part of Edmonton protocol and in other transplantation regimens Anti-IL2R Ab protects BB rat, but not NOD islet grafts IL2-Receptor + Cells increased at diagnosis of DM IL-2R+, CD4hi population harbors autoreactive T cells (mouse and man) Relative known toxicities of drugs are low

Mycophenolate Mofetil (MMF) Inhibits inosine monophosphate dehydrogenase (IMPDH) Inhibits de novo pathway of guanosine nucleotide synthesis –T and B cells need de novo pathway (other cell types use salvage pathway)

APC T cell MHC TCR IL-2 Blocking of activated T and B cell proliferation and antibody formation Does not block IL-1, IL-2 production MMF: Mode of action Greenbaum, C Benaroya Research Institute Seattle, WA

MMF (CellCept) An immunosuppressive medication that is most commonly used to prevent transplant rejection. Capsules, taken by mouth, twice a day for two years. Most common side effects: Diarrhea, vomiting, nausea, decreased white blood cells.

MMF Toxicities LeukopeniaLeukopenia GastrointestinalGastrointestinal Increased rate of viral infectionsIncreased rate of viral infections Lymphoproliferative disorder? No increase in multidrug regimens. No increase in single drug use (Psoriasis).Lymphoproliferative disorder? No increase in multidrug regimens. No increase in single drug use (Psoriasis). Cancer? (Psoriasis data – No).Cancer? (Psoriasis data – No).

Daclizumab (Zenapax) Humanized IgG monoclonal antibody that binds to the alpha subunit (CD25, p55alpha, Tac) of IL-2 receptor on the surface of activated lymphocytes Greenbaum, C Benaroya Research Institute Seattle, WA

Activated T cell α γ IL-2 ß Activated T cell α γ IL-2 ß DZB DZB: Mode of action Inhibit IL-2 mediated activation of lymphocytes Greenbaum, C;Benaroya Research Institute; Seattle, WA

DACLIZUMAB (DZB) An immunosuppressive medication, commonly used in patients receiving kidney transplants. IV infusion, twice during the study (Baseline and Week 2). Most common side effects: Diarrhea, vomiting, nausea, decreased white blood cells.

No increase in most frequently reported AEs in Zenapax-treated patients* % of Patients with Adverse Event Safety Results Tremor Headache Pain Posttraumatic Wound Healing Insomnia Musculoskeletal Pain * Pooled data. Placebo (n=293) Zenapax (n=336)

Eligibility Criteria Age 12-35Age New onset within 12 weeks of diagnosisNew onset within 12 weeks of diagnosis Presence of autoantibodies: GAA, IAA, ICA512 or ICAPresence of autoantibodies: GAA, IAA, ICA512 or ICA No evidence for Hepatitis B, C or HIVNo evidence for Hepatitis B, C or HIV No major illnesses or use of other immunosuppressive agentsNo major illnesses or use of other immunosuppressive agents

POPPI Study 3 arm study: MMF alone, MMF and 2 doses of DZB and placebo3 arm study: MMF alone, MMF and 2 doses of DZB and placebo 60 subjects per arm, 180 total, through TrialNet centers (6 initially)60 subjects per arm, 180 total, through TrialNet centers (6 initially) Type 1 diabetes (autoantibodies) within 12 weeks of diagnosisType 1 diabetes (autoantibodies) within 12 weeks of diagnosis Ages 12-35, without significant other diseaseAges 12-35, without significant other disease Outcomes: HbA1c, C-peptide, hypoglycemia, T cell assaysOutcomes: HbA1c, C-peptide, hypoglycemia, T cell assays Start Date: July 27, patients enrolled.Start Date: July 27, patients enrolled.

Potential Benefits of the Study Patient will be the most important part of a research team that is attempting to learn more about type 1 diabetes. Diabetes may be easier to manage. Less chance for long-term complications of diabetes.

Anti-CD3 Monoclonal Antibody in New-Onset Type 1 Diabetes Mellitus Kevan C. Herold, MD; William Hagopian, MD, PhD; Julie A. Auger, BA; Ena Poumian-Ruiz, BS; Lesley Taylor, BA, David Donaldson, MD; Stephen E. Gitelman, MD, David M. Harlan, MD; Danlin Xu, PhD; Robert A. Zivin, PhD; & Jeffrey A. Bluestone, PhD Herold K. et al., N Engl J Med 2002; 346:

hOKT3  1(Ala-Ala) Ala-Ala Binds to CD3 hOKT3  1(Ala-Ala) is a monoclonal antibody that binds to the CD3 (T cell receptor) on human T cells. The drug is a “humanized” antibody with a mutation in the Fc chain to prevent binding to the Fc receptor. Binding to the Fc receptor and crosslinking of the CD3 molecule is thought to activate T cells, cause release of cytokines, and account for the toxicity of OKT3.

Changes from Study Entry to 12 Months in the Total C-Peptide Response to Mixed-Meal Tolerance Testing Herold K. et al., N Engl J Med 2002; 346: Total Area under the C-Peptide Response Curve (nmol/l/4 hr) Monoclonal-Antibody Group Total Area under the C-Peptide Response Curve (nmol/l/4 hr) Control Group

A single course of h  (Ala-Ala) at dx of diabetes improves insulin secretion for over 2 years ** (p<0.0001**p<0.02)

Hemoglobin A1c levels in Drug treated and Control Subjects P< by RPANOVA ** ** p<0.01, * p<0.1 * *

Before treatment1 wk after treatment Induction of IL-10+CD4+ cells in vivo following Treatment with hOKT3g1(Ala-Ala) IL-10+, IFN-g- CD45RO+ CTLA-4- Some TGF-b+ CCR4+

The Ratio of CD4+ and CD8+ T-Cells in the Monoclonal-Antibody Group According to the Presence or Absence of a Response to Treatment Herold K. et al., N Engl J Med 2002; 346:

CFSE hOKT3g1(Ala-Ala) induces proliferation of CD8+ T cells in vitro CD4CD8 PHA hOKT3  (Ala-Ala) 5 ug/ml

CD25 CD3 engagement and signaling Postulated Induction of CD8+ regulatory T cells by hOKT3γ1(Ala-Ala) CD8 CD4 CD8+ T cell proliferation Inhibition of antigen-reactive CD4+ cells

Antigen Specific Therapy Magic bullet Approach Targets autoreactive cells Generates protective cells Spares rest of immune system Minimal Toxicity Timing may be critical to efficacy

Insulin Beta Cell Specific Predominant T-cell reactivity islets NOD Insulin expressed lymphoid tissue by dendritic and macrophage-like cells Thymic messenger RNA for insulin related to “protective” insulin allele Proinsulin expression in thymus prevents NOD diabetes

Effect of Insulin Injections on Diabetes & Insulitis Female NOD Mice Atkinson, Diabetes 1991

Prevention of Diabetes with B:9-23 Peptide “Immunization” Age in Weeks Percent Not Diabetic Tetanus control B:9-23 peptide D.Daniel,D.Wegmann. PNAS,1996

Altered Peptide Ligand APC T cell MHC TCR IFNγ APC T cell MHC TCR IL-4 Greenbaum, C;Benaroya Research Institute; Seattle, WA

Efficacy of NBI-6024 in animal models with ‘new onset’ Type I diabetes. Figure 2. NBI-6024 Treatment of NOD mice in a Preventive Paradigm Figure 3. NBI-6024 Treatment of NOD mice Near Onset of Disease

NBI New Onset Trial Altered Peptide Ligand B:9-23 Double-blind Phase I/II trial to test safety and efficacy of an altered insulin peptide ligandDouble-blind Phase I/II trial to test safety and efficacy of an altered insulin peptide ligand Forty Patients (12-40) were randomized to receive 5 doses of NBI-6024 (0.1mg, 1.0 mg, or 5.0mg) or placebo biweekly and monthly.Forty Patients (12-40) were randomized to receive 5 doses of NBI-6024 (0.1mg, 1.0 mg, or 5.0mg) or placebo biweekly and monthly. No side effects. No obvious benefit to subjects, but study was primarily a safety study.No side effects. No obvious benefit to subjects, but study was primarily a safety study. Evidence of immunologic effect on T cells was observed in a dose dependent fashion in adolescent groups particularly.Evidence of immunologic effect on T cells was observed in a dose dependent fashion in adolescent groups particularly.

ELISPOT analysis of Th1 and Th2 Responses to Insulin B(9-23)();NBI-6024 () or Medium only (); 0.1 mg Adolescent Tx’ed Patients from NBI ELISPOT analysis of Th1 and Th2 Responses to Insulin B(9-23) (▲); NBI-6024 ( ) or Medium only ( ); 0.1 mg Adolescent Tx’ed Patients from NBI  IFN-  IL-5 Patient #OPatient #P IFN-  IL-5 IFN  IL-5 IL-5

Recent and Ongoing Antigen-specific Immunotherapy Trials in T1DM Joslin Parenteral Insulin: “Delay” Schwabing Parenteral Insulin: “Delay” DPT-1 Parenteral:No Effect DIPP (intranasal):? Melbourne (intranasal):? DPT-1 Oral Insulin: Possible for subgroup Italy/France Oral Insulin:No Effect Maclaren Oral Insulin: ? NBI Neurocrine, BDC No effect B chain – Orban, Joslin? hGAD s.c. in alum (Diamyd) 20ug dose only Peptor Heat Shock Protein? Prediabetes New Onset

PREVENTION

Primary Prevention Xautoantibodies or diabetes as the endpoint Xavoidance of environmental agents ? Xinduction of autoantigen tolerance ? Rewers-BDC

Natural History of Type 1 Diabetes CELLULAR (T CELL) AUTOIMMUNITY LOSS OF FIRST PHASE INSULIN RESPONSE (IVGTT) (IVGTT) GLUCOSE INTOLERANCE (OGTT) HUMORAL AUTOANTIBODIES (ICA, IAA, Anti-GAD 65, IA 2 Ab, etc.) PUTATIVEENVIRONMENTALTRIGGER CLINICALONSET TIME BETA CELL MASS DIABETES “PRE”- DIABETES GENETIC PREDISPOSITION INSULITIS BETA CELL INJURY

Primary Prevention Trials DPT-1 - Parenteral - Ineffective - Oral Insulin – May be effective in subgroup DIPP - Nasal Insulin INIT - IntraNasal Insulin Trial ENDIT - Nicotinamide - Ineffective TRIGR - Casein Hydrolysate (Cow’s Milk Elimination) NIP - Nutritional Intervention to Prevent T1DM

DPT-1 Parenteral Study – Time to Diabetes By Treatment Number at Risk Survival Distribution Function P- Value= (Log Rank Test) InterventionObservation Years Followed STRATA: Intervention Observation Control Treated New Engl J Med 2002; 346:1679

ENDIT: Kaplan-Meier failure curve - European Nicotinamide Diabetes Intervention Trial (ENDIT) Group Lancet 2004; 363: 925–31

Rationale for Oral Insulin T H1 Cell s IFN- , IL-2 Destructive Cytokines T H2 Cells IL-4, IL-5, IL-10 TGF-  T H3 Cells Protective Cytokines

Oral Antigen Protocol Initial results appeared to suggest no effect of oral insulinInitial results appeared to suggest no effect of oral insulin Secondary analysis suggests that for original cohort there is delay in onset compared to placebo treated patiens.Secondary analysis suggests that for original cohort there is delay in onset compared to placebo treated patiens. A new trial to confirm these observations is being planned by TrialNetA new trial to confirm these observations is being planned by TrialNet Additional arms including intranasal insulin are being consideredAdditional arms including intranasal insulin are being considered

Nutritional Intervention to Prevent Type 1 Diabetes (NIP – Diabetes) Plan: Use of an omega 3 fatty acid (Docosahexanoic acid or DHA) to prevent the initial autoimmune process. DHA supplementation will begin in: the last trimester of pregnancy the first 6 months after birth It will be continued in medium or high risk infants for 3 years.

Dietary Intake – Western Diets The Ratio of n-6 to n-3 Fatty Acids in our diet: 1800’s = 1 or 2 (n-6) to 1 (n-3) Present = 20 or 30 (n-6) to 1 (n-3) High n-3: anti-inflammatory anti-thrombotic hypolipidemic vasodilatory (High n-6 has the opposite effect) (Am J. Clin Nutr. 70, , 1999)

III) Mechanisms of Action of Omega 3 Fatty Acids  Decrease AA in cell membranes  alters PGE 1 and 2 production (inflammatory prostaglandins)  Decrease pro-inflammatory cytokines TNF , IL-1 and IL6 (  efficacy of IL4 and IL10)  Decrease ICAM-1 on monocyte surfaces in humans fed 3g fish oil/dx 21 days (  chronic inflammation)  DHA and /or vit D may have important immune modulating effects in babies at risk for developing T1DM

Mechanistic Studies  Biomarker: Omega-3 FA will be measured  Reduction of inflammation CRP (  with DHA) (Am J Cardiol 88:1139,2001) inflammatory PGs: PGE-1 and 2 Inflammatory cytokines: IL-1 and IL-6  Islet cell antibodies

TrialNet Sites

TrialNet International Sites AustraliaAustralia United KingdomUnited Kingdom FinlandFinland Italy & GermanyItaly & Germany

NIDDKNIAIDNICHDNCRR ADA JDRF Sponsors

Type 1 Diabetes TrialNet Protocol Development Procedure Stage 1: Concept Proposal Stage 2: Draft Protocol Stage 3: Full Protocol Prioritization & Implementation

Types of Trials Phase 1 – Safety and/or Proof of Concept Phase 2 – New-Onset Diabetes Phase 2 – Prevention of Diabetes Natural History & Epidemiology

TrialNet Interventions New-Onset Diabetes –Anti-CD3 (via ITN collaboration) –Mycophenolate Mofetil +/- Anti-CD25 –Anti-CD20 –IL-2 plus Sirolimus – Phase 1 Safety Study Relatives At Risk –Natural History –Oral Insulin –Beta Cell Preservation (exenatide) – pilot study Newborns –Nutritional : Omega-3-Fatty Acids

Other TrialNet Studies Comparison of Mixed Meal Tolerance Test and Glucagon Stimulation Test for Stimulation of C- Peptide Reproducibility and Validation of T-Cell Assays for Monitoring of Diabetes Intervention Trials Collaboration with Type 1 Diabetes Genetics Consortium (T1DGC)

Key Elements of Successful Clinical Trials Prospective Randomized Controlled Statistical power Objective endpoints Risk/benefit to individual Cost benefit to society

What We Need Proven biomarkers for disease progression or improvement Better mechanistic assays Better rationale for moving potential interventions to RCTs The courage to study interventions with potential adverse side effects

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