DESIGN AND IMPLEMENTATION

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

3
Cellular Mechanics of Autoimmune Type 1 Diabetes
Target
b
b
NK
b
Tc1
b
CD4CD25
Effector Cells
b
b
b
B
MO
Tr1
Th1
Th2
Th3
Regulatory Cells
NKT
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Progression to Diabetes vs Number of
Autoantibodies (GAD, ICA512, Insulin)
Percent not Diabetic
Years of Follow-up
3 Ab n 41 17 8 1 2
Abs n 44 27 15 4 2 1 1 Abs n
93 23 14 10 6 4
Verge et al, Diabetes 45926-933, 1996
BDC
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Type 1a Diabetes An Autoimmune Disorder

• Autoantibodies to islet proteins insulin, GAD
  65, IA-2 (ICA512)
• T cell responses to islet proteins
• HLA association
• Immunosuppressive drugs can ameliorate the
  disorder ex. Cyclosporine
• Recurrence of autoimmunity in pancreas
  transplants between monozygotic twins

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

• Goal - induction of diabetes remission and
  preservation of C-peptide
• non-antigen-specific interventions
• antigen specific interventions

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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
  2000342381-9.

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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
  2000342381-9.

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b-Cell Function and Complications in theDiabetes
Control and Complications Trial
- Steffes MW, et al. Diabetes Care 26832836,
2003
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b-Cell Function and Hypoglycemia in theDiabetes
Control and Complications Trial
- Steffes MW, et al. Diabetes Care 26832836,
2003
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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

14
Metabolic Effects of AZA and Prednisone at 1 year
in New Onset T1DM
- Silverstein, et al. NEJM 1988, 319599-604
15
Accelerated Loss of C-Peptide with BCG
Vaccination at Onset
Fasting C-Peptide
Stimulated C-Peptide
Age

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Adapted from Allen, et al, Diabetes Care 1999,
221703-07
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Ongoing and Proposed Non-antigen Specific
Immunotherapy Trials in New Onset Type 1 DM

• 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 BjorkA Karlsson, Sweden
• Lisofylline i.v. - S Kirk, Virginia
• Vitamin Enicotinamide - P Pozzilli, Italy

• 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

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Preservation of Pancreatic Production of Insulin
(POPPI) Participating Centers
Existing Centers
New Centers

• The Barbara Davis Center
• Indiana University
• Stanford University
• University of Florida
• University of Minnesota
• Virginia Mason (Washington)

• Joslin Diabetes Center
• Columbia University
• UCSF
• Childrens Hospital of Los Angeles
• Toronto, Canada
• Milan, Italy and Munich, Germany

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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

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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)

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MMF Mode of action

• Blocking of activated T and B cell proliferation
  and antibody formation
• Does not block IL-1, IL-2 production

IL-2
Greenbaum, C Benaroya Research Institute Seattle,
WA
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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.

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MMF Toxicities

• Leukopenia
• Gastrointestinal
• Increased rate of viral infections
• Lymphoproliferative disorder? No increase in
  multidrug regimens. No increase in single drug
  use (Psoriasis).
• Cancer? (Psoriasis data No).

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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
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DZB Mode of action
Inhibit IL-2 mediated activation of lymphocytes
IL-2
DZB
IL-2
?
a
?
a
ß
ß
Activated T cell
Activated T cell
Greenbaum, CBenaroya Research Institute
Seattle, WA
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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.

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Safety Results
No increase in most frequently reported AEs in
Zenapax-treated patients
Musculoskeletal Pain
Insomnia
Wound Healing
Pain Posttraumatic
Headache
Tremor
0
20
40
60
80
100
of Patients with Adverse Event
Pooled data.
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Eligibility Criteria

• Age 12-35
• New onset within 12 weeks of diagnosis
• Presence of autoantibodies GAA, IAA, ICA512 or
  ICA
• No evidence for Hepatitis B, C or HIV
• No major illnesses or use of other
  immunosuppressive agents

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POPPI Study

• 3 arm study MMF alone, MMF and 2 doses of DZB
  and placebo
• 60 subjects per arm, 180 total, through TrialNet
  centers (6 initially)
• Type 1 diabetes (autoantibodies) within 12 weeks
  of diagnosis
• Ages 12-35, without significant other disease
• Outcomes HbA1c, C-peptide, hypoglycemia, T cell
  assays
• Start Date July 27, 2005. 23 patients enrolled.

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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.

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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 3461692-8.
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hOKT3g1(Ala-Ala)
Binds to CD3
hOKT3g1(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.
Ala-Ala
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Changes from Study Entry to 12 Months in the
Total C-Peptide Response to Mixed-Meal Tolerance
Testing
Monoclonal-Antibody Group
Control Group
Total Area under the C-Peptide Response Curve
(nmol/l/4 hr)
Total Area under the C-Peptide Response Curve
(nmol/l/4 hr)
Herold K. et al., N Engl J Med 2002 3461692-8.
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A single course of hOKT3g1(Ala-Ala) at dx of
diabetes improves insulin secretion for over 2
years



(p 34
Hemoglobin A1c levels in Drug treated and Control
Subjects




P p 35
Induction of IL-10CD4 cells in vivo following
Treatment with hOKT3g1(Ala-Ala)
Before treatment
1 wk after treatment
IL-10, IFN-g- CD45RO CTLA-4- Some TGF-b CCR4
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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 3461692-8.
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hOKT3g1(Ala-Ala) induces proliferation of CD8 T
cells in vitro
CD4
CD8
hOKT3g1(Ala-Ala) 5 ug/ml
PHA
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Postulated Induction of CD8 regulatory T cells
by hOKT3?1(Ala-Ala)
CD3 engagement and signaling
CD8 T cell proliferation
CD25
Inhibition of antigen-reactive CD4 cells
CD8
CD8
CD8
CD8
CD4
CD25
CD8
CD8
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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

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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

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Effect of Insulin Injections on Diabetes
Insulitis
Female NOD Mice
Atkinson, Diabetes 1991
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Prevention of Diabetes with B9-23 Peptide
Immunization
100
B9-23 peptide
80
Tetanus control
60
Percent Not Diabetic
40
20
0
0
10
20
30
40
50
60
Age in Weeks
D.Daniel ,D.Wegmann . PNAS,1996
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Altered Peptide Ligand
Greenbaum, CBenaroya Research Institute
Seattle, WA
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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
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NBI 6024-003 New Onset TrialAltered Peptide
Ligand B9-23

• Double-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.
• 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.

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ELISPOT analysis of Th1 and Th2 Responses to
Insulin B(9-23) (?) NBI-6024 (?) or Medium only
( ) 0.1 mg Adolescent Txed Patients from
NBI-6024-0003
?
Patient O
Patient P
IFN-g
IFN-g
IFNg
IFNg
IL-5
IL-5
IL-5
IL-5
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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 6024-0003 - Neurocrine, BDC No effect
• B chain Orban, Joslin ?
• hGAD s.c. in alum (Diamyd) 20ug dose only
• Peptor Heat Shock Protein ?

Prediabetes
New Onset
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PREVENTION
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Primary Prevention

• autoantibodies or diabetes as the endpoint
• avoidance of environmental agents ?
• induction of autoantigen tolerance ?

Rewers-BDC
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Natural History of Type 1 Diabetes
CELLULAR (T CELL) AUTOIMMUNITY
LOSS OF FIRST PHASE INSULIN RESPONSE (IVGTT)
GLUCOSE INTOLERANCE (OGTT)
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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
• (Cows Milk Elimination)
• NIP - Nutritional Intervention to
  Prevent T1DM

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DPT-1 Parenteral Study Time to Diabetes By
Treatment
1.0
0.9
0.8
0.7
0.6
Treated
Survival Distribution Function
0.5
0.4
Control
0.3
P- Value 0.796 (Log Rank Test)
0.2
Number at Risk
0.1
169 170
144 131
96 101
69 69
39 40
13 14
Intervention Observation
1
0.0
0
1
2
3
4
5
6
7
Years Followed
Observation
STRATA
Intervention
New Engl J Med 2002 3461679
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ENDIT Kaplan-Meier failure curve
- European Nicotinamide Diabetes Intervention
Trial (ENDIT) Group Lancet 2004 363 92531
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Rationale for Oral Insulin
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Oral Antigen Protocol

• Initial 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.
• A new trial to confirm these observations is
  being planned by TrialNet
• Additional arms including intranasal insulin are
  being considered

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• 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.

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Dietary Intake Western Diets

• The Ratio of n-6 to n-3 Fatty Acids in our diet
• 1800s 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, 560-569, 1999)

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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

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Mechanistic Studies

• Biomarker Omega-3 FA will be measured
• Reduction of inflammation
• CRP (?with DHA)
• (Am J Cardiol 881139,2001)
• inflammatory PGs PGE-1 and 2
• Inflammatory cytokines IL-1 and IL-6
• Islet cell antibodies

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TrialNet Sites
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TrialNet International Sites

• Australia
• United Kingdom
• Finland
• Italy Germany

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Type 1 Diabetes TrialNet Protocol Development
Procedure

• Stage 1 Concept Proposal
• Stage 2 Draft Protocol
• Stage 3 Full Protocol
• Prioritization Implementation 

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Types of Trials

• Phase 1 Safety and/or Proof of Concept
• Phase 2 New-Onset Diabetes
• Phase 2 Prevention of Diabetes
• Natural History Epidemiology

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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

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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)

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Key Elements of Successful Clinical Trials

• Prospective
• Randomized
• Controlled
• Statistical power
• Objective endpoints
• Risk/benefit to individual
• Cost benefit to society

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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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