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.
Cellular Mechanics of Autoimmune Type 1 Diabetes
Target
NK
b
CD4CD25
b
b
b
b
Tc1
b
Effector Cells
b
B
MO
Tr1
Th1
Th2
Th3
NKT
Regulatory Cells
Progression to Diabetes vs Number of Autoantibodies
(GAD, ICA512, Insulin)
Percent not Diabetic
100
80
3 Abs
2 Abs
1 Ab
60
40
20
0
0
2.5
5
7.5
10
12.5
15
Years of Follow-up
3 Ab n = 41
2 Abs n = 44
1 Abs n = 93
17
27
23
8
15
14
Verge et al, Diabetes 45:926-933, 1996
1
4
10
2
6
1
4
BDC
The Major Histocompatibility Complex
Class III
Class II
Human
Chromosome 6
DP
DQ
Antigen Processing
Genes
Mouse
Chromosome 17
Class I
Class II
K
I-A
DR
B
Complement
Proteins
Cytokines
Class III
I-E
Class I
C
A
Class I-like genes
and pseduogenes
Class I
D
L
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
Prevention of Type 1 diabetes
Primary:
1autoimmunity
1b-cell loss
1clinical diabetes
Complications
Tertiary
Autoimmunity
1b
b-cell loss
1c
1a
Genetic
susceptibility
Clinical
diabetes
Secondary
No autoimmunity
?
Clinical
remission
Secondary Prevention
 Goal - induction of diabetes remission
and preservation of C-peptide
 non-antigen-specific interventions
 antigen 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.
b-Cell Function and Complications in the
Diabetes Control and Complications Trial
- Steffes MW, et al. Diabetes Care 26:832–836, 2003
b-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
Imuran
Corticosteroids
Plasmapheresis
BCG (Denver)
Nicotinamide (DENIS)
Gluten-free diet (Italy)
Q fever vaccine s.c.
Nicotinamide and Vitamin E
- no lasting effect
- no lasting effect
- no lasting effect
- no lasting effect
- no effect
- no effect (At risk)
- no effect
- no effect
- no effect
Metabolic Effects of AZA and Prednisone
at 1 year in New Onset T1DM
Treated
Control
Significance
7.2 (20)
8.6 (20)
NS
Total Ins.
Ins. Indep.
0.42+0.27
15%
0.66+0.24
0%
P<0.001
NS
C-peptide
0.52nmol/ 0.26nmol/
L
L
50%
15%
P<0.03
HbA1c
Responder
(HbA1c<6.8)
- Silverstein, et al. NEJM 1988, 319:599-604
NS
Accelerated Loss of C-Peptide
with BCG Vaccination at Onset
Fasting C-Peptide
Age
Stimulated C-Peptide
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0.8
0.6
< 12
0.4
0.2
0
0
>=12
5
10
15
20
25
0
5
10
15
20
25
30
0
5
10
15
20
25
30
30
1.4
1.2
1
0.8
0.6
0.4
0.2
0
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
Adapted from Allen, et al, Diabetes Care 1999, 22:1703-07
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
Existing Centers
• The Barbara Davis
Center
• Indiana University
• Stanford University
• University of Florida
• University of
Minnesota
• Virginia Mason
(Washington)
New Centers
• Joslin Diabetes
Center
• Columbia University
• UCSF
• Children’s Hospital of
Los Angeles
• Toronto, Canada
• Milan, Italy and
Munich, Germany
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
MMF: Mode of action
• Blocking of activated T and B cell
proliferation and antibody formation
T cell
• Does not block IL-1, IL-2 production
IL-2
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
•
•
•
•
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).
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
DZB: Mode of action
Inhibit IL-2 mediated activation of lymphocytes
IL-2
IL-2
DZB
α ß γ
α ß γ
Activated T cell
Activated T cell
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.
Safety Results
No increase in most frequently reported AEs in
Zenapax-treated patients*
Musculoskeletal Pain
Insomnia
Wound Healing
Placebo (n=293)
Pain Posttraumatic
Zenapax (n=336)
Headache
Tremor
0
20
40
60
80
% of Patients with Adverse Event
* Pooled data.
100
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.
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; 346:1692-8.
hOKT3g1(Ala-Ala)
Binds to CD3
Ala-Ala
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.
Changes from Study Entry to 12 Months in the Total
C-Peptide Response to Mixed-Meal Tolerance Testing
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)
Monoclonal-Antibody Group
Herold K. et al., N Engl J Med 2002; 346:1692-8.
A single course of hOKT3g1(Ala-Ala) at dx of
diabetes improves insulin secretion for over 2
years
160
140
**
Drug
Control
**
**
AUC (pmol/ml/240min)
120
100
80
(p<0.0001
**p<0.02)
60
40
20
0
0
6
12
Month
18
24
Hemoglobin A1c (%)
Hemoglobin A1c levels in Drug treated
and Control Subjects
9.5
8.5
7.5
**
*
**
*
Drug treated
6.5
5.5
4.5
Control
0
6
12
Month
P<0.0001 by RPANOVA
** p<0.01, * p<0.1
18
24
Induction of IL-10+CD4+ cells in vivo following
Treatment with hOKT3g1(Ala-Ala)
Before treatment
1 wk after treatment
IL-10+, IFN-gCD45RO+
CTLA-4Some TGF-b+
CCR4+
hOKT3g1(Ala-Ala) induces proliferation of CD8+
T cells in vitro
CD8
CD4
hOKT3g1(Ala-Ala)
5 ug/ml
PHA
CFSE
Postulated Induction of CD8+ regulatory T cells by
hOKT3γ1(Ala-Ala)
CD3 engagement
and signaling
CD8+ T cell proliferation
CD25
CD8
CD8
CD8
CD8
CD25
CD8
CD8
Inhibition of
antigen-reactive
CD4+ cells
CD4
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
100
90
80
70
60
50
40
30
20
10
0
3
2.5
Insulitis Score
% Diabetes
Female NOD Mice
2
1.5
1
0.5
0
Placebo
Insulin
Placebo
Insulin
Atkinson, Diabetes 1991
Prevention of Diabetes with B:9-23
Peptide “Immunization”
Percent Not Diabetic
100
B:9-23 peptide
80
Tetanus control
60
40
20
0
0
10
20
30
40
50
60
Age in Weeks
D.Daniel ,D.Wegmann . PNAS,1996
APC
Altered Peptide Ligand
T cell
APC
T cell
IFNγ
IL-4
Greenbaum, C;Benaroya Research Institute; Seattle, WA
NBI 6024-003 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 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.
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-6024-0003
500
800
Patient #O
IFN-g
600
IFNg
Patient #P
450
700
IFN-g
400
IFNg
350
500
300
400
250
200
300
150
200
100
100
50
0
0
0
5
10
15
20
Weeks After Initiation
25
0
30
10
15
20
Weeks After Initiation
25
30
10
15
20
Weeks After Initiation
25
30
100
70
IL-5
90
60
IL-5
5
IL-5
80
70
50
IL-5
40
60
50
30
40
20
30
20
10
10
0
0
5
10
15
20
Weeks After Initiation
25
30
0
0
5
New Onset
Prediabetes
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
?
PREVENTION
Primary Prevention
autoantibodies or diabetes as the
endpoint
avoidance of environmental agents ?
induction of autoantigen tolerance ?
Rewers-BDC
Natural History of Type 1 Diabetes
PUTATIVE
ENVIRONMENTAL
TRIGGER
CELLULAR (T CELL) AUTOIMMUNITY
BETA CELL MASS
HUMORAL AUTOANTIBODIES
(ICA, IAA, Anti-GAD65, IA2Ab, etc.)
LOSS OF FIRST PHASE
INSULIN RESPONSE
(IVGTT)
GLUCOSE INTOLERANCE
GENETIC
PREDISPOSITION
INSULITIS
BETA CELL INJURY
(OGTT)
“PRE”DIABETES
CLINICAL
ONSET
DIABETES
TIME
Primary Prevention Trials
• DPT-1
- Parenteral - Ineffective
- Oral Insulin – May be effective in
subgroup
• DIPP
• INIT
- Nasal Insulin
- 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
1.0
Survival Distribution Function
0.9
0.8
0.7
0.6
Treated
0.5
0.4
Control
0.3
P- Value= 0.796
(Log Rank Test)
0.2
Number at Risk
0.1
169
170
0.0
0
144
131
96
101
69
69
39
40
13
14
1
2
3
4
5
1
6
Years Followed
New Engl J Med 2002; 346:1679
STRATA:
Intervention
Observation
Intervention
Observation
7
ENDIT: Kaplan-Meier failure curve
- European Nicotinamide Diabetes Intervention Trial (ENDIT) Group
Lancet 2004; 363: 925–31
Rationale for Oral Insulin
TH1
TH2
TH3
Cell
s
Cells
Cells
IFN-g, IL-2
Destructive
Cytokines
IL-4, IL-5, IL-10 TGF-b
Protective
Cytokines
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
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, 560-569, 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
• Australia
• United Kingdom
• Finland
• Italy & Germany
Sponsors
NIDDK
ADA
NIAID
NICHD
JDRF
NCRR
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 CPeptide
• 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
1-800-HALT-DM1 (1-800 – 425-8361)
www.diabetestrialnet.org