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Prevention Of Type 1 Diabetes Mellitus:
Current Status And Future Directions
ITAMAR RAZ
Hadassah University Hospital
Jerusalem Israel
© DeveloGen AG
1
Foreward on Type 1A Diabetes Mellitus …
 A chronic progressive autoimmune disease
 Inflammatory response targeted specifically
at beta cells in the islets of Langerhans
© DeveloGen AG
2
Preventing the
Progression of
Diabetes in Its
Early Stages
© DeveloGen AG
=
3
Interfering with the
autoimmune disease
Stages in Development of Type 1 Diabetes
GENETICALLY AT RISK
BETA CELL MASS
MULTIPLE ANTIBODY POSITIVE
LOSS OF FIRST PHASE
INSULIN RESPONSE
GENETIC
PREDISPOSITION
INSULITIS
BETA CELL INJURY
“PRE”DIABETES
DIABETES
TIME
Modified
© DeveloGen
AG from
NEJM, 1986
G. Eisenbarth,
NEWLY DIAGNOSED DIABETES
4
Initial Trials…
 Prednisone
 Prednisone with Azathioprine
 Anti Thymocyte Globulin
 Cyclosporine A
Elliot et al
Schernthaner
et al
© DeveloGen AG
Bougneres et al
5
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
© DeveloGen AG
6
AUTOANTIGEN
VACCINATION
Insulin
GAD
Diapep 277
T CELL
MODULATORY
APPROACH
Cyclosporin A
Prednisone
Azathioprine
Cytokines (IL-4, IL-10)
Anti CD3 Ab
Peptide MHC dimers
GAD
GAD
vaccine
NKT
a galactosy
l- ceramide
Diapep
277
p 277
Anti- CD3 Ab
Al
INNATE SYSTEM
MODULATION
a Galactosylceramide
Peptide 277 ( Diapep 277)
p277
TLR-2
Al
p 277
T
GAD
APC
MHC dimers
TCR
TLR-2
T
Anti- inflammatory
effect
b
Insulin
B9-23
© DeveloGen AG
Pro- inflammatory
effect
Immune
modulatory agent
TCR
7
Immune Modulation in Type 1 Diabetes
More questions than answers…
© DeveloGen AG
8
Proliferative T-cell Responses
Stimulation index (log)
100
GAD65
Hsp65 peptide
Crboxypeptidase H
Insulin
10
1
3
4
6
8
12
Age of mice (weeks)
© DeveloGen AG
9
15
© DeveloGen AG
10
© DeveloGen AG
11
© DeveloGen AG
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DPT-1 - Kaplan-Meier Curves according to Treatment-Group Assignment
NEJM 346:1685, 2002
© DeveloGen AG
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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.
© DeveloGen AG
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Insulin B9-23
 Diabetes Prevention Trial-1
1
 Sub cutaneous Insulin B chain peptide B:9-23
2
 vaccination with DNA encoding peptide B:9-23
1. Pozzilli et al
2. Liu et al; Ramiya et al
© DeveloGen AG
3. Urbanek-Ruiz et al
15
(in NOD mice)
3
(in NOD mice)
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
٢ A Vaccinia virus expressing GAD
٢ GAD derived peptides
© DeveloGen AG
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16
4
5
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
Transgenic plants expressing autoantigens fed to mice to
induce oral immune tolerance.
Ma et al. Nat Med. 1997
“a GAD-expressing transgenic plant, given as a dietary
supplement, inhibits the development of diabetes in the NOD
mouse…”
© DeveloGen AG
17
ous
Volume 346:1692-1698 May 30, 2002 Number 22
Next
Anti-CD3 Monoclonal Antibody in New-Onset
Type 1 Diabetes Mellitus
Kevan C. Herold, M.D., William Hagopian, M.D., Ph.D., Julie A. Auger,
B.A., Ena Poumian-Ruiz, B.S., Lesley Taylor, B.A., David Donaldson, M.D.,
Stephen E. Gitelman, M.D., David M. Harlan, M.D., Danlin Xu, Ph.D.,
Robert A. Zivin, Ph.D., and Jeffrey A. Bluestone, Ph.D.
Results Treatment with the monoclonal antibody maintained or improved
insulin production after one year in 9 of the 12 patients in the treatment group,
whereas only 2 of the 12 controls had a sustained response (P=0.01). The
treatment effect on insulin responses lasted for at least 12 months after
diagnosis. Glycosylated hemoglobin levels and insulin doses were also
reduced in the monoclonal-antibody group. No severe side effects occurred,
and the most common side effects were fever, rash, and anemia. Clinical
responses were associated with a change in the ratio of CD4+ T cells to CD8+
T cells 30 and 90 days after treatment.
© DeveloGen AG
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© DeveloGen AG
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Low dose linomide in Type I juvenile diabetes of recent onset:
a randomised placebo-controlled double blind trial.
Coutant R Saint-Vincent-de-Paul Hospital, Paris,France. Diabetologia. 1998
 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)
© DeveloGen AG
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An immunomodulatory effect?
TH-2 cell
IL-10
IL-4
P 277
linomide
hOKT31 TH-1 cell
© DeveloGen AG
21
GAD65
Proliferation Index
T cell Proliferative Responses to hsp60
5
4.5
4
5
3.5
blood donor group
3
2.5
2
1.5
1
P<0.001
P<0.01
type 1 diabetes group
1.7
type 2 diabetes group
1.6
0.5
0
© DeveloGen AG
T cell responses (SI)22
Hsp60 Promotes Inflammation in b-cell Autoimmunity
Stressed
b-cell
TNFa NO
Destruction of
b-cells by
autoimmune
responses
Autoimmune
disease
Stressed
b-cell
© DeveloGen AG
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insulin
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 diabetes1
© DeveloGen AG
1. Anderton
et al; van Haltren et al; van Eden et al
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Peptide p277: HSP60 positions 437-460
VLGGGVALLRVIPALDSLTPANED
60 kDa Heat Shock Protein (HSP60)
HSP60
p277
© DeveloGen AG
•Targeted by T-cells from diabetic NOD
mice and Type I IDDM patients.
•Vaccination with p277 arrests NOD
diabetes
25
HSP60 effects macrophages and T cells:
Pro- vs Anti-inflammation
macrophages
endothelial cells
HSP60
Activates pro-inflammatory
cytokines and matrix
metalloproteinase
T cells
Activates adhesion to FN and
intracellular signaling
Down-regulates chemokine
receptors and chemotaxis
Up-regulates expression of
adhesion molecules
Inhibits IFNg
TLR-4 dependent signaling
© DeveloGen AG
p277
TLR-2 dependent signaling
26
Innate Regulation of Inflammation
by HSP60 and p277
Ligand
HSP60
p277
© DeveloGen AG
Cell
TLR
Effect on Inflammation
M
4
Up-regulation
T
2
Down-regulation
T
2
Down-regulation
27
Effects of p277 on
p277-specific T-cells
T-cell receptor
p277
•Promotion of cell adhesion
•Inhibition of migration
P277-specific
clone
•Modulation of cytokine
secretion upon activation
TLR2
IL4
Th2
P277-specific
clone
Activated cells are
more sensitive
© DeveloGen AG
IL10
IL13
Long term effect
28
DiaPep277 Immunomodulation
P277
IL-4
Th2
T-cell
IL-10
MF
IL-1, NO
IFNg
b-cell
© DeveloGen AG
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DiaPep277 treatment prevents the progression of diabetes in NOD mice
Blood glucose (µmol/L)
Death
Death
Death
600
600
600
400
400
400
200
200
200
0
10
20
30
0
40
0
10
20
30
40
10
Age (weeks)
Treatment (arrow) of NOD mice with DiaPep277
© DeveloGen AG
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or Control
20
30
40
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).
Control
© DeveloGen AG
Treatment
31
DiaPep277 reduces IFNg secretion
by islet infiltrating T-lymphocytes
None
p278
4
IFN 
IL-4
IL-2
3
DiaPep277 2
Vehicle
1
0
10
20
30
Spot-forming cells per islet
© DeveloGen AG
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40
Dosing Scheme for Studies 420 and 431
© DeveloGen AG
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Full Preservation of Endogenous Insulin Secretion in
DiaPep277 Treated Patients (study 420)
Change in C-peptide AUC
nmolmin-1l-1
 Endogenous insulin secretion tested by I.V. 1mg glucagon stimulation.
 AUC calculated for time points 0-20min.
 Two-tailed unpaired t-test.
2.5
Placebo
DiaPep277
0.0
-2.5
-5.0
* P<0.05
-7.5
-10.0
-12.5
-15.0
*
2
4
6
* *
8
10
Months
© DeveloGen AG
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12
14
16
*
18
Change in Daily insulin Dose
Ukg-1day-1
Change in Daily Insulin Dose
0.4
DiaPep277®
Placebo
0.3
0.2
0.1
0.0
7
13
15
Months
© DeveloGen AG
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18
Percentage of patients with %HbA1c < 7.5
%Patients
100
Placebo
DiaPep277®
80
60
0
13
15
Months
© DeveloGen AG
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18
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.
Change in AUC
nmolmin-1l-1
-2.5
Diapep277->DiaPep277
DiaPep277->Placebo
Placebo->DiaPep277
-5.0
-7.5
-10.0
-12.5
© DeveloGen AG
37
Change in %HbA1c
Change in %HbA1c
7
6
5
4
3
2
1
0
-1
-2
-3
-4
420
431
DiaPep277
Placebo
DiaPep277->DiaPep277
DiaPep277->Placebo
Placebo->DiaPep277
7
13
15
18
12
Months
© DeveloGen AG
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Change in Daily insulin Dose
Ukg-1day-1
Change in Daily Insulin Dose
420
2.5
431
DiaPep277®
Placebo
DiaPep277->DiaPep277
DiaPep277->Placebo
Placebo->DiaPep277
2.0
1.5
1.0
0.5
0.0
7
13
15
18
12
Months
© DeveloGen AG
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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.
© DeveloGen AG
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Combined Analysis of Israeli Trials:
Basal Fasting C-peptide is Significantly Preserved
C-peptide nmol/l
0.50
Placebo
DiaPep277®
0.45
0.40
*
0.35
0.30
0.25
0
Months
N=52
© DeveloGen AG
13
p = 0.021 for change from start
41
Combined Analysis of Israeli Trials:
Stimulated Endogenous Insulin Secretion is Preserved
 DiaPep277-treated patients fully preserve glucagon-stimulated C-peptide
secretion (AUC)
13
Placebo
DiaPep277®
C peptide AUC
nmol/l/min
12
11
*
10
9
* p = 0.0031 from start
8
7
6
0
© DeveloGen AG
Months
13
42
Significant Difference in Beta-Cell Function
between DiaPep277-Treated and Placebo
The change from baseline in stimulated C-peptide AUC was significant.
Change in
AUCnmol/l/min
-0.00
DiaPep277®
Placebo
-0.05
-0.10
-0.15
p = 0.0188
-0.20
© DeveloGen AG
43
Combined Analysis of Adult Phase II Studies: Beta-cell Preservation in upper Thertile
Change in Stimulated C-peptide (AUC) by Treatment Dose
0.2
p=0.04
p=0.21
0
nmol/L
0
6
12
p=0.56
1.0mg
18
2.5mg
p=0.000
p=0.06
-0.2
0.2mg
p=0.50
-0.4
Placebo
-0.6
Time (months)
High responders: Patienrs with high44 beta-cell reserve at start of treatment
© DeveloGen AG
Opposite Trends in Glycemic Control
of DiaPep277 and Placebo Treated Patients
 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.
Change in % HbA1c
0.50
DiaPep277®
Placebo
0.25
0.00
-0.25
-0.50
-0.75
© DeveloGen AG
p = 0.3818
N=54
45
Kinetics of Th2 induced shift in
DiaPep277-treated patients
n=17
No of IL-10 producing cells
1000
n=15
IL-10 response to p277
n=14
100
10
1
0.1
0
6
12
DiaPep277
administration:
© DeveloGen AG
Time (months)
46
18
Change in Cytokine Profile
in DiaPep277 Treated Newly Diagnosed T1D Adults
800
600
400
DiaPep277
200
Placebo
0
Th1
© DeveloGen AG
Th2
47
Specific Intervention with DiaPep277TM Re-establishes
Balance in the Immune System
Health
Diabetes
+
-
Th1
Th2 Treg
© DeveloGen AG
48
Rx
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 Cpeptide secretion.
 DiaPep277 treated patients also showed trend for improved
glycemia at similar insulin requirement.
© DeveloGen AG
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