© DeveloGen AG 1

Prevention Of Type 1 Diabetes Mellitus:Current Status And Future Directions

ITAMAR RAZITAMAR RAZ

Hadassah University HospitalJerusalem Israel

© DeveloGen AG 2

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 3

Preventing the Progression of Diabetes in Its Early Stages

=Interfering with the autoimmune disease

© DeveloGen AG 4

LOSS OF FIRST PHASE LOSS OF FIRST PHASE INSULIN RESPONSEINSULIN RESPONSE

LOSS OF FIRST PHASE LOSS OF FIRST PHASE INSULIN RESPONSEINSULIN RESPONSE

TIMETIME

Stages in Development of Type 1 Diabetes B

ET

A C

EL

L M

AS

SB

ET

A C

EL

L M

AS

S

DIABETES

“PRE”-DIABETES

GENETICPREDISPOSITION

INSULITISBETA CELL INJURY

NEWLY DIAGNOSED DIABETES

MULTIPLE ANTIBODY POSITIVEMULTIPLE ANTIBODY POSITIVE

GENETICALLY AT RISK

Modified from G. Eisenbarth, NEJM, 1986

© DeveloGen AG 5

Initial Trials…

Prednisone

Prednisone with Azathioprine

Anti Thymocyte Globulin

Cyclosporine A

Elliot et alSchernthaner et alBougneres et al

FAILED

© DeveloGen AG 6

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 7

GAD

b

GAD

Insulin

APC

NKT

T

TCR

TLR-2

T

TCR

TLR-2

T CELL MODULATORY

APPROACHCyclosporin APrednisoneAzathioprineCytokines (IL-4 ,IL -10(Anti CD 3 AbPeptide MHC dimers

Anti- CD 3 Ab

MHC dimers

AUTOANTIGEN VACCINATION

InsulinGADDiapep 277

INNATE SYSTEM MODULATION

a GalactosylceramidePeptide 277 )Diapep 277(

GAD vaccine

GAD

Insulin B9-23

AlAl

p277 p277

p277

a galactosyl- ceramide

Diapep277

Pro- inflammatory effect

Anti- inflammatory effect

Immune modulatory agent

© DeveloGen AG 8

Immune Modulation in Type 1 Diabetes

More questions than answers…

© DeveloGen AG 9

Proliferative T-cell Responses

3 4 6 8 12 15

GAD65

Hsp65 peptide

Crboxypeptidase H

Insulin

1

10

100

Age of mice (weeks)

Sti

mu

lati

on

in

dex

(lo

g)

© DeveloGen AG 10

Gad?

Heat shock protein 60 (HSP60)?

Insulin B9-23 peptide ?

© DeveloGen AG 11

© DeveloGen AG 12

© DeveloGen AG 13

DPT-1 - Kaplan-Meier Curves according to Treatment-Group Assignment

NEJM 346:1685, 2002

© DeveloGen AG 14

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 15

Insulin B9-23

Diabetes Prevention Trial-11

Sub cutaneous Insulin B chain peptide B:9-232 (in

NOD mice)

vaccination with DNA encoding peptide B:9-233 (in

NOD mice)

1. Pozzilli et al2. Liu et al; Ramiya et al3. Urbanek-Ruiz et al

© DeveloGen AG 16

Glutamic Acid decarboxylase

١ reduction of severity of insulitis and prevention of onset of diabetes in NOD mice was achieved with:

٢ Injection of GAD651

٢ Injection of GAD 67 (isoform) 2

٢ Anti GAD monoclonal antibodies 3

٢ GAD-plasmid DNA/DNA vaccine/GAD antisense4

٢ A Vaccinia virus expressing GAD5

٢ GAD derived peptides6

1. Ramiya et al; Pleau et al; Petersen et al; Tisch et al 2. Elliott et al3. Menard et al4. Yoon et al;Balasa et al; Li et al5. Jun et al6. Tisch et al;Sai et al

© DeveloGen AG 17

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

© DeveloGen AG 18

Previous 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 19

© DeveloGen AG 20

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

© DeveloGen AG 21

An immunomodulatory effect?

TH-2 cell

TH-1 cell

IL-4IL-10

IL-4

IL-4

IL-10

IL-1

0

GAD65hOKT31

linomideP 277

© DeveloGen AG 22

T cell Proliferative Responses to hsp60

1.7

5

1.6

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

T cell responses (SI)

blood donor group

type 1 diabetes group

type 2 diabetes group

P<0.001 P<0.01

Pro

life

rati

on I

nd

ex

© DeveloGen AG 23

Stressed-cell

Stressed -cell

Hsp60 Promotes Inflammation in -cell Autoimmunity

GAD

Th1

IL-12IL-12

IL-12IL-12

CTL

hsp60

MacrophageDestruction of Destruction of -cells by -cells by autoimmune autoimmune responsesresponses

AutoimmuneAutoimmune diseasedisease

TNFTNFNONO

IFN ; TNF

Cell lysis

hsp60

hsp60

hsp60

hsp60

hsp60

insulin

© DeveloGen AG 24

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

1. Anderton et al; van Haltren et al; van Eden et al

© DeveloGen AG 25

60 kDa Heat Shock Protein (HSP60)

HSP60

p277

Peptide p277: HSP60 positions 437-460

VLGGGVALLRVIPALDSLTPANED

•Targeted by T-cells from diabetic NOD mice and Type I IDDM patients.

•Vaccination with p277 arrests NOD diabetes

© DeveloGen AG 26

HSP60 effects macrophages and T cells:

Pro- vs Anti-inflammation

HSP60macrophages

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

© DeveloGen AG 27

Ligand Cell TLR Effect on Inflammation

HSP60 M

T

4

2

Up-regulation

Down-regulation

p277 T 2 Down-regulation

Innate Regulation of Inflammation by HSP60 and p277

© DeveloGen AG 28

P277-specific clone

Th2

IL4

IL10

IL13

Long term effectActivated 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

© DeveloGen AG 29

DiaPep277 Immunomodulation

-cell

IL-1, NOIL-1, NOIFNIFN

Th1 T-cell

M

IL-4IL-4 IL-10IL-10

Th2T-cell

M

P277

© DeveloGen AG 30

DiaPep277 treatment prevents the progression of diabetes in NOD mice

Treatment (arrow) of NOD mice with DiaPep277 or Control

Death

Death

Death

Blo

od

glu

cose

(µ

mo

l/L)

Age (weeks)

600

400

200

010 20 30 40

600

400

200

010 20 30 40

600

400

200

010 20 30 40

© DeveloGen AG 31

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 Treatment

© DeveloGen AG 32

DiaPep277 reduces IFN secretionby islet infiltrating T-lymphocytes

0 10 20 30 40

Spot-forming cells per islet

1

2

3

4

IFNIL-4IL-2

None

p278

DiaPep277

Vehicle

© DeveloGen AG 33

Dosing Scheme for Studies 420 and 431

© DeveloGen AG 34

Full Preservation of Endogenous Insulin Secretion in DiaPep277 Treated Patients

(study 420)

2 4 6 8 10 12 14 16 18

-15.0

-12.5

-10.0

-7.5

-5.0

-2.5

0.0

2.5

DiaPep277Placebo

Months

Ch

ang

e in

C-p

epti

de

AU

C

nm

olm

in-1

l-1

* * * *

* 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.

© DeveloGen AG 35

Change in Daily Insulin Dose

7 13 15 180.0

0.1

0.2

0.3

0.4DiaPep277®Placebo

Months

Ch

ang

e in

Dai

ly i

nsu

lin

Do

se

Ukg

-1d

ay-1

© DeveloGen AG 36

Percentage of patients with %HbA1c < 7.5

0 13 15 1860

80

100PlaceboDiaPep277®

Months

%P

atie

nts

© DeveloGen AG 37

Partial Preservation in Extension Study: Need for Continuous Treatment

-12.5

-10.0

-7.5

-5.0

-2.5Diapep277->DiaPep277DiaPep277->PlaceboPlacebo->DiaPep277

Ch

ang

e in

AU

C

nm

olm

in-1

l-1 After a 6-18 no treatment period, patients were re-

randomized Patients on continued DiaPep277 treatment showed the

least drop in beta-cell function.

© DeveloGen AG 38

Change in %HbA1c

-4-3-2-101234567

7 13 15 18 12

DiaPep277

Placebo

Placebo->DiaPep277

DiaPep277->Placebo

420 431

DiaPep277->DiaPep277

Months

Ch

ang

e in

%H

bA

1c

© DeveloGen AG 39

Change in Daily Insulin Dose

7 13 15 18 120.0

0.5

1.0

1.5

2.0

2.5DiaPep277®

Placebo

DiaPep277->Placebo

DiaPep277->DiaPep277

Placebo->DiaPep277

420 431

Months

Ch

ang

e in

Dai

ly i

nsu

lin

Do

se

Ukg

-1d

ay-1

© DeveloGen AG 40

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 41

Combined Analysis of Israeli Trials:Basal Fasting C-peptide is Significantly

Preserved

0.25

0.30

0.35

0.40

0.45

0.50

DiaPep277®Placebo

0 13

*

p = 0.021 for change from start

Months

C-p

epti

de

nm

ol/

l

N=52

© DeveloGen AG 42

Combined Analysis of Israeli Trials:Stimulated Endogenous Insulin Secretion is

Preserved

6

7

8

9

10

11

12

13

0 13

PlaceboDiaPep277®

*

* p = 0.0031 from start

Months

C p

epti

de

AU

Cn

mo

l/l/

min

DiaPep277-treated patients fully preserve glucagon-stimulated C-peptide secretion (AUC)

© DeveloGen AG 43

Significant Difference in Beta-Cell Functionbetween DiaPep277-Treated and Placebo

-0.20

-0.15

-0.10

-0.05

-0.00DiaPep277®Placebo

p = 0.0188Ch

ang

e in

AU

Cn

mo

l/l/

min

The change from baseline in stimulated C-peptide AUC was significant.

© DeveloGen AG 44

Combined Analysis of Adult Phase II Studies: Beta-cell Preservation in upper Thertile

Change in Stimulated C-peptide (AUC) by Treatment Dose

-0.6

-0.4

-0.2

0

0.2

0 6 12 18

Time (months)

nm

ol/L

1.0mg

2.5mg

0.2mg

Placebo

p=0.04

p=0.000p=0.21

p=0.06p=0.56

p=0.50

High responders: Patienrs with high beta-cell reserve at start of treatment

© DeveloGen AG 45

Opposite Trends in Glycemic Controlof DiaPep277 and Placebo Treated Patients

-0.75

-0.50

-0.25

0.00

0.25

0.50

p = 0.3818

DiaPep277®Placebo

Ch

ang

e in

% H

bA

1c

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.

© DeveloGen AG 46

IL-10 response to p277

0.1

1

10

100

1000

0 6 12 18

Time (months)

No

of IL

-10

prod

ucin

g ce

lls

n=15 n=14n=17

DiaPep277

administration:

Kinetics of Th2 induced shift inDiaPep277-treated patients

© DeveloGen AG 47

Change in Cytokine Profile in DiaPep277 Treated Newly Diagnosed T1D Adults

0

200

400

600

800

Th1 Th2

Placebo

DiaPep277

© DeveloGen AG 48

Specific Intervention with DiaPep277TM Re-establishes Balance in the Immune System

Health Diabetes

Th1 Th2 Treg Th1 Th2 Treg

Rx

DiaPep277

+ -

© DeveloGen AG 49

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.