IMMUNOTHERAPIE DU DIABETE

AUTO-IMMUN

INSTITUT UNIVERSITAIRE

DE FRANCE INSERM U1013

Time

Natural history of type 1 diabetes

b cell function

(%)

EURODIAB registered 29 311 new cases of T1D in children before 15 years

of age between 1989-2003.

The overall annual increase was 3.9%,

New cases in Europe in 2005 : 15 000

(24%: 0-4 years, 35%: 5-9 years, and 41%: 10-14 years).

In 2020, the predicted number of new cases is 24 400, with a doubling in

numbers in children younger than 5 years.

Prevalence under age 15 years is predicted to rise from 94 000 in 2005, to

160 000 in 2020.

Doubling of new cases of type 1 diabetes in children younger than 5 years

and prevalent cases younger than 15 years will rise by 70%.

AN ALARMING EPIDEMIOLOGICAL TREND

Patterson et al. Lancet 2009, 373, 1999

30

Months

Re

mis

sio

n (

%)

Placebo (n = 127)

20

10

0 3 6 9 12 15 18 24

CsA (n = 155)

40

PLACEBO-CONTROLLED TRIAL OF CYCLOSPORIN A IN RECENTLY DIAGNOSED IDDM (CDF study)

EFFECT ON REMISSION RATE

G. Feutren et al. Lancet 1986; 2: 119-124

M. Pescovitz et al. New Engl J

Med 2009, 361: 2143-2152

RITUXIMAB IN T1D

One course

4 doses (375 mg/m2 )

1 year

p=0,0195

Over time p< 0,038

Over time p< 0,004

TWO STRATEGIES TO INDUCE IMMUNE

TOLERANCE

1. Administration of b-cell autoantigens

2. Use of monoclonal antibodies

mediating T cell signaling

Antigen Trial Reference

Oral Insulin New onset T1D Chaillous L et al.

The Lancet 356, 545, 2000

Oral Insulin New onset T1D Pozilli P et al.

Diabetologia 43, 1000, 2000

APL insulin B chain (NBI-6,024) New onset T1D Walter M et al.

Diabetes Care 32, 2036, 2009

Hsp60 p277 New onset T1D Raz I et al.

The Lancet 358, 1749, 2001

i.v. + s.c. insulin

DPT-1

Late preclinical T1D Skyler J et al.

New Engl J Med 346, 1685, 2002

Oral insulin

DPT1

Insulin-autoantibody positive

relatives

Skyler J et al.

New Engl J Med 346, 1685, 2002

Intranasal insulin Insulin-autoantibody positive

relatives

Näntö-Salonen K et al.

The Lancet 372, 1746, 2008

Alum formulated GAD Recent onset T1D Ludvigsson J et al.

New Engl J Med 359, 1909, 2008

AUTOANTIGEN TRIALS

- Expresses the unique ability to restore self-

tolerance in established autoimmunity

- The therapeutic effect initially demonstrated in

the experimental setting was successfully

transferred to the clinic

- The mode of action involves 'resetting' of active

tolerance

CD3 antibody therapy

ACTIVE TOLERANCE

Tolerance requiring active contribution of

antigen-specific activated T cells

Related concepts

dominant tolerance

infectious tolerance

immunoregulation

T-cell mediated suppression

CD4+

CD8+

CD8+

T cell

CD4+

T cell

CD4+

CD25+high

Foxp3+

CD4+

CD25-

Foxp3-

CD4+

CD25-

Foxp3-

CD4+

CD25+high

Foxp3+

CD4+ Th1

Effector

CD25-

Foxp3-

Tr1

Th2

Th3

Systemic Ag

Oral Ag

TGF-b

IL-4/ IL-10

IL-10/ TGF-b

Ag Stimulation

Pro-inflammatory

context

HETEROGENEITY OF CD4+ REGULATORY T CELLS

THYMUS -

- - -

CD4+

CD25+low

Foxp3+

Ag Stimulation

‘tolerogenic’

context

Cell contact

TGF-b, GITR, CTLA-4…

- IN AUTOIMMUNE PRONE

INDIVIDUALS

CD4

CD

25

CD4+CD25low

CD4+CD25high

CD4+CD25-

Diab

Diab/CD25low

Diab/CD25high

Weeks after transfer

% D

iabete

s

20

40

60

80

100

0 2 4 6 8 10

Both CD4+CD25high and CD4+CD25low T cells are

suppressive in vivo in NOD mice

0

2 0

4 0

6 0

8 0

1 0 0

%

r e m

i s s

i o n

( n = 2 0 )

( n = 1 6 )

( n = 1 6 )

( n = 1 7 )

p < 0 . 0 0 1

p < 0 . 0 0 5

T r e a t m e n t ( d 0 - d 5 )

2 4 6 8 1 0 1 2 W e e k s

A n t i - C D 3 C o n t r o l s

REMISSION OF ESTABLISHED DIABETES IN

CD3 ANTIBODY-TREATED NOD MICE

L. Chatenoud et al. Proc.Natl.Acad.Sci. USA 1994; 91: 123-127

-Belghith M et al. Nat Med 9:1202-1208, 2003

-You S et al. Immunol Rev 212:185-202, 2006

- You S et al. (2007) Proc Natl Acad Sci (U S A) 104: 6335-6340, 2007

-Chatenoud L & Bluestone JA. Nat Rev Immunol 7:622-632, 2007

-You S et al. Adv Immunol 100: 13-37, 2008

THERAPY OF ONGOING R-EAE USING CD3 ANTIBODIES

Day 0-4 Treatment

3.2

2.6

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10 11 13 14 15 17

Days Post Disease Induction

Mean

Cli

nic

al S

co

re

Control aCD3 F(ab')2

Rx

Day 6-10 Treatment

2.2

0.4

0

0.5

1

1.5

2

2.5

0 2 4 6 8 10 11 13 14 15 17

Days Post Disease Induction

Mean

Cli

nic

al S

co

re

Control aCD3 F(ab')2

Rx

1.2

2.8

1.6

2.6 2.6

0.4

0.6

0

0.5

1

1.5

2

2.5

3

3.5

0 4 8 11 14 17 21 25 29 33

Days Post Disease Induction

Me

an

Cli

nic

al S

co

re

Control aCD3 F(ab')2

Day 13-17 Treatment

Rx

Rat YTH 12.5

hIgG1

Reshaped

Humanized YTH 12.5 (CAMPATH 3)

Mutation Asn to Ala position 297

(prevents glycosylation)

S. Bolt et al European J Immunol, 1993, 23, 403

Treatment of established Type 1 diabetes with a

non-mitogenic CD3 antibody

Phase II trial – Multicenter – Placebo Controlled

Between 07/06/2000 and 07/03/2003

210 consecutive recent onset diabetic patients were screened

80 patients included

12-39 years old. positive for ICA and/or GAD Abs

insulin-treatment <4 weeks

8mg/day of ChAglyCD3 for 6 consecutive days

EBV IgG positive.

Keymeulen B. N. Engl. J. Med 2005, 352:2598-2608

0 6 12 18 24 36 48

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

IU/k

g/d

ay

Months

HbA1c

Insulin dose

p=0.004 p<0.001 p=0.001 p=0.001

n=31

n=31

n=29 n=31

n=31

n=27

n=28

n=33

n=33

n=31

n=32

n=33

n=32 n=33

0 6 12 18 24 36 48

5

6

7

8

9

Months

%

p=0.761 p=0.984 p=0.990 p=0.428

ChAglyCD3

Placebo

Keymeulen B. Diabetologia 2009, 53: 614-623

6 6.5 7 7.5

0.25 NS NS 0.01 0.01

0.5 NS NS NS

0.75 NS

HbA1C %

Insu

lin d

ose

U/K

g/d

ay

NS NS

NS*

*

NS NS

NS

12 MONTHS AFTER TREATMENT

NS: non significant difference between placebo and

CD3 antibody-treated patients (Chi Square)

Data from European Phase II trial using otelixizumab (Keymeulen B. N. Engl. J.

Med 2005, 352:2598-2608 )

Lancet. 2011; 378 :487-497

Phase III Study : teplizumab

AUTOIMMUNE DIABETES MULTIPLE SCLEROSIS D

ISA

BIL

ITY

GE

NE

TIC

PR

ED

ISP

OS

ITIO

N

OVERT HYPERGLYCEMIA

BETA CELL

AUTOANTIBODIES

BETA CELL

AUTOANTIBODIES

BETA CELL

AUTOANTIBODIES

BETA CELL

AUTOANTIBODIES

BENIGN

RELAPSING

REMITTING

SECONDARY CHRONIC

PROGRESSIVE

PRIMARY CHRONIC

PROGRESSIVE

LO

SS

OF

BE

TA

CE

LL

FU

NC

TIO

N

GE

NE

TIC

PR

ED

ISP

OS

ITIO

N

GE

NE

TIC

PR

ED

ISP

OS

ITIO

N

GE

NE

TIC

PR

ED

ISP

OS

ITIO

N

CD3 mAb Therapeutic Window

M1

M1

NOD 38% MFI=142

NOD-huCD3e 40% MFI=72

human PBMCs 75% MFI=244

NOD-huCD3e 36% MFI=119

mCD3

co

un

ts

17 <1

82

2 85

10

2 14

2

2 87

8

thymus

NOD NOD-huCD3e

<1 82

3 3

spleen

NOD NOD-huCD3e

44 1

54

13 <1

32

4 43

<1

13 <1

31

<1 53

55 55

huCD3

mC

D3

mCD4

mC

D8

huCD3

co

un

ts

Transgenic Human CD3e-NOD mice

0

20

40

60

80

100

0 5 10 15 20 25 30 35 40

weeks of age

% d

iab

ete

s

(n = 32)

NOD NOD-huCD3e

(n = 40)

islets with invasive infiltration

islets with peripheral infiltration

islets w/o infiltration

0

20

40

60

80

100

% i

nfi

ltra

ted

is

lets

6-wk diabetic Otelixizumab 12-wk

0

20

40

60

80

100

5 10 15 20

weeks after treatment

% r

em

iss

ion

NOD-huCD3e + Otelixizumab

NOD + F(ab’)2-2C11

anti-CD3

(n = 26)

(n = 23)

merge

CD3

FoxP3 Insulin merge

6-w

k

12-w

k

dia

be

tic

Ote

lixiz

um

ab

0

20

40

60

80

100

0 2 4 6 8

weeks after transfer

% d

iab

ete

s

CD25-CD62L- diabetic

CD25-CD62L- Otelixizumab

SPLEEN

6wk 16wk diab 2wk >8wk

remission

0

200

400

600

800

SP

U / 1

06 c

ell

s

IGRP 206-214

BLOOD

SP

U / 1

06 c

ell

s

0

200

400

600

6wk 16wk diab 2wk >8wk

remission

0

10

20

30 SPLEEN

PI B:15-23

0

BLOOD

100

200

300

6wk 16wk diab 2wk >8wk

remission

6wk 16wk diab 2wk >8wk

remission

BEFORE

TREATMENT

CD3Ab

TREATMENT

AFTER

TREATMENT

CONCLUSIONS

CD3 antibodies are effective at preserving b cell

function and decreasing insulin needs in newly

diagnosed Type 1 diabetic patients

The effect is long lasting

Patients are responsive to unrelated antigens

CD3 antibodies induce/ expand TGF-b dependent

adaptive Tregs

These results are a proof of concept and pave the way

for the use of CD3 antibodies as tolerance-promoting

agents in various T cell-mediated autoimmune

diseases, in transplantation and cell therapies

IDDM

Multiple sclerosis

Crohn's disease

Asthma

55

Incidence of prototype infectious diseases and immune disorders

75 85 95

100

50

Infectious diseases

%

100

200

Immune disorders

%

300

0

65

Hepatitis A

Rheumatic fever

Measles

Tuberculosis

55 75 85 95 65

JF Bach, N Engl J Med, 2002

5.3

1.9 (56-74)

3.78

1.69 24

0.96 (mortality or incidence)

2.2

5

1.1

2.24

3.94

3.3

1.72

Incidence of multiple sclerosis (per 100,000)

24 (77-85)

9.4

7.3 (66-89)

8.05 (54-86)

11.5 (67-90)

19.9

10.9 (85-88)

7.7

19.7

35.8 (87-99)

13.7

Incidence of IDDM (per 100,000)

Adapted from : www.eatlas.idf.org

Incidence per 100,000 children

(0 – 14years)

High (> 16)

Intermediate (8 – 16)

Low (0 – 8)

No estimate

Type 1 Diabetes (incidence in children 0 – 14 years)

Adapted from : www.atlasofms.org

Prevalence per 100,000 (2007)

High (> 60)

Intermediate (20 - 60)

Low (0 – 20)

No estimate

Multiple Sclerosis

Adapted from : Masoli et al. Allergy. 2004:59

Proportion of clinical asthma *

High (> 10%)

Intermediate (5.1% – 10%)

Low (0% – 5%)

No estimate

Childhood Asthma

Risk level

High

Intermediate

Low

Hepatitis A virus antibodies prevalence

Adapted from : wwwn.cdc.gov

IDDM incidence in children of migrants from Pakistan to Yorkshire

Staines A. (1997) and Bodansky H.J. (1992)

0

2

4

6

8

10

12

Pakistan Migrant's children

Yorkshire

Incidence of diabetes /10 5

CAUSAL LINK

PREVENTION OF IDDM IN NOD MICE

BY INFECTIOUS AGENTS

Bacteria streptococci

salmonella

mycobacteria (CFA, BCG, …)

Viruses LCMV

MHV

LDHV

Parasites schistosoma

pimworms

TGF-b antibody treatment abrogates

diabetes protection by OM-85

0

20

40

60

3 7 1 1 15 19 23 27

Age (weeks)

OM-85 anti-TGF-b

NOD OM-85

NOD controls

NOD OM-85

+ anti-TGF-b

% d

iabete

s

, , , , , , , ,

, , , , , ,

, , , , , ,

Immune response of dendritic

cell to TLR agonists

v v

v v

TLR4

TLR5

TLR2

Flagellin Endotoxins or LPS

Mycobacterial soluble

factor

Proteine MMLV

Mannan

Fibrinogen

HSP70, HSP60

Hyaluronan

Beta-defensin

Surfactant Protein A

Lipoproteins

Zymosan

P. Gingivalis LPS

Lipoteichoic acid

Lipoarabinomannan

HCMV

Trypanosoma cruzi GPI

KOmpA

HSP70

v v

v v TLR7

v v TLR9

TLR3

TLR1

TLR11

v v TLR8

v v TLR6

Bacterial DNA (CpG)

Herpes simplex virus 2

upec

Imidazoquinoline

dsARN

ssARN

Poly(I:C)

Lipopeptide

lipopeptide

IRF3 NF-kB

AP-1

Imidazoquinoline MyD-88 TRIF

IFN

TLR2, TLR3,TLR4 and TLR7 agonists

delay autoimmune diabetes onset

Dia

bete

s incid

ence (

%)

Age (weeks)

TLR2 TLR3 TLR4 TLR7

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25

Non treated

P40

p=0.0064

Treatment

0

10

20

30

40

50

60

70

80

90

100

5 15 25

Non treatedPoly(I:C)

p=0.000450

10

20

30

40

50

60

70

80

5 10 15 20 25

Non treatedLPS

p=0.00850

10

20

30

40

50

60

70

80

90

5 10 15 20 25

Non treated

R848

p=0.0143

Similar protective results were obtained with TLR agonists : synthetic lipopetide Pam3CSK4 (TLR2 agonist) or dsRNA

Poly(I)Poly(C) …

p <0.05

p <0.05

Thieblemont N et al. PLoS ONE 2010, 5(7): e11484

p <0.05

PROTECTION BY TLR AGONISTS

AND IMMUNE REGULATION

ALLERGIC ASTHMA IN NOD

MICE

Immunization

1, 10, 100 µg OVA

+ Alum i.p./ s.c.

D0 D10

Measurement of airway

hyperreactivity to

metacholine Sacrifice and collection of

Sera (cytokines, IgE)

BALFs (cells, cytokines)

Lungs (cytokines)

D7, D8, D9

OVA challenge

Eosin

ophils

in B

AL 1

05/m

l

OVA

p< 0.005

p< 0.005

Araujo LM et al. Eur.J.Immunol. 2004, 34: 327

Prevention of allergic asthma

by a TLR2 agonist

-2 -1 0 1 2 3 4 5 6 7 8 9

1.53.04.56.07.59.0

OVA/P40

Saline/Vehicle Saline/P40

OVA/Vehicle

Pe

nh

OVA Saline OVA Saline0

1500

3000

4500

6000

P40-treated Vehicle-injected

p=0,0357

Lung e

ota

xin

level (p

g/m

l)

OVA Saline OVA Saline0

50

100

150

200 p=0,0736

Lung IL-4

level (p

g/m

l)

P40-treated Vehicle

0

7

14

21

28

Eosinophils Neutrophils

Macrophages Lymphocytes

Saline SalineOVA OVABA

LF

cell c

ount

(10

5)

P40-treated Vehicle