Development of safe and effective oral tolerogen for Myasthenia gravisMyasthenia gravis

Sin-Hyeog Im, Ph.D.

Gwangju Institute of Science and Technology Gwangju Institute of Science and Technology (GIST)(GIST)

KOREAKOREA

Myasthenia Gravis

AChR

Anti-AChR

Ab

Induction of AChR-specific T cell tolerance

AChR-reactive CD4 T cellsCD4 T cells

AChR-reactive CD4 T cellsCD4 T cells

AChR-reactive B cellsB cells

Our Goal

Development of an Ag-specific immunotherapy for MG

Methods

Induction of lymphocyte tolerance to AChR

Mucosal tolerance Mucosal tolerance with recombinant proteins of non-

myasthenogenic derivatives of human AChR.

Induction of AChR-specific T cell tolerance; mucosal tolerance with recombinant hAChR

Oral treatment with recombinant hAChR suppresses ongoing chronic experimental MG

-20

-10

0

10

20H1-205

Weeks after injection7 8 9 10 11 12 13 14

Body weight change

Control(OVA)

0

1

2

3

4

7 8 9 10 11 12 13 14

Clinical score

Control(OVA)

H1-205Clin

ical

sco

re(m

ean)

J Clin Invest. 1999 Dec;104(12):1723-30

Bod

y w

eigh

t(d

elta

gra

m/m

ean

)

1 205

H1-205 (No fusion partner)

H 1-210 (Thioredoxin fusion)1 210

Trx

Role of tolerogen conformation in oral tolerance :

1: Torpedo AchR 2: GST-Hα 1~210

3: Trx-Hα 1~210 4: Hα 1~205J Immunol 2000. 165: 3599-3605

Mea

n cl

inic

al s

core

OVA

H1-205

0

1

2

3

0 1 2 3 4 5 6 7 8 9 10Weeks after immunization

TAChRStart of Oral administration of AChRs

H1-210Trx -Less native form of AChR Less native form of AChR

showed more protective effect showed more protective effect

Clinical trials with oral tolerogen

DiseaseDisease AntigenAntigen

Multiple Sclerosis (MS) Myelin Basic Protein (MBP)

Rheumatoid Arthritis (RA) Type II collagen

Type I Diabetes Insulin

Uveitis S-antigen

Transplant Rejection MHC molecules

No success >>>> Why ?

Immunogenicity of oral tolerogen ?Immunogenicity of oral tolerogen ?

Generation of BF-AChR from TrxH1-210

H1-210

67 ~ 76 (MIR) 129 ~ 145 (MAR)

Deletionprimer pair 1

Deletion primer pair 2

Thioredoxin

H1-210 Thioredoxin

- BF-AChR -

- TrxH1-210 -

Recovery of therapeutic effects

Immunochemical Characterization of BF-AChR

A) SDS-PAGE

1 2 3 4

50

40

30

20

kDa

Lane 1 : Torpedo AChRLane 2 : TrxH1-210Lane 3 : BF-AChRLane 4 : H1-205

B) mAb 198

1 2 3 4C) 125I-BTX

50

40

30

20

kDa

1 2 3 4

D) mAb 5.5

1 2 3 4Yi et al. Mol Imm 45 . 3748-3755

Removal of B cell epitopes reduces the reactivity to the AChR-reactive Abs from EAMG sera

Yi et al. Mol Imm 45 . 3748-3755

0

0.2

0.4

0.6

0.8

1

1.2

1.4

EAMG serum mAb198 mAb5.5

Trx-H1-210

BF-AChR

Trx

A)

SDS-PAGE

1 2 1 2

EAMG serumB)

* p < 0.001*

Re

lativ

e b

ou

nd

Ab

leve

l

Reduced proliferation of AChR-reactive T and B cells Reduced proliferation of AChR-reactive T and B cells by deletion of B-cell epitopes in AChRby deletion of B-cell epitopes in AChR

Yi et al. Mol Imm 45 . 3748-3755

0

0.50

1.0

1.5

2.0

2.5

3.0

0 2 4 8 16

Protein concentration (g/ml)

CP

M(x

10

3 )

OVA

Trx-H1-210

BF-AChRBF-AChRBF-AChRBF-AChR

B) B-cell proliferation assay

26.5%33.7%

42.1%

16.8%

C) C) B-cell dependent T-cell B-cell dependent T-cell proliferation assayproliferation assay

TAChR

Trx-H1-210 BF-AChR

OVANormal rat CD4+

CFSE

23.5%

A) Mixed lymphocyte proliferation

TAChR Trx-H1-210

BF-AChR OVA0

1

2

3

4

6

CP

M(x

10

3 )

*

**

* p < 0.03**p< 0.04

5

* p < 0.03**p< 0.05

*

**

Suppression of EAMG by BF-AChR

****

* * *

0

1

2

3

4

Mea

n cl

inic

al s

core

OVA

Trx-H1-210

BF-AChR

BF-AChR** p < 0.05

* p < 0.01

0 1 2 3 4 5 6 7 8Weeks after immunization with AChR

TAChR Start of Oral administration of AChRs

Yi et al. Mol Imm 45 . 3748-3755

WithoutWithout B cell epitopes

WithWith B cell epitopes

Effect of oral treatment with BF-AChR on AChR-specific IgG isotypes

Yi et al. Mol Imm 45 . 3748-3755

0

1

2

3

4

5

6

Total IgG IgG1 IgG2a IgG2b IgG2c

Re

lativ

e a

ntib

od

y tit

er

ind

ex

OVA

Trx-H1-210

BF-AChR

*

***

**

* p < 0.01

** p < 0.05

**

**

0

0.5

1

1.5

2

2.5

IL-2 IFN- IL-12 TNF-

OVA

TrxH1-210

BF-AChR

0

0.4

0.8

1.2

1.6

B7.1 B7.2 CD28 CD40L

0

2

4

6

8

Foxp3 IL10 TGF-

*

*

* p < 0.03** p < 0.05

**

*

*

*

**

*

*

*

Re

lativ

e e

xpre

ssio

n le

vel

Re

lativ

e e

xpre

ssio

n le

vel

*

Cytokines shift by oral-adminiatration of BF-AChRPro-inflammatory Anti-inflammatory

Yi et al. Mol Imm 45 . 3748-3755

1. Containing enough CD4 T-cell epitopes

2.2. Lack of pathogenic epitopes Lack of pathogenic epitopes CD8 T cell epitope (e.g., Type I diabetes) Lack of B cell epitope (MG, RA, Lupus)

3. No or less mucosal immunity

Criteria for Safe and Effective Oral Tolerogen

Acknowledgements

KoreaGIST Hwa-Jung Yi Chang-Suk Chae IRT Lab Members

Israel Prof. Sara Fuchs

(The Weizmann Institute of Science) Prof. Miriam C. Souroujon

(The Open University of Israel)

Greece Prof. Socrates J. Tzartos

(University of Patras) (Hellenic Pasteur Institute)

Jisim Island, KOREA. July. 2009

II RRTTmmunemmuneII RRTTmmunemmune

Safe and Effective Oral Tolerogen