1. Development of safe and effective oral tolerogen for Myasthenia gravis
Sin-Hyeog Im, Ph.D.
Gwangju Institute of Science and Technology (GIST)
KOREA

2. Myasthenia Gravis
Anti-
AChR Ab
AChR

3. Induction of AChR-specific T cell tolerance
AChR-reactive B cells
AChR-reactive
CD4 T cells
AChR-reactive
CD4 T cells

4. Our Goal Methods Development of an Ag-specific immunotherapy for MG
Induction of lymphocyte tolerance to AChR
Mucosal tolerance with recombinant proteins of non-myasthenogenic derivatives of human AChR.

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

6. Oral treatment with recombinant hAChR suppresses ongoing chronic experimental MG
Clinical score 1 2 3 4
Control
(OVA)
Clinical score
(mean) H a
1-205 7 8 9 10 11 12 13 14 B o d y w e i g h t c h a n g e - 2 1 H a 1 - 2 5
(delta gram/mean)
Body weight C o n t r o l ( O V A ) 7 8 9 1 2 3 4 W e e k s a f t e r i n j e c t i o n
J Clin Invest Dec;104(12):

7. Role of tolerogen conformation in oral tolerance :H a
1-210
Trx - 1 2 3
Less native form of AChR
showed more protective effect
OVA
Mean clinical score H a
1-205 1 2 3 4 5 6 7 8 9 10
Weeks after immunization
Start of Oral administration of AChRs
TAChR
1: Torpedo AchR
2: GST-Hα 1~210
3: Trx-Hα 1~210
4: Hα 1~205 1
205 H a
1-205 (No fusion partner)
1-210 (Thioredoxin fusion)
210
Trx
A previous study gave me a clue about this issues
This without fusion partnered recombinant AChR, its oral administration suppress the symptom of EAMG.
In contrast, fusion protein TRX conjugated AChR, its oral administration had no effect suppressing EAMG clinical symptoms.
The only difference is their spatial conformation between them.
The 3-dimensional differences were measured
by western-blotting and ELISA with conformation-specific affinity Abs and bungarotoxin.
As you see in this figure, Lane 3, TrxAChR have strong affinity to the conformation specific molecules
Whereas, Lane 4 without fusion partner AChR Halpha205 shows significant less affinity to the molecules.
In addition.
J Immunol :

8. Clinical trials with oral tolerogen
Disease Antigen
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 ?

9. Immunogenicity of oral tolerogen ?

10. Generation of BF-AChR from TrxHa1-210
Thioredoxin
Recovery of
therapeutic effects
D67 ~ 76 (MIR)
D129 ~ 145 (MAR)
- BF-AChR -
Accordingly, I devised and anticipated that removal of B-cell epitopes in TrxHa1-210
would recover its therapeutic effect for treatment of myasthenia gravis as an oral tolerogen.
I removed the two well known pathogenic B-cell epitopes MIR and MAR by site directed mutagenesis.
4. And I designated this recombinant AChR fragment as BF-AChR.
5. After purification and detoxification of them
Thioredoxin
Ha1-210
Deletion
primer pair 1
Deletion
primer pair 2

11. Immunochemical Characterization of BF-AChR
A) SDS-PAGE 50 40 30 20
kDa
B) mAb 198
Lane 1 : Torpedo AChR
Lane 2 : TrxHa1-210
Lane 3 : BF-AChR
Lane 4 : Ha1-205
C) 125I a-BTX 50 40 30 20
kDa
D) mAb 5.5
1. I investigated the immuno-chemical characteristics of them.
2. I resolved same amount of each recombinant AChRs in SDS-PAGE gel.
3. An transferred them into membrane and then western-blotting with conformation specific affinity Abs and toxin.
4. In figure A show they are equal amount on the gel
5. In figure B, Lane 3, BF-AChR have no band due to absence of mAb198 binding region
6. Because Conformation specific mAb198 are specific to B-cell epitope MIR
6. In figure B and D, lane2 and 3 have similar affinity to bungarotoxin and mAb5.5
7. Collectively, these data indicate BF-AChR have no dominant two B-cell epitopes
8. without change of its spatial conformation, comparing with its patent recombinant AChR TrxH1-210.
Yi et al. Mol Imm

12. Removal of B cell epitopes reduces the reactivity to the AChR-reactive Abs from EAMG sera
1.4
* p < 0.001
1.2 * 1
Trx-Ha1-210
Relative bound Ab level
0.8
BF-AChR
0.6
Trx
0.4
0.2
EAMG serum
mAb198
mAb5.5 B)
Furthermore, I confirmed whether the removed two regions are real B-cell epitopes in myasthenia gravis.
In order to do that, I measured the reactivity between BF-AChR and sera from EAMG, compared with TrxHa1-210 reactivity.
Because the AChR-Abs from EAMG sera are originated from AChR-specific autoreactive B-cells.
I coated each recombinant AChRs on ELISA plate
And I reacted the plate with EAMG sera and measured the affinity.
As you see in here, gray bar BF-AChR have significantly reduced affinity to EAMG sera.
SDS-PAGE
EAMG serum 1 2
Yi et al. Mol Imm 1 2

13. Reduced proliferation of AChR-reactive T and B cells
by deletion of B-cell epitopes in AChR
A) Mixed lymphocyte proliferation
0.50
1.0
1.5
2.0
2.5
3.0 2 4 8 16
Protein concentration (mg/ml)
CPM(x103)
OVA
Trx-Ha1-210
BF-AChR
B) B-cell proliferation assay *
* p < 0.03
**p< 0.05
* p < 0.03
**p< 0.04 6 5 **
CPM(x103) 4 3 ** 2 * 1
Trx-
Ha1-210
TAChR
BF-AChR
OVA
26.5%
33.7%
42.1%
16.8%
C) B-cell dependent T-cell proliferation assay
TAChR
Trx-Ha1-210
BF-AChR
OVA
Normal rat CD4+
CFSE
23.5%
Furthermore, I measured B-cell dependent T-cell proliferation with BF-AChR and other antigens.
In order to verify whether B-cell activation by presence or absence of B-cell epitopes
influence autoreactive T-cell activation as an Antigen-Presenting Cells.
I isolated CD4+ T cells and B-cells from chronic EAMG rats
And I stained CD4+ T cell with CFSE, a fluorescence for viable cell tracing (assay).
The stained CD4+ T cells were cocultured with isolated B-cell upon presence of individual AChR antigens or ovalbumin.
And 5days later I re-isolated CD4+ T cells and analyzed fluorescence intensity dilution by flowcytometry.
These percentage-numbers indicate diluted population of fluorescence intensity.
In this experiment, the number of diluted population are proportional to the B-cell dependent T-cell proliferation.
This panel, gray image shows the initial CFSE fluorescence intensity in CD4+T cells without intensity-dilution, it means no-proliferation.
In this panel, TAChR, positive control. Induced high proliferation of T-cell due to its plenty of B-cell epitopes
OVA, negative control, shows the lowest T cell proliferation due to its non-specificity.
BF-AChR shows much-less T-cell proliferation, compared with TrxHa1-210 due to its absence of B-cell epitopes.
It means that absence of B-cell epitopes in BF-AChR cause reduced B-cell dependent T cell activation.
And collectively, B-cell epitopes deletion might improve immunological properties as oral tolerogen for EAGM treatment.
Yi et al. Mol Imm

14. Suppression of EAMG by BF-AChR4
OVA
Trx-Ha1-210 3
BF-AChR
* p < 0.01
Mean clinical score
** p < 0.05 2
BF-AChR * * * ** 1 **
Finally, I applied BF-AChR other proteins for EAMG treatment.
After I week or 4weeks of EAMG induction
I started oral administration of AChRs and OVA
And measured clinical scores and AChR contents in muscles.
OVA and TrxHa1-210 have no therapeutic effects on EAMG treatment, in accordance with previous reports.
BF-AChR suppressed the clinical scores of EAMG rats, compared with OVA or TrxHa1-210 treated animals
And prevented loss of AChR contents and body weights. 1 2 3 4 5 6 7 8
Weeks after immunization with AChR
TAChR
Start of Oral administration of AChRs
Yi et al. Mol Imm

15. Without B cell epitopes
With B cell epitopes

16. Effect of oral treatment with BF-AChR on AChR-specific IgG isotypes1 2 3 4 5 6
Total IgG
IgG1
IgG2a
IgG2b
IgG2c
Relative antibody titer index
OVA
Trx-Ha1-210
BF-AChR * **
* p < 0.01
** p < 0.05
So next in order to find out the underlying mechanisms of suppression by BF-AChR
I investigated the AChR-specific humoral responses against AChR.
I isolated the sera from each treated group.
And I isotyped AChR specific IgGs by Elisa
As you see here and here and here in total IgG and IgG2a and 2b
BF-AChR treatment reduced total IgG and IgG2a and IgG2b against AChR,
In contrast, TrxHa1-210 didn’t do that.
And production of pathogenic IgG2a,b, which may activate complementary pathway which would destroy neuromuscluar junction.
Furthermore, It is reported that IgG2a and b production are affected by Th1 inflammatory cytokines (phenotypically skewing).
So, reduced IgG2a/b and indirectly say that BF-AChR treatment reduced Th1 typed inflammatory cytokines.
Yi et al. Mol Imm

17. Cytokines shift by oral-adminiatration of BF-AChR
Pro-inflammatory  Anti-inflammatory
2.5
0.4
0.8
1.2
1.6
B7.1
B7.2
CD28
CD40L 2
1.5 **
Relative expression level ** 1 * * * * *
0.5 *
IL-2
IFN-g
IL-12
TNF-a 2 4 6 8
Foxp3
IL10
TGF-b *
OVA
TrxHa1-210
BF-AChR *
Relative expression level *
* p < 0.03
** p < 0.05
Yi et al. Mol Imm

18. Criteria for Safe and Effective Oral Tolerogen
Containing enough CD4 T-cell epitopes
Lack of pathogenic epitopes
CD8 T cell epitope (e.g., Type I diabetes)
Lack of B cell epitope (MG, RA, Lupus)
No or less mucosal immunity

19. Acknowledgements Israel Greece Korea Prof. Socrates J. Tzartos
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)
Korea
GIST
Hwa-Jung Yi
Chang-Suk Chae
IRT Lab Members

20. Jisim Island, KOREA. July. 2009

21. Safe and Effective Oral Tolerogen