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

Myasthenia Gravis Anti- AChR Ab AChR

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

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.

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

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

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 2000. 165: 3599-3605

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 ?

Immunogenicity of oral tolerogen ?

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

Immunochemical Characterization of BF-AChR A) SDS-PAGE 1 2 3 4 50 40 30 20 kDa B) mAb 198 1 2 3 4 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 1 2 3 4 D) mAb 5.5 1 2 3 4 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 45 . 3748-3755

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 45 . 3748-3755 1 2

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 45 . 3748-3755

Suppression of EAMG by BF-AChR 4 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 45 . 3748-3755

Without B cell epitopes With B cell epitopes

Effect of oral treatment with BF-AChR on AChR-specific IgG isotypes 1 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 45 . 3748-3755

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 45 . 3748-3755

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

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

Jisim Island, KOREA. July. 2009

Safe and Effective Oral Tolerogen