Developing Immunotherapy for Autoimmune Diseases

Premkumar Christadoss, M.B.B.S.Department of Microbiology and Immunology

University of Texas Medical Branch301 University Blvd.

Galveston, Texas 77555-1070pchrista@utmb.edu

Generalized Myasthenia Gravis

MG

Neuromuscular Junction (NMJ)Neuromuscular Junction (NMJ)

Conti-Fine, B.M. et al. J. Clin. Invest. (116) 2843-22854, 2006Conti-Fine, B.M. et al. J. Clin. Invest. (116) 2843-22854, 2006

NMJ in MGNormal MG

Electronmicroscopy Study of NMJ of an MG Patient

Engel et al. Mayo clinic proc. 52:267, 1977

AChR is a transmembrane glycoprotein formed by five homologous subunits in the stoichiometry or .

The molecular weights of the subunits range between 45 and 55 kDa.

The subunit is Considered to be the highly immunogenic region.

Primary immunization:Primary immunization: 20 microgram AChR/CFA 20 microgram AChR/CFA

Boost: 20 microgram AChR/CFA Boost: 20 microgram AChR/CFA

28-30 days28-30 days

28-30 days28-30 days

Immunopathological evaluationImmunopathological evaluation

AChR SourceAChR Source

Monitor for clinical EAMG

EAMG inductionEAMG induction

MG in Mice

Normal

MG

Class II

Peptide(146-162)

CD4

TCR

B7CD 28

AChR

APC

IL-1, IL-12

CD4

NK IFN-IL

CD40L/CD40

AChR-specific memory T cell

Proliferation and Differentiation

IL-10, TNF-, IL-6,

IL-12, IL-18

AChR-specific memory B cells

AChR-Ab Plasma cells

C’ C’

Damage to the neuromuscular junction

Complement activation

Molecular Mechanisms of EAMG

B

C1 C4b C2a

C3 C5 MAC

CLASSICAL PATHWAY

MBL PATHWAY

ALTERNATIVE PATHWAY

(C5bC9)

C3b Bb C3bBb

MBLMASP1MASP2

COMPLEMENT PATHWAYS

-5

15

35

55

75

95

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49

Days after second immunization

Clin

ical

inci

den

ce p

erce

nta

ges

%

C3-/-

C3+/-

C3+/+

-5

15

35

55

75

95

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49

Days after second immunization

Clin

ical

inci

den

ce p

erce

nta

ges

%

C4-/-

C4+/-

C4+/+

AChR-immunized C3-/- and C4-/- mice are resistant to clinical EAMG

Tuzun -Christadoss.J. Immunol. 171:3847, 2003

0

1

2

3

4

45 75

Days after primary immunization

nM

-b

un

gar

oto

xin

b

ind

ing

sit

es

C3-/-

C3+/+C4-/-

C4+/+

** **

Serum anti-AChR antibody levels ofAChR-immunized mice

Tuzun- Christadoss.J. Immunol. 171:3847, 2003

C4+/+ C4-/- C3-/-

C3

IgG

MAC

IF Studies Reveal IgG Deposits But Not C3 or MAC Deposits at the NMJs of Mice with C3 or C4 Deficiency

RED -bungarotoxin binding (NMJ)GREEN C3, MAC or IgG deposits

Tuzun –Christadoss J. Immunol. 171:3847, 2003

Immune Intervention

Antigen/organ specific

I. AChR T cell epitope tolerance

II. AChR B cell epitope tolerance

Disease specific

I. Anti-Proinflammatory Cytokinea. Soluble TNFR (etanercept)b. IL1-Rac. Anti-IL-6

II. Blocking classical complement pathwaya. Anti C1q/C2/C4

Targeting Classical Complement Pathway

Anti-C1q Administration Suppresses EAMG

Tuzun-Christadoss, J.Neuroimmunol.174:157-167, 2006

Dual Effect of Anti-C1q

0

5

10

15

20

25

30

35

40

C3 IgG MAC

aC1q

** ** **0

5

10

15

20

25

30

35

40

C3 IgG MAC

aC1q

** ** **

non-specific IgG

* ; p<0.05

pe

rce

nta

ge

o

f d

ep

osi

ts

at

th

e

NM

J p

er

se

ctio

n (

%) IL-6

0

100

200

300

400

500

600

700

MediumMedium TAChRTAChR αα146146--162162

pg/

ml

pg/

ml

aC1qaC1qNS NS IgGIgG

****

IL-6

0

100

200

300

400

500

600

700

MediumMedium TAChRTAChR αα146146--162162

pg/

ml

pg/

ml

aC1qaC1qNS NS IgGIgG

****

* ; p<0.05

aC1q Ab non-specific IgG

PNA-binding spleen cells

0

5

10

15

20

25

30

35

40

CD4CD4 CD8CD8 CD19CD19

% l

ymp

h n

od

e ce

lls

aC1qaC1qIgGIgG **

0

5

10

15

20

25

30

35

40

CD4CD4 CD8CD8 CD19CD19

% l

ymp

h n

od

e ce

lls

aC1qaC1qIgGIgG **

* ; p<0.05

Tuzun-Christadoss, J.Neuroimmunol.174:157-167, 2006

Anti-C1q Ab Treats EAMG

0

50

100

150

200

250

300

aC1q(100) aC1q(10) isotype

gri

p s

tren

gth

(g

r)

before

after

**

0

50

100

150

200

250

300

350

aC1q(100) aC1q(10) isotype

gri

p s

tren

gth

(g

r)

**

*

*

0

0.4

0.8

1.2

1.6

aC1q(100) aC1q(10) isotype

clin

ica

l gra

de

s

***

0

0.4

0.8

1.2

1.6

2

aC1q(100) aC1q(10) isotype

clin

ica

l gra

de

s

*

***

***

B6 RIII

Tuzun-Christadoss, J.Neuroimmunol, 182: 167-176, 2007

B6

0

200

400

600

800

1000

1200

1400

none TAChR α146-162

pg

/ml

aC1q (100)

aC1q (10)

isotype

**** **

**

RIIIS/J

0

500

1000

1500

2000

2500

3000

3500

none TAChR α146-162

pg

/ml ** **

****

Anti-C1q Ab Treatment Suppresses AChR

and Dominant Peptide Specific IL-6 Production

0 20 40 60 80 100 120

TNF- p55p75

IL-6

IL-18

IL-12

IFN-

IL-10

IL-4

Normal

% clinical disease and anti-AChR antibodies

Anti-AChR Ab Disease

Effect of Cytokine Deficiencies in Effect of Cytokine Deficiencies in Clinical EAMGClinical EAMG

Gen

e D

eple

tio

n

Activation of B cells and generation of effector B cells.

Potentiates production of IgG anti-AChR antibodies (pathogenic)

Activates C3Promotes EAMG pathology

AChRspecific

TNF

IL-6

TNF

IL6

GC formationTh Th

B B

IL-6 and TNF in EAMG

Targeting ProinflammatorCytokines

A. Soluble Recombinant HumanTNFR (Etanercept)

Soluble TNFR (Etanercept) Treats EAMG

Christadoss and GoluszkoJ. Neuroimmunol, 122:186, 2002

Etanercept Treatment Fails to Suppress Serum Anti-AChR Ab

Christadoss and Goluszko, J. Neuro. Immunol. 122:186, 2002.

A pilot Trial of Etanercept in the Treatment of Steroid-Dependent MG *+

Mean change in QMG score from basline at 6 months Mean change in QMG score from basline at 6 months was - 2.9 (p=0.041).was - 2.9 (p=0.041).

Mean change in MMT at 6 months was - 8.4 (p=0.020).Mean change in MMT at 6 months was - 8.4 (p=0.020). Mean decrease in prednisone dose from baseline to end Mean decrease in prednisone dose from baseline to end

of study was 17.5 mg/48 hr dose (p=0.0084).of study was 17.5 mg/48 hr dose (p=0.0084). Etanercept was well tolerated, and no severe adverse Etanercept was well tolerated, and no severe adverse

reaction observedreaction observed

+ 11 patients enrolled; 8 completed, and 2 patients withdrawn due to disease + 11 patients enrolled; 8 completed, and 2 patients withdrawn due to disease worsening.worsening.

Immunological Effect of Etanercept

No reduction in plasma anti-AChR antibody.No reduction in plasma anti-AChR antibody.

Peripheral blood CD4 and B cell (CD19+) Peripheral blood CD4 and B cell (CD19+) counts rose steadily during the 24 week study.counts rose steadily during the 24 week study.

Patients who had higher increases in their Patients who had higher increases in their cytokine levels had a worse outcome.cytokine levels had a worse outcome.

Targeting ProinflammatorCytokines

b. Recombinant Human IL1-Ra

Activation of the Adaptive Immune System with IL-1

IL-1Ra Treatment Prevents Clinical EAMG

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 22 24 26

days after second AChR immunization

mean

clin

ical sco

re

IL-Ra 0.01mg/mouse, ip, 5 times per day

IL-Ra 0.001mg/mouse, ip, 5 times per day

placebo

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 22 24 26

days after second AChR immunization

clin

ical in

cid

en

ce (

%)

IL-Ra 0.01mg/mouse, ip, 5 times per day

IL-Ra 0.001mg/mouse, ip, 5 times per day

placebo

IL-1Ra treatmentstopped

IL-1Ra treatmentstopped

Yang –Christadoss, J. Immunol. 175:2018, 2005

Clinical severity

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

clin

ical g

rad

e

IL-1 Ra 0.01mg Placebo

Before After Before After

Serum anti-AChR Antibody

0

0.5

1

1.5

2

2.5

3

3.5

B

un

ga

roto

xin

bin

din

g s

ite

s(n

M)

IL-1 Ra 0.01mg Placebo

Before After Before After

Muscle AChR content

0

5

10

15

20

25

30

-B

TX

-bin

din

g s

ites

(p

M/g

)

IL-1 Ra 0.01mg Placebo

P<0.05

P<0.05

P<0.05

IL-1Ra Treats EAMGYang –Christadoss, J. Immunol. 175:2018, 2005

IL-1IL-1Ra

CD40L, OX40 Expression on T cells

Possible Consequence of Down Regulating IL-1 by IL-1Ra in Mice with

Clinical EAMG

Inflammatory cytokines IFN-IL-2, IL-1, IL-6, TNF-

Anti-AChR IgG , IgG1 and C3

Anti-AChR antibodies and complement mediated NMJ pathology

IL-6 in MG: Multiple Hit

IgG2b -C1qC4-C3-C5-9

IL-6

CD4

B

AChR Specific

C3

NMJ

IL-6 – A Danger Molecule in EAMG !

1.1. IL-6 deficient mice are resistant to EAMG and produce less C3IL-6 deficient mice are resistant to EAMG and produce less C3

2.2. C3 and FCC3 and FCγγRIII deficient mice are resistant to EAMG and RIII deficient mice are resistant to EAMG and produce less IL-6produce less IL-6

3.3. Amelioration of EAMG following anti-C1q treatment is Amelioration of EAMG following anti-C1q treatment is associated with reduced IL-6associated with reduced IL-6

-0.5

0

0.5

1

1.5

2

2.5

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

clin

ical sere

vit

y

anti-IL-6 Ab

PBS

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Ac

cu

mu

late

d c

lin

ica

l in

cid

en

ce

(%)

anti-IL-6 Ab

PBS

Anti-IL-6 Ab Treatment Reduces the Incidence of EAMG

Days after second immunization

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Anti-IL-6 Ab PBS

-B

T-b

ind

ing

sit

es (

nM

)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Anti-IL-6 Ab PBS

IgG

(O

D)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Anti-IL-6 Ab PBS

IgG

1 (O

D)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Anti-IL-6 Ab PBS

IgG

2b (O

D)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Anti-IL-6 Ab PBS

IgG

2c (O

D)

Anti-IL-6 Ab Treatment Suppresses Serum Anti-AChR IgG

and IgG2b Ab

0

1000

2000

3000

4000

5000

6000

T-AChR T146

antigen used in vitro

Lym

phoc

yte

prol

ifera

tion(

D cpm

) anti-IL-6 Ab

PBS

0

100

200

300

400

500

600

700

800

900

T-AChR T146

antigen used in vitro

IFN

- (p

g/m

l)

anti-IL-6 Ab

PBS

0

100

200

300

400

500

600

700

T-AChR T146

antigen used in vitro

IL-2

(pg

/ml)

anti-IL-6 Ab

PBS

0

20

40

60

80

100

120

140

160

180

T-AChR T146

antigen used in vitro

IL-6

(pg/

ml)

anti-IL-6 Ab

PBS

0

50

100

150

200

250

300

350

T-AChR T146

antigen used in vitro

IL-1

0 (p

g/m

l)

anti-IL-6 Ab

PBS

Anti-IL-6 Ab Treatment Suppresses AChR SpecificCytokine Production

AChR

Antigen presenting

cell

T helper

anti-AChR antibody

productionB cell

AChR

C1q

C3

Immune complex formation

C1qC1s

C1r

Classical complement pathway activation

Membrane attack complex formation

FcγRIII activation

Stimulation of

IL-6, C1q and C3 production

IL-6

Classical Complement Pathway and IL-6 in EAMG Pathogenesis

Balancing the Immune System to Treat Autoimmune Disease (MG)

IL-6, TNF

C3-C5b-C9

Disease

Healthy

Anti-AChR IgG

IL-6, TNF-normal level

Suppress anti-AChR and C5b-C9

AChR

Antigen presenting

cell

T helper

Suppressed anti-AChR antibody

productionB cell

AChR

C1q

C3

Immune complex formation

C1qC1s

C1r

Classical complement pathway activation

Membrane attack complex formation

FcγRIII activation

Stimulation of

IL-6, C1q and C3 production

IL-6

Targeting IL-6 and Classical Complement Pathway

MG lab in Galveston

MG Lab - GalvestonMG Lab - GalvestonErdem TuzunErdem Tuzun1,2 1,2

Shamsher SainiShamsher SainiAndrey BednovAndrey BednovBen Scott Ben Scott 33

Jing LiJing Li11

Iris WingrowIris Wingrow33

Huibin QiHuibin Qi

Xiarong WuXiarong Wu

1MG foundation Osserman/Sosin/McClure Post doctoral Fellows

1,2MDA Research Career Award Recipients

3 MG Foundation Henry Viets Fellow

Supported by NIH,MDA, AFM,and MG Foundation

CollaboratorsCollaboratorsHuan YangHuan YangBo WuBo WuStephen HiggsStephen HiggsTian Lin XioTian Lin XioGalen KaufmannGalen KaufmannMat MerigioliMat MerigioliJuli RowinJuli Rowin