interleukin-6; back to the future prof. tadamitsu kishimoto · socs-1 il-6 gp130 il-6r jaks ... one...

Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto

1The screen versions of these slides have full details of copyright and acknowledgements

1

Interleukin-6Back to the Future

Prof. Tadamitsu Kishimoto MD, Ph.D.Graduate School of Frontier Biosciences,

Osaka University

2

Before treatmentHT 107cm, BW 23 kg

18 months after treatmentHT 125.2cm, BW 34 kg

Humanized anti-IL-6R mAb therapy for JIA

• 5 y.o. Boy, Disease duration 1 years 2 months• Previous treatment ASA, PSL, mPSL, LDx, CsA, AZP• Complications:

– Growth retardation

– Compression fracture of T-spine due to osteoporosis

3



4

Interleukin-6

• B cell Stimulatory Factor 2 (BSF-2)

• Interferon b2 (IFN b2)

• 26kD protein

• Hybridoma Plasmacytoma Growth Factor (HPGF)

• Hepatocyte Stimulating Factor (HSF)

5

Em-IL-6 transgenic mouse (Fo 33)

6

Impaired immune and acute-phase responses in IL-6-deficient mice



7

8

Cytokine receptor systems

9

1. Dimerization of gp130

2. Activation of JAK-family tyrosine kinases

3. Tyrosine-phosphorylation of gp130 and recruitment of STAT3

4. Tyrosine-phosphorylation of STAT3and its dimerization

5. Gene activation

gp130 gp130

P-Y JAK Y-PJAK

SH2 P-Y Y-P SH2

STAT3 STAT3

SH2SH2

P-YY-P

Y Y



10

Ras

Ras

GTP

GDP GTP

GDP Pi

Raf

MEK

MAPK

Sos Grb

Inflammatory cytokines Acute phase proteins

Viruses(RSV, HIV-1, FIV,HBV)

(IL6, IL1, IL-8, TNF-a, G-CSF, Ets)

Transcriptional activation

Nucleus

NF-IL6

P P

NF-IL6

P PACATTGCACAATCT

NF-IL6(C/EBPb) induces acute phase proteins, cytokines and viruses

11

CXCR4

Host DNA

CD4

Quiescent T cell

Host DNA

CXCR4

CD4

Activated T cell

Productive HIV-1 infectionNon-productive HIV-1 infectionKinoshita & Taguchi, PNAS (2008)

NF-IL6 induces HIV-1 replication by inhibiting cytidine deaminase - APOBEC3G

DNA degradation by UNG

UGGACC Virus RNA

ACCTGG DNA (-)

AUUTGGTAAACC DNA (+)

DNA (-)

AUUTGG DNA (-)

APOBEC3G

* *

G/A hypermutation

UGGACC Virus RNA

ACCTGG DNA (-)

AUUTGGTCCACC DNA (+)

DNA (-)

AGGTGG DNA (-)

APOBEC3G

Viral DNAIntegration

Nuclear Entry

ReverseTranscription

NF-IL6 P

12

STAT3

Feedback regulation in IL-6 signaling

Activation Inhibition

Y YP P

PP

Y Y

SOCS-1

Y Y

Y Y

Acutephase

proteinsSOCS-1

IL-6 gp130

IL-6R JAKs



13

Aberrant production of IL-6 in cardiac myxoma cells

Patients suffer from autoimmune inflammatory symptoms

14

Dramatic increase in the concentration of synovial fluid IL-6 in RA patients

15

IL-6R

MRA

Intracellularregion

gp130

Signaltransduction

Geneexpression

Anti-IL6R antibody blocks IL-6 binding with the receptor

as well as neutralizes soluble receptors

IL-6

sIL-6R

Extracellularregion



16

• Code name actemra, generic name tocilizumab

Recombinant anti-human IL-6R monoclonal antibody

17

Anti-human IL-6R antibody (tocilizumab)JIA Castleman’s disease

(skin lesion)

RA

Control

Therapy

18

Anti-IL-6R antibody therapy of:

• Castleman’s disease

• Rheumatoid Arthritis

• Juvenile Idiopathic Arthritis



19

Castleman’s disease

• Lymphnode swelling with plasmacyte infiltration

• Hyper-g-globulinemia

• Increase in acute phase proteins

• Development into monoclonal gammopathy and multiple myelomas

20

21

Detection of KSHV/HHV8 in HIV positive- and negative-

Multicentric Castleman’s Disease (MCD)

Soulier et al., Blood 86:1275, 1995

• The KHSV/HHV8 genome can be detected in most Castleman’s disease-affected lymphnodes

• The HHV8 genome includes the viral IL-6 gene

• Viral IL-6 does not bind to the human IL-6R, but can directly bind to human GP130 which stimulates IL-6 production and induces various symptoms



22

Humanized anti-IL-6 receptor antibody (rhPM-1) therapy for Castleman’s disease

23

A therapy of Castleman’s diseaseby humanized anti-IL-6R Ab

The assessment of lymph nodes by Ga scintigraphy

Before therapy After therapy

24

CRP

02468

10(mg/dL)

SAA

0

200

400

600(µg/mL)

Hb

8

10

12

14(g/dL) Alb

2.5

3.0

3.5

4.0(g/dL)

IgG

2000

3000

4000

5000

6000

0 12 24 36 48 60Week

(mg/dL) T-CHO

100120140160180200

0 12 24 36 48 60Week

(mg/dL)

Anti-IL-6R Ab treatment improved laboratory abnormalities



25





26

27

p<0.001ControlDMARDs

Tocilizumab8mg/kg

ACR20 ACR50 ACR70

100

80

60

40

20

0

p<0.001

p<0.00189%

70%

47%

35%

14%6%

ACR response rate at week 52

% R

espo

nder

s

(September 2005 )



28

Pre and post radiographs

Control

PostPre

Tocilizumab

PostPre

29

Inhibition of RANK ligand expression by tocilizumab

IL-6+sIL-6R IL-6+sIL-6R+ actemra

30

Inhibition of TRAP-positive osteoclast formation by tocilizumab

IL-6+sIL-6R+ actemra

IL-6+sIL-6R



31

Disappearance of amyloid deposits in the colon by three injections of tocilizumab in a patient with AA amyloidosis

Before tocilizumab treatment Three months after treatment

32

The RADIATE study: Research on ActemraDetermining effIcacy after Anti-TNF failurEs

*

*

*

*

**10.1

30.4

50.0

3.8

16.8

28.8

1.35.0

12.4

0

10

20

30

40

50

60

Placebo + MTXn=160

TCZ 4 mg/kg + MTXn=163

TCZ 8 mg/kg + MTXn=175

ACR20 ACR50 ACR70

ACR response at 24w (%)

*p<0.0001 vs. placebo + MTX; **p=0.0002 vs. placebo + MTX

p<0.0001

p=0.0533

1.6

7.6

30.1

0

10

20

30

40

DAS28 <2.6 at 24w (%)

Placebo + MTX

TCZ 4 mg/kg + MTX

TCZ 8 mg/kg + MTX

To assess the efficacy and safety of tocilizumab (TCZ) in combination with methotrexate (MTX) vs. placebo with MTXin patients with an inadequate response to anti-TNFs (TNF-IR)

Paul Emery, et al., Ann. Rheum. Dis. 2008; 67: 1516-1523

33







34

Humanized Anti-IL-6R mAb therapy for JIA

Before treatmentHT 107cm, BW 23 kg

18 months after treatmentHT 125.2cm, BW 34 kg

• 5 y.o. Boy, Disease duration 1 years 2 months• Previous treatment ASA, PSL, mPSL, LDx, CsA, AZP• Complications:

– Growth retardation

– Compression fracture of T-spine due to osteoporosis

35

BackgroundSystemic-onset Juvenile Idiopathic Arthritis

• Poor QOL (spiking fever, arthritis, etc.)

• Growth retardation

• Osteoporosis

• Disease transition to Macrophage Activation Syndrome,and death (4~6%)

• Limited medications (high-dose corticosteroids)

36

Decrease in inflammation markers

0

10

20

30

0 10 20 30 40 50 60 70 80

CRP

(mg/dL)

Days

(mm/hr)

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70 80

ESR

Days



37

Decrease in fever episodes

Bod

y Te

mpe

ratu

re (o

C)

DaysPre-Study

38

Physicians’ assessment of disease activity

0

25

50

75

100

0 10 20 30 40 50 60 70 80

Physician's global assessment

Days

(0 –

100

mm

)

39

Efficacy responses during the double-blind and open-label extension phases

CPR=C-reactive protein; ACR Pedi=American College of Rheumatology Pediatric

ACR

pedi

30

resp

onse

(%

)Pa

tient

s wi

th n

orm

al

CRP

con

cent

ratio

n (%

)

Open label phase (weeks)

Double-blindphase (weeks)

Open label extension phase (weeks)

ACR

pedi

50

resp

onse

(%

)AC

R pe

di 7

0re

spon

se (

%)



40

Sustained response to anti-interleukin-6 receptor antibody, tocilizumab in two patients

with refractory relapsing polychondritis

21 months after treatmentOne year after treatment

41

Successful treatment of reactive arthritis with anti-interleukin-6

receptor antibody, tocilizumab

CRP(mg/dl)

Tocilizumab

DAS28-CRP

-200 -100 0 100 200

MMP3(ng/ml)

800

400

0

6

3

0

After treatment

6

3

0

42

Anti-interleukin-6 receptor antibody, tocilizumab ameliorates clinical symptoms

in polymyalgia rheumatica

01020

0

3

6

-600 -400 -200 0 2000

20

400

400

800

Tocilizumab

CRP(mg/dl)

MMP-3(ng/ml)

PMR-AS

PSL(mg/day)



4310

15

20

25

30

35

0

20

40

60

Tocilizumab 8mg/kg

0

0.5

1

0 2 4 6-2-4-6-8

15

20

25

30

20

40

60

80

mR

TSS

0.5

1

1.5

2

Tocilizumab 8mg/kg

HAQ

-DI

0 2 4 6-2-4-6month

Vesmeter hardness

month

Case 1 Case 2

The tocilizumab treatment ameliorated skin sclerosis

in two patients with systemic sclerosis

HAQ

-DI

mR

TSS

Vesmeter hardness

44

MR16-1, anti-interleukin-6 receptor antibody suppressed dermal thickening and hardness

in mouse model of scleroderma

C57BL6Day 0 Day 7 Day 14 Day 21 Day 28

Daily subcutaneous injection of 100μg of Bleomycin

MR16-1(2mg) iv. MR16-1(0.5mg) ip. assessmentIgG1 (2mg) iv.IgG1 (2mg) iv. IgG1 (0.5mg) ip.

*: p < 0.01

* : p < 0.01** : p = 0.04

Mast cell

ProtocolResults

myofibroblast (αSMA+cell)

Dermal thicknessDermal hardness (Vesmeter)

(n = 8)

A B

C

A : Cont Ab + PBS, B : Cont Ab + BLM,C : MR16-1 + PBS, D : MR16-1 + BLM

D

45 Japanese phase study MRA 009 JP submission DOSSIERMean±SEWeeks

The relationship between ACR response and serum IL-6 concentration with tocilizumab

0

50

100

150

0 4 8 12

IL-6

(pg/

ml)

Failure n=35ACR20 n=38ACR50 n=16ACR70 n=20



46

47

Suppression of ClI-induced arthritis with MR16-1

Cont Ab MR16-1 MR16-1

Day 0 Day 0 Day 14

48

Suppression of IL-17 production in mice treated with MR16-1

Lymph node cells(Stimulated with CII)

Serum



49

No suppressive effect on Th17 induction with TNFR-Fc

IFN-g

IL-1

7

TNFR-Fc d0-14 TNFR-Fc d21-

Lymph node cells Serum

-828 0 103 104 105 -525 0 103 104 105

-301

0 1

0210

310

410

5

-386

0 1

0210

310

410

5

350

300

250

200

150

100

50

0

Seru

m IL

-17

(pg/

ml)

50Days after immunization

Inci

denc

e (%

)

P<0.05

P<0.01

Rat IgG(n=18)

MR16-1(n=19)

MR16-1(anti-IL-6R mAb) treatment at day 0 reduces the incidence of EAE

0

20

40

60

80

100

0 5 10 15 20 25 30

P<0.001

51

MR16-1 treatment suppresses the development of Th17 and Th1 cells in lymph node

Rat IgG

MR16-1

analysis: LN at priming stage (day 8) of EAE



52

IL-6 KO mice are resistant to experimental autoimmune uveoretinitis (EAU)

WT

IL-6 KO

Histology

EAU Clinical Score

0 11 15 17 19 21 25 290

1

2

3

EAU

Clin

ical

Sco

re

WT

IL-6 KO

100 µm

100 µm

53

Defective Th17 development in IL-6 KO mice with EAU

Day 0 Day 10 Day 20

WT

IL-6 KO

Gate: CD4 T cells (Draining LN cells)

0.5

0.5

5.9

2.4

1.1

1.6

0.1

0.4

0.7

2.2

0.6

3.1IL-1

7

IFN-g

54

Both IL-17 KO mice and IFN-g KO mice develop EAU, but their disease is suppressed

by anti-IL-6R Ab treatment

Cont Ab

*

GKOEAU Clinical Score EAU Clinical Score after therapy

IL-17 KO GKOWT

NSNS

0

1

2

3

4

EAU

Clin

ical

Sco

re

Anti-IL-6R

*

IL-17 KO

0

1

2

3

EAU

Clin

ical

Sco

re

0

1

2

3

4

EAU

Clin

ical

Sco

re



55

Regulatory T cells are important for inhibiting EAU in IL-6 KO mice

Restored EAU development in IL-6 KO mice after Treg depletionEAU Clinical Score after Treg depletion

*

IL-6 KOGate: IRBP-specific CD4 T cells

30.0

Treg-depleted (aCD25)

Control (Rat IgG)

IL-6 KO

Treg-depleted (aCD25)

Control (Rat IgG)

0

1

2

EAU

Clin

ical

Sco

re

27.9

56

TNF-a, IL-1 and IL-23 together with TGF-bdo not induce Th17 cells

- IL-6 TNF-a IL-1 IL-23

TGF-b IL-6+TGF-b TNF-a +TGF-b IL-1+TGF-b IL-23+TGF-b

IL-17

IFN-g

103

102

101

100

103

102

101

100

100 101 102 103 100 101 102 103100 101 102 103 100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103

57

- IL-6 TNF-a IL-1 IL-23

TGF-b IL-6+TGF-b TNF-a +TGF-b IL-1+TGF-b IL-23+TGF-b

Foxp3

TNF-a, IL-1 and IL-23 do not inhibit Foxp3+ Treg cells

100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103



58

Rel

ativ

e ex

pres

sion

Aryl hydrocarbon receptor

- IL-6 TGF-b IL-6TGF-b

Aryl hydrocarbon receptor (Ahr) is specifically induced by IL-6 and TGF-b

0

5

10

15

20

25

30

35

40

59

• Similar to nuclear receptors

• Also known as dioxin receptor

• Exogenous ligands such as dioxin and flavonoids cause diverse toxic effects

• Transcriptional activation through protein interactions

• The natural ligand of Ahr is not well known(Gu Y-Z. et al., (2000). Annu. Rev. Pharmacol. Toxicol.40: 519-61)

• Also known as a ligand-dependent E3 ubiquitin ligase (Ohtake F. et al., (2007). Nature 446: 562-566)

• Ahr KO mice:– 40~50% of Ahr KO mice died

– Slower growth rate

– Normal proportions of lymphocytes in spleen, lymph nodes and thymus

Aryl hydrocarbon receptor (Ahr)

Gene (CYP1A1)

Cytoplasm

Ahr

ligand

mRNA

Protein Translation

XRE

TNGC GTG

Nucleus

Ahr

Arnt

60

IL-1

7(pg

/ml)

-IL-6

TGF-bIL-6 TGF-b

4 Days

a-CD3+a-CD28

Induction of IL-17 by TGF-b plus IL-6 is significantly reduced

in Ahr-deficient naïve T cells

0

1000

2000

3000

4000

5000

6000

7000

8000

WTAhr KO



61

- IL-6TGF-bIL-6 TGF-b - IL-6

TGF-bIL-6 TGF-b

Lysate IP:Ahr

IB:STAT5

IB:STAT1

IB:STAT3

IB:STAT6

IB:Ahr

a-CD3+a-CD28

Ahr specifically binds with STAT1 and STAT5, but not STAT3 nor STAT6

62

Distinct roles of the STAT family in Th17 differentiation

STAT5

STAT1

STAT3

63

-2

0

2

4

6

8

10

12

-10 10 30 50 70

WT KO

* * # # * * # * * **

* P<0.05 #P<0.01

Arth

ritic

sco

re

Days after immunization

Ahr gene deletion blocks the CIA development

WT Ahr KO



64

-2

0

2

4

6

8

10

12

14

0 20 40 60 80

Lck WT Lck Ht

* * * * * # # ##

Arth

ritic

sco

re

Days after immunization

T cell-specific deletion of Ahrameliorates the CIA development

Lck WT Lck Ht

* P<0.05 #P<0.01

650

50

100

150

200

250

RANKL

Pro-inflammatory cytokines ,RANKL and MMP3 in the sera of Ahr KO mice

0

20

40

60

80

100

120

IL-1β0

50

100

150

200

250

IL-6

0

100

200

300

400

500

600

MMP3

WTAhr KO

pg/m

l

pg/m

l

pg/m

l

ng/m

l

66

0

500

1000

1500

2000

2500

3000

IL-17

Cytokine production in the inguinal lymph node cells of Ahr KO mice

WTAhr KO

pg/m

l

01020304050607080

IL-10

pg/m

l

0

500

1000

1500

2000

2500

3000

IFN-gamma

pg/m

l

05

101520253035

IL-4

pg/m

l



67

68

CollaborationLaboratory for Immune Signal, National Institute of Biomedical Innovation

• Tetsuji Naka• Minoru Fujimoto• Satoshi Serata• Fumitaka Terabe

Department of Ophthalmology, Graduate School of MedicineOsaka University

• Nobuyuki Ohguro• Hiroshi Haruta• Satoshi Hohki

Laboratory of Immune Regulation, Graduate School of Frontier Biosciences, Osaka University

• Tadamitsu Kishimoto• Akihiro Kimura• Taisuke Nakahama• Ichino Chinen• Kazuya Masuda• Nguyen Nam Trung

69

interleukin-6; back to the future prof. tadamitsu kishimoto · socs-1 il-6 gp130 il-6r jaks ... one...

Documents