interleukin-6; back to the future prof. tadamitsu kishimoto · socs-1 il-6 gp130 il-6r jaks ... one...
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
2The screen versions of these slides have full details of copyright and acknowledgements
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Interleukin-6
• B cell Stimulatory Factor 2 (BSF-2)
• Interferon b2 (IFN b2)
• 26kD protein
• Hybridoma Plasmacytoma Growth Factor (HPGF)
• Hepatocyte Stimulating Factor (HSF)
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Em-IL-6 transgenic mouse (Fo 33)
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Impaired immune and acute-phase responses in IL-6-deficient mice
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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8
Cytokine receptor systems
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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
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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
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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13
Aberrant production of IL-6 in cardiac myxoma cells
Patients suffer from autoimmune inflammatory symptoms
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Dramatic increase in the concentration of synovial fluid IL-6 in RA patients
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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16
• Code name actemra, generic name tocilizumab
Recombinant anti-human IL-6R monoclonal antibody
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Anti-human IL-6R antibody (tocilizumab)JIA Castleman’s disease
(skin lesion)
RA
Control
Therapy
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Anti-IL-6R antibody therapy of:
• Castleman’s disease
• Rheumatoid Arthritis
• Juvenile Idiopathic Arthritis
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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Castleman’s disease
• Lymphnode swelling with plasmacyte infiltration
• Hyper-g-globulinemia
• Increase in acute phase proteins
• Development into monoclonal gammopathy and multiple myelomas
20
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
8The screen versions of these slides have full details of copyright and acknowledgements
22
Humanized anti-IL-6 receptor antibody (rhPM-1) therapy for Castleman’s disease
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A therapy of Castleman’s diseaseby humanized anti-IL-6R Ab
The assessment of lymph nodes by Ga scintigraphy
Before therapy After therapy
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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25
• Castleman’s disease
• Rheumatoid Arthritis
• Juvenile Idiopathic Arthritis
Anti-IL-6R antibody therapy of:
26
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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 )
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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28
Pre and post radiographs
Control
PostPre
Tocilizumab
PostPre
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Inhibition of RANK ligand expression by tocilizumab
IL-6+sIL-6R IL-6+sIL-6R+ actemra
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Inhibition of TRAP-positive osteoclast formation by tocilizumab
IL-6+sIL-6R+ actemra
IL-6+sIL-6R
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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
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Anti-IL-6R antibody therapy of:
• Castleman’s disease
• Rheumatoid Arthritis
• Juvenile Idiopathic Arthritis
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
12The screen versions of these slides have full details of copyright and acknowledgements
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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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 (
%)
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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)
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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47
Suppression of ClI-induced arthritis with MR16-1
Cont Ab MR16-1 MR16-1
Day 0 Day 0 Day 14
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Suppression of IL-17 production in mice treated with MR16-1
Lymph node cells(Stimulated with CII)
Serum
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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
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- 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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
21The screen versions of these slides have full details of copyright and acknowledgements
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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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
Interleukin-6; Back to the FutureProf. Tadamitsu Kishimoto
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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