Interleukin-6; Back to the Future Prof. Tadamitsu Kishimoto

Transcription

1 Interleukin-6 Back to the Future 1 MD, Ph.D. Graduate School of Frontier Biosciences, Osaka University Humanized anti-il-6r mab therapy for JIA Before treatment HT 17cm, BW 23 kg 18 months after treatment HT 125.2cm, BW 34 kg 2 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 1

2 Interleukin-6 B cell Stimulatory Factor 2 (BSF-2) Interferon b2 (IFN b2) 26kD protein Hybridoma Plasmacytoma Growth Factor (HPGF) Hepatocyte Stimulating Factor (HSF) 4 Em-IL-6 transgenic mouse (Fo 33) 5 Impaired immune and acute-phase responses in IL-6-deficient mice 6 2

3 7 Cytokine receptor systems 8 gp13 gp13 P-Y JAK Y-P JAK SH2 P-Y Y-P SH2 Y Y STAT3 STAT3 SH2 P-Y Y-P SH2 1. Dimerization of gp13 2. Activation of JAK-family tyrosine kinases 3. Tyrosine-phosphorylation of gp13 and recruitment of STAT3 4. Tyrosine-phosphorylation of STAT3 and its dimerization 5. Gene activation 9 3

4 NF-IL6(C/EBPb) induces acute phase proteins, cytokines and viruses Sos Grb GTP Ras GDP Pi NF-IL6 GDP Ras GTP Raf P P MEK MAPK Nucleus NF-IL6 Acute phase proteins Inflammatory cytokines (IL6, IL1, IL-8, TNF-a, G-CSF, Ets) Viruses (RSV, HIV-1, FIV,HBV) Transcriptional activation 1 P P ACATTGCACAATCT NF-IL6 induces HIV-1 replication by inhibiting cytidine deaminase - APOBEC3G Quiescent T cell CXCR4 Activated T cell CXCR4 CD4 UGGACC Virus RNA ACCTGG DNA (-) APOBEC3G AUUTGG DNA (-) DNA degradation by UNG * * TAAACC DNA (+) AUUTGG DNA (-) G/A hypermutation Host DNA CD4 UGGACC Host DNA Virus RNA ACCTGG DNA (-) APOBEC3G AGGTGG DNA (-) NF-IL6 P TCCACC DNA (+) AUUTGG DNA (-) Reverse Transcription Nuclear Entry Integration Viral DNA 11 Non-productive HIV-1 infection Productive HIV-1 infection Kinoshita & Taguchi, PNAS (28) Feedback regulation in IL-6 signaling IL-6 gp13 IL-6R P Y Y Y P Y JAKs Y Y STAT3 P P Y Y Activation Inhibition Acute phase proteins SOCS-1 SOCS

5 Aberrant production of IL-6 in cardiac myxoma cells Patients suffer from autoimmune inflammatory symptoms 13 Dramatic increase in the concentration of synovial fluid IL-6 in RA patients 14 Anti-IL6R antibody blocks IL-6 binding with the receptor as well as neutralizes soluble receptors IL-6 sil-6r MRA Extracellular region gp13 IL-6R Intracellular region Signal transduction 15 Gene expression 5

6 Recombinant anti-human IL-6R monoclonal antibody Code name actemra, generic name tocilizumab 16 Anti-human IL-6R antibody (tocilizumab) JIA Castleman s disease (skin lesion) RA Control Therapy 17 Anti-IL-6R antibody therapy of: Castleman s disease Rheumatoid Arthritis Juvenile Idiopathic Arthritis 18 6

7 Castleman s disease Lymphnode swelling with plasmacyte infiltration Hyper-g-globulinemia Increase in acute phase proteins Development into monoclonal gammopathy and multiple myelomas 19 2 Detection of KSHV/HHV8 in HIV positive- and negative- Multicentric Castleman s Disease (MCD) 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 GP13 which stimulates IL-6 production and induces various symptoms 21 Soulier et al., Blood 86:1275,

8 Humanized anti-il-6 receptor antibody (rhpm-1) therapy for Castleman s disease 22 A therapy of Castleman s disease by humanized anti-il-6r Ab The assessment of lymph nodes by Ga scintigraphy 23 Before therapy After therapy Anti-IL-6R Ab treatment improved laboratory abnormalities (mg/dl) (g/dl) 14 CRP Hb (µg/ml) (g/dl) 4. SAA Alb (mg/dl) IgG Week (mg/dl) T-CHO Week 8

9 Anti-IL-6R antibody therapy of: Castleman s disease Rheumatoid Arthritis Juvenile Idiopathic Arthritis ACR response rate at week 52 % Responders p<.1 35% 89% p<.1 7% p<.1 47% Control DMARDs Tocilizumab 8mg/kg % 6% ACR2 ACR5 ACR7 (September 25 ) 9

10 Pre and post radiographs Control Tocilizumab 28 Pre Post Pre Post Inhibition of RANK ligand expression by tocilizumab IL-6+sIL-6R IL-6+sIL-6R + actemra 29 Inhibition of TRAP-positive osteoclast formation by tocilizumab 3 IL-6+sIL-6R IL-6+sIL-6R + actemra 1

11 Disappearance of amyloid deposits in the colon by three injections of tocilizumab in a patient with AA amyloidosis 31 Before tocilizumab treatment Three months after treatment The RADIATE study: Research on Actemra Determining efficacy after Anti-TNF failures To assess the efficacy and safety of tocilizumab (TCZ) in combination with methotrexate (MTX) vs. placebo with MTX in patients with an inadequate response to anti-tnfs (TNF-IR) 6 5 ACR response at 24w (%) ACR2 ACR5 ACR7 5. * 4 DAS28 <2.6 at 24w (%) p< * 28.8 * 3 2 p= * 12.4 ** Placebo + MTX TCZ 4 mg/kg + MTX TCZ 8 mg/kg + MTX n=16 n=163 n=175 *p<.1 vs. placebo + MTX; **p=.2 vs. placebo + MTX Paul Emery, et al., Ann. Rheum. Dis.28; 67: Placebo + MTX TCZ 4 mg/kg + MTX TCZ 8 mg/kg + MTX Anti-IL-6R antibody therapy of: Castleman s disease Rheumatoid Arthritis Juvenile Idiopathic Arthritis 33 11

12 Humanized Anti-IL-6R mab therapy for JIA Before treatment HT 17cm, BW 23 kg 18 months after treatment HT 125.2cm, BW 34 kg 34 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 Background Systemic-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) 35 Decrease in inflammation markers (mg/dl) 3 CRP 2 (mm/hr) 12 ESR Days Days 12

13 Decrease in fever episodes Body Temperature ( o C) 37 Pre-Study Days Physicians assessment of disease activity 1 Physician's global assessment 75 ( 1 mm) Days Efficacy responses during the double-blind and open-label extension phases ACR pedi 3 response (%) ACR pedi 7 response (%) ACR pedi 5 response (%) 39 Patients with normal CRP concentration (%) Open label Double-blind Open label extension phase (weeks) phase (weeks) phase (weeks) CPR=C-reactive protein; ACR Pedi=American College of Rheumatology Pediatric 13

14 Sustained response to anti-interleukin-6 receptor antibody, tocilizumab in two patients with refractory relapsing polychondritis 4 One year after treatment 21 months after treatment Successful treatment of reactive arthritis with anti-interleukin-6 receptor antibody, tocilizumab CRP (mg/dl) MMP3 (ng/ml) Tocilizumab 6 DAS28-CRP After treatment Anti-interleukin-6 receptor antibody, tocilizumab ameliorates clinical symptoms in polymyalgia rheumatica 2 PSL 1 (mg/day) 6 CRP (mg/dl) MMP-3 (ng/ml) Tocilizumab 4 PMR-AS

15 The tocilizumab treatment ameliorated skin sclerosis in two patients with systemic sclerosis Tocilizumab 8mg/kg Case 1 Case 2 HAQ-DI mrtss month 4 2 Vesmeter hardness HAQ-DI mrtss Tocilizumab 8mg/kg month Vesmeter hardness Results Protocol MR16-1, anti-interleukin-6 receptor antibody suppressed dermal thickening and hardness in mouse model of scleroderma Dermal thickness Dermal Daily subcutaneous injection of 1μg of Bleomycin hardness A (Vesmeter) Day Day 7 Day 14 Day 21 Day 28 C57BL6 MR16-1(2mg) iv. IgG 1 (2mg) iv. MR16-1(.5mg) ip. IgG 1 (.5mg) ip. C B D assessment *: p <.1 myofibroblast (αsma+cell) Mast cell A : Cont Ab + PBS, B : Cont Ab + BLM, C : MR PBS, D : MR BLM (n = 8) 44 * : p <.1 ** : p =.4 The relationship between ACR response and serum IL-6 concentration with tocilizumab IL-6(pg/ml) Failure n=35 ACR2 n=38 ACR5 n=16 ACR7 n= Weeks Mean SE Japanese phase study MRA 9 JP submission DOSSIER 15

16 46 Suppression of ClI-induced arthritis with MR16-1 Cont Ab MR16-1 MR16-1 Day Day Day Suppression of IL-17 production in mice treated with MR16-1 Lymph node cells (Stimulated with CII) Serum 48 16

17 No suppressive effect on Th17 induction with TNFR-Fc Lymph node cells Serum IL TNFR-Fc d-14 TNFR-Fc d IFN-g Serum IL-17 (pg/ml) MR16-1(anti-IL-6R mab) treatment at day reduces the incidence of EAE Rat IgG (n=18) 1 P<.1 MR16-1 (n=19) Incidence (%) P<.1 P< Days after immunization MR16-1 treatment suppresses the development of Th17 and Th1 cells in lymph node Rat IgG MR analysis: LN at priming stage (day 8) of EAE 17

18 IL-6 KO mice are resistant to experimental autoimmune uveoretinitis (EAU) EAU Clinical Score EAU Clinical Score WT IL-6 KO Histology WT 1 µm IL-6 KO 52 1 µm Defective Th17 development in IL-6 KO mice with EAU WT Day Day 1 Day IL-6 KO IL IFN-g 53 Gate: CD4 T cells (Draining LN cells) EAU Clinical Score Both IL-17 KO mice and IFN-g KO mice develop EAU, but their disease is suppressed by anti-il-6r Ab treatment WT EAU Clinical Score NS IL-17 KO NS GKO EAU Clinical Score after therapy 4 GKO 3 EAU Clinical Score EAU Clinical Score * IL-17 KO * Anti-IL-6R Cont Ab 18

19 Regulatory T cells are important for inhibiting EAU in IL-6 KO mice Restored EAU development in IL-6 KO mice after Treg depletion EAU Clinical Score after Treg depletion 2 IL-6 KO Gate: IRBP-specific CD4 T cells IL-6 KO EAU Clinical Score 1 * Treg-depleted (acd25) Control (Rat IgG) Treg-depleted (acd25) 27.9 Control (Rat IgG) 1 3 TNF-a, IL-1 and IL-23 together with TGF-b do not induce Th17 cells - IL-6 TNF-a IL-1 IL TGF-b IL-6+TGF-b TNF-a +TGF-b IL-1+TGF-b IL-23+TGF-b IL-17 IFN-g TNF-a, IL-1 and IL-23 do not inhibit Foxp3+ Treg cells - IL-6 TNF-a IL-1 IL TGF-b IL-6+TGF-b TNF-a +TGF-b IL-1+TGF-b IL-23+TGF-b Foxp3 19

20 Aryl hydrocarbon receptor (Ahr) is specifically induced by IL-6 and TGF-b Aryl hydrocarbon receptor 4 Relative expression IL-6 TGF-b IL-6 TGF-b Ahr KO mice: 59 Aryl hydrocarbon receptor (Ahr) Similar to nuclear receptors Also known as dioxin receptor ligand 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 TNGC GTG (Gu Y-Z. et al., (2). Annu. Rev. Pharmacol. Toxicol. 4: ) XRE Also known as a ligand-dependent E3 ubiquitin ligase (Ohtake F. et al., (27). Nature 446: ) Cytoplasm Gene (CYP1A1) Nucleus 4~5% of Ahr KO mice died Slower growth rate Normal proportions of lymphocytes in spleen, lymph nodes and thymus Ahr Arnt Ahr Protein Translation mrna Induction of IL-17 by TGF-b plus IL-6 is significantly reduced in Ahr-deficient naïve T cells 8 7 WT AhrKO 4 Days IL-17(pg/ml) IL-6 TGF-b a-cd3+a-cd28 IL-6 TGF-b 2

21 Ahr specifically binds with STAT1 and STAT5, but not STAT3 nor STAT6 Lysate IP:Ahr - IL-6 TGF-b IL-6 - TGF-b IL-6 TGF-b IL-6 TGF-b a-cd3+a-cd28 IB:STAT5 IB:STAT1 IB:STAT3 IB:STAT6 61 IB:Ahr Distinct roles of the STAT family in Th17 differentiation STAT3 STAT1 STAT5 62 Ahr gene deletion blocks the CIA development 12 1 WT KO WT Ahr KO Arthritic score * * ## * * # * * ** Days after immunization 63 * P<.5 #P<.1 21

22 T cell-specific deletion of Ahr ameliorates the CIA development Lck WT Lck Ht Arthritic score * * * * * # ### Days after immunization Lck WT Lck Ht 64 * P<.5 #P<.1 Pro-inflammatory cytokines,rankl and MMP3 in the sera of Ahr KO mice pg/ml pg/ml pg/ml IL-1β ng/ml IL-6 WT Ahr KO 65 RANKL MMP3 Cytokine production in the inguinal lymph node cells of Ahr KO mice 66 pg/ml pg/ml IL-17 IFN-gamma pg/ml pg/ml IL-1 IL-4 WT Ahr KO 22

23 67 Collaboration Laboratory for Immune Signal, National Institute of Biomedical Innovation Tetsuji Naka Minoru Fujimoto Satoshi Serata Laboratory of Immune Regulation, Fumitaka Terabe Graduate School of Frontier Biosciences, Osaka University Tadamitsu Kishimoto Akihiro Kimura Taisuke Nakahama Ichino Chinen Kazuya Masuda Nguyen Nam Trung Department of Ophthalmology, Graduate School of Medicine Osaka University 68 Nobuyuki Ohguro Hiroshi Haruta Satoshi Hohki 69 23