Immunotherapy

Topics:Currently used immunotherapies

Monitoring response

Next generation: IL-15?

Pathogenesis: role of bone marrow

Synovial membrane in RA

Synovial fluid in RAcartilageHealthy joint Rheumatoid arthritis Macrophage-like synoviocytes Fibroblast-like synoviocytes Activated:Macrophage-like synoviocytesFibroblast-like synoviocytes, macrophages, T lymphocytes, B-lymphocytes , dendritic cells, NK cells, mast cells, angiogenesisActivated: Granulocytes, T-lymphocytes,B-lymphocytes, macrophagesNK-cells, plateletsDegraded: cartilage boneHealthy synovial membraneQuiescent:Modfied from Panayi et al. NEJM, 2001

Pathogenesis of Rheumatoid Arthritis different mechanisms and targets for immunotherapiesRANKLDenosumab (anti RANKL Ab)

Structure of anti-TNF agentsEtanercept(Enbrel)Infliximab(Remicade)Recombinant fusion protein: receptor/Fc fragment of IgG1Monoclonal AbAdalimumab(Humira) = mouse= humanModified: Hanauer SB. Gastroenterol Disord 2004;4(Suppl. 3):S18-24.IgG1FcIgG1Fc FabReceptor

InfliximabEtanerceptTNFaTNFbtransmembrane TNFaTNF receptorTNF receptorABCZiolkowska M, Maslinski WCurrent Opinion in Rheumatology, 2003, 15 (3)

Efficacy of anti-TNF therapy in RASmolen et al. Lancet 2007, 370, 1861IFXADAETARTXRTXABAABATOC

Roughly 50% of RA patients treated with anti-TNFa therapy are non-respondersHeterogeneity of RA

In non-responders, the role of TNFa is less pronounced

Identification of responders/non-responders

Other cytokines (IL-1, Anakinra, IL-6, Tocilizumab, IL-15..?), Cells:T-cells: CTLA4-Ig, Abatacept, B-cells: anti CD20 Ab, Rituximab

are more important for individual patients

IL-1 receptor antagonist (IL-1Ra)Signal transductionNo signalIL-1IL-1RaEffective blocking: IL-1Ra > IL-1

MRA (Tocilizumab) anti-IL-6 receptor antibodyMRA antibody binds to IL-6R alpha and indirectly prevents IL-6 signalingIL-6Rgp130sIL-6RsignalingIL-6Active complexIL-6IL-6Rgp130sIL-6RNo signalingIL-6No complex formationIL-6

Co-stimulation - termination stage:enhancement of CTLA4 expression during T-cell activation Induction (early phase)Termination (late phase)T cellAPCCTLA-4CD28 B7-1B7-2T cellAPCCTLA-4 B7-1B7-2CTLA-4CD28CTLA-4CTLA-4CD28B7-1B7-1CTLA4 binds B7 with higher (20X) affinity than CD28and blocks CD28-B7-1, B7-2 interaction Low CTLA4 expression can not block CD28-B7 interaction

Abatacept - structure

Extracellular domain of human CTLA4 receptorFc fragment of human IgG1 antibody

Biological effects of IL-15IL-15T-cell:ChemotaxisProliferationCytokine production (IFN-g, IL-17)Reduced apoptosisCytotoxicityOntogenyMaintenance of CD8+B-cell:MaturationIsotype switchActivationEndothelial cell:ICAM expressionAngiogenesis

Mast cell:ProliferationAnti-apoptotic(no b chain required)Muscle cell:DifferentiationAnabolic agentNK cell:CytotoxicityCytokine production (IFN-g)Ontogeny (no NK cells in IL-15KO mice)Neutrophil:ChemotaxisReduced apoptosisCytoskeleton rearrangementTrigger oxidative burst Synoviocyte:ProliferationCytokine productionIL-15/IL-15R- autocrine activation- proliferation -anti-apoptotic statusEpithelial cellTightens cell-cell interaction(no b-chain required)Monocyte/Macrophage:Activation,Induction: TNF-a, IL-1b....

Structure of IL-15 receptor antagonist (IL-15 mutant/Fcg2a, CRB-15)Q101DQ108DQ108DQ101Dhuman IL-15mutant (Q101D; Q108D)murine Fcg2aKim et al.1998. J. Immunol 160:5742

Short-term treatment with CRB-15 decreases the incidence and severity of collagen-induced arthritisFerrari-Lacraz et al. J. Immunol. In press (2004)treatment

CRB-15 is effective in preventing disease progression in mice with established arthritisFerrari-Lacraz et al. J. Immunol. In press (2004)treatment

HuMax-IL15 (anti-IL15 human Ab) Phase I/II RA

Encouraging short-term efficacyACR20 63%ACR50 38%ACR70 25%No safety or tolerability issues 30 patientsSix dose groups - 0.15 to 8 mg/kg and placeboTreatment once per week for 5 weeksMaximal response at weeks 7-11

Identification of patients responding/non-responding to anti-TNF therapy

RANKL, RANK and OPG regulates maturation, activation and survival of osteoclastsAdapted from Boyle WJ, et al. Nature 2003; 423: 337- 42.Receptor activator of NF-kB ligand - RANKL Receptor activator of NF-kB RANKOsteoprotegerin - OPG

Infliximab treatment normalizes serum OPG and RANKL levels in RA patientsOPG concentrations during time of treatmentRANKL concentrations during time of treatmentGroup 1: RA patients below 48 years old; n=10; mean age SEM 35.0 2.3 yearsGroup 2: RA patients over 48 years old; n=11; mean age SEM 56.5 2.0 yearsZiolkowska M. et al. Arthritis and Rheumatism, 2002, 46; 1744-53.

The EULAR response criteriaThe disease activity score (DAS28) including 28 joint counts for tenderness and swelling, ESR and the patient general health. The cumulative response score (CRS) over 62 weeks treatmentTime of infliximab + MTX treatment (weeks)026143022384662549 evaluations of response over 62 weeks of treatment

Changes of disease activity in subgroups of RA patients treated with infliximab plus MTXNS P < 0.0002 P < 0.0002P < 0.00002P < 0.05 vs P < 0.02 vs P < 0.007 vs Time of treatment (weeks)

Laboratory and clinical changes after anti-TNF treatment in RA patientsTime of treatment (weeks) Group 1- responders Group 2 non-responders

Different levels/changes of TNFa, RANKL and OPG in responders and non-responders to anti-TNFa treatment TNFa RANKL OPG Time of treatment (weeks)

P < 0.05, P < 0.001 - healthy vs group 1 or 2; # P < 0.05 - group 1 vs group 2; ! P < 0.05, !! P < 0.005, !!! P < 0.0003 - week 0 vs weeks 2-22

Relations between the cumulative response of RA patients during 62 weeks of anti-TNFa therapy and DAS28 or % of OPG changes from baseline

Group 1 responders Group 2 - nonresponders

Predictors of response to anti-TNFa treatment in RA patients Group 1- responders Group 2 non-responders

Wykres3

-36.0134003355

-423.29

-393.36

-22.01154163237.01

-63.99491094154.68

-15.14976958536.63

-45.20333680929.94

-59.76360502344.6

-44.27.25

-19.49860724234.1

-17.59371221288.11

-28.38235294127.6

-32.48667293827.99

-7.88840788846.62

9.04-16.4576457646

10.31-2.8073286052

8.92-30.2262819182

5.7716.1174355902

9.71-13.7090909091

7.544.7213622291

11.53-36.3546310054

9.21-33.4621044886

7.85-20.0258397933

7.691

10.07-28.4560350796

13.02-10.6888633754

11.114.0033130867

9.72.76816609

12.1434.2863475177

5.5639.8871877518

8.593.0456852792

11.864.1845493562

9.43-7.5183553598

11.71-26.7326732673

11.4725.1227259602

10.916.8426724138

Sum of DAS28 at 6 and 14 weeks

92% responders 5% non-responders

Group 1

Group 2

% of OPG change at 14 week

Arkusz1 (2)

2002- Figures - praca remicade; arkusz 1

DAS 28

02614223038465462

Group 16.173.443.073.093.023.212.993.153.123.21

Group 27.084.914.794.904.845.705.445.986.105.83

All patients6.724.344.124.194.134.734.494.884.944.79

Zmiany DAS

2614223038465462

Group 12.733.093.083.152.963.183.013.052.96OPG

Group 22.132.272.182.241.381.641.100.971.24Serum3

All patients2.362.592.532.591.992.241.841.781.93SF14.5

Clinical responsesRANKL

2614223038465462Serum262

Group 11.431.501.501.571.571.501.571.501.36SF588

Group 20.860.860.820.860.590.640.500.320.41

All patients1.081.111.081.140.970.970.920.780.78

Ratio

Serum12.8

Group 139%n=14number of patientsSF25.4

2614223038465462

good677887876

moderate877667677

no000000001

Group 261%n=22number of patients

2614223038465462

good231100100

moderate151316171413877

no565489131515

All patients - number of patients

2614223038465462All patientsGroup 1Group 2

good8108987976Time of treatment (weeks)

moderate232023232020131414

no565489141516

Group 1percent of patients

2614223038465462

good435050575750575043

moderate575050434350435050

no000000007

Group 2Time of treatment (weeks)

2614223038465462

good9145500500Good responseModerate responseNo response

moderate685973776459363232

no232723183641596868

All patients

2614223038465462

good222822252219251917

moderate645664645656363939

no141714112225394244

Stezenie OPG (pg/ml)% of OPG changes

0261422261422

Group 134652859266422172331Group 1-17.0-23.0-33.8-32.9

Group 236143484357533593429Group 2-1.72.5-3.9-0.8

Healthy22282228222822282228

sRANKL

01422

Group 1267213219

Group 2395315289 P < 0.05, P < 0.001- healthy versus group 1 or 2

Healthy229229229# P < 0.05 - group 1 versus group 2

* P < 0.05, ** P < 0.005, *** P < 0.0003 -

TNFweek 0 versus weeks 2-22

01422Lack of marks - not significant

Group 17710286

Group 2150178134

Healthy161616

Arkusz1 (2)

6.16642857147.07772727276.7208108108

3.43928571434.91090909094.3578378378

3.06785714294.78954545454.1451351351

3.08785714294.89863636364.2108108108

3.01785714294.83681818184.1413513514

3.215.69818181824.6591891892

2.99142857145.43590909094.4754054054

3.15285714295.88210526324.646875

3.125.95666666674.771875

3.20571428575.62611111114.5774193548

Group 1

Group 2

All patients

DAS28 (score)

Arkusz1

2.72714285712.1304545455

3.09428571432.2713636364

3.07857142862.1790909091

3.14857142862.2409090909

2.95642857141.3795454545

3.1751.6418181818

3.01357142861.2055555556

3.04642857140.9577777778

2.96071428571.3258823529

Group 1

Group 2

DAS28 improvement (score)

Arkusz2

00

00

00

00

00

00

00

00

00

Group 1

Group 2

Clinical response (score)

Arkusz3

000

000

000

000

000

Time of treatment (weeks)

**

***

***

*

#

#

0 2 6 14 22

Group 1

Group 2

Healthy

OPG levels (ng/ml)

000

000

000

***

**

*

*

#

#

Group 1

Group 2

Healthy

RANKL levels (pg/ml)

000

000

000

#

Group 1

Group 2

Healthy

TNF alpha levels (pg/ml)

019

029

Sum of DAS28 at 6 and 14 weeks

92% responders 5% non-responders

Group 1

Group 2

% of OPG change at 14 week

5

5

3.29

3.29

3.36

3.36

7.01

7.01

4.68

4.68

6.63

6.63

9.94

9.94

4.6

4.6

7.25

7.25

4.1

4.1

8.11

8.11

7.6

7.6

7.99

7.99

6.62

6.62

9.04

9.04

10.31

10.31

8.92

8.92

5.77

5.77

9.71

9.71

7.54

7.54

11.53

11.53

9.21

9.21

7.85

7.85

7.69

7.69

10.07

10.07

13.02

13.02

11.11

11.11

9.7

9.7

12.14

12.14

5.56

5.56

8.59

8.59

11.86

11.86

9.43

9.43

11.71

11.71

11.47

11.47

10.91

10.91

-36.013400335

-42

-39

-22.0115416323

-63.9949109415

-15.1497695853

-45.2033368092

-59.7636050234

-44.2

-19.4986072423

-17.5937122128

-28.3823529412

-32.4866729382

-7.8884078884

-16.4576457646

-2.8073286052

-30.2262819182

16.1174355902

-13.7090909091

4.7213622291

-36.3546310054

-33.4621044886

-20.0258397933

1

-28.4560350796

-10.6888633754

4.0033130867

2.76816609

34.2863475177

39.8871877518

3.0456852792

4.1845493562

-7.5183553598

-26.7326732673

25.1227259602

6.8426724138

sRANKL

RANKL (pg/ml)

00.2

01.3

OPG

OPG (ng/ml)

01.4

02.7

Ratio

Ratio OPG:sRANKL

ConclusionCombined values of :

DAS28 at weeks 6 and 14andReduction of serum OPG levels at week 14

may predict the efficacy of infliximab plus MTX long term treatment of RA patients.

Bone marrow oedema and subchondral cellular infiltrate in RA McQueen FM and Ostendorf B, AR&T 2006, 8:222

Bone marrow in animal models of RA During early stages of collagen induced arthritis (CIA) (before arthritis) enlargement of bone canals connecting bone marrow with synovium and traffic of mesenchymal cells was observed. Anti-TNF treatment blocks this effect.Marinova-Mutachieva et al. 2002, Arthritis Rheum 46:507

2.During ajuvant arthritis mycobacterial DNA spreads to bone marrow and spleen but not to the synovium. Ronaghy et al. J.Immunol. 2002, 168:51

3. Antigen induced arthritis can be transferred by bone marrow transplantation indicating abnormalities in this compartment.Kobayashi et al. J. Rheumatol. 2002, 29:1176

Bone marrow in RA Bone marrow: primary and secondary lymphoid organ

Efficient antigen presentation (exceeding lymph nodes) takes place in bone marrow M. Feuerer; Nat Med 2003; 1151-7

Involvement of subchondral bone marrow in rheumatoid arthritisBugatti et al., Arthritis Rheum 2005, 52: 3448

4. Bone marrow edema in rheumatoid arthritis McQueen and Ostendorf. Arthritis Res & Therapy 2006, 8: 222

5. Bone erosions and marrow edema reflect true bone marrow inflammation in rheumatoid arthritisJimenez-Boj at al. Arthritis Rheum 2007, 56: 1118

6.Naive recirculating B cells mature simultaneously in the spleen and bone marrow (mice)Cariappa et al. Blood 2007, 109: 2339

TOPICSProinflammatory cytokine microenvironment in RA bone marrow promotes osteoclast maturation and activation

Lymphocyte subpopulations in bone marrow: the role for IL-15 ?

Regulatory T-cells in bone marrow

Functional Toll-like receptors (TLR) expressed on B-cells in bone marrow

Levels of soluble RANKL and OPG in bone marrow plasmaLower OPG/RANKL ratio in RA than in OA suggests that osteoclast maturation/activation microenvironment in RA bone marrow contributes to faster bone degradation OPGRANKLOPG/RANKL ratio04000800012000160002400028000200000204080100140160120602001800100200300400600700500800n.s.P < 0.001RARARAOAOAOAOPG concentrations (pg/ml)RANKL concentrations (pg/ml)P < 0.001Radzikowska et al. in preparation

Elevated levels of TNFa in bone marrow plasma from RA in comparison to OA patientsOARAP=0,04Warnawin et al. in preparation

Elevated levels of IL-1 in bone marrow plasma from RA in comparison to OA patientsOARAP=0,03Warnawin et al. in preparation

Elevated levels of IL-15 in bone marrow plasma from RA in comparison to OA patientsP=0.01Warnawin et al. in preparation

Wykres1

760238.7

1304.5965.3

IL-15 pg/ml

Arkusz1

OARA

7601304.5

Arkusz1

0238.7

0965.3

IL-15 pg/ml

Arkusz2

Arkusz3

Levels of IL-6, sIL-6R and sgp130 in bone marrow isolated from RA and OA patientsLower level of sgp130 in RA compensate less IL-6 and sIL-6R than in OAThus, IL-6/IL-6Ra system forms better microenvironment in bone marrow to activate asteooclasts in RA IL-6 sIL-6R sgp130 sgp130/IL-6 sgp130/sIL-6R- RA- OA

Wykres5

195111

12355

sgp130

sgp130 (ng/ml)

Higher levels of sgp130 in RA than in OA

Arkusz1

RANKLOPGRANKL/OPGIL-6sIL-6Rsgp130

RA6110163284195195

OA4012311132123123

OPG

RA10

OA12

OPG/RANKL

RA163

OA311

IL-6

RA284

OA132

sIL-6R

RA195

OA123

sgp130

RA195

OA123

sgp130/sIL-6R

RA5.8

OA7.3

sIL-6R/IL-6

RA118

OA126

sgp130/IL-6

RA687

OA932

ala

Arkusz2

Fig.2Fig.5

Levels of RANKL and OPG in bone marrow isolated from RA and OA patientsLevels of IL-6, sIL-6R and sgp130 in bone marrow isolated from RA and OA patients

Fig.3Fig.6

Lower level of sgp130 in RA compensate less IL-6 and sIL-6R than in OA

Lower OPG/RANKL ratio in RA than in OA

Arkusz2

5.8

7.3

sgp130/sIL-6R

Ratio: sgp130/sIL-6R

Ratio: sgp130/sIL-6R

Arkusz3

118

126

sIL-6R/IL-6

sIL-6R/IL-6

Ratio: sIL-6R/IL-6

103.3

12

p

Conclusion:

RA bone marrow, rich in proinflammatory cytokines (TNF-a, IL-1 beta, IL-6, IL-15) and higher RANKL/OPG ratio provide microenvironment promoting inflammation and bone degradation

Lymphocyte subpopulations in RA bone marrow

Bone marrow obtained from RA patients contain more T-cells (CD3+) and fewer B-cells (CD19+) than from OA P=0.008P=0.02Warnawin et al. in preparation

Wykres2

3.26.11.62.8

20.850.90.3

OA

RA

Number of cells x 106/ ml of bone marrow

Arkusz1

OARAT cellsB cells

7601304.5OA3.22

RA6.10.85

Arkusz1

0238.7

0965.3

IL-15 pg/ml

Arkusz2

001.62.8

000.90.3

OA

RA

Number of cells x 106/ ml of bone marrow

Arkusz3

Expression of activation marker (CD25) on BM CD3+ T-cells

RA OA% CD3+CD4+CD25+p

RA patientOA patientPeripheral bloodBone marrowHigher expression of CD69 on CD4+ T cells in RA bone marrowRA patientOA patient0 %0 %17,2 %3,3 %RAOAMFI of CD69+ cells of CD4+ lymphocytesp

Expression of IL-15R on lymphocytes obtained from bone marrow of OA and RA patientsOARAAnti-IL-15R alphaAnti-IL-15R alphaCell numberCell numberWarnawin et al. in preparation

Surface expression of IL-15Ra chain on BM CD3+ cells OA RAp

IL-15 triggered proliferation of BM CD3+ T-cells (Ki-67 staining) OA RA% control

p

IL-15 triggers IL-17 production by BMMC (72h culture with IL-15)P

Are regulatory cells present in bone marrow?

What triggers expression of Foxp3/regulatory cells in bone marrow ?

P

K LPS 0,1mg LPS 1mg a-IL6 IL15 IL15/LPS a-IL6/IL15 a-IL6/LPSOA BMMCRA BMMC* *p

T-cell compartment in RA bone marrow

Activated, memory CD4+, CD8+ T-cells

Higher expression of IL-15 receptors

Increased proportions of Th17 and IL-17 production

Decreased number of FoxP3+, Treg

ObjectiveTo study the expression and functionality

of TLR9 in bone marrow B-cells

Higher density of CD27 on B cells from blood and bone marrow of rheumatoid arthritisPeripheral bloodBone marrowMFI of CD27 on CD19+CD27+ cellsMFI of CD27 on CD19+/-CD27++ cells

Higher expression of CD86 on B-cells (CD20+) from bone marrow than peripheral blood of RA patients

RA bone marrow-derived B-cellsexpress TLR9 at mRNA and protein levels Expression of mRNA encoding TLR9- RTGAPDH(196 bp)TLR9(260 bp)

bone marrow 1 2 3 4peripheral bloodExpression of TLR9 protein0,9TLR9 saponin+ saponinCD19TLR9 Isotype control2,942,1% of CD19+ cells expressing TLR9

% of CD19+ cells expressing CD86 or CD54 CpG-ODN, but not control GpC-ODN, in a dose dependent manner, enhance the expression of CD86and CD54 on B-cells in BMMC culture 60 hours of the cell cultureControl GpC-ODN: 30 mg/ml Isotype control 15 mg/ml 30 mg/mlAgonistic CpG-ODN:2,5 2,921,413,4 6,631,523,5 11,4 CD86 (B7.2)CD54 (ICAM-1)BMMC, Bone Marrow Mononuclear Cells

CpG-ODN induce differentiation of B lymphocytes into CD19+CD20+CD27high cellsCD19 PECD27 APCCD20 FITCIL-15 promotes differentiation of CpG-ODN-triggered CD19+CD20+CD27high B cells into CD19lowCD20lowCD138+ cells170 hours of the cell culture

CpG-ODN induce IL-6 and TNF-a secretionby bone marrow B-cells Untreated Control Agonistic control GpC-ODN CpG-ODN Untreated Control Agonistic control GpC-ODN CpG-ODNIL-6TNF-a60 hours of the cell culture IL-6 (% of untreated control) TNF-a (% of untreated control) p = 0,02 p = 0,02nsns

Wykres4

1000

105.5078869849.95233142

339.1198762152.6115042451

Arkusz1

K8080+IL-15IL-15

Smolarek37h158100211.3924050633513.9240506329341.1392405063

63,5h557100106.6427289048150.4488330341113.2854578097

0

K7880

Zych63h6310055.5555555556193.6507936508

K80

Podlasin2,5 dnia491100155.8044806517

76h

Kontrola (niestymulowane komrki)78 (30 mikrog/ml)80 (15 mikrog/ml)80 (30 mikrog/ml)

Malczewska61h12.82100155.4602184087695.5538221529795.631825273

K7880/3080/15

10055.55555556211.3924051

100193.6507937

100155.8044807

100155.4602184795.6318253695.5538222

K80/30K80/30Test t: z dwiema prbami zakadajcy nierwne wariancje

100211.3924051

100193.6507937rednia100rednia339.1198762K80/30

100155.8044807Bd standardowy0Bd standardowy152.6115042451rednia100339.1198762

100795.6318253Mediana100Mediana202.5215994Wariancja093161.0849117985

158334Tryb100TrybObserwacje44

63122Odchylenie standardowe0Odchylenie standardowe305.2230084902Rnica rednich wg hipotezy0

491765Wariancja prbki0Wariancja prbki93161.0849117985df3

9102KurtozaKurtoza3.8887778567t Stat-1.5668535435

SkonoSkono1.965101367P(T

Bone marrow CD20+ B-cells express TLR9

CpG stimulation enhance CD86 expression on bone marrow CD20+, but not CD20- B-cells in BMMC culture.

Bone marrow CD20+ B-cells respond better to CpG-ODN stimulation than their peripheral blood counterparts

Higher frequency of bacterial DNA in bone marrow samples from RA than OA patients

B-cell maturation in bone marrowTLR 9?

Functional TLR9 and CD20 are coexpressed on pre-B/immature B stages of B-cell maturation in bone marrow.

Bone marrow CD20+ B cells respond to TLR9 agonist stimulation more vigorously than peripheral blood CD20+ cells, thus they may represent the first line of responders to blood born bacterial DNA trafficking to bone marrow.

Higher level of bacterial DNA in bone marrow samples from RA than OA patients suggest the role of TLR9 in the initiation and/or perpetuation of inflammation in BM.

Thus: bone marrow represents important site for B-cell activation and may contribute to the pathogenesis of RA.Summary

ConclusionBone marrow is a secondary lymphoid organ that actively participates in the initiation and perpetuation of chronic inflammation in rheumatoid arthritis

Future therapies should take into account bone marrow as an important target site for RA

Contributors:

Tomasz BurakowskiMagdalena MassalskaEwa KontnyIryna Kril*Olena Plakhta*Anna RadzikowskaWeronika RudnickaEwa WarnawinMaria Ziolkowska

Clinic of Orthopaedy, Institute of RheumatologyPawel MaldykCezary Michalak

* Supported by EURO-RA grant from EU

Department of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland