Biological Biological DifferentiationDifferentiation

Part IPart IDubai, United Arab EmiratesDubai, United Arab Emirates

January 19th, 2009January 19th, 2009

Prof. Joachim R. KaldenProf. Joachim R. KaldenDirector emeritusDirector emeritus

Dept. of Internal Medicine IIIDept. of Internal Medicine IIIDiv. of Molecular ImmunologyDiv. of Molecular Immunology

Nikolaus-Fiebiger-CenterNikolaus-Fiebiger-CenterUniversity of Erlangen-NurembergUniversity of Erlangen-Nuremberg

OverviewOverview

Pharmacology of TNF inhibitorsPharmacology of TNF inhibitors

MOA of TNF inhibitorsMOA of TNF inhibitors

Differences in latent TB among TNF Differences in latent TB among TNF inhibitorsinhibitors

Dose creep in biologicsDose creep in biologics

ImmunogenicityImmunogenicity

10 years safety data10 years safety data

Structure of ENBRELStructure of ENBREL

Dimeric vs. monomeric sTNFR• Longer half-life• Higher affinity• More potent TNF- neutralizing activity (~1000X)

Mohler KM et al, J Immunol 151:1548–1561, 1993

Fc region ofFc region ofhuman IgGhuman IgG11

Extracellular domain ofExtracellular domain ofhuman p75 TNFRhuman p75 TNFR

CH3CH3 CH2CH2

SSSS

SSSS

SSSS

SSSS

SSSS

Mechanism of actionMechanism of actionEtanercept:Etanercept:

Binds soluble and Binds soluble and membrane TNF-membrane TNF- and and Lymphotoxin Lymphotoxin (LT- (LT- ) ) with high affinitywith high affinity

Competes with cell-Competes with cell-surface TNF receptors surface TNF receptors (TNFRs)(TNFRs)

In vitro, does not lyse In vitro, does not lyse cells expressing cells expressing membrane TNF-membrane TNF-

Neutralises TNF-Neutralises TNF- activityactivity

ENBREL® [package insert]. Seattle, WA; Immunex Corp. and Wyeth-Ayerst Laboratories, 2001.

Inhibitors of TNF:Inhibitors of TNF:Etanercept and the Etanercept and the

monoclonal antibodiesmonoclonal antibodies

TNF inhibitorsTNF inhibitors

Tracey et al. Pharmacology & Therapeutics 2008; 117: 244 – 79

TNF inhibition: Differences in TNF inhibition: Differences in mechanisms of actionmechanisms of action Soluble TNF receptorsSoluble TNF receptors

Human TNF receptor linked to Fc portion of IgGHuman TNF receptor linked to Fc portion of IgG Bind soluble and cell-bound TNF and LTBind soluble and cell-bound TNF and LT Complement dependant cytotoxicity (significantly lower Complement dependant cytotoxicity (significantly lower

as MAb)as MAb) ADCC activityADCC activity

TNF monoclonal antibodiesTNF monoclonal antibodies Chimeric and human versionsChimeric and human versions Variable (Fab) region binds to soluble and cell-bound TNFVariable (Fab) region binds to soluble and cell-bound TNF Do not bind LTDo not bind LT Bind soluble and cell-bound TNFBind soluble and cell-bound TNF Complement dependant cytotoxicityComplement dependant cytotoxicity ADCC activityADCC activity Induction of apoptosis and cell cycle arrest in trans-Induction of apoptosis and cell cycle arrest in trans-

membrane TNFalpha expressionmembrane TNFalpha expression

Suggested reasons for differences in Suggested reasons for differences in efficacy in Crohn’s disease related to efficacy in Crohn’s disease related to MAbMAb Variations in tissue penetration Variations in tissue penetration

Stability of the immune complexes with TNF Stability of the immune complexes with TNF

Induction of apoptosis of both monocytes and T Induction of apoptosis of both monocytes and T cellscells

Neutralisation of membrane TNF Neutralisation of membrane TNF

Antibody-dependent cell-mediated cytotoxicity Antibody-dependent cell-mediated cytotoxicity (ADCC) (ADCC)

Complement-dependent cytotoxicity (CDC) Complement-dependent cytotoxicity (CDC)

Reverse signalling via mTNFReverse signalling via mTNF

Interferring with granulomaformationInterferring with granulomaformation

Nesbitt et al. Inflamm Bowel Disease 2007; 13: 1323

Binding of TNFBinding of TNF

Proposed cell binding modelProposed cell binding model

Kohno et al. ACR 2005

Differences in binding Differences in binding characteristicscharacteristics

EtanerceptEtanercept InfliximabInfliximab AdalimumabAdalimumab CertolizumaCertolizumabb

ClassClass Receptor:Fc Receptor:Fc fusion proteinfusion protein MAbMAb MAbMAb MAb fragmentMAb fragment

Neutralisation Neutralisation and bindingand binding

sTNF low concsTNF low conc ++++++ ++++ ++++ No dataNo data

sTNF high concsTNF high conc ++++++ ++++++ ++++++ ++++++

tmTNF bindingtmTNF binding ++++ ++++++ ++++++ ++++++

tmTNF tmTNF neutralisationneutralisation ++++ ++++++ ++++++ ++++++

Reverse Reverse signallingsignalling

ApotosisApotosis ++/-++/- ++++++ ++++++ --

Cytokine Cytokine suppressionsuppression ++/-++/- ++++++ ++++++ ++++++

FcFcγγR bindingR binding

1:1 ratio*1:1 ratio* -- ++++ ++++ No dataNo data

>10:1 ratio*>10:1 ratio* -- -- -- No dataNo data

CDCCDC ++/-++/- ++++++ ++++++ --

ADCCADCC ++/-++/- ++++++ ++++++ --

* Drug:TNF ratioAdapted from Tracey et al. Pharmacology & Therapeutics 2008; 117: 244 – 79

Neutralisation of sTNF*Neutralisation of sTNF*

ETNCTZIFX

ADL

*Measured by cytoxicity to L929 cells

Nesbitt et al. Inflamm Bowel Disease 2007; 13: 1323

Binding to tmTNFBinding to tmTNF

ETNCTZIFX

ADL

IgGFab’ PEG

ETN, IFX, ADL and IgG detected with phycoerythrin-labeled anti-heavy and light chain polyclonal antiserum CTZ and Fab’ PEG detected with anti-Fab2

polyclonal antisera

Nesbitt et al. Inflamm Bowel Disease 2007; 13: 1323

Neutralisation of tmTNFNeutralisation of tmTNF

ETNCTZIFX

ADL

IgGFab’ PEG

Non-cleavable tmTNF-expressing NS0 clone 23 incubated with A549-Luc cells

Nesbitt et al. Inflamm Bowel Disease 2007; 13: 1323

Comparative stability of Comparative stability of tmTNF binding: infliximab vs. tmTNF binding: infliximab vs. etanerceptetanercept

100

80

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Time of Incubation (min)

% In

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imab

Bo

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None Infliximab Etanercept TNF

100

80

60

40

20

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0 20 40 60 80 100 120

Time of Incubation (min)

% E

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pt

Bo

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A B

Scallon B et al. J Pharmacol Exp Ther 2002; 301:418-26

SummarySummary

Nesbitt 2007Nesbitt 2007 Scallon Scallon 20022002 Khare 2006Khare 2006 Mitoma 2008Mitoma 2008

sTNF sTNF neutralisationeutralisationn

ETN>CTZ>IFX=ADLETN>CTZ>IFX=ADL

mbTNF mbTNF bindingbinding ETN=CTZ=IFX=ADLETN=CTZ=IFX=ADL

mbTNF mbTNF stabilitystability IFX>>>ETNIFX>>>ETN

mbTNF mbTNF neutralisationeutralisationn

ETN=CTZ=IFX=ADLETN=CTZ=IFX=ADL

CDCCDC IFX=ADL=ETN>>CTXIFX=ADL=ETN>>CTX=0=0 IFX>ETNIFX>ETN IFX=ADL>ETNIFX=ADL>ETN

ADCCADCC IFX=ADL>ETN>>CTZIFX=ADL>ETN>>CTZ=0=0

10:1 10:1 IFX>>ETNIFX>>ETN

40:1 IFX40:1 IFX≥ETN≥ETN

IFX=ADL=ETNIFX=ADL=ETN

ApoptosisApoptosis IFX=ADL>ETN>>CTZIFX=ADL>ETN>>CTZ=0=0 IFX=ADL>>ETNIFX=ADL>>ETN

Effects on mechanisms of Effects on mechanisms of immunity to TBimmunity to TB

The global problem of TBThe global problem of TB

The World Health Organisation (WHO) estimates The World Health Organisation (WHO) estimates that in 1999, there were 8.4 million new cases, up that in 1999, there were 8.4 million new cases, up from 8.0 million in 1997from 8.0 million in 1997

Estimated nearly 2 million deaths from tuberculosis Estimated nearly 2 million deaths from tuberculosis annuallyannually

Ranks second only to human immunodeficiency Ranks second only to human immunodeficiency virus (HIV) infection as an infectious cause of deathvirus (HIV) infection as an infectious cause of death

1.7 billion people, one third of the world's 1.7 billion people, one third of the world's population, are thought to be infected with population, are thought to be infected with Mycobacterium tuberculosis Mycobacterium tuberculosis

Developed countries are not protected from the Developed countries are not protected from the ongoing epidemic of tuberculosis occurring in the ongoing epidemic of tuberculosis occurring in the poor countries of the worldpoor countries of the world

Jasmer et al. NEJM 2002; 347: 1860-66

Host defence against TBHost defence against TB

Gardam et al. Lancet Infectious Diseases 2003; 3: 148 - 55

Effects on T cell activationEffects on T cell activation

Whole blood cultures of healthy tuberculin skin test Whole blood cultures of healthy tuberculin skin test + volunteers stimulated with TB culture filtrate + volunteers stimulated with TB culture filtrate antigen or PHAantigen or PHA

T cell activation, apoptosis, necrosis, cytokine T cell activation, apoptosis, necrosis, cytokine production, and gene expression profiles assessedproduction, and gene expression profiles assessed

Whole blood cultures infected with TB H37Ra and Whole blood cultures infected with TB H37Ra and growth assessedgrowth assessed

TNF blockers were added at therapeutic TNF blockers were added at therapeutic concentrationsconcentrations

ETN also studied at a supratherapeutic ETN also studied at a supratherapeutic concentration concentration

Activation (CD69), apoptosis (annexin V), and Activation (CD69), apoptosis (annexin V), and necrosis (7AAD) were measured by flow cytometrynecrosis (7AAD) were measured by flow cytometry

IFNγ and IL-10 measured by ELISAIFNγ and IL-10 measured by ELISASaliu et al. J Infect Dis 2006

Incidence of TB Is low in RA Incidence of TB Is low in RA patients receiving etanercept patients receiving etanercept therapy*therapy*

IndicatioIndication n n (%)n (%)

EtanerceEtanercept pt

Exposure Exposure in Pt-Yrin Pt-Yr

TB TB EventsEvents

Events/Events/100 100 Pt-YrPt-Yr

RA RA 4486 4486 (39.39) (39.39) 11,15811,158 22 0.0180.018

PsOPsO 4361 4361 (38.29)(38.29) 39653965 00 00

PsAPsA 1362 1362 (11.96)(11.96) 739739 00 00

ASAS 695 (6.10)695 (6.10) 664664 00 00

JIAJIA 486 (4.27)486 (4.27) 11291129 00 00

OverallOverall 11,39011,390 17,65517,655 22 0.0110.011

*Majority of patients in combined analysis were white, female, and enrolled in RA studies

Wajdula J et al. EULAR 2007

Data from adalimumab trials*Data from adalimumab trials*

11,439 RA patients (14,544 patient-years [pt-yrs] 11,439 RA patients (14,544 patient-years [pt-yrs] of exposure) in North America and Europe of exposure) in North America and Europe 3422 patients (5918.9 pt-yrs) North America 3422 patients (5918.9 pt-yrs) North America 8017 patients (8625.2 pt-yrs) Europe8017 patients (8625.2 pt-yrs) Europe

Before screening 7 cases TB (534 pt-yrs Before screening 7 cases TB (534 pt-yrs exposure; 0.013/pt-yr) exposure; 0.013/pt-yr)

After screening 27 cases TB (14,010 pt-yrs After screening 27 cases TB (14,010 pt-yrs exposure; 0.0019/pt-yr)exposure; 0.0019/pt-yr)

5 North America (rate 0.0008)5 North America (rate 0.0008)

22 Europe (rate 0.0027)22 Europe (rate 0.0027)

Ratio Europe to North America is 1:3.2 Ratio Europe to North America is 1:3.2 *Up to Dec 31 2004Perez. EULAR 2005: OP 0093

Rates of TB in France - RATIORates of TB in France - RATIO Case-control study found the following factors Case-control study found the following factors

predictive of TB in multivariate analysis: predictive of TB in multivariate analysis: ADA vs. ETA ADA vs. ETA HR: 10.05 HR: 10.05 [1.92-52.61], [1.92-52.61], PP=0.006=0.006

IFX vs. ETA IFX vs. ETA HR: 8.63 HR: 8.63 [1.38-53.78], [1.38-53.78], PP=0.02=0.02

Estimated incidence of tuberculosis (per 100,000 Estimated incidence of tuberculosis (per 100,000 pt/year)pt/year) French populationFrench population 8.7 8.7 TNFi treated patients TNFi treated patients 39.2 39.2 IFX or ADA IFX or ADA 71.571.5 ETN ETN 6.06.0

This large national prospective study clearly This large national prospective study clearly shows that the risk of TB is higher with shows that the risk of TB is higher with monoclonal antibodies than with the soluble monoclonal antibodies than with the soluble receptorreceptor

Tubach et al. Ann Rheum Dis 2008; 67(Suppl II):52; OP00143

0,0

0,1

0,2

0,3

0,4

0,5

févr-00 août-00 févr-01 août-01 févr-02 août-02 févr-03

Feb 2000 - Feb 2003

US

Rat

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er 1

,000

Act

ive

Pat

ient

s

Infliximab Safety Update: Tuberculosis

Reporting Rate per 1,000 Active PatientsTreated in Period

TB EducationProgram Initiated

PSUR 7, Feb 03.

TB Rates in Adalimumab Clinical TB Rates in Adalimumab Clinical StudiesStudies

1.3

0.08 0.13

0.0

0.5

1.0

1.5

2.0

EU North America EU

Rat

e pe

r 10

0 pt

-yrs

Pre-screening Post-screening

Data on file: Through July 2003

# cases 7 3

Exposure (pt-yrs) 534 3978

6

4447

Possible reasons for differences in Possible reasons for differences in reactivation of latent TB and efficacy in reactivation of latent TB and efficacy in CDCD Divergent effects on control of intracellular TB growth Divergent effects on control of intracellular TB growth

MAbs block T cell activation and IFNg production, while MAbs block T cell activation and IFNg production, while etanercept does not etanercept does not

Analysis of global gene expression by microarray shows Analysis of global gene expression by microarray shows differential effects of MAbs and ETNdifferential effects of MAbs and ETN

ETN affects TH1 genes, MAbs TH2ETN affects TH1 genes, MAbs TH2

These differential effects are not due to T cell apoptosis, These differential effects are not due to T cell apoptosis, CDC or ADCCCDC or ADCC

CTZ does not induce apoptosis, CDC or ADCC but cases of TB CTZ does not induce apoptosis, CDC or ADCC but cases of TB have been reported during clinical trials in RAhave been reported during clinical trials in RA

Irreversible binding of mbTNF by MAbs, including CTZ, but Irreversible binding of mbTNF by MAbs, including CTZ, but not ETN may be important by reducing cell-to-cell signalingnot ETN may be important by reducing cell-to-cell signaling

The high rate of TB reactivation by the TNF MAbs may The high rate of TB reactivation by the TNF MAbs may reflect their inhibition of both TNF and IFNgreflect their inhibition of both TNF and IFNg

Summary of TB dataSummary of TB data

Cases of TB reported with all TNF-targeted Cases of TB reported with all TNF-targeted therapiestherapies

Cases reported with ADL clinical trials, Cases reported with ADL clinical trials, even with screeningeven with screening

Cases reported with CTZ in clinical trialsCases reported with CTZ in clinical trials

Screening for TB not undertaken with ETN Screening for TB not undertaken with ETN clinical trials. Only 2 cases of TB reportedclinical trials. Only 2 cases of TB reported

Registries report significantly lower rates Registries report significantly lower rates of TB in patients treated with ETN than IFX of TB in patients treated with ETN than IFX or ADLor ADL

Opportunistic Infections*Opportunistic Infections*

227 559227 559> > 230 000230 000 Pt-yrs exposurePt-yrs exposure

----22 NocardiaNocardia

198 235198 235> 150 000> 150 000 # RA treated# RA treated

4444

9914142323161618181414

InfliximabInfliximab

----77

55551010331144

EtanerceptEtanercept

CoccidiodomycosisCoccidiodomycosis Systemic CandidiasisSystemic Candidiasis

CytomegalovirusCytomegalovirus AspergillusAspergillus Atypical mycobacteriaAtypical mycobacteria ListeriosisListeriosis HistoplasmosisHistoplasmosis Pneumocystis cariniiPneumocystis carinii

* To August 20021. FDA Arthritis Advisory Committee MeetingSafety update on TNF alpha inhibitors. March 4, 20032.Keystone, Advances in Targeted Therapies V,April,2003 .

Does dose escalation improve Does dose escalation improve clinical efficacy?clinical efficacy?

Why are we seeing dose Why are we seeing dose increases with infliximab ?increases with infliximab ?

Tachyphylaxis ? Anti-infliximab antibodiesTachyphylaxis ? Anti-infliximab antibodies

Sub-optimal dosingSub-optimal dosing

Concomitant meds: MTX, prednisoneConcomitant meds: MTX, prednisone

Transient disease flares / Ease of dose Transient disease flares / Ease of dose increases increases

PharmacokineticsPharmacokinetics

Infliximab Dose in 1248 Infliximab Dose in 1248 RA Patients After 1.5 YearsRA Patients After 1.5 Years

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2

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8

10

12

14

0.5 1

1.5 2

2.5 3

3.5 4

4.5 5

5.5 6

6.5 7

7.5 8

8.5 9

9.5 10

10.5 11

Infliximab Dose (mg/kg)

Pa

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nts

(%

)

Wolfe F, et al. Arthritis Rheum. 2003;48(9):S328.

1. 56% had dose increases. Average dose of 5 mg/kg.

2. Comparable to reported literature

3. Montreal cohort of 85 patients 46 (54%) had dose increases

Does Increasing Dose of Does Increasing Dose of Infliximab Lead to Better Infliximab Lead to Better Outcomes?Outcomes? 124 Patients from STURE database124 Patients from STURE database

44 patients treated with infliximab; dose 44 patients treated with infliximab; dose increased from 3 mg/kg to 5–7 mg/kg q8 increased from 3 mg/kg to 5–7 mg/kg q8 weeksweeks

44 patients treated with infliximab; dose 44 patients treated with infliximab; dose maintained at 3 mg/kgmaintained at 3 mg/kg

36 patients treated with etanercept36 patients treated with etanercept

Examined disease activity before and after Examined disease activity before and after dose increasedose increase

Van Vollenvoven RF, et al. Ann Rheum Dis. 2004;63:426-30.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Best DAS28before

DAS28 rightbefore

DAS28 right after Best DAS28 after

DA

S2

8

DAS28 Scores Before and After DAS28 Scores Before and After Dose EscalationDose Escalation

Van Vollenvoven RF, et al. Ann Rheum Dis. 2004;63:426-30.

IMMUNOGENICITYIMMUNOGENICITY

HACA / HAHAHACA / HAHA

AntibodiesAntibodies

Anti-idotype antibodies bind here

Anti-allotypic antibodies bind here

Anti-isotypic antibodies bind here

Incidence of Antibodies-to-Incidence of Antibodies-to-Infliximab (ATI) - Maintenance Infliximab (ATI) - Maintenance Studies*Studies*

Maintenance Studies

% of Pts without ATI% of Pts with ATI % of Patients Inconclusive†

*Patients. with evaluable samples

ACCENT I CD

n=514Week 72

16

2758

ACCENT IICD

n=258Week 54

17

52

31

ATTRACTRA

n=295Week 102

9

56

36

Antibody-to-Infliximab (ATI) Status

†Patients with long-lasting serum concentrations of infliximab and never ATI (+)

Integrated Safety Summary. Aug 09, 2002.

16

38

0

10

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30

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50

113 3

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10

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30

40

50

Infusions with Infusion Infusions with Infusion Reactions by Reactions by Antibody-to-Infliximab(ATI) Antibody-to-Infliximab(ATI) StatusStatus

ATTRACT through week 1021

ACCENT I through week 542

Positive*37 / 329

Negative*35 / 1224

Inconclusive*93 / 3092

Pro

port

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of I

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wit

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fusi

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Antibody-to-Infliximab Status Antibody-to-Infliximab Status

Pro

port

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of I

nfus

ions

wit

h In

fusi

on R

eact

ions

1 Internal data, Centocor.

Positive*42 / 254

Negative*55 / 656

Inconclusive*47 / 1470

*patients with evaluable samples

2 Lancet 2002; 359: 1541–49.

HACA and Patterns of Infliximab HACA and Patterns of Infliximab DosingDosing

TotalN = 52

Group AN = 19

Group BN = 11

Group CN = 22

Serum samples perpatient, mean (SD)

3 (1) 3 (1) 3 (1) 3 (1)

Patients with aninformative sample (%)

33 (63) 14 (74) 8 (73) 11 (50)

Patients with HACA, (%of patients with aninformative sample)

13 (39%) 4 (28%) 4 (50%) 5 (45%)

Maximum HACA levelin positive samples,mean (SD) [mcg/mlequivalents]

15 (9) 8 (5)1 15 (13)1 21 (4)1

Haraoui et al, in press

1P < 0.005, Group A vs. Group using two-tailed t-test.

Van Vollenhoven et al. SAT0198. EULAR 2006.

Secondary Loss of Efficacy with TNF-antagonist: STURE Registry

11.84.4adalimumab

27.521.516.811.45.41.4infliximab

12.49.86.85.63.51.9etanercept

6 years5 years4 years3 years2 years1 year

Probability of experiencing secondary loss of efficacy (%)

Conclusion:• Secondary loss of efficacy occurs to lesser degree with etanercept• Adalimumab data limited, some previous TNF treatment• Secondary loss of efficacy appear less during first years of treatment and greater with longer-term therapy

Adalimumab increasing starting dosepublished data

25% subjects adalimimab monotherapy and 11% of combination required dose escalation in the PREMIER1 study

25%

11%13%

0%

5%

10%

15%

20%

25%

30%

PREMIER -Monotherapy

PREMIER -Combination

Engel-Nitz et.al EULAR

2007

Ferdinand C. et al, Arthritis & Rheumatism, Vol. 54, Jan. 2006

THU0124 Safety and efficacy of over 10 years THU0124 Safety and efficacy of over 10 years of continuous etanercept therapy in patients of continuous etanercept therapy in patients with rheumatoid arthritis in North America with rheumatoid arthritis in North America and Europe and Europe