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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
60
40
20
0
0 20 40 60 80 100 120
Time of Incubation (min)
% In
flix
imab
Bo
un
d
None Infliximab Etanercept TNF
100
80
60
40
20
0
0 20 40 60 80 100 120
Time of Incubation (min)
% E
tan
erce
pt
Bo
un
d
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
e p
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
0
2
4
6
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
tie
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
20
30
40
50
113 3
0
10
20
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
ion
of I
nfus
ions
wit
h In
fusi
on R
eact
ions
Antibody-to-Infliximab Status Antibody-to-Infliximab Status
Pro
port
ion
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