22-09-08
TNF antagonists:
ADA and PK
Theo Rispens
Immunogenicity therapeutic
antibodies
Infliximab
Mouse
Chimeric
30% mouse
Immunogenicity:
Adalimumab
Humanized
3-5% mouse
Human
measurement of ADA
Antigen binding test (ABT)
prot. A
-  capture IgG from serum (Prot. A Sepharose)
-  wash out unbound material
-  incubate with radiolabeled adalimumab F(ab’)2
-  wash out unbound label
-  measure radioactivity
Prevent background of anti-hinge
antibodies
IVIG F(ab’)2
IFX F(ab’)2
anti-IFX
protein A Sepharose
Rispens et al., JIM 2011
anti-hinge
Rispens et al., JIM 2011
Development of Antidrug Antibodies
Against Adalimumab and Association
With Disease Activity and Treatment Failure
During Long-term Follow-up
Bartelds et al., JAMA April 2011
Long term clinical effects of ADA
272 adalimumab treated RA patients
3 year follow-up
Long term measurement of adalimumab levels, ADA
and disease activity.
Bartelds et al., JAMA April 2011
baseline characteristics
Total
Patient with AAA
Patients without AAA
n=272
n=76
n=196
Age, years
54 ± 12
53 ± 13
54 ± 11
Female, no. (%)
219 (81)
62 (82)
157 (80)
RF, no. (%)
196 (72)
57 (75)
139 (71)
Prior DMARDs
3.1 ± 1.4
3.4 ± 1.5*
3.0 ± 1.3*
MTX use, no. (%)
202 (74)
41 (54)*
161 (82)*
MTX dose (mg/wk)
25 (15‒25)
18 (10‒25)*
25 (15‒25)*
No DMARD, no. (%)
51 (19)
28 (37)*
23 (12)*
Disease duration
(years)
8 (3‒17)
12 (5‒18)*
8 (3-16)*
Erosive disease, no. (%)
201 (74)
63 (83)*
138 (70)*
ESR (mm/h)
23 (11‒42)
35 (18‒60)*
21 (11‒39)*
CRP (mg/L)
12 (5‒29)
19 (7‒46)*
11 (4‒22)*
DAS28
5.2 ± 1.2
5.5 ± 1.1*
5.1 ± 1.3*
Bartelds GM, et al. JAMA. 2011;305:1460‒1468 Adalimumab: Cohort 272 RA patienten
During 156 weeks of follow-up anti-adalimumab antibodies were detected
in 76 (28%) of patients
67% of ADA+ patients is positive already after 28 weeks
30
% ADA+
25
20
15
Antibodies against
adalimumab (%)
10
5
0
0
28
56
Bartelds et al., JAMA April 2011
84
112
140
Remission (DAS28 < 2.6) is associated with
absence of anti-drug antibodies
Remission probability
0.5
AAA -
0.4
0.3
p<0.0001
0.2
0.1
0.0
AAA+
0
50
100
150
200
Time in weeks
Gecorrigeerd voor MTX dosis, bezinking en CRP
(HR:3.6; 95%CI:1.8-7.2, p<0.0001)
Bartelds et al., JAMA April 2011
Anti-drug antibody formation to adalimumab and
infliximab measured with ABT is correlated with
diminished clinical efficacy
Drug interference
Assay comparison: Drug interference
-  Discrepancies found between assays:
-  In IgG4-specific assay, higher frequency of positive patients
compared to similar IgG-specific assay
-  Therefore, systematic comparison:
-  216 RA patients
-  sera after 28 weeks of treatment
-  Comparison between:
-  bridging elisa
-  IgG-specific antigen binding test
-  IgG4-specific antigen binding test
Anti-adalimumab measured in bridging ELISA and
antigen binding test
  different numbers of ADA+ patients in different assays
prot. A
7%
Hart et al., JIM 2011
13%
α-IgG4
22%
Relation between drug levels and ADA
  bridging elisa: detects ADA only if no adalimumab is detected
prot. A
α-IgG4
Hart et al., JIM 2011
Inhibition by adalimumab
-  serum with high anti-adalimumab levels
-  inhibition by adding increasing amounts of
adalimumab
Hart et al., JIM 2011
Rationalization of drug interference
prot. A
α-IgG4
prot. A
Natalizumab: IgG4
•  Treatment of Multiple Sclerosis
•  Directed against a4-integrin (VLA-4)
•  Interferes with lymphocyte migration
across blood-brain barrier
•  IgG4 antibody: Fab arm exchange
anti-lambda
natalizumab x serum IgG4
VLA-4
Labrijn et al. Nature Biotechnol. 2009
Antibodies to natalizumab at 12, 24, and 52 weeks
Vennegoor et al., Mult. Sclerosis 2013
Drug interference: mono vs bispecific IgG4
125I
natalizumab F(ab’)2
ADA
protein A
Sepharose
Rispens et al. J Pharm Biomed Anal 2013
Drug interference differs between assay formats
Moreover, drug interference may depend differentially
on drug characteristics
Dynamics of ADA formation to adalimumab and
drug-tolerant assays
Measurement of ADA in complex with drug
-  Can we increase recovery by acid treatment?
-  Prevention of re-association upon neutralization:
block adalimumab with rabbit anti-idiotype F(ab)
adalimumab
F(ab’)2
anti-adalimumab
prot. A
Ph-shift anti-Idiotype ABT (PIA)
adalimumab
patient anti-adalimumab
acid
rabbit anti-adalimumab
add excess rabbit anti-ada Fab
neutralize
prot. A
Can we increase recovery using acid treatment and
blocking agent?
Model system:
Rabbit anti-idiotype
ADA
ADA + acid treatment
ADA + drug + F(ab) + acid t.
ADA + drug + F(ab)
ADA + drug
• Add adalimumab to make
complexes
• Acid to dissociate complexes
• Excess of rabbit anti-idiotype
F(ab) to keep complexes
dissociated.
  Recovery is more efficient with a combination of acid
treatment and rabbit anti-idiotype F(ab)
Schouwenburg et al., JIM 2010
prot. A
Long term measurement with PIA
-  3 year follow up for 99 adalimumab-treated patients
47% of the patients develop ADA
>90% of ADA+ patients develop ADA in the first 6 months
van Schouwenburg et al. ARD 2013
Results gradients Prot A
adalimumab,
no ADA (in ABT)
adalimumab,
low ADA (in ABT)
  Most sera contain complexes
  Complexes are small
  These complexes are not rapidly cleared
no adalimumab,
high ADA (in ABT)
Tolerance
Long term follow-up with PIA allows research on transient ADA
production
Of the 53 patients positive for ADA at any time point, 17 are
transiently positive for ADA.
van Schouwenburg et al. ARD 2013
• Drug tolerant ADA assays using acid dissociation
• >90% of ADA+ patients develop ADA in the first 6
months
• Responses are diverse and include ‘transient’ and
‘persistent’ cases
• Drug tolerant ADA assays may provide
immunological insight, but offer limited clinical insight
Characterization of anti-adalimumab antiodies
Making human monoclonal antibodies
Schouwenburg et al., ARD 2012
All monoclonal antibodies are derived from
different precursor B-cells
data unpublished, available upon request (t.rispens@sanquin.nl)
Mabs underwent extensive somatic hypermutation
A
VH gene usage
Vκ gene usage
VH CDR3 length
data unpublished, available upon request (t.rispens@sanquin.nl)
FR CDR FR CDR
All mAbs bind to an overlapping epitope
ADA-bt
ADA
ADL
data unpublished, available upon request (t.rispens@sanquin.nl)
Competition of adalimumab binding: ADA from
patient sera
 A single monoclonal anti-adalimumab
inhibits binding of all serum antiadalimumab (50 pt tested so far)
+ IVIG F(ab)
+ anti-Id F(ab) 1.2
Schouwenburg et al., Ann Rheum Dis. 2012
mAbs neutralize adalimumab
TNF
IL-8
TNF + ADL
TNF + ADL + ADA
no IL-8
IL-8
data unpublished, available upon request (t.rispens@sanquin.nl)
TNF + ADL + ADA
Summary I
-  Antibody response to adalimumab:
-  antigen-driven:
-  extensive somatic hypermutation and affinity maturation
-  broad:
-  broad range of unrelated clones
-  restricted:
-  >98% of antibody response targets overlapping epitopes
Neutralization by ADA
Adalimumab: TNF inhibition
125I
adalimumab Fab
Adalimumab
data unpublished, available upon request (t.rispens@sanquin.nl)
Infliximab: potential epitopes
TNF
VH
VL
infliximab
non-human
germ-line
non-mouse
germ-line
adapted from Liang et al. JBC 2013
Infliximab: TNF inhibition
125I
infliximab Fab
Infliximab
data unpublished, available upon request (t.rispens@sanquin.nl)
certolizumab: TNF inhibition
125I
certolizumab
Certolizumab
data unpublished, available upon request (t.rispens@sanquin.nl)
correlation
WEHI assay – anti-infliximab RIA
data unpublished, available upon request (t.rispens@sanquin.nl)
•  ADA to anti-TNF antibodies are predominantly
neutralizing:
> 98% for adalimumab (16 pt)
> 90% for infliximab (25 pt)
> 97% for certolizumab (9 pt)
•  paratope is immunodominant?
•  No need for bioassays assessing neutralizing capacity
Drug level testing
PK assay
Anti-idiotype
adalimumab
TNF
Anti-TNF
Serum adalimumab concentrations
no AAA
Median adalimumab
concentration (MG/L)
AAA 13-100
AU/ml
AAA >100
AU/ml
14
12
10
*
8
6
4
2
0
t=0
t=16wk
Bartelds et al., JAMA April 2011
t=40wk
t=78wk
t=130wk
p<0.0001*
Drug levels vs anti-drug antibodies: a balance
16 or 28 weeks after start adalimumab
ABT vs PK
PIA vs PK
Detection of anti-drug-antibodies
(ADA)
Anti-TNF antibody levels
ADA production
Free anti-TNF agent
ADA-drug complexes
Free ADA
ADA detection method
ELISA
–
–
–
+
ABT
–
–
+/–
+
PIA
–
+/–
+
+
++
+
+/–
–
Pharmacokinetic assay
(TNF capture)
van Schouwenburg PA, et al. Nat Rev Rheumatol. 2013;9:164‒172 Concentration-effect curve (adalimumab/RA)
Each dot is mean of 20 patients
Pouw et al. ARD 2013 in press
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Smart testing for health benefits and cost-effective therapy.
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August 7, 2014 | 52
Drug levels (PK)
• Functional adalimumab levels: balance between drug
levels and ADA levels
• functional drug levels are correlated with clinical
efficacy
• Some non-responders with normal/high drug levels
• Optimal concentrations of adalimumab for RA are ca.
5-8 mg/L
Take home
•
neutralization leads to non-response
•
Quantity of ADA is relevant: balance ADA/drug
•
ADA assays are difficult to standardize
•
Drug-tolerant assays not useful for clinic
•
Drug level testing can be useful for clinical
management
Acknowledgements
Sanquin Research
Pauline van Schouwenburg
Margreet Hart
Reade
Gertjan Wolbink
Simone Kruithof
Margret de Koning
Els de Groot
Charlotte Krieckaert
Marieke van Ham
Margret Bartelds
Mieke Pouw
Gertjan Wolbink
Mike Nurmohamed
Diana Wouters
Lucien Aarden
Karin van Schie
Sanquin Diagnostics Services
Genmab
Desiree van der Kleij
Henk de Vrieze
Rob de Jong
Astrid van Leeuwen
Steven Stapel
Esther van Buren
Tom Vink