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Pre-existing antibodies to biologics: Predictive for treatment outcome?
Claus H. NielsenProfessor, Ph.D., M.D., M.Sc.
Institute for Inflammation ResearchDept. of Infectious Diseases and RheumatologyCopenhagen University Hospital Rigshospitalet
Appearance of serum sickness-like reactions during treatment with rituximab
• In a study on Sjögrens disease HACAs were formed in 4 of 8 patients, 3 of these developed SSLR (Pijpe J, Arthritis Rheum 2005)
• In children with ITP, RTX caused SSLRs in 5 out of 60 (Bennet et al. Blood 2006; Wang et al., J Pediatr 2005).
• In a study on Graves’ disease 3 of 10 patients developed SSLR (el Fassi et al.,J Endocrin Invest, 2011)
Arthralgia, skin eruptions, fever, lymphadenopathy
Serum sickness like reactions (SSLR)
Anti-IgG/IgA/IgM
Demonstration of circulating complement-opsonized immune complexes
IgM
-25
0
25
50
75
100
125
A
P=0.01
P= 0.01
#5
#3 #10M
FI
IgG
0
100
200
300
B
P= 0.008
P= 0.01
#5
#3#10
MF
I
C3
+ RTX
+ RTX
- RTX
0
100
200
C
P= 0.003
P= 0.04
P=0.004
#5
#3 #10
M
FI
An anti-C3-capture ELISA developed with anti-IgG/IgA/IgM confirmed
El Fassi et al. J Endocrinol Invest 2011; 34: e163-e167
Is there a pre-existing immune response to rituximab?
How can you develop antibody responseswhen no B cells are present?
Ongoing production of pre-existing antibodies by plasma cells?
Basic conundrum
Study on endometrial protein PP14: 81% of male sera were false-positive
Analyte
Coating Ab
Detecting Ab
False-positive reactions in two-site immunoassays
Coating Ab
Detecting Ab
False-positive reactions in two-site immunoassays
Cross-binding anti-mouse Ig Ab
Prevalence of antibodies causing false-positive reactions
Kricka et al. Clin Chem 1999; 45: 942-56
HAAAs: Human anti-animal antibodies
HAMAs: Human anti-mouse antibodies
HACAs: Human anti-chimeric antibodies
HAHAs: Human anti-human antibodies
Heterophilic antibodies
What are they?
HAAAs versus Heterophilic Antibodies
Heterophilic antibodiesImmunoglobulin Group I
Anti-Fab(85% of positive
samples)
Group IIAnti-Fc
(15% of positive samples)
Negative samples
Mouse +++ +++ -
Rat +++ - -
Goat +++ - -
Horse +++ +++ -
Rabbit - +++ -
Cow +++ +++ -
BSA (neg. Control) - - -
Hennig et al.
Anaphylactic reactions to cetuximabcaused by pre-existing IgE antibodies to the α-gal epitope
All humans have IgG antibodies specific for the oligosaccharide galactose-α-1,3-galactose, which is closely related to substances in the ABO blood group
Produced in mouse hybridoma cells
Chung et al., N Engl J Med 2008; 358;1109-17
Our assay
Coating: F(Ab’)2 of IFX, RTX or murine IgG
Proband serum/plasma
Anti-human γ-chainbiotin
HRP-straptavidin
IgG antibodies against mouse Fab, anti-IFX Fab and anti–RTX Fab are common in human plasma
T-helpercells
Anti-mouse Fab, anti-IFX Fab and anti–RTX Fab correlate
r2=0.90, p < 0.0001r2=0.90, p < 0.0001 r2=0.90, p < 0.0001
Mouse IgGcolumn
Anti-IF
X F(a
b')20
200
400
600
800
1000
mU
/L
IFX F(ab')2
Infliximab column
Anti-IF
X F(a
b')20
200
400
600
800
1000
mU
/L
IFX F(ab')2
Cross-reactivity between anti-IFX and anti-mIgG
Anti-m
ouse Ig
G
0
200
400
600
800
1000
mU
/L
Mouse IgG F(ab')2
Anti-m
ouse Ig
G
0
200
400
600
800
1000
mU
/L
Mouse IgG F(ab')2
29 patients with Crohn’s disease treated with infliximab
21 patients with ulcerative colitis treated with infliximab
Measurement of pre-existing anti-infliximab-Fab Absand clinical responses
Clinical study
Observational, retrospective, single center study
Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83
Pre-existing anti-IFX Fab IgG: Prevalence in IBD patients naïve to ‘biologics’
Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83
8
Pre-existing anti-IFX Fab IgG: Association with 1 year remission on IFX
Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83
9
Pre-existing anti-IFX Fab IgG:Levels associated with 1 year remission in CD
Anti-IFX Fab Ab cut-off
mU/l
Sensitivity
% [95%CI]
Specificity
% [95%CI]
Identification of patients in remission <439 100 [63–100] 67 [43–85]
Identification of patients not in remission ≥61 100 [84–100] 25% [3–65]
Optimal differentiation between patients in
remission or not (sensitivity=specificity)
233 75 [35–97] 76 [53–92]
Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83
Primary vs. secondary non-responders
Other modalitiesOther TNF-inhibitor
Primary non-responder
TNF-α inhibitor
Primary responder
Secondary non-responder
Continue TNF-α inhibitor
1/3
1/3
1/3
Probably patients without ”TNF-driven disease”
Ususally loss of response due to ADAsPre-existing antibodies indicate priming
10
Efficacy by anti-IFX Fab Abs
Crohn's disease
Primary non-response Loss of response Remisson
10
100
1000
10000p=0.397
p=0.05
p<0.01
n=8 n=7 n=8
A
308308
692
91
Pre
-exi
stin
g an
ti-IF
X F
ab A
b (m
U/l)
Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83
Antigen receptor (BCR)
B cell
Heterophilic BCR
Mouse IgG
Pre-existing T-cell responses may support development of drug-neutralizing antibodies
Th cell
T-helper cell with specificity for xenogenic peptide
TCRMHC II
Pre-existing T-cell responses may support development of drug-neutralizing antibodies
BCR recognizingidiotype of drug
Drug
B cell Th cell
T-helper cell with specificity for xenogenic peptide
TCRMHC II
10
All patients
Safety by anti-IFX Fab Abs
Steenholdt et al. Aliment Pharmacol Ther 2013; 37: 1172-83
”Positive samples”
Meta-analysis by Xu & Rup
31 studies: 1331 patients & 499 controls
What are the antigenic determinants?
Glycosylation
Xenogenic part
F(ab’)2
Origin of pre-existing antibodies?
Heterophilic antibodiesImmunoglobulin Group I
Anti-Fab(85% of positive
samples)
Group IIAnti-Fc
(15% of positive samples)
Negative samples
Mouse +++ +++ -
Rat +++ - -
Goat +++ - -
Horse +++ +++ -
Rabbit - +++ -
Cow +++ +++ -
BSA (neg. Control) - - -
Hennig et al.
Bovine IgGcolumn
1 10 1000.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
OD
490
nm
Dilution 1:x
Eluate
Cross-reactivity between cow IgG, mouse Fab and infliximab
1 10 1000.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
OD
490
nm
Dilution 1:x
Eluate
Anti-IFX Fab
Anti-mouse IgG Fab
Origin: Possibly vaccines
Diphte
ria to
xoid
I (u
sed u
ntil 1
990)
Tetan
us to
xoid
I (u
sed u
ntil 1
990)
Ovalb
umin
)(neg
ativ
e co
ntrol)
Bovine
IgG (p
ositiv
e co
ntrol)
Havrix
(Hap
atiti
s A)
Diphte
ria to
xoid
II (u
sed a
fter1
990)
Pneum
ococc
al p
olysa
cchar
ide
vacc
ine
Haem
ophilus
influ
enza
e b
Human
pap
illom
a vi
rus
Tetan
us to
xoid
II (u
sed a
fter1
990)
Conclusions regarding pre-existing antibodies
• exist against chimeric antibodies (IFX, RTX, CTX)
• are presumably ”heterophilic antibodies”
• apparently predict outcome of infliximab therapy in Crohn’s disease
• may cause IgG- or IgE-mediated infusion reactions
• may be induced by vaccination
Everybody at Institute for Inflammation Research
Thanks to:
Yaseelan Palarasah, Christina Aniol-Nielsen, Casper Steenholdt, Børge Teisner