Influence of immunogenicity on the efficacy of long-term treatment with infliximab in rheumatoid arthritis.
ABSTRACT To analyse the clinical relevance of the production of anti-infliximab antibodies (anti-infliximab Abs) in patients with RA undergoing infliximab treatment over a prolonged period of time.
Clinical characteristics, serum trough infliximab and antibody levels were evaluated in 85 RA patients treated with infliximab for a median of 4.42 (interval 0.4-10.2) years. DAS in 28 joints (DAS-28), EULAR response criteria and survival of treatment were assessed at 3 time points (6 months, 12 months and >4 years).
Antibodies against infliximab were detected in 28 (32.9%) patients and were present in all EULAR non-responder patients. Antibody levels were higher in EULAR non-responders throughout the study period (P = 0.05 at 6 months, P = 0.02 at 1 year, P = 0.003 at >4 years) compared with EULAR (good and moderate) responders. Nine (10.5%) patients, all of them with high-serum anti-infliximab Ab levels, developed infusion-related reactions. Patients with anti-infliximab Abs more often required increased infliximab doses (51.7%) (P = 0.032) and median survival time on treatment was shorter (4.15 vs 8.89 years) (P = 0.0006). MTX co-therapy was not associated with lower proportion of anti-infliximab Ab-positive patients, but those receiving both infliximab and MTX had lower levels of anti-infliximab Abs (P = 0.073) and longer survival (P = 0.015) on treatment.
The formation of anti-infliximab Abs during treatment with infliximab is associated with a loss of clinical response, the appearance of infusion reactions and discontinuation of treatment.
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ABSTRACT: OBJECTIVES: To compare the efficacy and safety of innovator infliximab (INX) and CT-P13, an INX biosimilar, in active rheumatoid arthritis patients with inadequate response to methotrexate (MTX) treatment. METHODS: Phase III randomised, double-blind, multicentre, multinational, parallel-group study. Patients with active disease despite MTX (12.5-25 mg/week) were randomised to receive 3 mg/kg of CT-P13 (n=302) or INX (n=304) with MTX and folic acid. The primary endpoint was the American College of Rheumatology 20% (ACR20) response at week 30. Therapeutic equivalence of clinical response according to ACR20 criteria was concluded if the 95% CI for the treatment difference was within ±15%. Secondary endpoints included ACR response criteria, European League Against Rheumatism (EULAR) response criteria, change in Disease Activity Score 28 (DAS28), Medical Outcomes Study Short-Form Health Survey (SF-36), Simplified Disease Activity Index, Clinical Disease Activity Index, as well as pharmacokinetic (PK) and pharmacodynamic (PD) parameters, safety and immunogenicity. RESULTS: At week 30, ACR20 responses were 60.9% for CT-P13 and 58.6% for INX (95% CI -6% to 10%) in the intention-to-treat population. The proportions in CT-P13 and INX groups achieving good or moderate EULAR responses (C reactive protein (CRP)) at week 30 were 85.8% and 87.1%, respectively. Low disease activity or remission according to DAS28-CRP, ACR-EULAR remission rates, ACR50/ACR70 responses and all other PK and PD endpoints were highly similar at week 30. Incidence of drug-related adverse events (35.2% vs 35.9%) and detection of antidrug antibodies (48.4% vs 48.2%) were highly similar for CT-P13 and INX, respectively. CONCLUSIONS: CT-P13 demonstrated equivalent efficacy to INX at week 30, with a comparable PK profile and immunogenicity. CT-P13 was well tolerated, with a safety profile comparable with that of INX. CLINICALTRIALS.GOV IDENTIFIER: NCT01217086.Annals of the rheumatic diseases 05/2013; · 8.11 Impact Factor
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ABSTRACT: To analyze sex differences in adverse drug reactions (ADR) to the immune suppressive medication in inflammatory bowel disease (IBD) patients. All IBD patients attending the IBD outpatient clinic of a referral hospital were identified through the electronic diagnosis registration system. The electronic medical records of IBD patients were reviewed and the files of those patients who have used immune suppressive therapy for IBD, i.e., thiopurines, methotrexate, cyclosporine, tacrolimus and anti-tumor necrosis factor agents (anti-TNF); infliximab (IFX), adalimumab (ADA) and/or certolizumab, were further analyzed. The reported ADR to immune suppressive drugs were noted. The general definition of ADR used in clinical practice comprised the occurrence of the ADR in the temporal relationship with its disappearance upon discontinuation of the medication. Patients for whom the required information on drug use and ADR was not available in the electronic medical record and patients with only one registered contact and no further follow-up at the outpatient clinic were excluded. The difference in the incidence and type of ADR between male and female IBD patients were analyzed statistically by χ(2) test. In total, 1009 IBD patients were identified in the electronic diagnosis registration system. Out of these 1009 patients, 843 patients were eligible for further analysis. There were 386 males (46%), mean age 42 years (range: 16-87 years) with a mean duration of the disease of 14 years (range: 0-54 years); 578 patients with Crohn's disease, 244 with ulcerative colitis and 21 with unclassified colitis. Seventy percent (586 pts) of patients used any kind of immune suppressive agents at a certain point of the disease course, the majority of the patients (546 pts, 65%) used thiopurines, 176 pts (21%) methotrexate, 46 pts (5%) cyclosporine and one patient tacrolimus. One third (240 pts, 28%) of patients were treated with anti-TNF, the majority of patients (227 pts, 27%) used IFX, 99 (12%) used ADA and five patients certolizumab. There were no differences between male and female patients in the use of immune suppressive agents. With regards to ADR, no differences between males and females were observed in the incidence of ADR to thiopurines, methotrexate and cyclosporine. Among 77 pts who developed ADR to one or more anti-TNF agents, significantly more females (54 pts, 39% of all anti-TNF treated women) than males (23 pts, 23% of all anti-TNF treated men) experienced ADR to an anti-TNF agent [P = 0.011; odds ratio (OR) 2.2, 95%CI 1.2-3.8]. The most frequent ADR to both anti-TNF agents, IFX and ADA, were allergic reactions (15% of all IFX users and 7% of all patients treated with ADA) and for both agents a significantly higher rate of allergic reactions in females compared with males was observed. As a result of ADR, 36 patients (15% of all patients using anti-TNF) stopped the treatment, with significantly higher stopping rate among females (27 females, 19% vs 9 males, 9%, P = 0.024). Treatment with anti-TNF antibodies is accompanied by sexual dimorphic profile of ADR with female patients being more at risk for allergic reactions and subsequent discontinuation of the treatment.World Journal of Gastroenterology 12/2012; 18(47):6967-73. · 2.55 Impact Factor
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ABSTRACT: The objectives of this study are to evaluate the effect of anti-drug antibodies on the clinical efficacy and withdrawal rate of the anti-TNFα biologics in patients with rheumatic diseases. Consecutive patients with rheumatic diseases recently commenced on anti-TNFα biologics were recruited. Serum samples were collected for assay of drug level and antibody titer against the corresponding biologics. Comparison of the clinical efficacy and drug retention rate was performed between patients with and without anti-drug antibodies. Fifty-eight Chinese patients were studied (64 % women; age 47.8 ± 12.9 years; disease duration 6.7 ± 6.4 years). The proportion of patients using infliximab (IFX), adalimumab (ADA), and etanercept (ETN) was 41, 28, and 31 %, respectively. Antibodies against IFX, ADA, and ETN were demonstrated in 12(50 %), 5(31 %) and 0(0 %) patients, respectively. Patients who developed anti-drug antibodies had significantly lower levels of the corresponding drugs (IFX level: 0.004 ± 0.01 vs 3.81 ± 3.49 μg/ml; p = 0.002; ADA level: 0.0 vs 7.6 ± 8.3 μg/ml; p = 0.008). Anti-drug antibody-positive patients had a significantly higher cumulative drug withdrawal rate due to inefficacy (64.7 and 71.8 % vs 10.3 and 10.3 % at month 12 and month 24, respectively; p < 0.001). In rheumatoid arthritis and psoriatic arthritis, non-responders was significantly more frequent in antibody-positive patients (54 vs 13 %; p = 0.01). In spondyloarthritis, the improvement in ankylosing spondylitis disease activity score was significant in patients without antibodies (3.89 ± 0.82 to 2.22 ± 0.86; p = 0.01) but not in those with anti-drug antibodies (3.40 ± 1.67 to 3.23 ± 1.40; p = 0.73). We concluded that the presence of neutralizing antibodies is associated with lower serum levels of the anti-TNFα biologics, leading to lower efficacy and higher withdrawal rate.Clinical Rheumatology 07/2013; · 2.04 Impact Factor
Influence of immunogenicity on the efficacy
of long-term treatment with infliximab in
Dora Pascual-Salcedo1, Chamaida Plasencia2, Susana Ramiro1, Laura Nun ˜o2,
Gema Bonilla2, Daniel Nagore3, Ainhoa Ruiz del Agua3, Antonio Martı ´nez3,
Lucien Aarden4, Emilio Martı ´n-Mola2and Alejandro Balsa2
Objective. To analyse the clinical relevance of the production of anti-infliximab antibodies (anti-infliximab
Abs) in patients with RA undergoing infliximab treatment over a prolonged period of time.
Methods. Clinical characteristics, serum trough infliximab and antibody levels were evaluated in 85 RA
patients treated with infliximab for a median of 4.42 (interval 0.4?10.2) years. DAS in 28 joints (DAS-28),
EULAR response criteria and survival of treatment were assessed at 3 time points (6 months, 12 months
and >4 years).
Results. Antibodies against infliximab were detected in 28 (32.9%) patients and were present in all EULAR
non-responder patients. Antibody levels were higher in EULAR non-responders throughout the study
period (P=0.05 at 6 months, P=0.02 at 1 year, P=0.003 at >4 years) compared with EULAR (good
and moderate) responders. Nine (10.5%) patients, all of them with high-serum anti-infliximab Ab levels,
developed infusion-related reactions. Patients with anti-infliximab Abs more often required increased
infliximab doses (51.7%) (P=0.032) and median survival time on treatment was shorter (4.15 vs 8.89 years)
(P=0.0006). MTX co-therapy was not associated with lower proportion of anti-infliximab Ab-positive pa-
tients, but those receiving both infliximab and MTX had lower levels of anti-infliximab Abs (P=0.073) and
longer survival (P=0.015) on treatment.
Conclusion. The formation of anti-infliximab Abs during treatment with infliximab is associated with a loss
of clinical response, the appearance of infusion reactions and discontinuation of treatment.
Key words: Rheumatoid arthritis, Infliximab therapy, Immunogenicity, Efficacy, Long-term treatment.
Since the approval of the first therapeutic mAb against
TNF 15 years ago, the use of biological drugs in clinical
practice has grown constantly . The treatment of RA,
diseases, which are usually refractory to conventional
treatments, has improved considerably since combination
regimens of these new biological drugs and the classical
DMARDS were introduced .
Infliximab is a chimeric (mouse?human) mAb antagonist
to TNF, and was the first antibody-based therapy to be
introduced to treat patients with RA. Today its use has
become more generalized and it is being administered
to a growing number of patients at an early stage of dis-
ease, mainly because of its clinical efficacy and retarding
effects on joint destruction . Although the efficacy of
this drug as a treatment for patients with active RA has
been widely demonstrated [3, 4], some RA patients initially
respond to treatment but subsequently their responsive-
ness decreases . One of the alleged reasons for this
phenomenon is immunogenicity associated with the drug
itself. Infliximab can induce the formation of neutralizing
1Immunology Unit, IdiPAZ, RIER,2Rheumatology Department, IdiPAZ,
RIER, Hospital La Paz, Madrid,3Proteomika, S.L., Parque
Tecnolo ´gico, Derio, Spain and4Autoimmune Diseases Unit, CLB
Sanquin Amsterdam, Amsterdam, The Netherlands.
Correspondence to: Dora Pascual-Salcedo, Immunology Unit,
Hospital La Paz, Paseo de La Castellana 261, 28046 Madrid, Spain.
Submitted 17 November 2010; revised version accepted
22 February 2011.
! The Author 2011. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: firstname.lastname@example.org
Advance Access publication 22 March 2011
SCIENCE by guest on February 19, 2013
antibodies , resulting in loss of efficacy and appearance
of side effects such as infusion-related reactions [6, 7].
The induction of antibodies against the drug has been
described in about half of the patients receiving repeated
infliximab monotherapy; as a consequence, immune sup-
pression by concomitant administration of MTX is recom-
mended [4, 8, 9].
An antibody response to the drug often appears be-
tween the third and sixth month . As long as the rela-
tive amount of the anti-drug antibodies is lower than the
serum trough level of infliximab, the drug can provide a
clinical benefit . However, when the endogenous pro-
duction of antibodies exceeds the amount of drug in the
serum, the latter is cleared from the circulation [11, 12],
the therapy is rendered ineffective and free antibodies to
the drug can be measured in the patient’s serum. The
accelerated clearance of infliximab complexed to antibo-
dies may result in decreased pharmacological availability
[13, 14] and ultimately the loss of therapeutic effective-
ness of infliximab . Therefore, it is very likely that the
equilibrium between infliximab and antibody response will
regulate the overall effectiveness of the drug .
Most publications on the relationship between the pres-
ence of anti-infliximab antibodies (anti-infliximab Abs) and
clinical response in RA patients focus on the first period of
administration, with a maximum of 3 years follow-up .
Mulleman et al.  studied patients for >6 years, but only
monitored the infliximab concentration. However, Wolbink
and co-workers [11, 15] reported that development of the
immune response against infliximab is a gradual process
that may change over time because continuation of treat-
ment may either induce immune tolerance or stimulate
further antibody formation.
In this study, we present data on 85 RA patients under-
going infliximab treatment at the Rheumatology Unit of La
Paz University Hospital since the end of 1999 (>10 years).
Infliximab and anti-infliximab Ab levels were measured
in order to assess the clinical relevance of infliximab
immunogenicity throughout the course of the therapy.
Patients and sera
A total of 85 consecutive patients with RA, without previ-
ous biological treatment were included. Patients were
enrolled at the Department of Rheumatology of La Paz
University Hospital to receive infliximab therapy. This
was a retrospective observational study, approved by
the Hospital La Paz Ethics Committee and patients signed
an informed consent form according to the Declaration of
Helsinki. Serum samples (a total of 1451) were collected at
the time of infusion, stored frozen and only thawed for the
purpose of this study. The retrospective study period
covers the years 1999 until 2010. All patients fulfilled the
ACR 1987 revised criteria for RA and all of them had evi-
dence of active disease, as indicated by a 28-joint DAS
(DAS-28) at inclusion of 5.49 (1.2) [mean(S.D.)]. At first all
patients were given i.v. infusions of 3mg/kg infliximab at
0, 2, 6 and every 8 weeks thereafter. After 14 weeks of
treatment, the rheumatologist was allowed to increase the
infliximab dosage to 5mg/kg depending on the observed
clinical response. Every 6 months, disease activity using
the DAS-28 and European League Against Rheumatism
(EULAR) response criteria  was measured to assess
clinical response. Six months, 1 year and >4 years [mean
(S.D.) 5.9 (2) years] were chosen from the study as repre-
sentative time points for patients’ clinical response.
Infusion reactions were defined as any event appearing
during infusion requiring either arrest of drug infusion or the
administration of parenteral medication.
Blood samples were collected at baseline and just
before each infusion at 2, 6 and every 8 weeks thereafter,
so that a maximum of 6?8 samples per year were obtained
from each patient. Precise timing is required to compare
results, because with a longer time interval serum inflixi-
mab may become undetectable due to normal drug
pharmacokinetics, and not as a consequence of IC forma-
infliximab and anti-infliximab Abs were measured. At base-
line, infliximab and anti-infliximab Ab concentrations in all
patients were <10ng/ml and 50 AU/ml, respectively.
Serum infliximab assay
Serum infliximab levels were determined by a sandwich
ELISA, as described by Wolbink et al.  using a poly-
clonal anti-infliximab Ab . Briefly, microtitre plates were
coated with 2mg/ml mouse monoclonal anti-TNF antibody
(CLB/7) (Sanquin, Amsterdam, The Netherlands) and then
incubated with 0.01mg/ml recombinant human TNF-a
(Peprotech, Rocky Hill, USA). Serial dilutions of serum sam-
ples and standard curve (0.1?50ng/ml infliximab) were
made in high performance ELISA (HPE) buffer (Sanquin).
Bound infliximab was detected with biotinylated affinity
purified rabbit immunoglobulin G (IgG) to Fab regions of
infliximab,and the reaction
streptavidin?polyperoxidase (polyHRP) (Sanquin). The de-
tection limit of the assay was 1ng/ml infliximab. Cut-off
values were established with sera from 150 healthy blood
donors and 100 RA patients who had never received inflix-
imab (of whom 70% were RF positive) to exclude any
background signal that might have been caused by RF
or other auto-antibodies present in RA patient sera.
Serum infliximab levels >10ng/ml (mean+6 S.D. control
group) were considered positive.
Anti-infliximab Ab assay
Anti-infliximab Abs were detected by a two-site (bridging)
ELISA, which takes advantage of the monovalency of the
two arms of IgG subclasses 1, 2 and 3, to crosslink the
infliximab coated on plates to biotinylated infliximab
[11, 19]. Polystyrene plates (Nunc A/S, Roskilde, Denmark)
were coated with infliximab (0.5mg/ml) overnight. The fol-
lowing day, serial dilutions of samples (starting at 1/10)
and a standard curve (0.48?250AU/ml) diluted in HPE
were incubated for 1h with shaking. A standard curve
was constructed using a patient serum that showed a
high titre of anti-infliximab Ab (mainly IgG1) previously
titrated in arbitrary units per millilitre by one of the authors
Dora Pascual-Salcedo et al.
by guest on February 19, 2013
of this study (L.A., data not shown). After washing, 10ng/ml
infliximab biotinylated by standard procedures (Pierce,
Rockford, IL, USA), was added. Bound labelled infliximab
was detected by incubation with polyHRP (1:10000) in
PBS. The reaction was developed with tetramethylbenzi-
dine (TMB)/H2O2in 0.11M acetic acid buffer pH=5.5 and
stopped with 2M H2SO4. Washing steps were made in
0.01M PBS 0.02% Tween 20. The assay detection limit
was 2AU/ml and the cut-off for the presence of anti-
infliximab Ab in patient sera was established at 50AU/ml
(mean+6 S.D.) with the same control group used for the
measurement of free infliximab. A linear dose-response
curve for inhibition was obtained when positive samples
for anti-infliximab Abs were pre-incubated with infliximab.
Antibodies to CCP (aCCP) were measured by ELISA
(Eurodiagnostica, Malmo ¨, Sweden), and RF was mea-
sured by nephelometry (Siemens, Marburg, Germany)
with cut-off values of 25 and 9UI/ml, respectively.
Descriptive statistics were provided using the mean, S.D.,
median (Mdn) and interquartile range (IQR). Statistical
analysis was performed using the Statistical Package for
the Social Sciences version 10.0 (SPSS, Chicago, IL,
USA). Frequency data were compared by the Pearson’s
chi-square and Fisher’s exact tests. Differences in quan-
titative values between groups were analysed using
Mann?Whitney U and Wilcoxon non-parametric tests.
Time course data were analysed using the Kaplan?Meier
method. Statistical significance was calculated using the
log-rank test and P<0.05 was considered statistically
A total of 85 RA patients were enrolled in the study, of
whom 69 were women, with a mean (S.D.) age of 53.8
(14.2) years at the beginning of infliximab treatment.
Demographic and clinical characteristics are shown in
Table 1. All patients received 3mg/kg infliximab at base-
line; however, 44 (51.8%) patients needed a gradual inflix-
imab dose escalation by either increasing the dose to
5mg/kg and/or shortening the interval between infusions,
due to an inadequate response.
Clinical response and association with levels of
infliximab and anti-infliximab Ab
At baseline, all patients had active disease as indicated by
a mean (S.D.) DAS-28 of 5.49 (1.26) with no differences in
DAS-28 values between patients that subsequently did
[5.75 (1.28)], or did not [5.37 (1.25)] develop anti-infliximab
Ab (P=0.204). Anti-infliximab Abs were detected in serum
samples from 28 (32.9%) patients, in all cases with un-
detectable serum trough infliximab levels. These antibo-
dies appeared most frequently after the fourth infusion
[Mdn 16 (range 14?79) weeks]; although in four patients
the appearance of anti-infliximab Ab was delayed for >1
year. In most patients, antibody titres did not disappear,
but increased during treatment and were only modulated
by an increase in the dose of infliximab. Patients with
antibodies against infliximab had higher DAS-28 values
at 6-month follow-up (16 out of 49 patients), 1 year (7
out of 31 patients) and >4 years (11 out of 47 patients)
[4.85 (1.24) vs 3.67 (1.12), P=0.004; 4.95 (1.24) vs 3.13
(1.17), P=0.002; 4.00 (1.35) vs 3.46 (1.22), P=0.004, re-
spectively]. Similar results were found for ?DAS-28 from
baseline [1.10 (0.93) vs 1.73 (1.03), P=0.044; 1.24 (0.86)
vs 1.92 (0.72), P=0.061; 0.57 (1.86) vs 1.98 (1.26),
P=0.025] at the three time points, respectively.
Patients classified as responders were mainly patients
with no detectable anti-infliximab Ab levels (Fig. 1). Only
24% of EULAR (good and moderate) responders (n=75)
showed anti-infliximab Ab vs 100% of non-responder
patients (n=10) (P<0.001). Serum trough infliximab
levels (Mdn, IQR) were higher in EULAR responders
(good and moderate) than in EULAR non-responder pa-
tients at 6 months (992, 46?2960 vs 0, 0?60ng/ml,
P=0.005), 1 year (1792, 384?3904 vs 0, 0?555ng/ml,
0?2672ng/ml, P=0.101), respectively (Fig. 2A). Serum
anti-infliximab Ab concentration (Mdn, IQR) was higher
in non-responders than in responders at 6 months (208,
0?1087AU/ml vs 0, 0?100AU/ml, P=0.054), 1 year
(60, 12?8924 vs 0, 0?0AU/ml, P=0.018) and >4 years
(791, 0?6303 vs 0, 0?0, P=0.003), respectively (Fig. 2B).
(1536, 220?3456 vs0,
Survival of infliximab treatment
A total of 45 (53.5%) out of 84 patients interrupted inflix-
imab therapy, with a median survival rate of 5.75 (95%
TABLE 1 Demographic and clinical characteristics of
85 RA patients
At study inclusion
Age at onset, mean (S.D.), years
Gender: female, n (%)
aCCP positive, n (%)
RF positive, n (%)
During the study
Concomitant anti-rheumatic therapy
MTX alone, n (%)
MTX+other DMARDs, n (%)
Other DMARDs,an (%)
None, n (%)
Concomitant use of
glucocorticoids, n (%)
Time under infliximab,
mean (interval), years
Infliximab discontinued, n/total (%)
Patients with acquired
drug resistance, n (%)
aOther DMARDs: LEF, SSZ, HCQ and AZA.bThe evolution of
one patient was missed because she moved to another
Immunogenicity influences treatment with infliximab in RA
by guest on February 19, 2013
CI 4?7.5) years on the drug. The number of patients that
discontinued infliximab therapy (n=45) was significantly
higher among those who developed anti-infliximab Ab
[23 (82.1%) out of 28 vs 22 (39.3%) out of 57;
P<0.001]. Median survival time on infliximab treatment
was 4.15 (95% CI 2.78?5.53) years in patients with anti-
infliximab Ab vs 8.89 (95% CI 6.7?11) years for those with-
out antibodies (P=0.0006; Fig. 3A). Among patients who
developed anti-infliximab Ab, median survival time on
infliximab treatment was longer (P=0.022) in patients
receiving concomitant MTX (n=22; 4.52 years, 95% CI
3.8?5.23 years) than in those not receiving MTX (n=6;
1.06 years, 95% CI 0?3.79 years) (Fig. 3B).
Twenty-three (82.1%) of 28 patients who developed
anti-infliximab Ab discontinued infliximab treatment. In
four out of the five remaining patients, anti-infliximab
FIG. 2 Serum trough infliximab (A) and anti-infliximab Ab levels (B) in RA patients responding (R) (good and moderate)
(n=38 at 6 months, n=27 at 1 year, n=39 at >4years) and not responding (non-R) (n=11 at 6 months, n=4 at 1 year,
n=8 at >4 years) by EULAR criteria, to infliximab treatment. Data are shown as box plots, where the boxes represent the
25th to 75th percentiles, and the lines outside the boxes represent the 10th and 90th percentiles.
FIG. 1 Relationship between the presence of anti-infliximab Ab and EULAR response in RA patients treated with inflix-
imab. Good: DAS-28 decrease >1.2 with an attained DAS-28 <3.2 Moderate: DAS-28 decrease 41.2 and 50.6 with an
attained DAS-28 53.2 and 45.1. No response: DAS-28 decrease <0.6 with an attained DAS-28 >5.1.
Dora Pascual-Salcedo et al.
by guest on February 19, 2013
Ab concentration decreased below detection levels after
dosage escalation to 5mg/kg, with a consequent clinical
improvement, and the fifth patient continued treatment be-
cause her clinical response was good, despite remaining
antibodies in circulation.
Modulation of anti-infliximab Ab levels by drug
In 44 (51.7%) of the 85 patients, an acquired resistance to
the drug was observed necessitating either an increased
dosage of infliximab or a reduced time interval between
infusions to achieve a clinical improvement. This drug re-
sistance was higher in patients with anti-infliximab Ab [19
(67.9%) out of 28] than in those without anti-infliximab Ab
[25 (43.9%) out of 57] (P=0.032). Two kinds of response
on dose escalation were observed in our cohort. Type I:
anti-infliximab Ab disappeared after dose increase to
5mg/kg coinciding with measurable infliximab serum
trough levels and a DAS-28 decrease. In these cases,
anti-infliximab Ab could be detected again if the dose of
infliximab was subsequently reduced, with a simultaneous
clinical worsening (Fig. 4A). Type II: anti-infliximab Ab did
not disappear after drug escalation (Fig. 4B), reaching
high levels, which in some patients were associated with
development of infusion-related reactions (three patients).
Relation between infusion-related reactions and
Infusion-related reactions were recorded in nine patients,
all of whom had detectable anti-infliximab Ab. Anti-
infliximab Ab levels [Mdn (IQR)] at the time of infusion
reaction were higher in the patients who developed reac-
tions [20565 (5000?30625) AU/ml] than in those patients
with detectable anti-drug antibodies, but without infusion-
related reactions [10152 (491?8162)AU/ml] (P=0.041;
Influence of combined therapy with MTX on
anti-infliximab Ab presence
Sixty-nine (81.1%) patients received MTX [7.5?25mg/weekly,
mean (S.D.) 15 (4.96) mg/weekly] concomitantly with inflix-
imab. MTX was subcutaneously and orally administered in
19 and 50 patients, respectively. We did not find a lower
proportion of patients developing anti-infliximab Ab in as-
sociation with the use of MTX (32% with MTX vs 37%
without MTX, P=0.77). However, in patients receiving
MTX who did make antibodies (n=22), maximal levels
[Mdn (IQR)] tended to be lower than in those with antibo-
dies on infliximab monotherapy (n=6) [3414 (808?7426)
AU/ml with MTX vs 21250 (7049?47656) AU/ml without
It is widely accepted that immunogenicity of biological
drugs such as infliximab is the main cause of loss of clin-
ical response in the treatment of RA [10, 13, 18, 20]. In this
study, we have analysed the clinical significance of free
infliximab and anti-infliximab Ab concentration in serum in
a cohort of 85 Spanish RA patients treated for >4 years.
Our findings indicate that one-third of RA patients develop
antibodies and this is correlated with clinical response.
The variable incidence of anti-drug antibodies reported
in earlier literature was mainly methodological and related
to the method used to measure the antibodies .
sitive method to detect all antibody isotypes [5, 10, 18, 19,
21], but has the drawback of the use of radioactivity. The
FIG. 3 Kaplan?Meier curves for survival on infliximab therapy of RA patients. (A) Patients who either developed (—?) or
did not develop (— —) anti-infliximab Ab. (B) Patients who developed anti-infliximab Ab and are treated with (—?) or
without MTX (...).
Immunogenicity influences treatment with infliximab in RA
by guest on February 19, 2013
bridging ELISA employed in this worksuffers the disadvan-
tage that it fails to measure IgG4 antibodies. However, this
is not believed to represent a major problem as isolated
IgG4 antibodies usually do not occur in the absence of
other IgG isotypes  and the bridging ELISA approach
for measuring anti-drug antibodies has been validated by
its use in several other studies [11, 13, 20].
Serum levels of anti-infliximab Ab strongly correlate with
the clinical response as DAS-28 was significantly lower
in those patients without anti-infliximab Ab at all time
points. According to the EULAR response criteria, 100%
of non-responder patients at any studied time point
showed anti-infliximab Ab vs only 24% of responders.
Whereas most previously published studies were per-
formed over relatively short periods of time (41 year)
[5?7], we have extended the analysis over >4 years,
since we believe that immunogenicity rates can be under-
estimated if studies are restricted to <1 year. Moreover,
during our long-term follow up, we have seen that patients
with detectable levels of anti-infliximab Ab had to discon-
tinue treatment earlier than those who did not develop
As reported in previous literature, we have found that
serum trough infliximab levels inversely correlate with
the presence of antibodies against the drug and with the
clinical response [10, 14, 17]. One could, therefore, argue
FIG. 4 Modulation of serum infliximab and anti-infliximab Ab levels, as well as clinical response, with infliximab dose
changes. Infliximab dose ranges between 3 and 5mg/kg. (A) A ‘type I’ representative patient in whom anti-infliximab
Ab levels are inhibited only by a high infliximab concentration. Lowering infliximab dose results in the appearance of
antibodies and DAS-28 increase. (B) A ‘type II’ representative patient in whom anti-infliximab Abs do not disappear
after infliximab dose increase, with a poor clinical response (DAS-28).
FIG. 5 Maximum antibody levels in patients with
anti-infliximab Ab who developed or did not develop
an infusion-related reaction, defined as in ‘Material
and methods’ section.
Dora Pascual-Salcedo et al.
by guest on February 19, 2013
that there is no need to monitor both the drug and
anti-drug antibodies, as complexes are formed between
antibodies and infliximab . However, undetectable or
low levels of infliximab before the appearance of antibo-
dies may indicate that the patient will develop a high titre
of antibodies following the subsequent infusion. In fact, in
our cohort one patient developed an infusion-related re-
action during the fourth infusion, after a sharp decrease in
circulating infliximab, but before antibodies could be
The development of antibodies to infliximab occurred
mainly in the first 4 months of treatment, although it can
be delayed in patients with an early drug escalation, be-
cause only when the immune system makes sufficient
antibodies to overcome the infliximab concentration is
antibody detection possible. Detection of infliximab and
anti-infliximab Ab levels can be used to customize treat-
ment and help to avoid unnecessary therapy. As we have
shown, patients differ in their clinical response to an incr-
eased dose of infliximab. In some patients, an improve-
ment in DAS-28 was seen to coincide with measurable
serum trough infliximab levels and loss of anti-infliximab
Ab, which can reappear after dose decrease because
of clinical improvement. In another group of patients,
the antibodies remained in the circulation despite a drug
dosage increase to maximum levels. These patients did
not show clinical improvement and had a higher risk of
developing infusion-related reactions. Another important
utility of biopharmaceuticals monitoring by means of
drug and anti-drug Ab determinations has been clearly
exposed in a recent publication by Jamnitski et al. .
The authors show that among patients who discontinued
treatment with a first TNF-a inhibitor, those who had de-
veloped antibodies against the drug achieved a signifi-
cantly better clinical response after switching to another
anti-TNF (etanercept) than patients without antibodies.
Authors argue that immunogenicity monitoring is needed
in order to differentiate patients who will benefit from
a change in anti-TNF therapy from those who show no
Patients receiving infliximab treatment show a high rate
of infusion-related reactions [5, 20]. In our cohort, all pa-
tients with infusion-related reactions had anti-infliximab
Ab at high titres. These data support the view that ele-
vated titres of anti-infliximab Abs are associated with
increased risk of infusion reactions, probably because of
the formation of large antibody complexes. These com-
plexes are removed with difficulty by the liver and spleen,
and are associated with the occurrence of serious adverse
It has been reported that combined therapy with inflix-
imab and MTX is inversely associated with the formation
of anti-infliximab Ab . In our study, 81% of patients
received concomitant MTX with infliximab and 32% of
them developed anti-infliximab Ab. A similar number of
patients developed antibodies when they were treated
with infliximab alone or together with other DMARDs.
These results, although similar to the findings reported
by Haraoui et al. , were different from those observed
in previous studies, which suggested that combined ther-
apy reduced the number of patients developing anti-drug
antibodies , probably due to differences in adminis-
tration guidelines. However, in our study we observed
that patients who developed anti-infliximab Ab continued
on anti-TNF treatment significantly longer if they were
receiving concomitant therapy with MTX, probably be-
cause the immunosuppression is associated with the pro-
duction of lower antibody levels and the effectiveness of
MTX itself on disease activity. This fact encourages us to
recommend the routine use of MTX concomitantly with
In conclusion, the formation of anti-infliximab Ab is asso-
ciated with a poor clinical response and with the appear-
ance of infusion reactions. Long-term follow-up shows
that levels of these antibodies may be modulated by incr-
easing drug concentration, which suggests that they may
be used to monitor the appropriate therapeutic regime.
Moreover, they are associated with the discontinuation
of treatment over time.
Rheumatology key messages
. Immunogenicity of infliximab is associated with loss
of clinical response and appearance of infusion
. Detection of anti-infliximab Ab can be used to cus-
tomize treatment and help to avoid unnecessary
. Patients with anti-infliximab Ab discontinue inflixi-
mab treatment earlier than those who did not de-
The authors are grateful to Jesu ´s Dı ´ez for providing help
in the statistical analysis, to Maria Teresa Lo ´pez for her
handling of data files and to Gijs Jochems, Simon
Santacruz and Charlotte Krieckaert for critical reading of
the manuscript and helpful discussions.
Funding: This study was supported by grants from
Fundacio ´n Mutua Madrilen ˜a Cod IdiPaz:PI-668 and from
Progenika SL, Cod IdiPaz:PI-751.
Disclosure statement: D.N., A.M. and A.R.d.A are full-time
employees of Proteomika, S.L. A.B. has received fees
from Roche, Schreing-Plough, Wyeth, Abbott, BMS and
UCB. L.A. serves as a consultant to Algonomics, Genmab
and ArgenX and has received speaker honoraria from
Hoffman LaRoche and Pfizer. E.M.-M. is a consultant
and a member of a speakers’ bureau for Pfizer, MSD,
UCB and Abbott. All other authors have declared no
conflicts of interest.
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