Download The Next Generation of Monitoring Adalimumab Drug and Antibody

The Next Generation of
Monitoring Adalimumab
Drug and Antibody Levels
I
nflammatory bowel diseases (IBDs) are chronically relapsing
intestinal inflammatory conditions, of which the most common
are Crohn’s disease (CD) and ulcerative colitis (UC). Their
etiopathogenesis has not been clearly elucidated, but accumulating
evidence suggests that IBD represents an aberrant mucosal immune
response to commensal gut organisms in a genetically susceptible
host.1-3 Genes that contribute to IBD susceptibility appear to control
several pathways crucial for intestinal mucosal homeostasis,
including the function of the epithelial barrier and regulation of innate
and adaptive immunity.1 The initial breakdown of the intestinal
epithelial barrier results in increased bacterial translocation through
the lamina propria, where microbial antigens encounter an underlying
network of antigen-presenting cells. These cells, which are part of the
innate immune system, recognize repetitive motifs on microbial
antigens and present antigen fragments to T lymphocytes, leading to
secretion of multiple soluble mediators, a majority of which are
cytokines and include tumor necrosis factor alpha (TNF), interferon
gamma, interleukin-13, and interleukin-17. Subsequent activation of
other cell types (eg, endothelial cells) results in an escalating cycle of
cellular trafficking, mediator production, uncontrolled intestinal
inflammation, and tissue injury.3
TNF is a proinflammatory cytokine that is abundantly expressed
in the gastrointestinal tract of patients with IBD.4,5 Secreted
primarily by activated monocytes and macrophages, TNF amplifies
mucosal inflammation through several mechanisms, including
disruption of the epithelial barrier, induction of apoptosis in villous
epithelial cells, and secretion of chemokines from intestinal
epithelial cells.6 Its proinflammatory activity also extends to
upregulating the expression of adhesion molecules and
inflammatory mediators on other cell types involved in inflammation,
including endothelial cells, neutrophils, macrophages, and immune
T and B cells. Because of its central role in IBD pathogenesis, TNF
is a logical target for therapeutic intervention and the development
of TNF-directed therapies has been a major advance in the treatment
of IBD.
Monoclonal antibodies targeting TNF have proven highly effective
in managing CD and UC. The anti-TNF agents—infliximab (IFX;
Remicade®, Janssen Biotech, Horsham, PA), a chimeric monoclonal
antibody against TNF,7-9 adalimumab (ADA; Humira®, Abbott
Laboratories, Abbott Park, IL), a fully human anti-TNF monoclonal
antibody,10,11 and certolizumab pegol (Cimzia®; UCB, Inc., Smyrna,
GA), a humanized anti-TNF Fab’ monoclonal antibody fragment linked
to polyethylene glycol12,13 — have demonstrated efficacy for induction
and maintenance of remission in patients with moderate-to-severe
CD and UC.
Despite the substantial improvement in outcomes that anti-TNF
agents have been able to provide for patients with IBD, response is not
universal. More than one third of patients do not respond to induction
therapy (primary nonresponse)10,12 and, even among initial
responders, response wanes over time in 20% to 60% of patients
(secondary nonresponse).8,9,11,13,14 These therapeutic failures have
not been adequately addressed and pose a challenge to clinicians.
Hypotheses for treatment failure that are being actively investigated
include the potential role of inadequate serum levels of the anti-TNF
agent, consumption of the drug owing to high inflammatory disease
burden, and development of immunogenicity (ie, formation of
antibodies to the anti-TNF therapy) during the treatment course.15,16
Because of marked interindividual differences in drug pharmacokinetics,
bioavailability, and immunogenicity, there is increasing recognition
that optimizing treatment according to individual patient needs may
provide a more rational therapeutic approach than universal use of a
standard regimen derived from large clinical trials in nonuniform
patient cohorts.15 Emerging evidence indicates that higher serum drug
levels are associated with longer durations of response and that
development of antibodies-to-IFX or -ADA (ATIs or ATAs, respectively)
is associated with a diminished or shorter response,17-21 suggesting
that monitoring anti-TNF serum levels and anti-drug antibody status
to inform treatment decisions may result in better outcomes for
patients undergoing anti-TNF therapy. A recent review of clinical
scenarios using anti-TNF serum levels and anti-drug antibody status
to make treatment decisions demonstrated that anti-TNF monitoring
in clinical practice has potential in optimizing individual treatment
algorithms.22 However, the clinical utility of such testing has been
limited by the sensitivity of currently available test methodologies,
including the interference of anti-TNF drug levels with measurement
of anti-drug antibodies.
This monograph focuses on adalimumab, one of three anti-TNF
agents available for treatment of CD and UC, by presenting the following:
■ A brief overview of the efficacy, safety, and tolerability of ADA in
patients with lBD.
■ Available evidence summarizing the interrelationship between
serum ADA levels, ATAs, and disease activity in patients who lose
response during ADA therapy.
■ A description of a newly available assay—PROMETHEUS®
AnserTM ADA (Prometheus Laboratories, San Diego, CA)—for the
measurement of serum ADA and ATA concentrations, including
validation of the assay, its technical advantages over enzymelinked immunosorbent assay (ELISA) and electrochemiluminescence
immunoassay (ECLIA), and its clinical utility for informing the
overall treatment of patients with IBD.
1
Adalimumab
ADA is a subcutaneously administered, recombinant human
immunoglobulin G1 (IgG1) monoclonal antibody specific for human
TNF, with human-derived heavy and light chain variable regions and
human IgG1 kappa constant region (Figure 1).23,24 It is considered
“fully human” because the coding gene sequences do not contain
elements cloned from other species.25 Even though ADA is a fully human
antibody and is less immunogenic than IFX (a human-murine chimera
containing 25% murine sequences), the unique antigen-binding site on
the variable region can render it immunogenic and capable of provoking
an immune response (Figure 2).24,25 The resulting formation of ATAs
may reduce the clinical effectiveness of the drug. ADA is approved for
the treatment of IBD and several rheumatologic conditions. In CD, ADA
is indicated for reducing signs and symptoms and inducing and
maintaining clinical remission in adults with moderate to severe disease
who have responded inadequately to conventional therapy, have
lost response to IFX, or have intolerance to IFX.23 In UC, ADA is
indicated for inducing and sustaining clinical remission in adult
patients with moderate to severe UC who have had an inadequate
response to immunosuppressants such as corticosteroids, azathioprine,
or 6-mercaptopurine, but the effectiveness of ADA in patients with loss
of response or intolerance to TNF blockers has not been established.23
Overview of Clinical Studies of ADA
A number of pivotal trials establishing the safety and efficacy of
ADA for treating patients with IBD resulted in approval of this agent for
CD and UC. These studies are reviewed in brief, and the main results
are presented here.
The safety and efficacy of multiple doses of ADA were assessed in
three randomized, double-blind, placebo-controlled studies,
CLASSIC I, GAIN, and CHARM (described below),10,11,26 in adult
patients with moderate to severe CD (Crohn’s Disease Activity index
[CDAI] ≥220 and ≤450), the majority of whom were receiving
concomitant conventional therapy. Induction of clinical remission
(defined as CDAI <150) was evaluated in two of these studies,
CLASSIC I (Clinical Assessment of Adalimumab Safety and Efficacy
Studied as Induction Therapy in Crohn’s Disease 1)10 and GAIN
(Gauging Adalimumab Efficacy in Infliximab Nonresponders).26 In the
CLASSIC I study,10 299 TNF-naïve patients were randomized to
receive either placebo at weeks 0 and 2, ADA 160 mg at week 0 and
80 mg at week 2, ADA 80 mg at week 0 and 40 mg at week 2, or ADA
40 mg at week 0 and 20 mg at week 2. At week 4, clinical remission
was observed in 36% of patients in the ADA 160/80 mg group,
compared with 12% in the placebo group (P=.001). In the GAIN
study,26 ADA induction therapy (160 mg at week 0 and 80 mg at
week 2) in 325 randomized patients who had lost response to or
were intolerant of IFX resulted in significantly higher rates of
remission at 4 weeks, compared with placebo (P<.001).
The CHARM study (Crohn’s Trial of the Fully Human Antibody
Adalimumab for Remission Maintenance)11 evaluated the maintenance
of clinical remission with ADA in 854 patients with active CD who
received induction therapy (ADA 80 mg at week 0 and 40 mg at
week 2) and were then randomized at week 4 to receive ADA 40 mg
every other week, ADA 40 mg every week, or placebo for 52 weeks.
A response (defined as ≥70-point decrease in CDAI) was achieved in
499 of 854 patients (58%) at week 4. Among responders, rates of
Figure 1. Structure of infliximab (IFX) and adalimumab (ADA). IFX and ADA are monoclonal antibodies that bind tumor necrosis factor alpha
(TNF-α). IFX is a human-murine chimera that joins the variable regions of a mouse antibody to the constant region of human immunoglobulin
G1 (IgG1), and ADA is a human IgG1 antibody. [Reprinted with permission from Anderson.24]
Infliximab
Mouse
variable
region
Adalimumab
TNF-α
Human
variable
region
Human lgG1
2
clinical remission were higher after maintenance therapy with ADA
dosing every other week and once weekly than with placebo at both
week 26 (40% and 47% vs 17%, respectively; P<.001) and week 56
(36% and 41% vs 12%, respectively; P<.001). Maintenance therapy
with weekly dosing of ADA 40 mg offered no advantage over dosing
every 2 weeks.11
In UC, two randomized, double-blind, placebo-controlled studies,
ULTRA 1 and 2 (Ulcerative Colitis Long-Term Remission and
Maintenance with Adalimumab 1 and 2),27,28 showed that ADA could
induce and maintain clinical remission in patients with moderate to
severe UC (Mayo score 6–12 on a 12-point scale and an endoscopy
subscore of ≥ 2 on a 0 to 3 scale), including those naïve to TNFblocker therapy and those secondarily unresponsive or intolerant to
another TNF blocker. ULTRA 127 assessed the induction of clinical
remission (defined as Mayo score ≤ 2 with no individual subscore
>1) at week 8 in 390 patients naïve to TNF-blocker therapy who
were randomized to receive ADA or placebo at weeks 0, 2, 4, and 6.
ADA was administered in one of two regimens: 160 mg at week 0,
80 mg at week 2, and 40 mg at weeks 4 and 6 or 80 mg at week 0
and 40 mg at weeks 2, 4, and 6. Clinical remission was achieved in
18.5% of patients in the ADA 160/80 mg group, 10.0% in the ADA
80/40 mg group, and 9.2% in the placebo group. The remission rate
achieved by the ADA 160/80 group was significant vs placebo
(P=.031).27
The second study, ULTRA 2,28 evaluated both induction and
maintenance of clinical remission with ADA in 494 patients, 40% of
whom had prior exposure to another TNF blocker. Patients were
stratified based on prior anti-TNF exposure and randomized to receive
ADA (160 mg at week 0, 80 mg at week 2, and 40 mg every other
week through week 52) or placebo. Primary end points were the
proportions of patients achieving clinical remission at weeks 8 and
52. Remission was achieved in 16.5% of ADA-treated patients by
week 8 vs 9.3% of placebo-treated patients (P=.019). At week 52,
remission rates were 17.3% vs 8.5% (P=.004), respectively. Remission
rates in patients who were anti-TNF naïve significantly favored ADA
vs placebo at both week 8 (21.3% vs 11%, respectively; P=.017) and
week 52 (22% vs 12.4%, respectively; P=.029). In patients with a
history of prior anti-TNF exposure, no difference in remission rates
between ADA and placebo were noted at week 8 (9.2% vs 6.9%,
respectively; P=.559) but were significant at week 52 (10.2% vs 3%,
respectively; P=.039).28
Safety concerns with anti-TNF agents, including ADA, include an
increased risk of serious infections resulting in death or
hospitalization (including tuberculosis, bacterial sepsis, and invasive
fungal and other opportunistic infections), lymphoma, reactivation of
hepatitis B virus, new onset or exacerbation of central nervous system
demyelinating disease, cytopenia, worsening of congestive heart
failure, and formation of autoantibodies.23 Fatal hepatosplenic T-cell
lymphoma has been reported in adolescent and young adults
with IBD treated concomitantly with anti-TNF agents and
immunosuppressants. The main adverse events reported more
frequently with ADA than with placebo in controlled clinical trials
across all approved indications were injection site reaction, infection,
formation of autoantibodies, and liver enzyme elevations. In general,
the safety profile of ADA was similar across approved indications, and
no safety concerns unique to the use of ADA in IBD were identified.23
Figure 2. Formation of antibodies with infliximab and adalimumab. (A) Human antimouse antibodies are formed in the presence of chimeric
monoclonal antibodies, and (B) human antihuman antibodies are formed in the presence of humanized monoclonal antibodies. The antibodies
may be neutralizing or non-neutralizing. [Reprinted with permission from Anderson.24]
A
B
3
Table 1. Summary of Published Studies on Antibodies to Adalimumab, Adalimimab Levels, and Clinical Outcomes in Crohn’s Disease and
Ulcerative Colitis
Study
Regimen and
Follow-up
Patients
With ATA
ADA Serum
Levels
(μg/mL)
Impact of
ATA on
ADA Levels
Impact of
ADA Levels
on Efficacy
NR
NR
3 of 7 ATA+ patients
(43%) in remission
at wk 24; 2 of 7
patients (29%) in
remission at wk 56
NR
NR
NR
NR
NR
Impact of ATA
on Efficacy
N
Indication
Hanauer et al
(CLASSIC I) 10
299
Moderate to
severe CD; naïve
to anti-TNF therapy
ADA 40/20 mg,
80/40 mg, or
160/80 mg or
placebo at wk 0
and 2; follow-up
4 wk
2 patients developed
ATAs—Placebo: 1
ATA+ at wk 0;
ADA 160/80: 1 ATA+
at wk 2 and ATI–
at wk 4
NR
At wk 4 —
40/20 mg: 2.79±1.48;
80/40 mg: 5.65±3.06;
160/80 mg:
12.61±5.25
Sandborn et al
(CLASSIC II)14
269
Moderate to severe
CD; clinical
remission at wk 0
(wk 4 of CLASSIC I)
and wk 4
In remission:
ADA 40 mg eow
or ADA 40 mg/wk
or placebo;
follow-up 56 wk
7 of 269 patients
(2.6%) developed
ATAs
NR
Sandborn et al
(GAIN) 26
325
Moderate to severe
ADA 160/80 mg or
CD; loss of response placebo at wk 0 and
or intolerance to IFX 2; follow-up 4 wk
Wk 4—12.6
No patient in ADA
group ATA+ at wk 4;
measurable ADA
serum levels precluded
determination of ATA
West et al 29
30
Active luminal or
fistulizing CD;
loss of response or
intolerance to IFX
ADA 160/80 at
wk 0 and 2 and
then 40 mg eow
(retrospective
study); median
ADA treatment
duration 318 d
5 (17%)
NR
4 of 5 ATA+ patients
(80%) were ADA
nonresponders;
ATA+ was related to
ADA nonresponse
(OR 13.1; 95% CI
1.7–99.2; P = .006)
NR
NR
Karmiris et al 21
168
Luminal or fistulizing
CD or arthritic
manifestations;
loss of response or
intolerance to IFX
ADA 160/80 mg or
80/40 mg at wk 0
and 2 or ADA
40 mg eow; median
follow-up 20 mo
9.2%
Higher levels with
induction dose of
160/80 mg than
80/40 mg
No relationship with
short-term response;
11/11 (100%) ATA+
patients had low
trough levels of
ADA and
discontinued ADA
Median trough
ADA
concentrations
lower in ATA+
patients (P < .001)
Lower ADA trough
concentrations (short and
long term) in patients
who discontinued ADA;
correlation of response
to dose escalation with
increase in serum
ADA levels
Bodini et al 30
22
CD; IFX naïve;
remission with ADA
Maintenance
ADA therapy
(dosage NR);
follow-up 2 yr
9%
Remission, 8.1
(6.7–9.2); disease
activity, mild 5.8
(5.2–6.1), moderate
3.9 (3.2–4.7),
severe 1 (0.1–2.6)
[P < .01 for all]
NR
No effect
Higher ADA trough
levels in those
remaining in remission
throughout follow-up
(P < .01); lower ADA
trough levels associated
with loss of response
and discontinuation
Afif et al 31
20
Moderate to severe
UC; IFX naïve or
loss of response or
intolerance to IFX
ADA 160/80 mg
at wk 0 and 2 and
then 40 mg eow;
follow-up 24 wk
NR
NR
Clinical response
to ADA in 5 ATI+
patients (63%) vs 1
ATI– patient (20%)
NR
NR
Ferrante et al 32
50
Moderate to severe
UC; loss of
response, infusion
reactions, or
intolerance to IFX
ADA 160/80 mg
at wk 0 and 2 and
then 40 mg eow;
median follow-up
23 mo
NR
NR
Successful dose
escalation associated
with median ADA level
increase from 4.75 to
7.95 (P=.023)
NR
26 patients 52%
achieved durable
clinical response
Sandborn et al
(ULTRA 2) 28
494
Moderate to severe
UC
160/80 mg at wk 0
and 2 and then
40 mg eow or
matching placebo;
follow-up 52 wk
2.9%, all receiving
ADA monotherapy
NR
Median trough ADA
levels in remitters vs
nonremitters: wk 8
11.4±5.2 vs 8.5±4.4;
wk 32 10.6±5.6 vs
7.0±4.0; wk 52:
10.8 ± 7.5 vs 6.2 ± 4.2
NR
Remitters at wk 8 and
52 had higher median
trough ADA levels than
nonremitters
Crohn’s Disease
Ulcerative Colitis
ADA, adalimumab; ATA, antibodies-to-adalimumab; ATI, antibodies-to-infliximab; CD, Crohn’s disease; CLASSIC I and II, Clinical Assessment of Adalimumab Safety and Efficacy
Studied as Induction Therapy in Crohn’s disease I and II; eow, every other week; IFX, infliximab; GAIN, Gauging Adalimumab Efficacy in Infliximab Nonresponders study; NR, not reported;
UC, ulcerative colitis; ULTRA 2, Ulcerative Colitis Long-term Remission and Maintenance with Adalimumab 2 study.
4
Clinical Significance of ADA Levels
and ATA Formation in IBD
Unlike IFX, ADA has been investigated in only a limited number of
studies that evaluated clinical outcomes in IBD in relation to ADA trough
levels and the formation of ATAs, owing to the lack of commercially
available assays to measure these parameters. These studies are
detailed below, and select studies are summarized in Table 1.
In the CLASSIC I trial, 299 patients with moderate to severe CD
who were naïve to prior anti-TNF treatment were randomized to receive
placebo or one of three induction regimens of ADA subcutaneously at
weeks 0 and 2 (160/80 mg, 80/40 mg, or 40/20 mg).10 Rates of
remission (defined as CDAI score <150) at week 4 were significantly
higher in the two highest-dose ADA groups (36% and 24%,
respectively) than in the placebo group (12%; P=.004). Injection site
reactions occurred in 38%, 24%, and 26% of patients in the ADA
160/80 mg, 80/40 mg, and 40/20 mg groups, respectively, compared
with 16% in the placebo group. Mean ADA serum concentrations
reflected the dosing regimen and were higher among patients
receiving the highest doses of ADA (12.61 ± 5.25, 5.65±3.06, and
2.79±1.48 μg/mL, respectively). Results from CLASSIC I demonstrate
that serum ADA concentrations achieved with the 160/80 mg dose
were effective for inducing remission. Only 1 patient in the ADA group
developed ATAs, but the authors acknowledged that the short 4-week
study may have underestimated the likelihood of developing
antibodies.10 Additionally, the technical limitations of ELISA/ECLIA
related to the measurement of ATAs in the presence of ADA increases
the rate of inconclusive results, rendering them less useful in terms of
informing patient management.
In the CLASSIC II study, which evaluated the long-term efficacy of
ADA treatment, 276 participants from CLASSIC I received open-label
ADA 40 mg at weeks 0 (week 4 of CLASSIC I) and 2.14 Patients (n=55)
who achieved remission at weeks 0 and 4 were re-randomized to
receive ADA 40 mg weekly, ADA 40 mg every other week, or placebo
up to week 56. Re-randomized patients with nonresponse or with
disease flare could switch to open-label treatment with ADA 40 mg
every other week or weekly, if indicated. Patients not in remission at
weeks 0 and 4 also received open-label treatment with ADA 40 mg
every other week or weekly, if indicated. Among 55 patients
randomized at week 4, remission rates at week 56 were significantly
higher for the groups receiving ADA 40 mg every other week (15/19,
79%) and ADA 40 mg weekly (15/18, 83%), compared with the group
receiving placebo (8/18, 44%; P<.05 for each ADA group vs
placebo). In the open-label cohort of 204 patients not in remission,
93 (46%) were in remission at week 56. Blood samples from 7 of 269
patients tested (2.6%) were positive for ATAs. Of the ATA-positive
patients, 3 were in remission at week 24 and 2 were in remission at
week 56.14
Data from CLASSIC I and CLASSIC II were analyzed by Li et al33
to evaluate correlations between serum ADA concentrations and
clinical remission and to determine if there is a threshold concentration
that can reliably predict remission. Serum trough levels of ADA,
available for 258 patients, showed a considerable overlap between
patients in remission and those not achieving remission. Logistic
regression analysis showed a small but statistically significant
correlation between ADA levels at week 4 (CLASSIC I) and remission
(P=.01). In CLASSIC II, serum ADA concentrations did not correlate
with remission status at weeks 4, 24, or 56 (P=.102, P=.123, and
P=.091, respectively). Threshold analysis did not reveal a serum ADA
concentration that was predictive of remission. Thus a dose-exposure
relationship was identified for ADA induction, but overlap between
groups during maintenance therapy suggests that ADA levels may be
of limited value in making therapeutic decisions.33
The GAIN study evaluated the efficacy of ADA induction (160 mg
at week 0 and 80 mg at week 2) or placebo in 325 patients with
moderate to severe CD who had either lost response or were intolerant
of IFX.26 At week 4, remission was achieved in 21% of patients
receiving ADA compared with 7% of those receiving placebo (P<.001).
The mean ADA concentration in ADA-treated patients at week 4 was
12.6 ± 6.0 μg/mL, which was almost identical to the level achieved
with ADA 160/80 mg in TNF-naïve patients in CLASSIC I. None of the
159 patients treated with ADA tested positive for ATAs, but this
determination may have been compromised by the presence of
measurable ADA in serum,26 which interferes with ELISA/ECLIA
determination of ATAs because of their technical limitations.
A limited number of studies have assessed outcomes after ADA
treatment in relation to ADA levels and formation of ATAs. A small
retrospective study by West et al29 was the first to demonstrate that the
immunogenicity of ADA negatively influenced the response to ADA
treatment. Thirty patients with CD previously treated with IFX were
subsequently treated with ADA 160 mg at week 0, 80 mg at week 2,
and 40 mg every 2 weeks thereafter. The overall clinical response was
77% (23/30 patients). ATAs were detected in 5 patients (17%), of
whom 4 were nonresponders. The presence of ATAs was related to a
nonresponse to ADA (odds ratio [OR] 13.1; 95% confidence interval
[CI] 1.7– 99.2; P=.006). There was no relationship between the
presence of ATAs and clinical response in patients with fistulizing
disease, whereas ATAs were detected in 75% (3/4) of nonresponders
and in only 5.6% (1/17) of responders, and the presence of ATAs
was associated with nonresponse in patients with luminal disease
(OR 13.5; 95% CI 1.9–98.5; P=.01). The presence of ATAs was not
related to receipt of concomitant immunosuppression or to a
requirement for dose escalation. ATIs were measurable in 57% of
patients; serum levels of ATIs were significantly increased in ADA
nonresponders compared with responders (163.0 ± 58.6 vs
5
Figure 3. Relationship between antibodies-to-adalimumab (ATA)
and adalimumab (ADA) trough serum concentrations at different
time points. [Reprinted with permission from Karmiris et al.21]
11.1
(n=46)
Median ADA trough serum
concentration (μg/mL)
12
10
8
6
4
2
0
6
ATA (+)
ATA (–)
8.9
8.8
(n=53)
(n=37)
6.1
(n=58)
2.1
(n=9)
0.6
(n=8)
Week 4
0.1
(n=8)
Week 12 Week 24
Time points
0.02
(n=3)
Week 54
Figure 4. Rates of continuation and discontinuation at month 6
based on trough levels at week 4 (A), week 12 (B), and week 24 (C).
Top and bottom of box indicate 75th and 25th percentiles,
respectively; the band within the box indicates the 50th percentile.
[Reprinted with permission from Karmiris et al.21]
ADA trough concentration
wk 4 (μg/mL)
A
ADA trough concentration
wk 12 (μg/mL)
Mann-Whitney P =.04
25
20
15
10
6.2
5
0
B
30
3.8
Continue
(n=43)
Discontinue
(n=24)
Mann-Whitney P =.0 16
20
10
8.9
1.6
0
C
ADA trough concentration
wk 24 (μg/mL)
520.4 ± 244.0 AE/mL, respectively; P=.01), but no relationship was
observed between the presence of ATAs and ATIs. This study was
limited by its retrospective design, small size, and measurement of
ATAs at varying time points.29
A prospective observational cohort study by Karmiris et al 21
evaluated the effect of trough serum ADA concentrations and the
presence of ATA during long-term treatment with ADA in 209 patients
with CD who lost responsiveness to IFX. Overall, 70.5% of patients
(105/149) responded to ADA by week 4 and 61.5% (96/156) showed
a sustained clinical benefit. This study reported a higher rate of ATA
development — 9.2% of patients — than previous studies. Median
trough concentrations of ADA were lower in patients who developed
ATAs than in patients who did not at both week 4 (2.1 vs 6.1 μg/mL,
respectively; P<.02) and consistently at all subsequent time points
(Figure 3). Over a median follow-up of 20.4 months, lower trough
serum ADA concentrations were associated with higher rates of
discontinuation (Figure 4). In the 59 (45.4%) patients who
discontinued ADA therapy, median trough serum ADA concentration
at the time of discontinuation was 3.2 μg/mL. Trough serum ADA
concentrations were significantly higher (>6.2 μg/mL at week 4)
throughout the study in those patients who continued on therapy
compared with those who discontinued (<3.8 μg/mL at week 4).
Thus, low trough ADA concentrations were associated with higher
early and late discontinuation rates, and a great majority of patients
with undetectable serum ADA concentrations also displayed ATAs.21
Bodini et al 30 reported findings of an open-label study that
prospectively investigated the relationship of clinical outcomes to
ADA concentrations and ATA in IFX-naïve patients (N=22) who had
achieved remission and received maintenance treatment with ADA
over a 2-year period. Clinical activity was assessed using the HarveyBradshaw Index (HBI) score and C-reactive protein (CRP) levels.
Continue
(n=46)
40
Discontinue
(n= 15)
Mann-Whitney P =.03
30
20
10
0
8.9
0.4
Continue
(n=32)
Discontinue
(n= 13)
ADA discontinuation by wk 24
Ten patients (45%) who had sustained clinical remission (HBI <5) at
2 years had significantly higher ADA trough levels than patients with
any level of active disease. Median (range) ADA trough concentrations
at 2 years were 8.1 μg/mL (6.7–9.2 μg/mL) in patients in remission,
5.8 μg/mL (5.2–6.1 μg/mL) in patients with mild disease, 3.9 μg/mL
(3.2–4.7 μg/mL) in patients with moderate disease, and 1 μg/mL
(0.1–2.6 μg/mL) in patients with severe disease (P<.01 via ANOVA).30
Similar to the findings observed by Karmiris et al,21 the 4 patients who
lost response and discontinued therapy in this study had significantly
lower ADA levels than patients who continued receiving ADA (2.1 vs
6.7 μg/mL, respectively; P<.01). ATAs were detected in 9% of patients.
In this small study, lower ADA concentrations were associated with
loss of response, relapse, and discontinuation of treatment.30
To date, the management of loss of response to anti-TNF agents
in IBD has included two strategies: one based on results from the
determination of anti-TNF drug levels and the presence of anti-drug
antibodies and the other based on empiric dose-escalation. The cost
implications of these two strategies in patients with CD and secondary
nonresponse were unknown until recently. Using a decision analytic
model, Velayos et al34 investigated the outcomes of these strategies by
comparing two simulated cohorts of patients with CD and examining
outcomes in terms of the yield and cost of quality-adjusted life-years
(QALYs) gained over a 1-year period.
Results of the model showed that the testing strategy resulted in
a cost reduction of $5396 for each QALY gained compared with the
cost of each QALY gained using the empiric strategy.34 Drug level and
anti-drug antibody testing resulted in lower proportions of patients
receiving high-dose biologics (41% vs 54%, respectively), fewer
months receiving high-dose biologic therapy (39% vs 53%,
respectively), and more months receiving no biologic therapy (34% vs
19%, respectively) compared with those receiving empiric dose
escalation. Thus, the testing strategy provided cost benefits in the
biologic therapy management of CD by avoiding the use of this therapy
in patients who were unlikely to benefit from it.34
In UC, a 24-week uncontrolled trial assessed the benefit of ADA
(160 mg at week 0, 80 mg at week 2, and 40 mg thereafter every 2
weeks) in 20 patients who were IFX-naive or who had lost response or
developed intolerance to IFX.31 At week 8, the rate of clinical response
(defined as a decrease in the Mayo score >30% from baseline and a
decrease of ≥3 points, with a decrease in the rectal bleeding subscore
[RBS] ≥1 point or an RBS of 0 or 1) was 25%, and the rate of clinical
remission (defined as a Mayo score <2 and no individual score >1)
was 5%. Eight of 13 patients (63%) previously treated with IFX were
positive for ATIs. A clinical response to ADA was achieved in 5
(63%) of the ATI-positive patients but in only 1 (20%) ATI-negative
patient, suggesting that patients who fail IFX secondary to
immunogenicity may be more likely to respond to another TNF
inhibitor than those who lose response for another reason.31
The effect of serum ADA levels on long-term efficacy of ADA
(induction with 160/80 mg at 0 and 2 weeks, respectively, followed by
maintenance with 40 mg every other week) in patients with UC was
studied in 50 patients previously treated with IFX.32 A short-term clinical
response (week 4) was obtained in 68% of patients. Dose escalation
was required in 38 (76%) patients either within 6 weeks of initiation
(n=20) or because of loss of response after a median of 16 weeks
(n=18). In total, 26 (68%) patients responded to dose escalation.
Increases in median ADA serum levels from 4.75 to 7.95 μg/mL were
noted in patients for whom dose escalation was successful but were
not seen in patients with worsening disease. Short-term response and
response to dose escalation were associated with colectomy-free
survival. The large proportion of patients responding to dose escalation
suggests that higher ADA doses may be required in UC.32
Despite being fully humanized, ADA is not associated with a
complete lack of immunogenicity. This is not surprising because any
exogenous protein has the capacity to induce an immune response.35
The commonly used ELISA and ECLIA methods for detecting ATAs are
limited by the interference caused by the presence of drug in
serum.21,26 Fortunately, a more accurate and sensitive assay has now
been developed that overcomes the limitations of these
methodologies. As a result, this new assay has the potential to open
new doors in the management of IBD by informing treatment
decisions for patients receiving ADA.
Current Assays for Determining ADA
Levels and Formation of ATA
As noted earlier, the incidence of ATA formation in patients treated
with ADA for CD or UC is largely unknown. One of the factors
contributing to this uncertainty is lack of standardization and
methodologic limitations of assays available up to now. In clinical
trials in IBD, rates of ATA development after ADA treatment have been
reported as high as 17%29 in a retrospective study and 9.2% when
assessed prospectively 21; however, because of drug interference with
the assay, this is probably an underestimation. As an example, assay
limitations in the UC trials allowed ATAs to be detected only when
serum ADA levels were < 2 μg/mL; in these patients (25% of the total
UC patient population), the rate of detection of ATAs was 20.7%.23
In the past, the most commonly used assay to measure antibodies
to anti-TNF agents is a solid-phase (double-antigen or sandwich)
ELISA in which antibodies in a serum sample are captured on a solid
phase coated with a specific anti-TNF agent and detected by binding
of biotinylated or peroxidase-coupled anti-TNF.18,21,23 However,
limitations may compromise the accuracy and precision of these
assays. Measurable anti-TNF drug levels can cause interference and
lead to inconclusive results and a potential underestimation of
antibodies,35 a problem encountered with the detection of ATAs.21,26
This is especially problematic if serum is collected shortly after
administration of the anti-TNF agent; most studies tried to minimize
this problem by evaluating trough samples (measured just before the
next infusion of anti-TNF). However, the long half-life of ADA may
even compromise this strategy. Another confounder is formation of
immune complexes between ADA and ATA and their rapid clearance in
vivo, which may underestimate both ADA and ATA concentrations and
lead to false-negative results.36-38 Matrix effects may lead to epitope
masking, which prevents detection of functionally monovalent
immunoglobulins, such as IgG4, and leads to false-negative results
with bridging ELISA. A particular problem with solid-phase ELISAs is
false-positive findings caused by nonspecific binding of other
7
immunoglobulins, rheumatoid factors, or complement factors to the
Fc segment of the anti-TNF antibody.16 ELISA is similar in
performance to ECLIA, although the latter uses an
electrochemiluminescent label 39 and may be associated with greater
sensitivity.40 However, detection of low-affinity anti-drug antibodies
remains problematic with ECLIA owing to sample dilution and a final
wash step; ECLIA is also associated with significantly higher costs
than ELISA.40
To address the shortcomings of currently available assays, a new
homogeneous mobility shift assay was developed to quantify anti-TNF
antibodies and anti-TNF levels simultaneously in serum samples from
patients with IBD. The development, analytical validation, and clinical
use of the PROMETHEUS® Anser™ ADA Assay (Prometheus
Laboratories, San Diego, CA) for detection of ADA and ATA levels in
serum are described in the following sections.
PROMETHEUS ® ANSERTM ADA ASSAY
The PROMETHEUS® AnserTM ADA Assay is a new, proprietary,
nonradiolabeled, fluid-phase assay for detection of ADA and ATA
levels. This assay offers several improvements over ELISA/ECLIA;
these are summarized in Table 2. The assay has a higher tolerance
for ADA in the serum sample compared with current ELISAs and can
measure both ADA and ATAs in the same sample.
Analytical Validation of PROMETHEUS® AnserTM
ADA Assay
The relative ATA concentration was defined as 100 U/mL, equal to
1:100 dilutions. To validate the standard curve, the performance
characteristics of the ATA calibration standards were assessed over
29 runs.41 The coefficient of variation (CV) was < 20% for
concentrations > 0.031 U/mL, and the dynamic range of the assay
was 2 orders of magnitude. The calculated limit of detection (LOD)
was 0.026 U/mL from the 29 standard curve runs. The lower and
upper limits of quantitation were the lowest and highest amounts of an
analyte in a sample that could be quantitatively determined with
suitable precision and accuracy. Complete analytical validation was
performed with the quality control standards by multiple analysts
using different instruments on different days. For the PROMETHEUS®
Anser™ ADA Assay, intra-assay precision had a CV of <3% and
accuracy of <13% error; interassay precision had a CV of <9% and
accuracy of <18% error.
The performance characteristics of the PROMETHEUS® AnserTM
ADA Assay standard curve were similarly assessed in 29 experimental
runs by multiple analysts using different instruments over different
days.41 The CV for all concentrations except the lowest was <25%,
and the dynamic range was 2 orders of magnitude. The intra-assay
precision was <20% and the accuracy was <3%, whereas the
interassay precision was <12% and the accuracy was <22%.
Cut points for ATA and ADA values with the PROMETHEUS®
Anser™ ADA Assay were determined from 100 serum samples
collected from ADA-naïve, healthy subjects.41 The calculated cut point
for the ATA assay was 0.549 U/mL, with only one sample containing
a higher ATA level (100% clinical sensitivity; 99% clinical specificity).
Using the same serum samples, the cut point for the ADA assay was
0.676 μg/mL (100% clinical sensitivity; 97% clinical specificity).
Further, if a threshold of 1.7 U/mL for the ATA assay or 1.6 μg/mL for
the ADA assay is applied, the clinical specificity is increased to 100%
[data on file, Prometheus Laboratories, San Diego, CA].41
Table 2. Methods for Monitoring Antibodies-to-Adalimumab Levels in Patient Serum Samples
Bridge ELISA
PROMETHEUS® AnserTM ADA Assay
Nonspecific background interference
High
Low
Sensitivity
Low
High
Possibility of false-positive or false-negative
High
Low
IgG4 antibody detection
No
Yes
Ig isotope identification
No
Yes
Poor
Good
No
No
Tolerance of drug in the sample
Use and disposal of radioactive material
ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; IgG4, immunoglobulin G4.
8
Nonspecific binding of serum substances, which interferes with
the accuracy of solid-phase ELISAs, is likely to be minimized in the
fluid-phase PROMETHEUS® AnserTM ADA Assay. The potential
interference by common endogenous serum components in both ATA
and ADA assays was examined by measuring the recovery of ADA and
ATAs from spiked quality control samples. Neither assay showed
significant interference from physiologic levels of immunoglobulin,
rheumatoid factor, hemolyzed serum, or lipemic serum or from the
presence of azathioprine (≤10 μM) or methotrexate (≤2 mM).41
Clinical Validation of PROMETHEUS® AnserTM
ADA Assay
The ELISA assay method has been used in a research setting to
measure the presence of ATA in the serum from patients treated with
ADA.21,23 The clinical performance of the PROMETHEUS® Anser™
ADA Assays for measuring ADA and ATAs was assessed with serum
samples from 100 patients (with CD, UC, rheumatoid arthritis, or
plaque psoriasis) who had received standard ADA therapy for ≥3
months and had achieved and subsequently lost response.41 ADA
levels were below the cut point of 0.68 μg/mL in 26% of samples and
above 20 μg/mL in 22% of samples. The mean (± standard deviation)
ATA level was 4.64±19.20 U/mL, which was significantly higher than
the levels recorded in healthy control serum (0.33±0.07 U/mL;
P < .00001). Based on an ATA cut point of 0.55 U/mL, 44% of samples
were considered ATA positive. ADA levels were inversely related to the
presence of ATAs — 68% of serum samples were ATA positive when
their ADA levels were lower than the cut point, and 18% were ATA
positive when ADA levels were above 20 μg/mL (Figure 5).41
Initial studies evaluated the clinical usefulness of the
PROMETHEUS® Anser™ ADA Assay for examining relationships
between ATAs, serum ADA levels, and clinical outcomes in patients
with IBD. Wolf et al42 performed a cross-sectional study to evaluate
mechanisms for loss of response to ADA in 49 patients with CD.
Detectable serum ADA levels (median 7.2 μg/mL; range 0.73 – 31.54
μg/mL) were present in 86% (42/49) of patients, and detectable ATAs
were present in 47% (23/49). This ATA-positive group would not
likely have been detected using standard ELISA/ECLIA testing, because
the presence of ADA in the serum would have interfered with ATA
identification. Given that ADA has a mean terminal half-life that ranges
from 10 to 20 days, if given every 2 weeks, the drug will almost always
be present to some degree in the serum of these patients, making
ELISA/ECLIA ATA measurements much less useful. ATAs were present
in 6 of 7 (86%) patients with undetectable serum ADA. The ADA levels
were inversely associated with the occurrence of ATA—the median
ADA concentration in ATA-negative samples was 9.78 μg/mL,
Figure 5. Inverse relationship between adalimumab (ADA)
concentration and antibodies-to-adalimumab (ATA) occurrence in
serum samples from patients with secondary nonresponse after
treatment with ADA. [Reprinted with permission from Wang et al.41]
100
80
ATA Positivity (%)
Potential Interference from Serum Components
or ADA
60
40
20
0
0–0.68
0.69–10.00 10.01–20.00
>20.00
Adalimumab (μg/mL)
compared with 4.9 μg/mL in ATA-positive samples (P = .022). This
study revealed a high incidence of ATA positivity in patients with loss
of response to ADA, as well as an inverse association of ADA levels
with ATA generation.42
The PROMETHEUS® AnserTM ADA Assay was used in another
cross-sectional study to determine the relationship between ADA and
ATAs and inflammation markers and clinical symptoms in patients with
IBD.43 Patients were recruited based on use of ADA and not on disease
status. In 54 patients, 22.2% (12/54) had detectable ATAs (defined as
≥1 U/mL) and 90.7% (49/54) had detectable ADA (≥1 μg/mL). An
ADA concentration ≥5 μg/mL was associated with reduced CRP level
(P = .001). Detectable ATA was positively associated with elevated CRP
(P = .002) and this correlation was independent of ADA concentration.
Patients with low ADA levels (< 5 μg/mL) or with detectable ATA had
more active disease (P = .01). This study underscored the high
prevalence of ATA in IBD patients treated with ADA, as well as the
influence of ATA and ADA levels on the inflammatory response. The
correlation of active disease with detectable ATA or low ADA levels
suggests the clinical usefulness of these assessments.43
The impact of dosing strategies on levels and immunogenicity of
ADA was examined by Ben-Bassat et al44 in 57 patients with IBD who
received ADA induction therapy followed by three maintenance
regimens: 40 mg every other week, dose escalation to 40 mg every
week, or dose escalation to 80 mg every other week. Rates of steroidfree clinical remission (HBI ≤ 2) and normalization of CRP levels
(< 5 μg/L) were assessed in relation to detectable ADA levels and ATA
formation. Serum samples were obtained prior to or at the midinterval
9
of maintenance therapy. After a median follow-up of 20.8 months,
rates of steroid-free remission were similar between all three
maintenance groups (69.2%, 66.6%, and 75.1%, respectively).
Median serum ADA levels were similar between the two escalation
groups (20.2 μg/mL with 80 mg every other week and 19.1 μg/mL
with 40 mg every week) and somewhat higher than with 40 mg every
other week (12.5 μg/mL). Overall, 14% of patients had detectable
ATAs, with comparable rates of ATA formation between the three
maintenance groups (15.3%, 16.6%, and 12.5%, respectively).
Median ADA levels were higher among patients in remission than
among those with uncontrolled disease (23.8 and 4.9 μg/mL,
respectively; P < .001) and also higher for patients with normal CRP
levels than among those with CRP levels > 5 μg/L (22.9 vs 10.5
μg/mL; P < .001). Patients who failed treatment had a higher rate of
ATA formation (62.5%) and lower median ADA levels (4.2 μg/mL)
independent of the maintenance regimen. This study showed both
dose escalation regimens to be similar with respect to ADA levels and
immunogenicity. Regardless of dosing strategy, treatment failure was
associated with lower ADA levels and a higher rate of ATA formation.44
The PROMETHEUS® AnserTM ADA Assay was also used to evaluate
the relationship of serum ADA levels and ATAs with indices of serologic
and endoscopic mucosal inflammation in another cross-sectional
study in IBD.45 Of 66 patients with CD or UC in the study, 62 (94%)
had detectable ADA levels and 18 (27%) had detectable ATA levels.
An ADA cut point of 5 μg/mL was the best predictor of elevated CRP
levels (receiver operator characteristics curve AUC=0.71). The mean
ADA level was significantly higher among patients without ATAs than
among those with ATAs (12.5 vs 5.7 μg/mL, respectively; P = .001)
and was also higher among patients with mucosal healing than
among those with macroscopic mucosal inflammation (13.3 vs 8.5
μg/mL, respectively; P = .02). ADA levels were also higher in patients
using an immunomodulator concomitantly than in those receiving
ADA monotherapy (14 vs 9 μg/mL, respectively; P=.026). The mean
CRP level was higher among patients with ATAs than among those
without ATAs (12 vs 2.1 mg/dL, respectively; P = .002). Patients with
detectable ATAs were more likely to have ADA levels <5 μg/mL (OR
8.6; 95% CI 2.3–31.8; P < .001), display macroscopic mucosal
inflammation (OR 3.8; 95% CI 1.1–13.2; P = .03), require steroids
(OR 3.7; 95% CI 1.1–12.9; P = .03), and to have used IFX previously
(OR 3.9; 95% CI 1.0–15.2; P = .04). Thus, both ADA and ATA levels
correlated with inflammatory activity.45
10
Conclusions
Adalimumab is effective in reducing disease in TNF-naïve and
TNF-experienced patients with CD or UC, but maintaining long-term
response and remission has proven clinically challenging.
Increasingly, attention has focused on the immunogenicity of
adalimumab and other anti-TNF agents to explain the loss of clinical
efficacy over time.17,18,21,46 Although completely humanized,
adalimumab is not devoid of immunogenicity, and anti-idiotypic
antibodies directed at the variable binding region do develop in
adalimumab-treated patients. The presence of antibodies to
adalimumab may lead to ineffective subtherapeutic levels of
adalimumab and contribute to loss of response by increasing drug
clearance or blocking the drug effect. Information on the frequency of
anti-adalimumab antibody formation and its influence on response to
adalimumab therapy in patients with IBD is limited, but emerging data
increasingly highlight the interdependence of the formation antibodies
to adalimumab and adalimumab levels in the therapeutic
response.21,47,48 Because of the increasing clinical relevance of antiadalimumab antibodies and adalimumab levels, monitoring these
levels during therapy should therefore be integrated into the
management of patients receiving adalimumab.
The fluid-phase PROMETHEUS® AnserTM ADA Assay offers several
advantages over the solid-phase bridge ELISA/ECLIA assays used for
measuring antibodies and anti-TNF levels in serum.41,49 The
PROMETHEUS® AnserTM ADA Assay displays high sensitivity,
precision, and accuracy. Importantly, the initial acid dissociation step
of the PROMETHEUS® AnserTM ADA Assay increases tolerance for free
adalimumab in the sample, allowing detection of low levels of ATAs in
the presence of levels of adalimumab as high as 20 μg/mL. By avoiding
multiple washing steps, the PROMETHEUS® AnserTM ADA Assay can
detect antibodies of low affinity, and lack of stereoscopic hindrance
allows all immunoglobulin isotypes and subclasses of IgG to be
detected. Nonspecific binding is limited because the antigen-antibody
reaction occurs in the liquid phase, thereby reducing false-positive
results.41,49 The validation data, as well as preliminary studies in IBD
patients losing responsiveness to adalimumab, indicate that the assay
is effective in measuring ATA in the presence of serum
adalimumab.41,43,44 By providing a reliable measure of circulating
adalimumab levels and immunogenicity, the PROMETHEUS® AnserTM
ADA Assay may be more useful than prior methodologies in informing
treatment decisions for optimal clinical outcomes.
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