close

Вход

Забыли?

вход по аккаунту

?

Neutralization of interferon-╨Ю┬▒ inducible genes and downstream effect in a phase I trial of an antiinterferon-╨Ю┬▒ monoclonal antibody in systemic lupus erythematosus.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 60, No. 6, June 2009, pp 1785–1796
DOI 10.1002/art.24557
© 2009, American College of Rheumatology
Neutralization of Interferon-␣/␤–Inducible Genes and
Downstream Effect in a Phase I Trial of an Anti–Interferon-␣
Monoclonal Antibody in Systemic Lupus Erythematosus
Yihong Yao, Laura Richman, Brandon W. Higgs, Christopher A. Morehouse,
Melissa de los Reyes, Philip Brohawn, Jianliang Zhang, Barbara White, Anthony J. Coyle,
Peter A. Kiener, and Bahija Jallal
overexpression of messenger RNA for BAFF, TNF␣,
IL-10, IL-1␤, GM-CSF, and their respective inducible
gene signatures in whole blood and/or skin lesions, we
observed a general trend toward suppression of the
expression of these genes and/or gene signatures upon
treatment with anti-IFN␣ mAb.
Conclusion. IFN␣/␤-inducible gene signatures in
whole blood are effective pharmacodynamic biomarkers
to evaluate anti-IFN␣ mAb therapy in SLE. Anti-IFN␣
mAb can neutralize overexpression of IFN␣/␤-inducible
genes in whole blood and lesional skin from SLE
patients and has profound effects on signaling pathways
that may be downstream of IFN␣ in SLE.
Objective. Type I interferons (IFNs) play an important role in the pathogenesis of systemic lupus
erythematosus (SLE). This phase Ia trial was undertaken to evaluate the safety, pharmacokinetics, and
immunogenicity of anti-IFN␣ monoclonal antibody
(mAb) therapy in SLE. During the trial, we also examined whether overexpression of an IFN␣/␤-inducible
gene signature in whole blood could serve as a pharmacodynamic biomarker to evaluate IFN␣ neutralization
and investigated downstream effects of neutralizing
IFN␣ on BAFF and other key signaling pathways, i.e.,
granulocyte–macrophage colony-stimulating factor
(GM-CSF), interleukin-10 (IL-10), tumor necrosis factor ␣ (TNF␣), and IL-1␤, in SLE.
Methods. Affymetrix Human Genome U133 Plus
2.0 microarrays were used to profile whole blood and
lesional skin of patients receiving standard therapy for
mild to moderate SLE. Selected IFN␣/␤-inducible proteins were analyzed by immunohistochemistry.
Results. With the study treatment, we observed
anti-IFN␣ mAb–specific and dose-dependent inhibition
of overexpression of IFN␣/␤-inducible genes in whole
blood and skin lesions from SLE patients, at both the
transcript and the protein levels. In SLE patients with
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by severe immune system
defects and production of autoantibodies that lead to
inflammation and tissue damage (1). SLE symptoms
range from a mild rash to life-threatening nephritis and
central nervous system disease. Current SLE therapies
are aimed at control of symptoms and do not address the
underlying causes of disease, and may entail risk of
serious adverse effects (2). Novel therapies that address
disease pathogenesis more directly and with less toxicity
are greatly needed.
Type I interferons (IFNs) have been implicated
in autoimmune diseases (3–7), including SLE (1), and of
note, evidence from gene expression profiling studies
implicates type I IFNs in SLE (8–11). An especially
important research observation is that elevated levels of
IFN␣ are observed in the serum of a subset of SLE
patients (8,12–16).
To treat SLE by lowering IFN␣ levels, we have
developed a fully human IgG1␬ monoclonal antibody
(mAb) that binds to a majority of the subtypes of human
IFN␣ and inhibits IFN-mediated signaling. A singledose, double-blind, placebo-controlled phase Ia trial
ClinicalTrials.gov identifier: NCT00299819.
Yihong Yao, PhD, Laura Richman, DVM, PhD, Brandon W.
Higgs, PhD, Christopher A. Morehouse, MS, Melissa de los Reyes, BS,
Philip Brohawn, BS, MBA, Jianliang Zhang, PhD, Barbara White,
MD, Anthony J. Coyle, PhD, Peter A. Kiener, DPhil, Bahija Jallal,
PhD: MedImmune, Gaithersburg, Maryland.
Drs. Yao, Richman, Higgs, Zhang, Coyle, Kiener, and Jallal,
and Mr. Morehouse, Ms de los Reyes, and Mr. Brohawn own stock or
stock options in MedImmune, which is a subsidiary of AstraZeneca
International. Dr. White owns stock options in AstraZeneca.
Address correspondence and reprint requests to Yihong Yao,
PhD, One MedImmune Way, Gaithersburg, MD 20878. E-mail:
YaoY@MedImmune.com.
Submitted for publication March 24, 2008; accepted in revised
form March 2, 2009.
1785
1786
(MI-CP126) was conducted to study anti-IFN␣ mAb
treatment in patients with mild to moderate SLE with
cutaneous involvement who were receiving standard-ofcare therapy (17). The primary objective of the MICP126 trial was to evaluate the safety and tolerability of
intravenously administered anti-IFN␣ mAb, over a dose
escalation range of 0.3–30 mg/kg, as compared with
placebo in adult SLE patients. Other objectives, reported here, were to evaluate pharmacodynamic effects
of anti-IFN␣ mAb and, additionally, to study the effects
of IFN␣ neutralization on downstream signaling pathways in SLE.
Pharmacodynamic biomarkers are needed in
early-phase clinical trials to demonstrate that a potential
therapeutic molecule has altered its intended target,
based on the proposed mechanism of action, at clinically
achievable concentrations (18). Ideal pharmacodynamic
biomarkers are sensitive, easy to measure, and present in
easily accessible tissue, and correlate with disease activity in target tissue (18). Whole blood provides an easily
accessible surrogate tissue for monitoring drug pharmacodynamics, especially when pharmacokinetic parameters are routinely measured in the peripheral blood to
circumvent difficulties in assessing drug concentration at
the disease site. Free IFN␣ protein in the serum of SLE
patients would be the most reasonable choice as a
pharmacodynamic marker for evaluating anti-IFN␣
therapy in SLE. However, our internal studies, as well as
studies by other investigators (8,12,13), have demonstrated that only a fraction of SLE patients have measurable IFN␣ protein in the serum. IFN␣-inducible
genes, in contrast, are directly downstream of the drug
target, are overexpressed in whole blood from the
majority of SLE patients, and their expression can be
quantitatively measured with microarray or TaqMan
quantitative real-time polymerase chain reaction
(PCR)–based assays (9–11,19).
In previous work, we used microarray transcript
profiling and TaqMan quantitative real-time PCR to
demonstrate overexpression of messenger RNA
(mRNA) for type I IFN family members and a large
panel of IFN␣/␤-inducible genes in whole blood from
SLE patients. We found that the IFN␣/␤ signaling
pathway was the most highly activated signaling pathway
in SLE whole blood (19). We defined algorithms for
using cytokine-inducible gene signature scores to evaluate cytokine activity in whole blood and at disease sites
in inflammatory and autoimmune diseases (19,20). We
also developed a panel of potential pharmacodynamic
biomarkers for anti-IFN␣ mAb, comprising 21 IFN␣/␤inducible genes that are overexpressed in whole blood
from SLE patients (19).
YAO ET AL
In this study, we tested this 21–IFN␣/␤-inducible
gene signature as a pharmacodynamic biomarker for
determining whether anti-IFN␣ mAb neutralizes IFN␣
in a specific and dose-dependent manner. In addition,
we investigated the effects of anti-IFN␣ mAb on signaling pathways for interleukin-10 (IL-10) (21), granulocyte–
macrophage colony-stimulating factor (GM-CSF) (22),
and tumor necrosis factor ␣ (TNF␣) (23,24), all of which
exhibit elevated protein levels in SLE serum. We also
examined effects of anti-IFN␣ mAb on B lymphocyte
stimulator/BAFF, which is overexpressed in SLE and is
a therapeutic target (25,26); B cell autoantibody production and effector functions are considered so crucial to
SLE that BAFF inhibitors are under development to
decrease B lymphocyte populations and thereby allow
healthy B cells to regenerate to normal levels after
treatment.
PATIENTS AND METHODS
SLE patients and controls. The MI-CP126 trial was a
multicenter randomized (2:1), double-blind, placebocontrolled, single-dose, dose-escalation study in patients with
SLE, with an open-label extension. The primary objectives
were determination of safety and pharmacokinetics. Other
trial objectives reported herein included assessment of the
effects of anti-IFN␣ mAb on pharmacodynamic markers and
assessment of its effects on disease activity. Adults (age ⱖ18
years) who met the American College of Rheumatology criteria for SLE (27,28) were enrolled in the trial. Stable background SLE treatment with acetaminophen, nonsteroidal antiinflammatory drugs, antimalarial agents, and/or prednisone
ⱕ20 mg/day (or equivalent) was allowed. Patients receiving
cyclophosphamide, azathioprine, methotrexate, mycophenolate mofetil, cyclosporine, prednisone ⬎20 mg/day (or equivalent), immunoglobulin, blood products, investigational drugs,
or antiviral therapies were excluded. Also excluded were
patients with active or chronic infection, recent vaccination
with live attenuated viruses, recent herpes zoster virus infection, history of severe herpesvirus infection, active central
nervous system lupus, clinically significant cardiac, cerebrovascular, liver, or renal disease, or history of cancer. Most of the
patients were middle-aged white women with mild to moderately active SLE with cutaneous involvement.
The MI-CP126 trial was conducted in accordance with
the Declaration of Helsinki, and the study protocol was
approved by the institutional review board at each site. All
patients provided written informed consent before studyrelated procedures were performed.
Subjects were treated with anti-IFN␣ mAb in single
escalating intravenous doses of 0.3, 1.0, 3.0, 10.0, or 30.0 mg/kg.
All patients were followed up for 84 days. A total of 62 patients
were enrolled in the trial (3 patients participated in both the
blinded and the open-label portions). The ages of patients
ranged from 23 to 80 years, and the female:male ratio was
⬃20:1. Whole blood samples for IFN␣-inducible gene expression profiling were collected in PAXgene RNA tubes (PreAnalytiX, Hilden, Germany) on study day 0 (before dosing) and on
ANTI-IFN␣ TREATMENT OF SLE
postdosing days 1, 2, 4, 7, 14, 28, and 84. Skin biopsy samples
were collected on study day 0 and on postdosing day 14. Skin
biopsy specimens were preferentially obtained from involved
skin, with followup specimens obtained from near the original
biopsy site when possible.
Control whole blood samples were obtained from 24
healthy donors enrolled internally (MedImmune) (ages 26–56
years; female:male ratio 5:1). The majority of these donors
were white. Blood was collected in PAXgene RNA tubes. All
healthy donors provided written informed consent.
Total RNA extraction, microarray processing, and
microarray data analysis. The Human Genome U133 Plus 2.0
array platform (Affymetrix, Santa Clara, CA) was used to
evaluate the effects of anti-IFN␣ mAb in whole blood from the
62 SLE patients and in lesional skin from the 16 patients from
whom skin biopsy samples were collected. The general procedures for sample processing and data analysis for microarray
studies have been described previously (19,20).
Other methods. Whole blood from healthy donors was
stimulated ex vivo. Cytokine gene signature scores were calculated. Neutralization of IFN␣/␤-inducible genes in whole
blood from patients with SLE was measured, and genes
affected by anti-IFN␣ mAb were ranked. Details on these
procedures, as well as on immunohistochemistry analysis and
other experimental methods, are available online at http://
www.medimmune.com/translationalscience/data/MI-CP126A&R-2009.
RESULTS
Anti-IFN␣ mAb neutralizes overexpression of
IFN␣/␤-inducible genes in whole blood from SLE patients in a dose-dependent manner. To evaluate whether
anti-IFN␣ mAb affects its target in SLE patients prior to
their receiving anti-IFN␣ mAb treatment, we profiled
whole blood from 62 SLE patients (3 patients were
enrolled in both the blinded and the open-label portions
of the trial). Whole blood samples from all patients were
collected before dosing and 1, 2, 4, 7, 14, 28, and 84 days
postdosing.
We first evaluated the expression of IFN␣/␤inducible genes in SLE. Because IFN␣/␤ protein levels
were difficult to measure in SLE patients, IFN␣/␤
activity in whole blood was evaluated using IFN␣/␤inducible gene signature scores (Figure 1A). The scores
indicated that IFN␣/␤-inducible genes were overexpressed in whole blood from a majority of the SLE
patients enrolled in the trial, and that the gene signature
score in patients was significantly increased compared
with the score in 24 healthy controls (mean and median
scores in patients 8.4 and 5.4, respectively; both P ⬍ 0.01
versus controls).
The magnitude of overexpression of the IFN␣/␤inducible gene signature in whole blood allowed us to
categorize patients with SLE as having high, moderate,
or weak overexpression of IFN␣/␤-inducible genes (19).
1787
It is likely that more accurate assessment of drug target
neutralization would be obtained in patients with high or
moderate overexpression (gene signature score of ⱖ4).
On study day 0, whole blood samples from 37 of the 62
patients (60%) exhibited high or moderate overexpression of the IFN␣/␤-inducible gene signature. Using this
group of 37 patients, we monitored the pharmacodynamic effect of anti-IFN␣ mAb on target neutralization
in SLE. Ten of the 37 patients received placebo, and the
remaining 27 received anti-IFN␣ mAb in varying single
doses (0.3 mg/kg [n ⫽ 5], 1.0 mg/kg [n ⫽6], 3.0 mg/kg
[n ⫽ 6], 10.0 mg/kg [n ⫽ 6], or 30.0 mg/kg [n ⫽4]).
Data on the anti-IFN␣ mAb target neutralization
values on days 1, 2, 4, 7, 14, 28, and 84 postdosing,
calculated using the gene signature scores for 21 IFN␣/
␤-inducible genes in each SLE patient as previously
described (19), are available online at http://
www.medimmune.com/translationalscience/data/MICP126-A%26R-2009/. Overall, the IFN␣/␤-inducible
gene signature scores in SLE patients who received
placebo yielded target neutralization values that oscillated around baseline values for 84 days following
treatment. Patients treated with 0.3 mg/kg anti-IFN␣
mAb exhibited a substantial decrease in IFN␣/␤inducible gene signature scores in the first 2 days
postdosing (57% and 46% mean target neutralization on
day 1 and day 2 post–anti-IFN␣ mAb treatment, respectively). Although IFN␣/␤-inducible gene signature
scores in this group gradually recovered over time, on
day 84 postdosing we still observed average neutralization of 25%. Furthermore, with increases in the antiIFN␣ mAb dose from 3 mg/kg to 10 mg/kg to 30 mg/kg,
there was a dose-dependent increase in target neutralization, especially in the early days posttreatment (days
1, 2, and 4). These data provide evidence that overexpression of IFN␣/␤-inducible gene signatures in whole
blood from SLE patients could serve as a potential
pharmacodynamic biomarker for the evaluation of antiIFN␣ mAb therapy in SLE (19).
The gene signature scores for IFN␣/␤-inducible
genes in whole blood as calculated using the list of 21
IFN␣/␤-inducible genes (static list) were compared with
scores calculated using alternative lists, i.e., the 25 most
highly overexpressed IFN␣/␤-inducible genes in each
patient (dynamic lists, potentially differing between patients). The correlation coefficient between results obtained with the 2 score calculation methods was 0.95,
suggesting that either algorithm was sufficient to capture
the magnitude of overexpression of the IFN␣/␤inducible genes in whole blood from patients with SLE.
Data on both scores in each individual patient, as well as
on the genes used in the calculation in an individual
1788
YAO ET AL
Figure 1. Five cytokine-inducible gene signature scores in whole blood (WB) from systemic lupus erythematosus
(SLE) patients and from normal controls. A, Relative expression of the interferon-␣/␤ (IFN␣/␤)–inducible gene
signature in whole blood from 62 SLE patients before anti-IFN␣ monoclonal antibody (mAb) treatment and from
24 normal controls. A high IFN␣/␤-inducible gene signature was defined as a score of ⱖ10, and a moderate signature
was defined as a score of ⱖ4 and ⬍10. Thirty-seven patients (60%) had a moderate or high IFN␣/␤-inducible gene
signature score. B, Relative expression of the gene signature for 4 cytokine-inducible genes (granulocyte–
macrophage colony-stimulating factor [GM-CSF], interleukin-10 [IL-10], IL-1␤, and tumor necrosis factor ␣
[TNF␣]) in whole blood from the SLE patients before anti-IFN␣ mAb treatment and from normal controls. The
gene signature score is calculated as the median fold change, using the 15 most highly induced cytokine-inducible
genes (as determined using each cytokine in a separate ex vivo stimulation experiment), as measured with the Human
Genome U133 Plus 2.0 array platform (Affymetrix, Santa Clara, CA). Horizontal bars show the medians. Signature
scores for the IFN␣/␤-inducible and IL-10 genes were significantly different between patients and controls (P ⬍
0.01).
patient, are available online at http://www.medimmune.
com/translationalscience/data/MI-CP126-A%26R2009/. In this representative patient, 19 of the 21 “static”
genes were included among the 25 most overexpressed
highly IFN␣/␤-inducible genes, further demonstrating
the similarity between the static list and dynamic list
approaches.
The mean (and SEM) target neutralization values calculated at each time point posttreatment, for each
dose level, were calculated using the static list of 21
IFN␣/␤-inducible genes (available online at http://
www.medimmune.com/translationalscience/data/MICP126-A%26R-2009/). Dose-dependent target neutralization was again demonstrated, as evaluated using a
criterion of ⬎50% neutralization at any time point. To
provide a statistical summary of the differences in target
neutralization between placebo-treated patients and pa-
tients treated with anti-IFN␣ mAb at each dose level
across time, Hotelling’s T2 test was applied. This multivariate analog to Student’s t-test accounts for the correlation structure between the time points posttreatment.
The neutralization values for each dose were compared
with those obtained with placebo, separately, using data
from days 1–14 postdosing, since the half-life of antiIFN␣ mAb is within the range of 14–20 days.
From the pairwise comparisons of target neutralization values between patients treated with anti-IFN␣
mAb at each of the 5 dose levels and patients treated
with placebo, the effect of anti-IFN␣ mAb at the 3
mg/kg, 10 mg/kg, and 30 mg/kg doses was found to be
significantly different from that of placebo (P ⫽ 0.03,
0.01, and 0.02, respectively); target neutralization values
in patients treated with anti-IFN␣ mAb at 0.3 mg/kg or
1 mg/kg were not significantly different from values in
ANTI-IFN␣ TREATMENT OF SLE
1789
Figure 2. Dose responses to anti-IFN␣ mAb therapy in whole blood from SLE patients with overexpression of the
IFN␣/␤-inducible gene signature as determined by the gene signature score. A, Neutralization of 21 IFN␣/␤-inducible
genes from day 0 (pretreatment) to day 84 (posttreatment), averaged for each study cohort (placebo [red], anti-IFN␣
mAb 0.3 mg/kg [blue], anti-IFN␣ mAb 1 mg/kg [green], anti-IFN␣ mAb 3 mg/kg [orange], anti-IFN␣ mAb 10 mg/kg
[black], and anti-IFN␣ mAb 30 mg/kg [pink]). The fraction of neutralization on each study day was subtracted from 1
for each patient separately. Values that exceed 1 from this formula represent increased transcript levels of
IFN␣/␤-inducible genes in whole blood following treatment (mostly observed in placebo-treated patients). Values are
the mean ⫾ SEM. B, Principal components analysis (PCA) plots obtained using a static list of 21 IFN␣/␤-inducible
genes. PCA showed that placebo treatment did not cause significant change in the IFN␣/␤-inducible gene signature in
whole blood from SLE patients. Blue dots represent the 24 normal subjects; red dots represent SLE patients before
treatment (day 0). Left plot shows results obtained on day 1 after placebo treatment (cyan); center plot shows results
obtained on day 14 after placebo treatment (black); right plot shows results obtained on day 84 after placebo treatment
(yellow). See Figure 1 for other definitions.
placebo-treated patients (P ⫽ 0.17 and P ⫽ 0.10, respectively).
Specificity of neutralization of the IFN␣/␤inducible gene signature by anti-IFN␣ mAb. The IFN␣/
␤-inducible gene signature was not significantly altered
in whole blood from SLE patients treated with placebo
(Figure 2A), and the neutralization observed in the
anti-IFN␣ mAb–treated patients was dose dependent.
These findings indicate that the target neutralization
observed in SLE patients following anti-IFN␣ mAb
treatment is likely drug specific. A single-factor analysis
of variance was used to evaluate the differences in
IFN␣/␤-inducible gene signature scores across all time
points. There was not a significant difference in the
IFN␣/␤ gene signature score in the placebo-treated
patients (n ⫽ 17) between any days pre- or postdosing
(P ⫽ 0.94). Each pairwise time point comparison was
also assessed using Tukey’s honest significant difference
test; again, no pairwise significant differences were
observed. Similar results were obtained (P ⫽ 0.31) in
analyses including only the placebo-treated patients who
had a baseline IFN␣/␤-inducible gene signature score of
ⱖ4 (n ⫽ 10). Figure 2B shows principal components
analysis (PCA) plots obtained using the 21 IFN␣/␤inducible genes in whole blood from SLE patients on
days 1, 14, and 84 following placebo treatment. Placebo
1790
treatment did not result in significant changes in either
direction in the IFN␣/␤-inducible gene signatures in
whole blood from SLE patients at any time following
treatment.
A heat map and PCA plot depicting target neutralization in whole blood from a representative SLE
patient treated with a single-dose intravenous injection
of 30 mg/kg anti-IFN␣ mAb are available online at
http://www.medimmune.com/translationalscience/data/
MI-CP126-A%26R-2009/. In this patient, strong neutralization (81%) was observed on day 1 postdosing, and
peak neutralization (98%) occurred on day 4; neutralization diminished in subsequent measurements. Substantial target neutralization (58%) was still observed on
day 84 postdosing. PCA showed that the patient’s IFN␣/
␤-inducible gene signature was significantly decreased
on day 1 following drug treatment, was decreased further, to a level comparable with that in healthy controls,
on day 4, and then rose steadily from day 14 to day 84
postdosing. We ranked all genes that were neutralized
by anti-IFN␣ mAb or changed in placebo-treated patients and found that IFN␣/␤-inducible genes comprised
93 of the top 100 probes neutralized by anti-IFN␣ mAb
on day 7 postdosing. In contrast, only 1 of the top 100
probes neutralized with placebo treatment on day 7
postdosing was an IFN␣/␤-inducible gene. The difference was significant (P ⬍ 0.01 by 2-sample [2-tailed]
proportions test), suggesting that the effect of anti-IFN␣
mAb was drug-specific in the SLE patients. Similar results
were observed on days 1, 2, 4, 14, and 28 postdosing.
Neutralization of IFN␣/␤-inducible genes at the
disease site is confirmed by transcript profiling and
immunohistochemistry. To examine whether target neutralization in whole blood correlated with target neutralization at disease sites, we profiled skin lesions from the
16 SLE patients from whom skin biopsy samples were
available, using microarrays. Skin lesion specimens were
collected on day 0 predosing and day 14 postdosing.
Forty-two of the 50 most highly overexpressed genes in
lesional skin from SLE patients were IFN␣/␤-inducible
genes, consistent with the observation that the overwhelming majority of genes overexpressed in whole
blood from patients with SLE are IFN␣/␤-inducible
genes.
A list of the 50 most highly overexpressed genes
in lesional skin from these 16 SLE patients, and the
pretreatment IFN␣/␤-inducible gene signature scores in
skin lesions and whole blood from each individual
patient, are available online at http://www.medimmune.
com/translationalscience/data/MI-CP126-A%26R2009/. Overall, expression patterns of IFN␣/␤-inducible
genes were similar in whole blood and skin lesions. In 13
YAO ET AL
of the 16 patients, IFN␣/␤-inducible gene signature
scores in whole blood and skin were either both above
the cutoff for defining presence of the signature (i.e.,
ⱖ4) or both below the cutoff (P ⬍ 0.05 by Fisher’s exact
test). These similar trends of overexpression of IFN␣/␤inducible genes in whole blood and skin lesions from
patients with SLE provided further scientific rationale
for using whole blood as a surrogate tissue to measure
the pharmacodynamics of anti-IFN␣ mAb. We also
compared target neutralization trends in skin and whole
blood from SLE patients. Of the 8 patients who exhibited positive IFN␣/␤-inducible gene signatures in both
skin and whole blood, 7 showed a similar trend of target
neutralization in both whole blood and skin lesions on
day 14 after receiving either anti-IFN␣ mAb treatment
or placebo. These results provided evidence that antiIFN␣ mAb is able to neutralize its target in disease
tissue.
To determine whether the highly overexpressed
IFN␣/␤-inducible genes in lesional skin were associated
with similar changes in protein expression, we performed immunohistochemistry analyses to assess the
presence of 3 IFN␣/␤-inducible proteins, hect domain
and RCC1-like domain 5 (HERC-5), interferon-induced
protein 15 (ISG-15), and chemokine (CXC) motif ligand
10 (IP-10). These proteins were chosen based on strong
overexpression of their respective mRNA at disease sites
and the availability of immunohistochemistry reagents.
Skin lesions from the same biopsy samples as were used
in the whole-genome array analysis were also used for
immunohistochemistry. Immunohistochemistry characterization of the cellular infiltrates (plasmacytoid dendritic cells [pDCs], myeloid DCs [mDCs], and CD4⫹
cells) allowed us to compare numbers of IFN-producing
cells and inflammatory cells in paired biopsy samples of
lesional skin, obtained predosing and on day 14 postdosing.
In selected patients whose paired biopsy specimens were evaluated, lesional skin contained increased
numbers of CD4⫹ cells and exhibited significant upregulation of HERC-5, IP-10, and ISG-15 proteins in the
dermis. In contrast, skin biopsy samples from normal
donors did not contain appreciable numbers of pDCs or
mDCs, and did not stain for HERC-5, IP-10, or ISG-15
(results not shown).
Figure 3 shows amelioration of SLE skin lesions
and decreases in CD4⫹ cell infiltrates and cells expressing IFN-inducible proteins on day 14 postdosing, in a
patient who was treated with 10 mg/kg anti-IFN␣ mAb.
Immunohistochemical analysis of paired biopsy specimens from lesional skin (day 0 predosing and day 14
postdosing) (Figure 3A) demonstrated significant de-
ANTI-IFN␣ TREATMENT OF SLE
1791
Figure 3. Effects of anti-IFN␣ mAb treatment in a single SLE patient who showed a response to treatment with 10 mg/kg anti-IFN␣ mAb. A,
Immunohistochemical analyses of paired biopsy specimens from lesional skin (obtained predosing [day 0] and on day 14 postdosing). BDCA2 is a
specific marker for plasmacytoid dendritic cells (DCs), CD83 is a marker for myeloid DCs, and CD4 is present on T cells and DCs. Overexpression
of hect domain and RCC1-like domain 5 (HERC-5), interferon-induced protein 15 (ISG-15), and chemokine (CXC) motif ligand 10 (IP-10) that
was observed in lesional skin on day 0 was decreased on day 14 (original magnification ⫻ 300). A principal components analysis (PCA) plot of data
from the same SLE patient, obtained using the 21 IFN␣/␤-inducible genes in the skin lesion compared with a normal population (blue), is also
shown; data were obtained before dosing (red) and on day 14 (black). B, Resolution of a skin lesion in the patient following anti-IFN␣ mAb
treatment. C, Heat map representation of the neutralization of 21 IFN␣/␤-inducible genes in whole blood from the SLE patient following anti-IFN␣
mAb treatment. Columns left-to-right correspond to day 0 and days 1, 7, 14, and 28 following treatment; rows correspond to the 21 static
IFN␣/␤-inducible genes in this patient (predosing). See Figure 1 for other definitions.
creases in CD83 and CD4 staining after treatment.
Staining for ISG-15, HERC-5, and IP-10 proteins was
also significantly reduced on day 14 postdosing (Figure
3A), consistent with observed substantial decreases in
levels of mRNA for these respective genes. The PCA
plot showing overexpression of the 21 IFN␣/␤-inducible
genes in the skin lesion from this SLE patient, compared
with normal controls, indicated substantial neutralization of the IFN␣/␤-inducible gene signature on day 14
following anti-IFN␣ mAb treatment. As seen in Figure
3B, the skin lesion also resolved after administration of
anti-IFN␣ mAb.
A heat map representation of the neutralization
of the 21–IFN␣/␤-inducible gene signature in whole
blood from this anti-IFN␣ mAb–treated SLE patient is
shown in Figure 3C. Strong target neutralization was
observed in whole blood from day 1 to day 28 postdosing, consistent with the substantial target neutralization
observed in the skin lesion. Similar decreases in levels of
inflammatory cells and IFN-inducible proteins were not
1792
YAO ET AL
Figure 4. Effects of anti-IFN␣ mAb on cytokine signaling pathways in whole blood from SLE patients predosing and on day 14 postdosing. Each
row indicates the overexpression of a different cytokine-inducible gene signature; each vertical column of symbols represents an individual patient.
Elevated levels of cytokine gene signatures in each patient are represented by colors approaching red, while colors approaching blue represent low
cytokine gene signature values. See Figure 1 for definitions.
observed in either placebo-treated patients or anti-IFN␣
mAb–treated patients with no substantial target neutralization in the skin lesion (data available online at
http://www.medimmune.com/translationalscience/data/
MI-CP126-A%26R-2009/).
Effects of anti-IFN␣ mAb on BAFF, other cytokines, and their pathways in patients with SLE. After
demonstrating that gene signature scores enabled measurement of the effects of anti-IFN␣ mAb on IFN␣/␤inducible genes, we next evaluated the effects of antiIFN␣ mAb on the signaling pathways of other cytokines
of interest in whole blood from SLE patients. In these
experiments, cytokine-inducible gene signature scores
were measured using panels of cytokine-inducible genes
specific to each cytokine studied. The cytokine-inducible
gene signatures were used to evaluate the effect of these
cytokines, since the proteins were difficult to measure,
similar to the case with IFN␣ protein as described above.
Prior to anti-IFN␣ mAb treatment, IFN␣/␤inducible gene signature scores in whole blood (Figure
1A) demonstrated high levels of overexpression in most
of the SLE patients. Similarly, prior to anti-IFN␣ mAb
treatment, cytokine-inducible gene signature scores for
GM-CSF, TNF␣, IL-1␤, and IL-10 indicated elevated
levels of expression in whole blood in some SLE patients
(Figure 1B). Figure 4 shows the effects of anti-IFN␣
mAb and placebo on cytokine signaling pathways in SLE
whole blood as measured by cytokine-inducible gene
signature scores predosing and on day 14 postdosing. In
some patients, suppression of other cytokine-inducible
genes was observed along with neutralization of IFN␣/
␤-inducible genes by anti-IFN␣ mAb. Patient 41, for
example, had exceptionally high baseline levels of
IFN␣/␤, GM-CSF, TNF␣, and IL-1␤ activity, as demonstrated by cytokine-inducible gene signature scores (Figure 4).
The PCA plots in Figure 5 show the effects of
anti-IFN␣ mAb on IFN␣/␤-, IFN␥-, TNF␣-, and IL-1␤–
inducible genes in whole blood from this patient on days
1, 14, and 84 postdosing. The overexpressed IFN␣/␤inducible gene signature exhibited a rapid decrease on
day 1 postdosing, remained low on day 14, and increased
substantially on day 84 (Figure 5A). A similar trend in
the IFN␥-inducible gene signature in patient 41 was
observed following anti-IFN␣ mAb treatment (Figure
5B). Both the TNF␣- and the IL-1␤–inducible gene
signatures decreased to levels comparable with those in
healthy controls on day 1 postdosing, then recovered
significantly on day 14 and were maintained near the
day-14 level on day 84 postdosing (Figures 5C and D).
Ex vivo IFN␣ stimulation study of healthy donor
whole blood indicated that BAFF is inducible by IFN␣.
Prior to treatment with anti-IFN␣ mAb, significant
overexpression of BAFF mRNA was observed in whole
ANTI-IFN␣ TREATMENT OF SLE
1793
Figure 5. Principal components analysis (PCA) plots of data from SLE patient 41 (treated with a single dose of 10 mg/kg anti-IFN␣ mAb) and a
normal population (blue), obtained using the 15 (25 for IFN␣/␤) most highly overexpressed cytokine-inducible genes in whole blood from the
patient. A, IFN␣/␤. B, IFN␥. C, TNF␣. D, IL-1␤. Data on the SLE patient were obtained before dosing (red) and on days 1 (cyan), 14 (black), and
84 (yellow) postdosing. See Figure 1 for other definitions.
blood from some SLE patients as compared with normal
controls, as measured by TaqMan quantitative real-time
PCR. Data on relative expression levels of mRNA for
BAFF, GM-CSF, IL-10, TNF␣, and IL-1␤ in whole
blood and lesional skin from SLE patients versus controls are available online at http://www.medimmune.
com/translationalscience/data/MI-CP126-A%26R2009/. Anti-IFN␣ mAb treatment suppressed BAFF
mRNA expression in blood (data not shown), as well as
expression of mRNA for BAFF, GM-CSF, and TNF␣ in
lesional skin (Figure 6). Changes in expression levels of
IFN␣/␤-inducible genes in SLE skin lesions were posi-
1794
YAO ET AL
Figure 6. Relative expression of mRNA for BAFF, GM-CSF, and TNF␣ in lesional skin
from SLE patients before dosing (day 0) and 14 days after treatment with placebo (A) or
anti-IFN␣ mAb (B). Expression was measured by TaqMan quantitative real-time polymerase chain reaction and compared with that in pooled samples from normal donors. Only
patients who had ⬎2-fold overexpression of the transcripts either predosing or postdosing
(or both) are included. One patient treated with placebo exhibited a reduction in expression
of mRNA for BAFF, GM-CSF, and TNF␣ in lesional skin, along with a reduction in
IFN␣/␤-inducible genes. See Figure 1 for definitions.
tively correlated with the changes in mRNA for GM-CSF,
TNF␣, and BAFF (␳ ⫽ 0.67, 0.92, and 0.82, respectively).
DISCUSSION
We are currently exploring the use of an antiIFN␣ monoclonal antibody as therapy for SLE. As part
of this effort, we are testing a genomic approach to
develop pharmacodynamic biomarkers that would aid in
monitoring anti-IFN␣ mAb activity in clinical trials and
to inform dosage selection in subsequent trials.
In this study, we have shown that neutralization
of a gene signature comprising 21 IFN␣/␤-inducible
genes in whole blood from SLE patients can serve as a
pharmacodynamic biomarker for assessing anti-IFN␣
mAb activity. Our studies demonstrate that this biomarker possesses many characteristics of an ideal biomarker
(18). It is sensitive and specific. In SLE whole blood, it is
stable and can be quantitatively measured with multiple
assays. This static 21-gene signature panel captures the
overexpression of the IFN␣/␤-inducible genes in whole
blood from SLE patients and allows the categorization
of patients as having high, moderate, or weak overexpression of IFN␣/␤-inducible genes. Using these 21
genes, we have demonstrated a specific and dosedependent neutralization of IFN␣ by anti-IFN␣ mAb.
This pharmacodynamic marker enables measurement of
ANTI-IFN␣ TREATMENT OF SLE
biologic activity of anti-IFN␣ mAb in an easily accessible
surrogate tissue, whole blood. In addition to allowing
sampling at multiple time points in a relatively noninvasive and cost-effective manner, whole blood is the surrogate tissue most frequently used to monitor pharmacokinetics in clinical trials. Thus, pharmacokinetic/
pharmacodynamic modeling using IFN␣/␤-inducible
gene signature scores along with other factors (such as
clinical benefit) can guide dose scheduling for anti-IFN␣
mAb in future SLE trials.
We also used lists of the 25 most overexpressed
IFN␣/␤-inducible genes in individual SLE patients to
evaluate the magnitude of overexpression of IFN␣ in
these patients. This approach should enable more accurate evaluation of the activation of the IFN␣/␤ signaling
pathway in patients in whom the most highly overexpressed IFN␣/␤-inducible genes are not included in the
21-gene panel. Use of a static 21-gene panel is statistically more robust than use of a patient-specific dynamic
set of 25 genes, and is more feasible than use of a
dynamic panel as a potential diagnostic marker to
predict response to anti-IFN␣ mAb treatment in individual SLE patients. Notably, in analyses of whole blood
from the 62 SLE patients, the correlation between the
IFN␣/␤-inducible gene signature scores obtained using
21 genes and those obtained using 25 genes was very
strong (r ⫽ 0.95) (scores in individual patients available
online at http://www.medimmune.com/translational
science/data/MI-CP126-A&R-2009).
It is important to note that the IFN␣/␤-inducible
gene signatures had not returned to initial baseline
levels at the last time point assessed (day 84 postdosing),
even with the lowest dose of anti-IFN␣ mAb (0.3
mg/kg). Pharmacokinetic data indicated that drug availability in the serum of the patients was very low on day
84 in the 0.3 mg/kg–treated cohort (half-life of antiIFN␣ mAb was ⬃18 days). The sustained neutralization
of IFN␣/␤-inducible genes on day 84 was more significant at a higher drug dose, particularly at the 10 mg/kg
and 30 mg/kg doses, where drug was still available in the
serum (based on pharmacokinetic data). These changes
were observed despite the limited number of SLE
patients eligible for pharmacodynamic evaluation (4–6
in each anti-IFN␣ mAb treatment cohort).
These studies also showed that anti-IFN␣ mAb
neutralization of IFN␣ observed in whole blood as
surrogate tissue adequately represented IFN␣ neutralization occurring at disease sites (in this case, skin
lesions in patients with mild to moderate SLE). In most
cases, microarray analysis data on SLE skin lesions
paralleled the data obtained with whole blood. Further,
immunohistochemical analysis revealed that treated pa-
1795
tients whose symptoms responded to therapy all showed
significant decreases in levels of inflammatory and selected IFN␣/␤-inducible proteins. These changes were
not observed in the majority of the placebo-treated
patients evaluated by immunohistochemistry. It should
be noted, however, that the sample size was too small to
draw statistically robust conclusions based on data from
the MI-CP126 trial. Also, obtaining quality skin biopsy
specimens pre- and post–anti-IFN␣ mAb treatment
posed some technical challenges. However, our preliminary data are encouraging, and we will continue to
generate more data in ongoing trials in which patients
with severe SLE will also be enrolled.
While we focused on using IFN␣/␤-inducible
genes to evaluate the pharmacodynamic effect of antiIFN␣ mAb in SLE, the power of whole-genome microarray analysis for such studies allowed us also to
examine the effect of anti-IFN␣ mAb on other signaling
pathways in SLE. In the MI-CP126 trial, we evaluated
how anti-IFN␣ mAb treatment affected BAFF and
other signaling pathways (GM-CSF, IFN␥, IL-10, TNF␣,
and IL-1␤) in the periphery and lesional skin of patients
with SLE. These signaling pathways were activated in
selected patients, as evidenced by overexpression of
their transcripts and/or cytokine-inducible genes in peripheral blood. The transcripts of these signaling molecules and/or their inducible gene signatures showed
trends for change similar to those observed with IFN␣/
␤-inducible genes, which suggests that these pathways
may reside downstream of IFN␣ in SLE, at least in some
patients. Understanding the broader importance of this
observation with regard to SLE pathogenesis and therapy will require further research.
It should be noted that the patients in this trial
represent only one subpopulation of SLE patients, specifically those with mild to moderate SLE with skin
involvement. The presence and usefulness of this 21–
IFN␣/␤-inducible gene signature is currently being
tested in patients with more severe skin involvement and
other manifestations of disease (i.e., lupus nephritis,
neurologic abnormalities). More patients are being evaluated in the subsequent trial so that statistically more
meaningful conclusions about this pharmacodynamic
gene signature biomarker can be drawn. It remains to be
determined whether this gene signature could be used to
prospectively identify patients who are most likely to
benefit from anti-IFN␣ mAb therapy and to help optimize their dosing. Current and future work aims to
further evaluate potential correlations between overexpression of the IFN␣/␤-inducible gene signature in
whole blood from SLE patients and clinical benefits of
1796
YAO ET AL
anti-IFN␣ mAb, to develop models for predicting patient response to treatment.
ACKNOWLEDGMENTS
We would like to thank Anmarie Boutrin, Nancy
Huddy, and Martha Wester for technical assistance, Jiaqi
Huang and Robert Georgantas III for critical review of the
manuscript, and Eric Phan, Krystal Bowers, and Denise Dawson for clinical trial sample management.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Yao had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Yao, Richman, Morehouse, White,
Kiener, Jallal.
Acquisition of data. Yao, Richman, Morehouse, de los Reyes, Brohawn.
Analysis and interpretation of data. Yao, Higgs, Morehouse, Zhang,
Coyle, Kiener, Jallal.
REFERENCES
1. American College of Rheumatology Ad Hoc Committee on
Systemic Lupus Erythematosus Guidelines. Guidelines for referral
and management of systemic lupus erythematosus in adults.
Arthritis Rheum 1999;42:1785–96.
2. Vasoo S, Hughes GR. Theory, targets and therapy in systemic
lupus erythematosus. Lupus 2005;14:181–8.
3. Smith PL, Lombardi G, Foster GR. Type I interferons and the
innate immune response: more than just antiviral cytokines. Mol
Immunol 2005;42:869–77.
4. Conlon KC, Urba WJ, Smith JW II, Steis RG, Longo DL, Clark
JW. Exacerbation of symptoms of autoimmune disease in patients
receiving ␣-interferon therapy. Cancer 1990;65:2237–42.
5. Fattovich G, Giustina G, Favarato S, Ruol A. A survey of adverse
events in 11,241 patients with chronic viral hepatitis treated with
alfa interferon. J Hepatol 1996;24:38–47.
6. Funk J, Langeland T, Schrumpf E, Hanssen LE. Psoriasis induced
by interferon-␣. Br J Dermatol 1991;125:463–5.
7. Okanoue T, Sakamoto S, Itoh Y, Minami M, Yasui K, Sakamoto
M, et al. Side effects of high-dose interferon therapy for chronic
hepatitis C. J Hepatol 1996;25:283–91.
8. Kirou KA, Lee C, George S, Louca K, Peterson MG, Crow MK.
Activation of the interferon-␣ pathway identifies a subgroup of
systemic lupus erythematosus patients with distinct serologic features and active disease. Arthritis Rheum 2005;52:1491–503.
9. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann
WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc
Natl Acad Sci U S A 2003;100:2610–5.
10. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, Banchereau
J, et al. Interferon and granulopoiesis signatures in systemic lupus
erythematosus blood. J Exp Med 2003;197:711–23.
11. Han GM, Chen SL, Shen N, Ye S, Bao CD, Gu YY. Analysis of
gene expression profiles in human systemic lupus erythematosus
using oligonucleotide microarray. Genes Immun 2003;4:177–86.
12. Hua J, Kirou K, Lee C, Crow MK. Functional assay of type I
interferon in systemic lupus erythematosus plasma and association
with anti–RNA binding protein autoantibodies. Arthritis Rheum
2006;54:1906–16.
13. Bengtsson AA, Sturfelt G, Truedsson L, Blomberg J, Alm G,
Vallin H, et al. Activation of type I interferon system in systemic
lupus erythematosus correlates with disease activity but not with
antiretroviral antibodies. Lupus 2000;9:664–71.
14. Dall’Era MC, Cardarelli PM, Preston BT, Witte A, Davis JC Jr.
Type I interferon correlates with serological and clinical manifestations of SLE. Ann Rheum Dis 2005;64:1692–7.
15. Hooks JJ, Moutsopoulos HM, Geis SA, Stahl NI, Decker JL,
Notkins AL. Immune interferon in the circulation of patients with
autoimmune disease. N Engl J Med 1979;301:5–8.
16. Pascual V, Farkas L, Banchereau J. Systemic lupus erythematosus:
all roads lead to type I interferons. Curr Opin Immunol 2006;18:
676–82.
17. Wallace DJ, Petri M, Olsen N, Kirou K, Dennis G, Yao Y, et al.
MEDI-545, an anti-interferon alpha monoclonal antibody, shows
evidence of clinical activity in systemic lupus erythematous [abstract]. Arthritis Rheum 2007;56 Suppl 9:S526–7.
18. Adjei AA. Incorporating biomarkers into early phase clinical trials
[abstract]. Ann Oncol 2006;17 Suppl 3:iii21.
19. Yao Y, Higgs BW, Morehouse C, de los Reyes M, Trigona W,
Brohawn P, et al. Development of potential pharmacodynamic and
diagnostic markers for anti-IFN-␣ monoclonal antibody trials in
systemic lupus erythematosus. Hum Genomics Proteomics, 2009.
URL: http://www.sage-hindawi.com/getarticle.aspx?doi⫽10.4061/
2009/374312.
20. Yao Y, Richman L, Morehouse C, de los Reyes M, Higgs BW,
Boutrin A, et al. Type I interferon: potential therapeutic target for
psoriasis? PLoS ONE 2008;3:e2737.
21. Chun HY, Chung JW, Kim HA, Yun JM, Jeon JY, Ye YM, et al.
Cytokine IL-6 and IL-10 as biomarkers in systemic lupus erythematosus. J Clin Immunol 2007;27:461–6.
22. Fiehn C, Wermann M, Pezzutto A, Hufner M, Heilig B. Plasma
GM-CSF concentrations in rheumatoid arthritis, systemic lupus
erythematosus and spondyloarthropathy. Z Rheumatol 1992;51:
121–6. In German.
23. Borodin AG, Baranov AA, Kliukvina NG, Abaitova NE, Nasonov
EL. Clinical and pathogenic significance of tumor necrosis factor-␣ in systemic lupus erythematosus. Ter Arkh 2002;74:32–5. In
Russian.
24. Palucka AK, Blanck JP, Bennett L, Pascual V, Banchereau J.
Cross-regulation of TNF and IFN-␣ in autoimmune diseases. Proc
Natl Acad Sci U S A 2005;102:3372–7.
25. Morimoto S, Nakano S, Watanabe T, Tamayama Y, Mitsuo A,
Nakiri Y, et al. Expression of B-cell activating factor of the tumour
necrosis factor family (BAFF) in T cells in active systemic lupus
erythematosus: the role of BAFF in T cell-dependent B cell
pathogenic autoantibody production. Rheumatology (Oxford)
2007;46:1083–6.
26. Stohl W, Metyas S, Tan SM, Cheema GS, Oamar B, Xu D, et al.
B lymphocyte stimulator overexpression in patients with systemic
lupus erythematosus: longitudinal observations. Arthritis Rheum
2003;48:3475–86.
27. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield
NF, et al. The 1982 revised criteria for the classification of systemic
lupus erythematosus. Arthritis Rheum 1982;25:1271–7.
28. Hochberg MC, for the Diagnostic and Therapeutic Criteria Committee of the American College of Rheumatology. Updating the
American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus [letter]. Arthritis
Rheum 1997;40:1725.
Документ
Категория
Без категории
Просмотров
4
Размер файла
1 004 Кб
Теги
systemic, erythematosus, antibody, tria, phase, antiinterferon, inducible, lupus, effect, downstream, monoclonal, neutralization, genes, interferon
1/--страниц
Пожаловаться на содержимое документа