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.
Systemic Lupus Erythematosus Clinical Studies
Neutralization of interferon-α/β–inducible genes and downstream effect in a phase I trial of an anti–interferon-α monoclonal antibody in systemic lupus erythematosus†
Article first published online: 28 MAY 2009
Copyright © 2009 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 60, Issue 6, pages 1785–1796, June 2009
How to Cite
Yao, Y., Richman, L., Higgs, B. W., Morehouse, C. A., de los Reyes, M., Brohawn, P., Zhang, J., White, B., Coyle, A. J., Kiener, P. A. and Jallal, B. (2009), Neutralization of interferon-α/β–inducible genes and downstream effect in a phase I trial of an anti–interferon-α monoclonal antibody in systemic lupus erythematosus. Arthritis & Rheumatism, 60: 1785–1796. doi: 10.1002/art.24557
ClinicalTrials.gov identifier: NCT00299819.
- Issue published online: 28 MAY 2009
- Article first published online: 28 MAY 2009
- Manuscript Accepted: 2 MAR 2009
- Manuscript Received: 24 MAR 2008
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
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-IFNα monoclonal antibody (mAb) therapy in SLE. During the trial, we also examined whether overexpression of an IFNα/β-inducible gene signature in whole blood could serve as a pharmacodynamic biomarker to evaluate IFNα neutralization and investigated downstream effects of neutralizing IFNα on BAFF and other key signaling pathways, i.e., granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), tumor necrosis factor α (TNFα), and IL-1β, in SLE.
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 IFNα/β-inducible proteins were analyzed by immunohistochemistry.
With the study treatment, we observed anti-IFNα mAb–specific and dose-dependent inhibition of overexpression of IFNα/β-inducible genes in whole blood and skin lesions from SLE patients, at both the transcript and the protein levels. In SLE patients with overexpression of messenger RNA for BAFF, TNFα, IL-10, IL-1β, 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-IFNα mAb.
IFNα/β-inducible gene signatures in whole blood are effective pharmacodynamic biomarkers to evaluate anti-IFNα mAb therapy in SLE. Anti-IFNα mAb can neutralize overexpression of IFNα/β-inducible genes in whole blood and lesional skin from SLE patients and has profound effects on signaling pathways that may be downstream of IFNα in SLE.
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 IFNα are observed in the serum of a subset of SLE patients (8, 12–16).
To treat SLE by lowering IFNα levels, we have developed a fully human IgG1κ monoclonal antibody (mAb) that binds to a majority of the subtypes of human IFNα and inhibits IFN-mediated signaling. A single-dose, double-blind, placebo-controlled phase Ia trial (MI-CP126) was conducted to study anti-IFNα mAb treatment in patients with mild to moderate SLE with cutaneous involvement who were receiving standard-of-care therapy (17). The primary objective of the MI-CP126 trial was to evaluate the safety and tolerability of intravenously administered anti-IFNα 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-IFNα mAb and, additionally, to study the effects of IFNα 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 IFNα protein in the serum of SLE patients would be the most reasonable choice as a pharmacodynamic marker for evaluating anti-IFNα 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 IFNα protein in the serum. IFNα-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 IFNα/β-inducible genes in whole blood from SLE patients. We found that the IFNα/β 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-IFNα mAb, comprising 21 IFNα/β-inducible genes that are overexpressed in whole blood from SLE patients (19).
In this study, we tested this 21–IFNα/β-inducible gene signature as a pharmacodynamic biomarker for determining whether anti-IFNα mAb neutralizes IFNα in a specific and dose-dependent manner. In addition, we investigated the effects of anti-IFNα mAb on signaling pathways for interleukin-10 (IL-10) (21), granulocyte–macrophage colony-stimulating factor (GM-CSF) (22), and tumor necrosis factor α (TNFα) (23, 24), all of which exhibit elevated protein levels in SLE serum. We also examined effects of anti-IFNα 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
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
SLE patients and controls.
The MI-CP126 trial was a multicenter randomized (2:1), double-blind, placebo-controlled, 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-IFNα mAb on pharmacodynamic markers and assessment of its effects on disease activity. Adults (age ≥18 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 ≤20 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 study-related procedures were performed.
Subjects were treated with anti-IFNα 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 IFNα-inducible gene expression profiling were collected in PAXgene RNA tubes (PreAnalytiX, Hilden, Germany) on study day 0 (before dosing) and on 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-IFNα 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).
Whole blood from healthy donors was stimulated ex vivo. Cytokine gene signature scores were calculated. Neutralization of IFNα/β-inducible genes in whole blood from patients with SLE was measured, and genes affected by anti-IFNα 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-CP126-A&R-2009.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Anti-IFNα mAb neutralizes overexpression of IFNα/β-inducible genes in whole blood from SLE patients in a dose-dependent manner.
To evaluate whether anti-IFNα mAb affects its target in SLE patients prior to their receiving anti-IFNα 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 IFNα/β-inducible genes in SLE. Because IFNα/β protein levels were difficult to measure in SLE patients, IFNα/β activity in whole blood was evaluated using IFNα/β-inducible gene signature scores (Figure 1A). The scores indicated that IFNα/β-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 IFNα/β-inducible gene signature in whole blood allowed us to categorize patients with SLE as having high, moderate, or weak overexpression of IFNα/β-inducible genes (19). 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 ≥4). On study day 0, whole blood samples from 37 of the 62 patients (60%) exhibited high or moderate overexpression of the IFNα/β-inducible gene signature. Using this group of 37 patients, we monitored the pharmacodynamic effect of anti-IFNα mAb on target neutralization in SLE. Ten of the 37 patients received placebo, and the remaining 27 received anti-IFNα 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-IFNα mAb target neutralization values on days 1, 2, 4, 7, 14, 28, and 84 postdosing, calculated using the gene signature scores for 21 IFNα/β-inducible genes in each SLE patient as previously described (19), are available online at http://www.medimmune.com/translationalscience/data/MI-CP126-A%26R-2009/. Overall, the IFNα/β-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-IFNα mAb exhibited a substantial decrease in IFNα/β-inducible gene signature scores in the first 2 days postdosing (57% and 46% mean target neutralization on day 1 and day 2 post–anti-IFNα mAb treatment, respectively). Although IFNα/β-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 anti-IFNα 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 IFNα/β-inducible gene signatures in whole blood from SLE patients could serve as a potential pharmacodynamic biomarker for the evaluation of anti-IFNα mAb therapy in SLE (19).
The gene signature scores for IFNα/β-inducible genes in whole blood as calculated using the list of 21 IFNα/β-inducible genes (static list) were compared with scores calculated using alternative lists, i.e., the 25 most highly overexpressed IFNα/β-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 IFNα/β-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 patient, are available online at http://www.medimmune.com/translationalscience/data/MI-CP126-A%26R-2009/. In this representative patient, 19 of the 21 “static” genes were included among the 25 most overexpressed highly IFNα/β-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 IFNα/β-inducible genes (available online at http://www.medimmune.com/translationalscience/data/MI-CP126-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 patients treated with anti-IFNα 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 anti-IFNα mAb is within the range of 14–20 days.
From the pairwise comparisons of target neutralization values between patients treated with anti-IFNα mAb at each of the 5 dose levels and patients treated with placebo, the effect of anti-IFNα 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-IFNα mAb at 0.3 mg/kg or 1 mg/kg were not significantly different from values in placebo-treated patients (P = 0.17 and P = 0.10, respectively).
Specificity of neutralization of the IFNα/β-inducible gene signature by anti-IFNα mAb.
The IFNα/β-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-IFNα mAb–treated patients was dose dependent. These findings indicate that the target neutralization observed in SLE patients following anti-IFNα mAb treatment is likely drug specific. A single-factor analysis of variance was used to evaluate the differences in IFNα/β-inducible gene signature scores across all time points. There was not a significant difference in the IFNα/β 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 IFNα/β-inducible gene signature score of ≥4 (n = 10). Figure 2B shows principal components analysis (PCA) plots obtained using the 21 IFNα/β-inducible genes in whole blood from SLE patients on days 1, 14, and 84 following placebo treatment. Placebo treatment did not result in significant changes in either direction in the IFNα/β-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-IFNα 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 IFNα/β-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-IFNα mAb or changed in placebo-treated patients and found that IFNα/β-inducible genes comprised 93 of the top 100 probes neutralized by anti-IFNα mAb on day 7 postdosing. In contrast, only 1 of the top 100 probes neutralized with placebo treatment on day 7 postdosing was an IFNα/β-inducible gene. The difference was significant (P < 0.01 by 2-sample [2-tailed] proportions test), suggesting that the effect of anti-IFNα mAb was drug-specific in the SLE patients. Similar results were observed on days 1, 2, 4, 14, and 28 postdosing.
Neutralization of IFNα/β-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 IFNα/β-inducible genes, consistent with the observation that the overwhelming majority of genes overexpressed in whole blood from patients with SLE are IFNα/β-inducible genes.
A list of the 50 most highly overexpressed genes in lesional skin from these 16 SLE patients, and the pretreatment IFNα/β-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%26R-2009/. Overall, expression patterns of IFNα/β-inducible genes were similar in whole blood and skin lesions. In 13 of the 16 patients, IFNα/β-inducible gene signature scores in whole blood and skin were either both above the cutoff for defining presence of the signature (i.e., ≥4) or both below the cutoff (P < 0.05 by Fisher's exact test). These similar trends of overexpression of IFNα/β-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-IFNα mAb. We also compared target neutralization trends in skin and whole blood from SLE patients. Of the 8 patients who exhibited positive IFNα/β-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-IFNα mAb treatment or placebo. These results provided evidence that anti-IFNα mAb is able to neutralize its target in disease tissue.
To determine whether the highly overexpressed IFNα/β-inducible genes in lesional skin were associated with similar changes in protein expression, we performed immunohistochemistry analyses to assess the presence of 3 IFNα/β-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 up-regulation 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-IFNα mAb. Immunohistochemical analysis of paired biopsy specimens from lesional skin (day 0 predosing and day 14 postdosing) (Figure 3A) demonstrated significant decreases 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 IFNα/β-inducible genes in the skin lesion from this SLE patient, compared with normal controls, indicated substantial neutralization of the IFNα/β-inducible gene signature on day 14 following anti-IFNα mAb treatment. As seen in Figure 3B, the skin lesion also resolved after administration of anti-IFNα mAb.
A heat map representation of the neutralization of the 21–IFNα/β-inducible gene signature in whole blood from this anti-IFNα 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 observed in either placebo-treated patients or anti-IFNα 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-IFNα 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-IFNα mAb on IFNα/β-inducible genes, we next evaluated the effects of anti-IFNα 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 IFNα protein as described above.
Prior to anti-IFNα mAb treatment, IFNα/β-inducible gene signature scores in whole blood (Figure 1A) demonstrated high levels of overexpression in most of the SLE patients. Similarly, prior to anti-IFNα mAb treatment, cytokine-inducible gene signature scores for GM-CSF, TNFα, IL-1β, and IL-10 indicated elevated levels of expression in whole blood in some SLE patients (Figure 1B). Figure 4 shows the effects of anti-IFNα 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 IFNα/β-inducible genes by anti-IFNα mAb. Patient 41, for example, had exceptionally high baseline levels of IFNα/β, GM-CSF, TNFα, and IL-1β activity, as demonstrated by cytokine-inducible gene signature scores (Figure 4).
The PCA plots in Figure 5 show the effects of anti-IFNα mAb on IFNα/β-, IFNγ-, TNFα-, and IL-1β–inducible genes in whole blood from this patient on days 1, 14, and 84 postdosing. The overexpressed IFNα/β-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 IFNγ-inducible gene signature in patient 41 was observed following anti-IFNα mAb treatment (Figure 5B). Both the TNFα- and the IL-1β–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 IFNα stimulation study of healthy donor whole blood indicated that BAFF is inducible by IFNα. Prior to treatment with anti-IFNα mAb, significant overexpression of BAFF mRNA was observed in whole 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, TNFα, and IL-1β in whole blood and lesional skin from SLE patients versus controls are available online at http://www.medimmune.com/translationalscience/data/MI-CP126-A%26R-2009/. Anti-IFNα mAb treatment suppressed BAFF mRNA expression in blood (data not shown), as well as expression of mRNA for BAFF, GM-CSF, and TNFα in lesional skin (Figure 6). Changes in expression levels of IFNα/β-inducible genes in SLE skin lesions were positively correlated with the changes in mRNA for GM-CSF, TNFα, and BAFF (ρ = 0.67, 0.92, and 0.82, respectively).
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
We are currently exploring the use of an anti-IFNα 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-IFNα 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 IFNα/β-inducible genes in whole blood from SLE patients can serve as a pharmacodynamic biomarker for assessing anti-IFNα 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 IFNα/β-inducible genes in whole blood from SLE patients and allows the categorization of patients as having high, moderate, or weak overexpression of IFNα/β-inducible genes. Using these 21 genes, we have demonstrated a specific and dose-dependent neutralization of IFNα by anti-IFNα mAb. This pharmacodynamic marker enables measurement of biologic activity of anti-IFNα 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 IFNα/β-inducible gene signature scores along with other factors (such as clinical benefit) can guide dose scheduling for anti-IFNα mAb in future SLE trials.
We also used lists of the 25 most overexpressed IFNα/β-inducible genes in individual SLE patients to evaluate the magnitude of overexpression of IFNα in these patients. This approach should enable more accurate evaluation of the activation of the IFNα/β signaling pathway in patients in whom the most highly overexpressed IFNα/β-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-IFNα mAb treatment in individual SLE patients. Notably, in analyses of whole blood from the 62 SLE patients, the correlation between the IFNα/β-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/translationalscience/data/MI-CP126-A&R-2009).
It is important to note that the IFNα/β-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-IFNα 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 anti-IFNα mAb was ∼18 days). The sustained neutralization of IFNα/β-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-IFNα mAb treatment cohort).
These studies also showed that anti-IFNα mAb neutralization of IFNα observed in whole blood as surrogate tissue adequately represented IFNα 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 patients whose symptoms responded to therapy all showed significant decreases in levels of inflammatory and selected IFNα/β-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-IFNα 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 IFNα/β-inducible genes to evaluate the pharmacodynamic effect of anti-IFNα mAb in SLE, the power of whole-genome microarray analysis for such studies allowed us also to examine the effect of anti-IFNα mAb on other signaling pathways in SLE. In the MI-CP126 trial, we evaluated how anti-IFNα mAb treatment affected BAFF and other signaling pathways (GM-CSF, IFNγ, IL-10, TNFα, and IL-1β) 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 IFNα/β-inducible genes, which suggests that these pathways may reside downstream of IFNα 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–IFNα/β-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-IFNα mAb therapy and to help optimize their dosing. Current and future work aims to further evaluate potential correlations between overexpression of the IFNα/β-inducible gene signature in whole blood from SLE patients and clinical benefits of anti-IFNα mAb, to develop models for predicting patient response to treatment.
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- PATIENTS AND METHODS
- 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.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
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.
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