Development of Potential Pharmacodynamic and Diagnostic Markers for Anti-IFN-α Monoclonal Antibody Trials in Systemic Lupus Erythematosus

Abstract

To identify potential pharmacodynamic biomarkers to guide dose selection in clinical trials using anti-interferon-alpha (IFN-α) monoclonal antibody (mAb) therapy for systemic lupus erythematosus (SLE), we used an Affymetrix human genome array platform and identified 110 IFN-α/β-inducible transcripts significantly upregulated in whole blood (WB) of 41 SLE patients. The overexpression of these genes was confirmed prospectively in 54 additional SLE patients and allowed for the categorization of the SLE patients into groups of high, moderate, and weak overexpressers of IFN-α/β-inducible genes. This approach could potentially allow for an accurate assessment of drug target neutralization in early trials of anti-IFN-α mAb therapy for SLE. Furthermore, ex vivo stimulation of healthy donor peripheral blood mononuclear cells with SLE patient serum and subsequent neutralization with anti-IFN-α mAb or anti-IFN-α receptor mAb showed that anti-IFN-α mAb has comparable effects of neutralizing the overexpression of type I IFN-inducible genes as that of anti-IFNAR mAb. These results suggest that IFN-α, and not other members of type I IFN family in SLE patients, is mainly responsible for the induction of type I IFN-inducible genes in WB of SLE patients. Taken together, these data strengthen the view of IFN-α as a therapeutic target for SLE.

Figure 1

Representative heat map visualizing the overexpression of IFN-α/β-inducible gene signature, granulocyte signature, and underexpression of T-cell and B-cell signature in WB from 41 SLE patients () compared with WB from 24 healthy donors (). IFN = interferon; SLE = systemic lupus erythematosus.

Figure 2

Magnitude of overexpression of IFN-α/β-inducible gene signature in WB of 41 SLE patients in the initial study as measured by the median fold change of the 25 most overexpressed IFN-α/β-inducible genes (IFN-α/β-inducible gene signature score) in individual SLE patients. The horizontal bars represent the median values. Patients whose IFN-α/β-inducible gene signature score was >10 were considered to have high IFN-α/β-inducible gene signatures; those with scores between 4 and 10 were considered to have moderate IFN-α/β-inducible gene signatures, whereas those with scores < 4 were considered to have weak IFN-α/β-inducible gene signatures. IFN = interferon; SLE = systemic lupus erythematosus.

Figure 3

IFN-α/β-inducible genes in WB of SLE patients can be used to separate SLE patients with IFN-α/β-inducible gene signature from healthy normal controls. (a) Three-dimensional PCA plot of WB from 41 SLE patients in the initial study using the 110 upregulated IFN-α/β-inducible transcripts upregulated in WB of SLE patients compared with those from 24 healthy donors. (b) PCA plot of WB from 54 SLE patients in the prospective study using the same 110 upregulated IFN-α/β-inducible transcripts confirmed the overexpression of IFN-α/β-inducible gene signatures in SLE patients. (c) PCA plot of WB from 95 SLE samples in both discovery and prospective study using the 21 upregulated IFN-α/β-inducible gene panel in SLE patients compared with 24 healthy donors. Each point represents one sample (blue points: healthy normal controls; red points: SLE patients). IFN = interferon; PCA = principal components analysis; SLE = systemic lupus erythematosus.

Figure 4

TaqMan QRT-PCR confirmed the overexpression of IFN-α/β-inducible genes in WB of SLE patients. (a) Relative fold changes of 15 IFN-α/β-inducible genes (out of the 40 assayed) in SLE patients were compared with healthy donors (P < .05 for all). Averages of relative mRNA levels of genes in the pooled RNA from 24 healthy donors were scaled to 1 based on TaqMan QRT-PCR assays. Horizontal bars represent average fold change. (b) TaqMan QRT-PCR validation of overexpression of the 21-gene panel of IFN-α/β-inducible genes in WB of SLE patients as determined by whole genome array. The relative overexpression of 21 IFN-α/β-inducible genes in 2 SLE patients is shown via (left) microarray and (right) TaqMan assays. Correlation coefficients (r) between TaqMan QRT-PCR and microarray were 0.986 and 0.989 for patient X and Y, respectively. IFN = interferon; QRT-PCR = quantitative real-time reverse transcriptase polymerase chain reaction; SLE = systemic lupus erythematosus.

Figure 5

Relative expression of mRNAs and median fold changes (horizontal bars) of (a) type I IFN-α subtypes, (b) other members of the type I IFNs and IFN-α receptors, and (c) TNF-α, IFN-γ, and IFN-γ receptors in WB of SLE patients compared with healthy controls (P ≤ .05 for all). Averages of relative mRNA levels of these cytokines and their receptors in WB from 24 healthy donors were scaled to 1 based on TaqMan QRT-PCR assays. IFN = interferon; QRT-PCR = quantitative real-time reverse transcriptase polymerase chain reaction; SLE = systemic lupus erythematosus; TNF-α = tumor necrosis factor.

Figure 6

Representative heat map demonstrating anti-IFN-α, -IFNAR, and -IFN-γ mAb effects on healthy donor PBMC stimulated with serum from 1 SLE patient. Lane 1: SLE patient serum only; Lane 2: SLE patient serum plus reference antibody; Lane 3: SLE patient serum plus 10 μg/mL anti-IFN-γ mAb; Lanes 4–6: SLE patient serum plus increasing concentrations of anti-IFN-α mAb (0.1, 1, and 10 μg/mL); Lane 7: SLE patient serum plus 10 μg/mL anti-IFNAR mAb. Color represents relative neutralization (inhibition) of overexpression of individual genes upregulated by soluble mediators in the serum of an SLE patient. The red color represents no neutralization, and green represents neutralization of overexpression of individual genes. IFN = interferon; IFNAR = interferon associated receptor; PBMC = peripheral blood mononuclear cells; SLE = systemic lupus erythematosus.

Figure 7

Venn diagram illustrating the three primary analyses used in the selection process of 21 candidate PD markers for anti-IFN-α mAb therapy in SLE: (1) 807 IFN-α/β-inducible transcripts determined from ex vivo stimulation of healthy donor WB with 10 IFN-α subtypes and IFN-β (cyan region); (2) 110 transcripts found to be both overexpressed in WB of SLE patients and IFN-α/β-inducible in WB of healthy donors (combination of blue, yellow, and red regions); (3) 161 transcripts identified by ex vivo stimulation to be induced by SLE patient sera and subsequently neutralized by an anti-IFN-α mAb (combination of green, yellow, and red regions). The intersection of these three analyses provided a list of 77 transcripts, which were ranked by magnitude and prevalence across SLE patients (i.e., percentage of SLE patients with a fold change of at least 2) and the top 21 unique genes were chosen. IFN = interferon; SLE = systemic lupus erythematosus.

Figure 8

Stratification of 35 SLE patients into groups expressing low ((a) green), moderate ((b) gray), and high ((c) red) IFN-α/β-inducible gene signaturebased onmedian fold change across the 21-gene panel of IFN-α/β-inducible genes. Kernel density estimates (i.e., histograms or frequency plots) for each SLE individual are calculated and graphed using the fold change foreach of the 21 genes from each SLE patient on the log₂ scale to provide a representation of the distribution of 21 gene fold change values.The vertical dashed lines partition the 3 classes of IFN-α/β-inducible gene signature scores: 7 individuals with a weak IFN-α/β-inducible gene signature = median fold change <1.91 (0.93 on log₂ scale); 8 individuals with a moderate IFN-α/β-inducible gene signature = median fold change between 1.91 and 5.53; and 20 individuals with a strong IFN-α/β-inducible gene signature = median fold change >5.53 (2.47 on log₂ scale). IFN = interferon; SLE = systemic lupus erythematosus.