Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple immune system abnormalities, including production of autoantibodies that can lead to inflammation and tissue damage 1. SLE symptoms can range from a mild rash to life-threatening nephritis and central nervous system disease. These disease manifestations cause a significant burden of illness and can cause reduced physical function, loss of employment, lower health-related quality of life, and a lifespan shortened by about 10 years 2. Increased hospitalizations and side effects of medications including chronic corticosteroid and other immunosuppressive treatments add to the disease burden in SLE 2. No new treatment for SLE has been approved by the US Food and Drug Administration in about 50 years since hydroxychloroquine was approved for use in discoid lupus and SLE; otherwise, the existing standard of care for SLE consists of off -label medications.

The disease pathogenesis of SLE includes activation of innate immunity, with increased production of type I interferons, including IFNα, and increased plasmacytoid and myeloid dendritic cells in involved tissue 345678. Specific immunity, including both humoral and cellular immune systems, is activated. Autoantibodies are universally present and may precede development of clinically apparent disease 9. SLE-associated autoantibodies include anti-dsDNA, anti-nucleosomes, anti-RNP (ribonucleoprotein complex), and anti-Sm antibodies. Immune complexes containing anti-dsDNA or anti-RNP antibodies can activate type I interferon production 34. After internalization through Fc receptors, autoantibody-containing immune complexes bind endosomal Toll-like receptors 7 and 9, and stimulate production of type I interferon. Type I interferon stimulates myeloid dendritic cell maturation, which promotes loss of tolerance and generation of autoreactive T cells and B cells, autoantibody production, immune complex formation and further production of type I interferon, creating a self-perpetuating cycle of autoimmunity 51011.

Type I interferons include 14 IFNα family members, IFNβ, IFNτ, IFNκ and IFNω 12. This cytokine family regulates immune functions of cellular components of both innate and adaptive immune systems, including dendritic cells, T cells, B cells, and natural killer cells. For example, type I interferons promote dendritic cell maturation, memory CD8⁺ T-cell proliferation, natural killer-cell activation, and B-cell differentiation 513. Type I interferons also enhance the expression of immunologically important molecules such as MHC class I, CD38, interleukins such as BLyS, IL-6, IL-10 and IL-15, and multiple chemokines 14151617.

Emerging data indicate a role for type I interferons in disease pathogenesis in SLE. Genetic polymorphisms associated with type I interferon pathways are associated with susceptibility to SLE 1819. Treatment with IFNα has been associated with the development of autoantibodies and new or worsening clinical features of the SLE 2021. Higher IFNα levels and type I interferon activity are associated with greater disease activity in SLE 37. Patients with high anti-dsDNA antibody titers, lupus nephritis, and progressive skin rashes have high serum levels of IFNα 3. In addition, patients with acute skin involvement tend to have elevated IFNα in blood and skin 7.

These clinical observations in humans are supported by data that show a key role for type I interferon in animal models of SLE. IFNα can induce glomerulonephritis in normal mice and accelerates the onset of the spontaneous autoimmune disease of NZB/W mice 22. Autoimmune-predisposed mice deficient in the IFNα/β receptor exhibit significantly reduced anti-erythrocyte autoantibodies, hemolytic anemia, anti-DNA autoantibodies, kidney disease, and mortality 23. Furthermore, IFNα kinoid vaccine has been shown to induce neutralizing antibodies to prevent clinical manifestations in a lupus flare murine model 24.

Together, these human and animal data support the hypothesis that inhibiting type I interferon may reduce disease activity in SLE.

Molecular signatures or mRNA expression patterns in the periphery and disease tissues have been used to identify novel therapeutic targets and to predict patients that might respond favorably to therapeutic interventions 25262728. One distinct observation in SLE is the robust and prevalent overexpression of type I interferon-inducible mRNAs (type I interferon signature) in the blood and involved tissues of patients. Increased expression of mRNAs induced by type I interferon is prominent in peripheral blood mononuclear cells and in whole blood (WB) in approximately 60% of SLE patients, and is associated with greater disease activity 293031323334. Type I interferon is the most activated signaling pathway and type I interferon-inducible mRNAs are the most overexpressed mRNAs in WB of patients with SLE 35. Skin biopsies from a subset of patients with SLE also show increased expression of a type I interferon signature 363738. Expression of type I interferon-inducible IP-10/CXCL10 mRNAs is increased in SLE patients with active central nervous system symptoms 39. Selected type I interferon-inducible mRNAs (IFI44, IFI27, and IFI44L) and type I interferon-inducible proteins (IFI27, STAT1) are overexpressed in the synovium of SLE patients with arthritis 40.

Detection of type I interferon proteins by ELISA has yielded positive results in only a small population of SLE patients 41, whereas detection of overexpression of the type I interferon signature is positive in about 60% of patients overall. This discrepancy suggests the type I interferon signature is the more sensitive measurement to detect increased type I interferon activity in SLE. The type I interferon-signature thus provides a potential pharmacodynamic (PD) marker to evaluate inhibition of the molecular target and to help guide dose selection for anti-type I interferon therapies in SLE.

In addition to SLE, overexpression of the type I interferon signature has been reported in the peripheral blood of patients with a variety of inflammatory and autoimmune diseases that include dermatomyositis, polymyositis, multiple sclerosis, rheumatoid arthritis, scleroderma and Sjögren's syndrome 4243444546. The presence of increased numbers of plasmacytoid dendritic cells and overexpression of type I interferon-inducible mRNAs and/or proteins has also been reported at the disease tissues in some of the diseases 4748.

Figure 1 shows a heatmap of the relative expression of 807 type I interferon-inducible mRNAs (identified by ex vivo stimulation of healthy donor WB with type I interferon proteins, as described in 35) in WB of 106 SLE patients, 22 dermatomyositis patients, 20 polymyositis patients, and 66 rheumatoid arthritis subjects compared with 24 normal healthy control individuals. For each autoimmune disease (blocks of columns), the subjects (individual columns) are sorted, from left to right, from those showing the greatest overexpression of the 807 type I interferon-inducible mRNAs (defined as the median fold change for each of the 807 mRNAs versus healthy controls; rows) to those with the least expression. When compared with the 24 normal healthy control individuals, a subpopulation of patients within each of these autoimmune diseases demonstrated overexpression of the type I interferon signature in WB.

The evidence that the type I interferon signature is highly overexpressed in peripheral blood and tissue in multiple autoimmune diseases raises an important question as to whether type I interferon is involved in the pathogenesis of these diseases, and whether the activation of this signaling pathway is shared among the diseases. One key issue that needs to be addressed is whether activation of the type I interferon signaling pathway is concordant in WB and disease tissues of patients with these autoimmune diseases. Further more, it remains to be seen whether targeting type I interferon could benefit a subgroup of patients with distinct rheumatic diseases where the different disease pathogenesis and manifestations are different.

Sifalimumab is a fully human IgG_1κ monoclonal antibody that was generated by Medarex in mice transgenic for human immunoglobulin genes. The type I interferon signature was chosen as a potential PD marker to test whether sifalimumab inhibited its intended target, IFNα, in early clinical trials in SLE. This choice was made because increased expression of type I interferon-inducible mRNAs is a direct molecular consequence of increased expression of IFNα proteins, and because type I interferon-induced mRNAs are easy to measure by either RT-PCR or microarray-based technologies and demonstrate concordant overexpression in blood and involved tissue in SLE 38.

A three-tiered approach was taken to develop a panel of type I interferon-inducible mRNAs as potential PD markers for sifalimumab in SLE. First, the prevalence and magnitude of the overexpression of type I interferon mRNAs was determined in WB of SLE patients. WB samples from healthy donors were stimulated ex vivo with each of 10 IFNα subtypes or IFNβ, and then mRNAs (n = 807) were identified that were uniformly upregulated.

Secondly, mRNA expression was measured in the WB from 41 SLE patients and compared with mRNA expression in the WB of 24 normal healthy controls, using significance analysis of microarrays and the false discovery rate. Overall, 110 of the overexpressed mRNAs in the WB of these SLE patients were type I interferon-inducible, with type I interferon-inducible mRNAs among the most overexpressed mRNAs in the WB of SLE patients. The type I interferon signaling pathway was the most activated pathway in WB of these SLE patients. These results were confirmed in an independent set of 56 SLE WB samples.

Finally, mRNAs were identified whose expression was both stimulated when healthy donor peripheral blood mononuclear cells were exposed ex vivo to SLE patient sera and inhibited by sifalimumab in a dose-dependent manner in the same culture system. Overall, 77 type I interferon-inducible mRNAs were identified as overexpressed in each of the three different experiments. A subset of 21 of these 77 mRNAs was selected to be used as candidate PD biomarkers for sifalimumab in SLE, based on the magnitude and prevalence of overexpression in SLE 35. In the end, this three-tiered approach yielded a small, robust panel of type I interferon-inducible mRNAs that could be used in a high-throughput PD biomarker assay to evaluate target inhibition by sifalimumab in SLE trials.

The 21-mRNA panel demonstrated many desirable characteristics for a PD biomarker. Overexpression of the mRNAs within this panel provides a sensitive and specific marker for overexpression of type I interferons within WB of SLE patients 3538. Elevated expression versus normal expression of this mRNA panel was a consistent characteristic of an individual SLE patient, in the absence of therapeutic intervention with sifalimumab 29303132333435, which allows normalization of expression of this PD biomarker to be interpreted as treatment effect. Expression of the mRNAs within this panel can be measured quantitatively by multiple assays 35, allowing flexibility of the approach during clinical testing.

The level of expression of this mRNA panel calculated as a type I interferon signature score (Figure 2) in an individual subject provides a continuous measurement that can be used to classify subpopulations of SLE patients. The distribution of this type I interferon signature score in the WB of SLE patients exhibited a bimodal distribution, with relatively few SLE patient scores falling between subpopulations with normal levels and increased levels of expression (moderate and high over expression) of the type I interferon signature (Figure 2). This ability to differentiate between SLE patients with increased and normal expression of the type I interferon signature suggests potential utility of the type I interferon signature as a possible diagnostic marker to identify a subpopulation of SLE patients with increased expression of type I interferon. This hypothesis remains to be validated in clinical trials.

The safety and tolerability of sifalimumab were evaluated in a phase 1 trial in 62 mild-to-moderately active adult SLE subjects who were receiving standard-of-care therapy 3849. The trial evaluated intravenously administered sifalimumab over a dose range of 0.3 to 30.0 mg/kg, compared with placebo. The phase 1 trial integrated translational research to evaluate PD effects of the drug and downstream effects of inhibiting IFNα in SLE.

Sifalimumab caused specific and dose-dependent inhibition of overexpression of the type I interferon signature in WB of treated SLE subjects (Figure 3). Concordant overexpression of the type I interferon signature was observed in the majority of subjects who had matched WB and lesional skin biopsies 38. Seven out of eight patients who exhibited positive overexpression of the type I interferon signature in WB and skin lesions showed a similar trend of target neutralization in both sites at day 14 post sifalimumab or placebo treatment 38. Inhibition of expression of both type I interferon-inducible mRNAs and protein occurred in skin lesions of SLE subjects, as measured by TaqMan quantitative RT-PCR and immunohistochemistry in subjects treated with sifalimumab. Reductions of plasmacytoid dendritic cells, myeloid dendritic cells, CD4⁺ T cells and dendritic cells were detected in the lesional skin of SLE patients, along with reduced levels of type I interferon-inducible mRNAs and proteins in patients who had skin biopsies that responded to sifalimumab treatment (Figure 4). These changes were not observed in the majority of the placebo-treated patients or in some subjects who did not respond to sifalimumab treatment, as evaluated by immunohistochemistry 38. In a small number of SLE subjects with overexpression of mRNAs of B-cell activating factor belonging to the TNF family and activation of TNFα, IL-10, IL-1β and granulocyte-macrophage colony-stimulating factor signaling pathways in WB or skin lesions - as assessed by both TaqMan quantitative RT-PCR and microarray - a trend toward inhibition of the expression of these mRNAs was observed in response to sifalimumab treatment.

Results from this phase 1 trial showing dose-dependent inhibition of the type I interferon signature in the WB of SLE patients confirmed the mechanism of action of sifalimumab. The combination of concordant inhibition of the type I interferon signature in WB and involved skin and a reduction of type I interferon-inducible protein expression in skin in a phase 1a trial support further testing of sifalimumab in SLE. Genentech has recently shown in a phase I trial a similar dose-dependent inhibition of the type I interferon signature in the peripheral blood of SLE patients that followed either a single dose or repeat doses of rontalizumab, a humanized IgG1 monoclonal antibody that also inhibits human IFNα 50. These results con firmed the potential usefulness of using the type I interferon signature as a PD marker to evaluate activity of anti-IFNα therapy in SLE. Furthermore, these studies showed that expression of the type I interferon signature in WB reflects involved tissue in SLE, and raised the possibility of testing the type I interferon signature as a potential predictive biomarker to identify a subset of SLE patients who may preferentially respond to anti-IFNα treatment.

dsDNA: double-stranded DNA; ELISA: enzy me-linked immunosorbent assays; IFN: interferon; IL: interleukin; PCR: polymerase chain reaction; PD: pharmacodynamic; RT: reverse transcriptase; SLE: systemic lupus erythematosus; TNF: tumor necrosis factor; WB: whole blood.

All authors are employees of MedImmune.