November 1, 2010 (Vol. 30, No. 19)

New Assay Evaluates Neutralizing Anti-IFN-β in Individuals Afflicted with Multiple Sclerosis

Groundbreaking news concerning multiple sclerosis (MS) appeared in the mainstream media earlier this year when CNN and others reported that Chander Raman and colleagues had identified two unique forms of MS, underscoring the unforeseen complexity of this disease. Their landmark publication focused on the role of T helper-1 (Th1) versus T helper 17 (Th17) cells in mediating MS progression.

Both of these Th cell types have been implicated in promotion of MS in the past, and the current study examined their individual roles in the response to IFN-β therapy. Using an established murine model of MS, the authors showed that successful responses to exogenous IFN-γ administration depended upon the presence of Th1-induced interferon gamma (IFN-γ). When MS was mediated by Th17-derived interleukin 17 (IL-17), and serum IFN-γ levels were low, IFN-β therapy was not protective.

Notably, this study went on to show that, prior to treatment, a subset of human MS patients exhibited high serum levels of IL-17 and IFN-β, which were shown to correlate with a subsequent poor response to IFN-β therapy. Therefore, serum IFN-β and IL-17 levels may be predictive biomarkers of the unique Th17-mediated form of MS.

In fact, the authors suggest that IFN-β has contrasting effects in different contexts: beneficial under Th1 conditions and detrimental under Th17 conditions. Most importantly, this may explain why some individuals do not respond appropriately to IFN-β therapy.

Nonresponders may produce endogenous IFN-β to counteract Th17-mediated inflammation rendering them less responsive to exogenous IFN-β. Alternatively, IFN-β may, in fact, act as a pro-inflammatory cytokine during Th17-mediated disease, which may explain the fact that IFN-β treatment can actually exacerbate MS in some cases.

If confirmed in additional human studies, these findings could significantly increase mechanistic understanding of MS disease development. Furthermore, this may eventually lead to improved approaches for treating MS patients. In particular, it is plausible that if the serum levels of IFN-β and IL-17 continue to show correlation with MS disease classification and response to IFN-β therapy, a simple blood test could be used to better direct treatment options in the future.

Although it will likely be some time before this data can be used to tailor therapeutic regimens for patients presenting with different MS subtypes, there are more immediate implications for monitoring IFN-β responsiveness in patients with an elevated IL-17/IFN-β versus IFN-γ profile. Specifically, this new phenotypic category of MS may be associated with previously identified genetic markers, or in cases where there is a development of neutralizing antibodies to the IFN-β therapeutic protein.

Bridging studies using retrospective samples collected over time from IFN-β responders and nonresponders would be useful to determine if these latest findings are associated with specific gene polymorphisms and/or the development of neutralizing antibodies against IFN-β. Not only would these studies be of interest to scientists analyzing the mechanisms of MS development and disease progression, they may eventually provide guidance to physicians for clinical protocols as well.

A great deal of research has been conducted in an attempt to determine the role of anti-IFN-β antibody development by MS patients, and the subsequent effects on their responsiveness to IFN-β therapy. The two types of anti-IFN-β antibodies produced are defined in functional terms.

Binding antibodies (BAbs) include any antibodies that can bind to IFN-β. Neutralizing anti-IFN-β antibodies (NAbs) are a subset of BAbs and have the ability to inhibit the biological activity of IFN-β protein, thereby potentially decreasing a patient’s responsiveness to IFN-β therapy.

In fact, several studies have shown the presence of NAbs in the serum of some MS patients who have failed to respond to IFN-β therapy. So, in addition to a skewed IL-17/IFN-β serum cytokine ratio, the presence of NAbs, therefore, suggests another mechanism by which patients may fail to respond to IFN-β therapy.

Research into the development and functional consequences of NAbs has produced a wide range of mixed results. PBL InterferonSource has developed an assay that helps analyze NAbs in MS patients. Even within the population of patients who respond positively to IFN-β therapy, NAb production represents a major concern since it may diminish therapeutic efficacy and, thus, warrants close monitoring. 

Choosing an Assay Format

Neutralizing antibodies to IFN-β would be expected to block the function of the cytokine. IFN-β is a secreted cellular protein that inhibits viral replication. Consequently, building a robust NAb assay actually begins with the development of a sensitive functional activity assay.

Viral challenge assays have become the primary method used to determine the amount of IFN-β in an unknown sample. Briefly, cells are exposed to the putative IFN-containing sample, incubated, and subsequently challenged with a cytopathic virus. Control cells that are challenged with a virus in the absence of IFN-β pretreatment undergo death, a process referred to as the cytopathic effect (CPE).

IFN-β pretreatment of cells prevents viral replication, thereby blocking CPE in a dose-dependent manner (Figure 1). After a sensitive antiviral assay has been developed, then anti-IFN-β antibodies produced in animals can be screened to identify candidates to be used as positive controls in NAb screening assays.

Figure 1. In this CPE assay, encephalomyocarditis (EMC) virus was used to kill the human lung carcinoma cell line A549. Cell density and EMC virus dilution were optimized to yield a good differential between the cell control (no virus) and the virus control (no IFN-β). Under these conditions, IFN-β protects the cells in a dose-dependent manner. The EC50 for IFN-β generally ranges from 0.5 to 2 units/mL.

NAb Control Antibody Candidates

During assay development, sensitivity, precision, and robustness are important parameters. However, the most challenging aspect of NAb assay development is preparation of a potent NAb to serve as a positive control. Since NAb activity cannot be determined a priori, the only way to evaluate candidates is through rigorous screening.

In Figure 2, a panel of polyclonal antibodies (PAb) was examined for their neutralization potential. When NAbs are present in a particular sample, the protective effects of IFN-β are blocked and CPE is observed.

Figure 2. IFN-β-1a dose-dependency on the neutralizing effects of anti-IFN-β Abs (Part I): In one format, PAbs were serially diluted and screened against a constant concentration of IFN-β-1a (10 IU/mL). Several candidate PAbs were identified by this approach. All PAbs displayed neutralizing activities in a dose-dependent manner, with PAb 31410-1 displaying best-fit neutralization. Since these antibodies were produced in rabbits using highly purified IFN-β, they were not capable of inhibiting other interferons when screened in specificity assays. Likewise, the antibodies were tested alone for any effects on the antiviral assay, and they all showed no interference. Thus these antibodies, particularly 31410-1, displayed ideal characteristics for use as a NAb assay control antibody.

Assay Precision

Many different parameters can affect the precision and reproducibility of a biological assay. Consequently, it is important to establish a robust assay that performs consistently in the hands of multiple investigators. The Table shows the results of one such study.

The results reported in this study highlight the testing of a panel of anti-IFN-β antibodies displaying varying neutralizing capacities. Of these, PAb 31410-1 exhibited the most potent neutralization capability and lowest CV rate. Our results also demonstrated that the NAb assay is highly reproducible, and produces comparable neutralization titers even when used by different investigators.

Inter-operator reproducibility is critical, as this would allow for direct comparison of patient results between multiple operators within one or more diagnostic laboratories. These initial studies are promising and warrant further expanded studies with MS patient serum samples to better determine the assay sensitivity, precision, and false-positive rate.

Clearly, the immune response in MS patients is not uniform. Individual variations in the types of T helper cells present, and the levels and potency of neutralizing anti-IFN-β antibodies present in patients can vary greatly and potentially impact the efficacy of IFN-β therapy.

The ability to accurately predict the likelihood of a given patient response to IFN-β therapy (i.e., not in the high IL-17/IFN-β MS subtype) would be of enormous clinical benefit given the high costs and potential side effects of continuous IFN-β therapy. Thus, having an assay that precisely and reproducibly evaluates the levels of neutralizing anti-IFN-β present in patient serum samples is a critical step toward achieving this goal.

NAb activity and assay precision

Ronald Jubin, Ph.D. ([email protected]), is director of research and development at PBL InterferonSource.

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