Interferon beta (IFN-beta) have been used for treating many disorders with widespread clinical effects. However, like many other proteins, all IFN species are potentially immunogenic, particularly those produced by recombinant gene technology.
The reported frequencies and titers of anti-IFN antibodies varies significantly depending on various diseases, as does IFN preparations and administration, and the type of method used for detection of antibodies. Antibody-mediated decreased bioactivity of IFN (ADB) is increasingly recognised as a condition in which the clinical effect of prolonged continuous treatment with IFN is decreased or abolished. Wieslab has chosen to use a method that measures neutralizing antibodies (NAbs) against biological active interferon which is considered therapeutic significant.
IFN-beta is well known for the treatment of multiple sclerosis (MS). Presence of NAbs against IFN- beta has been shown to be associated with reduced treatment response, increased number of relapses, increased MRI activity and an increased disease progression.
All IFN-beta-based drugs can be tested:
Avonex (IFN-beta 1a)
Betaferon (IFN-beta 1b)
Rebif (IFN-beta 1a)
The treated patient cannot switch to alternative IFN-alpha treatment if NAbs against IFN-beta have developed since NAbs cross-react between the drugs.
The assay is based on an in vitro reporter cell (iLite), which tests whether the antibodies in patient serum can prevent IFN-beta from signaling via IFN-alpha receptor 1 (IFNAR1) expressed on the cell surface (see figure below). The signals from IFNAR1 activate a reporter gene (Firefly Luciferase) and the effect of the produced enzyme can be measured in an instrument. The method is very stable and repeatable and based on a cell cycle arrest and the cells can therefore be used straight from the freezer. Any NAbs cross-react between the drugs within the same interferon subgroup.
Test results are given as positive or negative.
The is a strong need for clinically useful, inexpensive and standardized screening assays, but attempts in this direction have failed despite the recommendation by WHO to use biological assays. A major reason for the lack of standardisation is that it is difficult and costly to perform complex antiviral neutralisation assays in the clinical setting. Some of these tests have not been validated for clinical relevance and therefore cannot provide very useful information to the clinician.
Several years of use of antiviral neutralisation assays have contributed to the conception of, and distinction between, “non-neutralizing binding antibodies” (BAb) and “neutralizing antibodies” (NAb) occurring in patients receiving IFN therapies. However, the concept has been further complicated by the frequent use of BAb assays that may give false positive results.
The concept of non-neutralizing BAb has considerably confused the interpretation of the clinical relevance of BAb to IFN-beta in MS patients. There are several reasons for this. One is the indiscriminate use of assays for BAb that is known to give false positive results, or report therapeutically irrelevant antibodies with low affinity. In such cases, the clinician assumes that a patient without specific antibody response to IFN-beta, and therefore without NAb, has developed “non-neutralizing” BAb to the interferon, which is misleading.
Binding assays such as ELISAs require the use of (mono) specific anti-human IFN-beta-antibodies, and do not give information of the bioactivity of the tested interferons. Unfortunately, the specificity and accuracy of these tests is often a problem, especially when testing biological samples, where soluble cytokine receptors and other factors can influence the test result and give a false positive result. Thus, using tests that measure the bioactivity of IFN-beta is highly relevant.
Cytopathic Effect or CPE assay is the most traditional bioassay which is based on measuring the natural anti-viral effect of IFN-beta by the ability of a sample containing IFN-beta to protect cells from exposure of otherwise toxic levels of encephalomyocarditis (EMC) virus.
Another well-known method is MxA assay based on measuring MxA mRNA in IFN-beta treated cell cultures which gives a correlation to IFN-beta levels in the sample. These methods are often complicated and difficult to use for routine diagnostic use.
Sampling should be done prior to the injection/infusion of the drug during the day of scheduled drug administration. The sample should be taken when the concentration of the drug is expected to be as low as possible (the "Cmin at steady state" or "trough" levels). "Cmin" corresponds to the minimum drug concentration at "steady state" when the variation of drug concentration in the serum is the lowest and provides the highest sensitivity for detection of NAbs. At detectable levels of the drug, the presence of NAbs is considered to be of low therapeutic significance.