ASSAYS FOR ANTIBODIES
Assays are probably an important factor influencing the reported incidence of antibody induction by therapeutic proteins. In the published studies with interferon a-2 in patients with viral infections the incidence of antibody induction varied from 0% to more than 60% positive patients. This variation must be assay related. Evaluations of the performance of different test laboratories with blind panel testing showed a more than 50-fold difference in titers found in the same sera. Thus, any reliable comparison between different groups of patients when looking for a clinical effect of antibodies or studying factors influencing immunogenicity can only be done if the antibody quantification is done with in a well-validated assay in the same laboratory.
There is obviously a lack of standardization of assay methodology. There are also only a few reference and/or standard antibody preparationsavailable. Recently, a number of white papers have appeared mainly authored by representatives of the biotechnology industry in the United States (Mire-Sluis et al., 2004). Although the area of biotechnology-derived therapeutics is still too much in development to formulate a definite assay methodology, there is a growing consensus on the general principles.
There is agreement that a single assay is not sufficient to evaluate the immunogenicity of a new protein drug, but a number of assays need to be used in conjunction. Most antibody assay strategies are based on a two-tier approach: a screening assay to identify the antibody positive sera followed by further characterization such as whether the antibo-dies are neutralizing and what is the titer, affinity and isotype.
In general, the screening assay is a binding assay, mostly an ELISA type of assay with the radioimmune-precipitation methodology as an alternative. Binding antibodies have mostly no biolo-gical consequences. However, assays for the more biologically important neutralizing antibodies are in general cumbersome and expensive. Thus, screening with a binding assay to select the positive sera for the neutralizing assay saves time and money.
Screening assays are designed for optimal sensitivity to avoid false negatives. For new proteins defining an absolute sensitivity is impossible because of the lack of positive sera. An alternative approach is to set the cut-point for the assay at a 5% false-positive level using a panel of normal human sera and/or untreated patient sera representative of the groups to be treated.
The assay for neutralizing antibodies is in general a modification of the potency assay for the therapeutic protein product. The potency assay is in most cases an in vitro cell-based assay. A predefined amount of product is added to the serum and a reduction of activity evaluated in the bioassay.
An important caveat in interpreting the neutra-lization assay results is the possible presence of inhibitors of the products other than antibodies (e.g., soluble receptors) in human serum, or factors stimu-lating the bioassay which may compensate for the neutralizing activity. To overcome these problems, patient serum should also be tested as control. IgG-depleted serum should also be tested for neutralizing activity to identify neutralizing factors other than antibodies. Further characterization of the antibodies may include evaluation of Ig isotype and affinity.