Protein-based biologic agents have the potential to induce a variety of immune responses from the body, ranging from very minor to life threatening. One such adverse immune response is generation of neutralizing antibodies (NAbs), which can bind to the biotherapeutic drug product administered and prevent it from performing its intended biologic function, in turn negatively impacting the product’s efficacy and safety profile.
While standard immunoassays can be used to detect drug-specific antibodies including NAbs, they cannot distinguish NAbs from any other antibodies found. So what can we use to determine if any antibodies detected by an immunoassay in fact have a neutralizing capability?
Cell-Based Assays are Ideal to Detect NAbs
Although complex, cell-based neutralizing antibody assays are the industry’s preferred method to detect the presence of NAbs because they best mimic the mechanism by which the NAbs exert their effect within a living biological system. These assays assess intracellular signaling events triggered by the drug product within the cell line or the ligand inhibited by the drug product, and may also include study of extracellular binding events at the cell surface. While there are factors that would justify use of a non-cell-based NAb assay, such as a drug product with blocked ligand activity or limited choice of appropriate cell lines, US FDA and European Medicines Agency (EMA) both generally consider cell-based assays more reflective of the situation actually taking place in the body and recommend them whenever possible.
Considerations for Cell-Based NAb Assay Design
The development of cell-based NAb assays requires thorough planning and thoughtful assay design in order to produce optimal results in a cost-efficient manner. For example, the selection of a direct or indirect assay should be determined with an understanding of the biotherapeutic drug product’s mechanism of action, or results will not be sufficient to understanding NAb effects on clinical results.
Identifying the positive control antibody is also vital of developing a cell-based NAb assay – one that is capable of neutralizing the drug of interest to assess the assay’s sensitivity. Negative control, ligand control, and drug control are also significant in the validation of NAb assays. Each control is used to help validate a different aspect of the assay and each provides information that will be important in the study.
Perhaps above all, it is critical to select a suitable, stable cell line that produces a specific response to the drug product, and that the platform used provides the appropriate sensitivity level and dynamic range needed to measure that specific response. At the end of the day, the ability to detect NAbs relies on the scientist’s ability to detect a change in the assay-specific cellular response, and therefore the choice of endpoint readout is of high importance. Cellular responses can be categorized as either early (receptor binding; receptor phosphorylation; detection of cAMP or ATP; signal transduction-specific protein phosphorylation) or late (cellular proliferation or apoptosis; cytokine production; reporter gene expression events) and can include a myriad out of outcomes such as phosphorylation of intracellular substrates, calcium mobilization, proliferation, or cell death.
A Tiered Approach to NAb Detection Using Cell-Based Assays
When using a cell-based assay to evaluate NAbs as a part of an immunogenicity study, our team here at BioAgilytix uses a tiered approach. First, the sample is screened using a ligand binding immunoassay to detect the presence of any anti-drug antibodies (ADAs). Positively screened samples are re-analyzed in a confirmatory assay, and confirmed positive ADA samples may be further characterized for their neutralizing capacity, binding affinity, isotyping, and other characteristics. When it comes specifically to NAb analysis, we determine the most appropriate format based on mechanism of action and all the other factors noted above, using our extensive cross-platform experience to develop cell proliferation, biomarker, gene expression, gene reporter, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement dependent cytotoxicity (CDC) assays that meet NAb detection requirements with high specificity, sensitivity, and precision.
Examples of NAb assays developed or validated at BioAgilytix include:
- Reporter Assays (Luminescence)
- Growth Factor Stimulated Phosphorylation (MSD)
- Growth Factor Stimulated Proliferation (Celltiter Glo)
- Compliment Dependent Cytotoxicity (MTS)
- IFN-gamma release (ELISA)
- Labeled drug binding (FACS)
- Competition binding (ELISA)
The Value of Cell-Based Assays
Because they use live cells, cell-based assays are able to produce results that are more biologically relevant or that have physiological relevance – which is key to understanding how NAbs impact an administered biotherapeutic. Although using cell lines adds layers of complexity, it provides a clearer picture of what is actually occurring in the body and how to address it, and with the right cell-based assay expertise you can gain a deeper understanding of drug product safety and efficacy.
In our upcoming blogs, we will take a closer look at the use of cell-based potency assays for lot release testing under GMP, and how BioAgilytix is utilizing cell-based assays to bring new insights at the discovery phase of development.
If you would like more information on BioAgilytix’s cell-based assay capabilities, speak to one of our scientists today.