Cell-Based NAb Assays
So, how do we deal with NAbs when it comes to the industrial production of biotherapeutic agents?
The bioanalytical community adopts a tiered approach to tackle the problem of NAbs. Firstly, any study samples in question are first screened and confirmed for the presence of binding antibodies via an established immunoassay. If the sample tests positive for binding antibodies, it is then examined for any drug-specific neutralizing ability in a NAb assay. Standard immunoassays such as enzyme-linked immunosorbent assays (ELISA), electrochemiluminescent (ECLIA), and other assays can detect anti-drug antibodies, nevertheless fail to discriminate whether these antibodies are binding or neutralizing in nature. To detect NAbs, we would require a more specialized, in vitro, mammalian cell-based assay or non-cell-based competitive ligand-binding assays.
While non-cell-based assays are also in practice, the European Medicines Agency (EMA) and Food and Drug Administration (FDA) have explicitly indicated that cell-based assays shall be used whenever possible. This recommendation stems from the fact that cell-based assay platforms most accurately mimic the mechanisms by which NAbs may exert their effect in vivo. This distinction becomes even more significant in the case of therapeutic proteins that resemble an endogenous protein (for example, erythropoietin) where autoimmunity is a major concern resulting from the cross-reactivity of the NAbs that can critically impact patient safety.
Since cell-based assays use live cells to offer a relevant representation of a real-life model, they allow for tracking of dynamic variables such as the number of cells and their behavior. These assays can be used to assess a variety of biochemical and functional outcomes including:
- cell proliferation
- apoptosis and cell death
- signal transduction
- specific enzyme activity
- reporter gene activity
- antibody and complement-dependent cytotoxicity
A functional read-out of compound activity can be derived from the live cell-based assays which makes them more biologically and physiologically relevant.
With all their salient features, cell-based NAb assays can be difficult to develop and validate because the type of assay (indirect vs direct) and their format can depend on the complete knowledge of a drug’s mechanism of action and agonistic or antagonistic properties. Since each assay needs to be target-specific, sensitive, minimally affected by components in the matrix, reproducible, and able to detect the NAb in the presence of the biologic agent (i.e. drug tolerance), the assay development process becomes even more cumbersome.
Direct assays are used for drug products that show their effect directly on a cell. For instance, the responses such as receptor phosphorylation, increase in cytoplasmic ATP, cAMP, mRNA synthesis, phosphorylation of cytoplasmic proteins via specific cell-signaling pathways, cytokine production, or cellular proliferation can be targeted by direct assays. These assays determine the NAb activity based on the principle that in its presence, the NAb either hinders or abrogates the otherwise detectable responses based on the aforementioned signaling pathways.
On the other hand, when a drug exerts its effect by blocking the binding of a ligand to a specific cell-surface receptor, indirect assays are used. Usually, indirect assays are deployed for monoclonal antibodies or soluble receptors. If NAbs are present and active, it is expected that there is an alteration of a particular cellular response associated with that cell surface receptor.
With these fundamental understandings of cell-based NAb assays, we can make informed decisions to develop a suitable assay tailored for our needs. This is particularly helpful to choose a proper cellular response (endpoint method) which needs to be specific and offer sufficient sensitivity to measure the cellular response mediated by the drug of interest. Another important factor is to determine the assay cut point- the response level at which a sample is either positive or negative. With cell lines determined and assay endpoints chosen, assay parameters like drug concentration, cell density, ligand concentration must be optimized. Finally, the NAb assay needs to be validated to confirm its analytic reproducibility, specificity, and sensitivity, among other important assay performance characteristics.
In a nutshell, cell-based NAb assays are one of the best procedural techniques for studying the new drug candidates and their biological effects. Understanding the basic principles and features of these essays can hence empower us to choose the suitable platform for us to adopt and utilize the strategies we need to develop a proper NAb assay.
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