Neutralizing Antibody Assay Development – a key consideration for immunogenicity packages

by Jennifer Sales

Therapeutic application of biological drugs is often accompanied by an unwanted immune reaction from the patient, which can result in the production of Anti-Drug Antibodies (ADAs). These ADAs can cause a spectrum of clinical effects, ranging from benign to fatal. It is therefore imperative to assess new products for their immunogenic potential, and its subsequent effect on the products ’s pharmacokinetics, pharmacodynamics, safety and efficacy. Due to the possibility of severe clinical side-effects, regulatory agencies require a risk-based approach to immunogenicity evaluation (1,2). This should include a multi-tiered testing strategy, to be implemented during clinical trials, more information on which can be found here (3).   

However, the mere presence of ADAs is not the only factor that should be taken into account when considering the clinical impact of immunogenicity. One subclass of ADAs, the Neutralising Antibodies (NAbs), not only bind to the therapeutic product, but also inhibit its in vivo function. Therefore, the extent of this neutralisation capacity as well as the titre of the Nab should be assessed, as a low titre Nab with a strong neutralising action may have a greater clinical impact than a higher titre Nab that only slightly mitigates the therapeutic product’s mechanism of action (MoA).  

The selection of an assay format to measure the action of NAbs is considered to be somewhat more complicated than the other levels of the tiered immunogenicity approach. NAb assays can typically be classed as either cell-based or non-cell-based (also sometime referred to as Competitive Ligand Binding (CLB) assays). Although cell-based assays have historically been preferred by regulatory agencies, proper justification may allow for deviations from this format. The final selection should by informed by three principal considerations: i) the therapeutic MoA of the drug product, ii) the evidence of desirable assay performance characteristics, and iii) the predicted risk of immunogenicity. While the immunogenic risk should still inform the schedule for sampling (4), the therapeutic MoA carries the most weight when deciding upon an assay format, and should be considered in the first instance. A summary of generalised MoAs and the appropriate assay format for each is shown in Table 1.

Table 1: Recommended assay formats by MoA for various therapeutic protein classes // mAb = monoclonal antibody; MoA = mechanism of action

Cell-based assay formats

Cell-based assays give a more accurate picture of the entirety of physiological effect of NAbs in biological systems, rather than only a snapshot of the NAb-drug interaction. These assays can be further classified, depending upon whether the drug directly targets a cell surface molecule (direct assay, Fig. 1a), or binds to an extracellular ligand that would otherwise induce a cellular response (indirect assay, Fig. 1b). For both assay formats, the choice of endpoint to be detected is a crucial factor (5). As a variety of downstream events may occur, such as receptor phosphorylation, cytokine release, cell proliferation or death. Care should be taken to select a process that shows a robust and measurable response, and fits with desirable assay parameters such as sensitivity, specificity and matrix/drug tolerance. Cell-based assays should particularly be considered for products including agonists, monoclonal Abs (including those with effector function) against cellular receptors, and Ab-Drug Conjugates (ADCs).
 

Non-cell-based assay formats

Alternatives to cell-based assays, such as CLB formats, may be considered for NAb assessment if a) the MoA of the biologic product targets a soluble ligand or b) a reliable cell-based assay cannot be developed, for example due to lack of a suitable cell line or susceptibility of the cells to matrix interference. It has also been shown that in some cases CLB assays outperform cell-based assays in parameters such as sensitivity, dynamic range, and precision (6). CLB assays rely on the detection of either the drug or a labelled ligand binding to its cognate receptor, which can either be sourced as a recombinant protein or from a cell membrane and can again be classified as either direct or indirect (Fig. 1c, 1d). A reliable supply of this target molecule is one of the critical considerations when designing a CLB assay. 

The alternative form of non-cell-based assay is used to assess NAbs against Enzyme Replacement Therapies (ERTs). Although these have a relatively high risk, as any NAbs could cross react with similar endogenous proteins, their MoA can be measured with an enzymatic assay, which does not require a cellular component (7). Other drugs with MoAs measurable by non-cell-based assays include mAbs against humoral targets and antagonists against soluble receptors.

Figure 1: the basic principles behind direct and indirect cell-based or Competitive Ligand Binding (CLB) assay formats to detect the presence of NAbs. Modified from Wu et al. 2016.

Once the MoA and risk assessment of the therapeutic have been taken into account, any assay should undergo thorough validation to ensure it meets the performance characteristics as stated by the regulatory authority guidelines (8). The potential role of immunogenicity in pharmacodynamics, pharmacokinetics, clinical safety and efficacy, and the choice of assays to assess it, can thus have a critical impact on the success of regulatory submissions. 

Our team of regulatory and bioanalytical experts will be happy to help you overcome your challenges in developing and validating assays for immunogenicity assessment. If you would like us to guide you, feel free to contact us and streamline your development program. 


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