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.
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.
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.
EMA, Guideline on immunogenicity assessment of therapeutic proteins, European Medicines Agency. Retrieved from https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-immunogenicity-assessment-therapeutic-proteins-revision-1_en.pdf 2017, Committee for Medicinal Products for Human Use (CHMP) London, UK.
FDA, Immunogenicity Testing of Therapeutic Protein Products–Developing and Validating Assays for Anti-Drug Antibody Detection (2019), FDA, US. Retrieved from https://www.fda.gov/media/119788/download 2019: Center for Drug Evaluation and Research (CDER) and Center for Biologics Evaluation and Research (CBER), Food and Drug Administration.
Biopharma Excellence, Fit for purpose immunogenicity assays: what to consider during assay development and validation? (2021) https://www.biopharma-excellence.com/news/2021/3/2/fit-for-purpose-immunogenicity-assays-what-to-consider-during-assay-development-and-validation
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Wu B, Chung S, Jiang X, McNally J, Pedras-Vasconcelos J, Pillutla R, White JT, Xu Y, and Gupta S. Strategies to Determine Assay Format for the Assessment of Neutralizing Antibody Responses to Biotherapeutics. The AAPS Journal, Vol. 18, No. 6, November 2016
Wu BW, Gunn GR, Shankar G. Competitive ligand-binding assays for the detection of neutralizing antibodies. In: Tovey MG, editor. Detection and quantiﬁcation of antibodies to biopharmaceuticals: practical and applied considerations. Hoboken: Wiley; 2011. p. 175–92.
Wang J, Lozier J, Johnson G, Kirshner S, Verthelyi D, Pariser A, et al. Neutralizing antibodies to therapeutic enzymes: considerations for testing, prevention and treatment. Nat Biotechnol. 2008;26(8):901–8.
EMA, Guideline on bioanalytical method validation. Retrieved from https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf 2011, Committee for Medicinal Products for Human Use (CHMP), London.