Overall approach: stepwise immunogenicity assessment
Figure 3. Flowchart depicting the steps involved in assessing biologicals and biosimilars’ immunogenicity. If immunogenicity is established, the ADAs are titrated to determine the likelihood of an adverse reaction, or the dose at which the drug loses efficacy modified from the FDA guidance of immunogenicity testing 2019 document21).
Development of appropriate assays to detect and measure ADAs is important for assessing immunogenicity of therapeutic protein products during both preclinical and clinical studies. A multi-tiered ADA testing approach has been recommended by the U.S. Food and Drug Administration (FDA) (Figure 3) and widely used by therapeutic developers21.
A complete ADA assessment process entails the following steps: First, a screening assay is employed to detect the presence of ADAs in all relevant isotypes in treated patients. Samples that tested as positive (found to contain ADAs) are also put through confirmatory assays to eliminate false positives and demonstrate that the ADAs present in the samples are specific to the therapeutic protein. Positives are further scrutinized with neutralization, titration, and other assays that characterize the biophysical properties (e.g., titer, isotype, cross-reactivity, epitope specificity, etc.) of each samples’ ADAs. Immunogenicity is assessed by considering all of the collected pharmacokinetics, pharmacodynamics, efficacy, and safety data.
ADA assay techniques
Several biochemical and bioanalytical techniques have been developed for applications in different stages of ADA testing. ADA screening and confirmatory assays include the enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence (ECL) immune assay, radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), and surface plasmon resonance (SPR). These tests have the advantages of robustness, sensitivity, and compatibility with readily available reagents and instruments. However, it is worth mentioning that the latter are only as robust as their standards.
Neutralization assays measure NAb activity through non-cell-based or cell-based experiments (Figure 4). The former (e.g., competitive ligand binding assays, and enzymatic activity assays) directly measure the binding between a surface-immobilized drug and target or vice versa. In contrast, cell-based bioassays have various outcomes such as phosphorylation of intracellular substrates, cell proliferation, cell death, or production of a secondary protein22. Therefore, cell-based assays tend to provide a more representative readout of the therapeutic’s in vivo mechanisms of action and are preferentially recommended by regulatory agencies such as the FDA21.
According to the FDA, an ideal ADA assay is expected to reliably detect ADAs with sensitivity, specificity, selectivity, precision, and reproducibility21. Thus, it is critical to implement assays with reliable, reproducible, and relevant standards, in addition to setting up the appropriate assay formats, and utilizing the right instruments for the job in order to meet regulatory requirements.
Positive control antibodies can make or break an ADA assay
Figure 4. Illustration showing the basic principles of direct non-cell-based and cell-based assay formats to detect the presence of NAbs.
Positive control reagents that mimic the ADA response to the drug are indispensable to immunogenicity testing. These reagents typically consist of animal (e.g., rabbits)-derived pooled polyclonal antibodies (pAb) or human monoclonal antibody (mAb) reference panels against the target protein drug.
Such controls are important to determine tolerance and alerting to potential toxicity. But perhaps most importantly, they dictate the very validity and sensitivity of the assay. For instance, the FDA defines sensitivity of an ADA assay by the lowest concentration at which a positive control antibody consistently produces a positive result21. As such, positive control antibodies with higher affinity generally correlate with better sensitivity in contrast to lower affinity positive controls in the same assay.
To capture the complete immunogenic potential of a drug, an assay must assess the entire ADA response to said drug. But the latter can be difficult to accomplish when endogenously similar proteins exist, or when some of the drug remains in circulation. Such forms of interference lead to drug tolerance, which is defined in the lab as the maximum quantity of free drug that still results in a positive ADA readout. Beyond this limit, the assay may yield a negative readout that would falsely indicate the drug is not being bound by anti-drug antibodies (Figure 5).
Interference testing often requires different known amounts of positive control antibodies spiked into ADA-negative control samples in the absence or presence of different quantities of the protein therapeutic. Interference testing can thus be used to determine whether the biological drug will interfere with ADA detection21.
Unlike some other reagents and even assays that can be obtained from commercial sources (e.g., ELISA kits), control antibodies need to be tailored to each assay, and therefore require custom development. Due to the difficulty of acquiring enough human ADA for continued use, ‘surrogate’ positive controls are most frequently used. Such surrogates are pooled pAbs prepared from antiserum of immunized animals like rabbits. Alternatively, affinity-purified mouse or rat anti-human mAbs have been used6.
Figure 5. Illustration depicting how interference between free drug and ADA impedes proper interpretation of ADA assays – in this case, an ELISA.
Limitations of animal-derived positive controls
Animal-derived standards are prone to many challenges including issues in characterizing which epitope(s) is targeted by the ADA response as animal-derived and human ADAs typically have different binding patterns. Furthermore, due to the finite and variable nature of animal pAb production, new batches still require validation and characterization prior to clinical testing. And yet, use of individual mAbs may omit the inherent complexity of the ADA response, thus failing to provide clinically meaningful results.
Toward development of antibody reference panels
Another long-standing challenge of ADA assays is the lack of available relevant immunological reagents to the species being clinically assessed. The latter contributes to the paucity in ADA assay reproducibility that the scientific field has yet to overcome. In 2015, the first monoclonal antibody reference panel against recombinant human erythropoietin (EPO), a drug for treatment of anemia related to progressive kidney failure, was developed by the World Health Organization (WHO)23. A repertoire of nine human mAbs against human EPO were identified with defined characteristics including affinities (low to high), isotypes, epitopes, and neutralizing activities (available from NIBSC).
The development of this reference panel started with immunization of transgenic mice with human EPO. Upon serum titer screening, mice that tested positive were used for hybridoma generation, followed by further ELISA screening. Then hybridoma sequencing was performed to derive the sequence of each selected mAb for subsequent characterization24 (Figure 6). The performance of the antibody panel has been evaluated using different assays including ELISA, SPR, ECL, RIP, and biolayer-interferometry (BLI)23,25. Some platforms were able to detect the diverse repertoire of ADAs in the panel, clearly indicating the suitability and need for reference standards for improved performance in ADA assays.
Figure 6. Process and techniques used by the WHO for developing antibody reference panels against EPO24.
Generating antibody reference panels presents an important step forward for the standardization of immunogenicity testing to facilitate the monitoring of the safety and efficacy of therapeutic protein products across different assay formats. Now, reference reagents, with established ranges, are recommended by the European Medicines Agency (EMA)26 and are under extensive development by clinical groups and regulatory agencies27.
Challenges of antibody reference panels
Despite the compelling benefits, human mAb reference panels can be tedious, costly, and time-consuming to generate using the conventional techniques such as aforementioned transgenic mice and hybridoma sequencing technologies. The repertoire of ADAs, which is polyclonal, needs to be detected and profiled to mimic the immune response to the drug as closely as possible. However, none of these techniques provides a direct and efficient tool to identify an adequate number of reference ADAs in an appropriate timeframe. For timely approval of a protein therapeutic, ADA assays should be developed and validated within 18 months of filing with the FDA. This can be a significant hurdle for many scientists in the industry.