Gene therapy has emerged as a groundbreaking approach for treating previously incurable diseases, from rare genetic disorders to more common conditions. Ensuring the safety and efficacy of these novel therapeutics requires robust validation of immunogenicity assays that determine whether a patient has pre-existing immunity to the adenovirus-associated virus (AAV) vectors commonly used in gene therapy, which can significantly impact treatment efficacy and safety. In an ever-evolving regulatory landscape, it is becoming increasingly important to understand how to properly develop and validate these assays for long-term success.
The regulatory requirements for gene therapy assays have evolved considerably since the first approvals. Luxturna®, approved in 2017, did not require a companion diagnostic (CDx). HEMGENIX® was approved in 2022 with a single-site laboratory-developed test (LDT) for its neutralizing antibody (NAb) assay. However, more recent approvals have been associated with requirements or post-market commitments for CDx assays that measure antibodies against the viral vectors used in these therapies (see Table 1).
Table 1. Regulatory requirements for recently approved gene therapies
This shift reflects both an increasing understanding of the importance of pre-existing immunity in predicting treatment outcomes and a growing emphasis on the importance of planning ahead for potential CDx requirements at the earliest stages of development.
The regulatory pathway for a clinical trial assay depends primarily on its intended use. If the assay is used for purely exploratory or secondary endpoints and no medical decisions are made using the assay data, a fit-for-purpose validation under good practice guidelines (GxP) standards is sufficient. However, if the results of the assay determine whether a patient receives treatment, the regulatory burden increases substantially.
The critical determination for the level of validation required is whether the assay is considered non-significant or significant risk (see Figure 1).
Figure 1. Regulatory requirements for a clinical trial assay
A non-significant risk determination allows for Clinical Laboratory Improvement Amendments (CLIA) validation, which includes studies such as reference range determination, analytical sensitivity, linearity (for semi-quantitative assays), precision/reproducibility, and sample stability assessment. A significant risk designation requires more extensive validation (more studies, samples, and replicates) under design control. Depending on the final outcome of legal proceedings concerning laboratory-developed tests (LDTs) in the US, an Investigational Device Exemption (IDE) submission and approval may be required for the assay. In this case, study designs should be discussed with the FDA through the Pre-IDE Q-submission process. Even if an IDE is not required, keep in mind that In-Vitro Device Regulation (IVDR) requirements in the EU require analytical validation at least as rigorous as what would be required for an IDE in the US, so it is important to keep those requirements in mind for clinical trials with sites in the EU.
The significant risk assessment is based on how the assay will be used in the context of the investigational therapeutic study. Key questions include:
It is worth noting that an assay initially deemed non-significant risk may later be reclassified as significant risk, reinforcing the importance of planning for potential regulatory changes.
When developing immunogenicity assays for gene therapies, several key considerations can impact long-term success.
First is the choice between a total antibody (TAb) assay, which measures binding antibodies, and a neutralizing antibody (NAb) assay, which measures only those antibodies that functionally inhibit viral transduction.
NAb assays are cell-based functional assays that typically take multiple days to complete and can process fewer samples per plate compared to TAb assays. They also tend to have higher variability and are more prone to interference from both endogenous and exogenous factors. TAb assays, on the other hand, are binding immunoassays that can be completed in a day and offer higher throughput, greater sensitivity, and better precision.
The correlation between these assay types is important but imperfect. On the one hand, only a subset of binding antibodies detected in TAb assays will neutralize the virus. On the other hand, some non-antibody factors may inhibit viral transduction in NAb assays, potentially leading to false positives that may not be clinically relevant. Thus, assay selection should be informed by empirical data specific to the AAV serotype and assay design, keeping in mind that the type of assay matters less than its clinical relevance.
Another crucial consideration is the assay format, as changing formats late in the development process will trigger significant rework. Qualitative assays, which yield a positive or negative response based on signal relative to background, allow for testing more samples per plate but provide less information about the degree of immunity. Meanwhile, semi-quantitative assays provide titer values that can be correlated with efficacy and allow for setting data-driven enrollment cutoffs but require more plate space for titration and fit fewer samples per plate.
When developing immunogenicity assays, there is often a temptation to maximize sensitivity by using the lowest possible minimum required dilution (MRD). However, this approach can compromise assay performance, particularly for cell-based NAb assays. Instead, developers should focus on other approaches to improve sensitivity, such as optimizing reagent concentrations and incubation times, using more sensitive detection platforms, or selecting sensitive substrates.
The choice and management of critical reagents is another key consideration. Changes in capsid expression systems (like switching from HEK293 to baculovirus), detection platforms, or matrix types can trigger additional development, validation, and clinical bridging studies. Consequently, it is essential to plan for the entire life cycle of critical reagents, with quality requirements becoming more stringent as development progresses (see Table 2).
Table 2. Lot and quality requirements for reagents by phase
Developers should also consider how to handle residual clinical samples, which can be invaluable as sources of biological matrix for difficult-to-source rare disease, starting material for preparing validation samples, or samples for clinical bridging studies, if required. Before using residual clinical samples, it is important to ensure proper patient consent and stability, with a stability testing regimen and storage in multiple aliquots to minimize freeze-thaw cycles.
Given that the validation requirements for gene therapy assays vary significantly based on their intended use and that risk assessment may change during development, it is advisable for developers to design their assays with a potential pivot to IVD or CDx in mind.
A core concept in diagnostic assays is the clinical cutoff, the result that drives medical decisions. It is essential to be thoughtful about the selection of the clinical cutoff, as changing later will require extensive additional analytical and clinical validation. For a semi-quantitative assay, this is a specific titer. For a qualitative assay, the clinical cutoff is essentially the limit of detection (LoD). Assay validation must specifically address this cutoff and include validation of samples both above and below it.
It is also important to understand the distinction between controls and validation samples as their different applications are subject to different requirements. Controls are used on every plate to determine acceptability. Ideally, positive controls are comprised of manufactured material to minimize lot-to-lot variability. Negative controls are typically a biological matrix, pre-tested to ensure no antibody response in the assay.
Validation samples, however, must be human samples, either from healthy donors with pre-existing immunity or from post-treatment samples. For IDE or CDx compliance, there are specific regulatory requirements to create a panel that can be used for analytical validation. For qualitative assays, the panel requires samples near the LoD, including negative, low positive, and moderate/high positive samples. For semi-quantitative assays, the panel requires samples near the cutoff for enrollment.
Strategies that can be employed to future-proof gene therapy assays include:
Figure 2. Time to IDE with and without early implementation of design control
Gene therapy assay development and validation require careful planning and consideration of current and future regulatory requirements. To stay compliant in this evolving landscape, developers should select a diagnostic partner with the right scientific, regulatory, and quality credentials who can support all phases from exploratory assay through CDx.
Precision for Medicine has developed immunogenicity assays for a broad range of AAV capsids. As expert contributors to the AAPS recommendations for gene therapy immunogenicity assay development, we ensure that every assay is fit-for-purpose, phase-appropriate, and compliant with the most rigorous regulatory standards. With a global footprint and a track record of more than 250 IVD and CDx regulatory filings, Precision for Medicine has supported gene therapy programs across AAV serotypes, assay types, and therapeutic areas. Precision for Medicine also developed, validated, and serves as the sole site for the HEMGENIX® global LDT.
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