Viral vectors have become the most common method for delivering gene therapy; however, pre-existing humoral immunogenicity can potentially render a gene therapy ineffective.
Viral vectors—primarily adeno-associated virus (AAV)—have become the most common method for delivering gene therapy (GT). Although these vectors provide numerous benefits, pre-existing humoral immunogenicity—which has an estimated prevalence of more than 50%—can potentially render a GT ineffective. That raises the risk of administering treatments without any benefit to many individuals. Consequently, many developers of GT exclude individuals with pre-existing antibodies from treatment. In cases in which clinical trial sponsors choose to exclude patients with pre-existing antibodies to a GT product, the FDA recommends consideration of “contemporaneous development of a companion diagnostic” to detect such antibodies.
For a clinical trial, that question hinges on whether the assay is used to determine enrollment eligibility. Early-phase trials typically do not require a diagnostic, though pivotal trials may require an assay that has undergone regulatory review. For approval and commercialization, a companion diagnostic (CDx) will likely be required if antibody response will be a criterion for GT treatment.
For those who would develop a CDx, both of the “classic” antibody detection methods—the total antibody (TAb) immunoassay and the neutralizing antibody (NAb) assay—come with important considerations (Table 1). Essentially, TAb assays look for antibodies that bind to the viral vector, whereas NAb assays assess whether the antibodies that are present stop the GT from transducing target cells. As cell-based assays, NAb assays are more complicated and often more variable than TAb. NAb assays also are considered less sensitive than TAb and have lower throughput. With TAb assays, the drug product or an empty capsid is used to capture the antibodies; NAb assays require a reporter vector, which is commonly a version of the GT encoding luciferase. Notably, both types of assays require positive controls, with human-derived material needed to build a diagnostic assay.
Table 1. Methods to Detect Antibodies to Gene Therapy Vectors
TAb and NAb are not mutually exclusive; a subset of binding antibodies also neutralize, but even if an antibody binds but does not neutralize, it could adversely affect the GT product. Both types of antibodies can potentially affect Fc-mediated clearance or activate complement.
The 2 most common methods of building a TAb assay are either through direct antigen capture or through bridging (Figure 1). The former involves binding an AAV capsid to a plate, where it captures any anti-AAV antibodies that are present in the serum; a conjugated anti-species antibody then detects the anti-AAV antibodies. The bridging format is a species- and isotype-independent approach in which an antibody forms a bridge between a biotinylated capsid and another capsid conjugated to a signaling moiety (such as ruthenium); the complex is immobilized to a streptavidin assay plate.
Figure 1. Constructing a TAb Assay
Measurement of transduction of cells through a reporter vector is a common format in NAb assays. A luciferase construct allows measurement of transduction using readily available laboratory equipment. As Figure 2 illustrates, if a luciferase signal is visible in the cells after a period of incubation, it is a sign that the GT has transduced the cells. If neutralizing antibodies are present, inhibition of transduction is visualized by loss of luciferase signal.
Figure 2. Overview of a Luciferase-Based NAb Assay
Whichever assay one chooses, it is best to have empirical data. The type of assay is less important than correlating the degree of immune response with efficacy, as that can accurately determine which individuals may benefit from treatment.
Both TAb and NAb assays can either be qualitative or semi-quantitative. The former determines a positive or negative response based on signal relative to background, whereas the latter uses a titration approach to calculate a semi-quantitative result. Because titration of samples is not necessary with a qualitative assay, it can fit a large number of samples per plate. However, the qualitative approach is more conservative in that it could exclude all patients who have a positive response, even if it is a low-level or weak response.
The semi-quantitative approach has the advantage of correlating a numerical titer value to efficacy or adverse events. It also enables setting a data-driven cutoff for enrollment. However, titration requires considerable space for samples, limiting the number of samples per plate.
Both qualitative and semi-quantitative assays have performance verification challenges. These are greater with NAb than with TAb assays, as the former involves many more steps, and the cell line and live reporter virus may introduce additional biological variation.
When developing a diagnostic, a panel of samples must be prepared to evaluate assay performance.The panel should include samples within 20% above and below the cutoff. However, for assays that have a 20% or more variability, this poses a significant challenge. Therefore, it may be necessary to space the panel members slightly above or below the 20% cutoff range. That underscores the importance of a defined release process, such as testing each sample independently, to ensure an accurate result.
Table 2 outlines some of the performance verification requirements tied to the purpose of the assay. For companies seeking an Investigational Device Exemption (IDE), the assay may need additional verification depending on the risk determination. Recent reports of adverse events in certain gene therapy programs increase the possibility that the assay carries a significant risk determination. An in vitro diagnostic (IVD) submission for a CDx requires a higher level of design verification as well as validation studies that meet the standards of the Clinical and Laboratory Standards Institute (CLSI), which should be followed for diagnostics.
Table 2. Assay Performance Verification Requirements
The pyramid in Figure 3 summarizes some of the key implementation considerations for immunogenicity diagnostics.
Figure 3. Key Implementation Considerations for Immunogenicity Diagnostics
Starting at the bottom of the pyramid, it is important to budget sufficient time, given the typical timelines for CDx development (greater than 1 year following development of a prototype for TAb assays and 1.5 years for NAb assays). The TAb assay timeline is shorter than for NAb because the experiments are quicker, sample throughputs are higher, and there is no requirement for cell banking. Regardless of assay type, one should thoroughly characterize the assay prior to design verification studies to reduce the risk of unexpected results during those studies. Following design verification, the timeline for the final phase of CDx development, clinical validation, depends on the schedule of the associated clinical study.
The shorter timeline makes TAb particularly attractive to gene therapy sponsors that seek to develop a CDx. Nevertheless, regulatory agencies could request to see a NAb assay instead of a TAb, forcing the company to scramble to assemble the pertinent data and revise the timeline. Early discussions with regulatory agencies, coupled with timely collection of data and assessment of long-term assay needs, can help to expedite development. The sooner one can ascertain the regulatory landscape, the more efficient the process.
Continuing up the pyramid, the assay cutoff is the level of immunogenicity deemed clinically acceptable; verification/validation must be centered around this level, which should be supported by clinical data.
Design control should begin as soon as one determines the need for a diagnostic; this process (outlined in Table 3) is crucial for managing assay development and demonstrating adequate performance to support the assay’s intended use.
Table 3. Key Actions in the Design Control Process
Planning ahead for critical reagents is another key consideration, especially if Good Manufacturing Practices (GMP)-level reagents are needed. At a minimum, reagents should include a capsid for a TAb assay or a reporter vector and a cell line for a NAb assay. Either assay type also requires a negative matrix and a positive control. The needs for reagents vary by development phase. For an early-phase clinical trial assay, a single reagent lot is sufficient, and does not need to comply with GMP. A pivotal clinical trial assay generally requires two or three lots (unless one lot is available in sufficient quantity) and does not require GMP reagents for immunogenicity assays. Requirements for an IVD are more stringent: three reagent lots and compliance with GMP, or “GMP-like” with rigorous documentation and testing.
One should also retain specimens from early-phase trials to support IVD design verification studies. Pre-treatment specimens are useful for demonstrating background immunogenicity, whereas post-treatment specimens can help generate human-derived design verification samples. In either case, the sponsor should obtain informed consent to cover use of specimens for subsequent research studies and ensure sufficient sample volume.
The pivotal trial design should incorporate retention of early-phase specimens for diagnostic validation. Extra aliquots of serum from early trial participants can enable assessment of preexisting immunity in a rare disease population, but without those early specimens, such understanding can be elusive.
Even when there is a clear need for a CDx to an AAV-based GT, the company may be uncertain whether it should be NAb or TAb. It may thus make sense to prepare both assay types in parallel and collect results with both assays to determine possible correlation to efficacy in early phase trials. This can be costly, but it may save time.
Ultimately, the choice between NAb and TAb boils down to correlation with efficacy, as noted above. Sponsors that perform the due diligence of data collection, assay cutoff determination, design control, reagent planning, specimen retention, and timely discussions with regulators will likely find themselves proceeding down a faster regulatory pathway.
Precision for Medicine has the capabilities and expertise to help sponsors determine an effective path forward, design and validate immunogenicity assays, and develop these assays into companion diagnostics with full regulatory support.