Screening Assays for Anti-AAV Antibodies in Support of Gene Therapies: TAb or NAb?

Adeno-associated virus (AAV) vectors are a leading platform for gene delivery for the treatment of a variety of human diseases. Immunogenicity assessments are critical for predicting the safety and efficacy of gene therapies, often serving as inclusion/exclusion criteria for clinical trials. While multiple assay formats measuring different endpoints—specifically binding versus neutralizing antibody activity—are available for assessing immunogenicity, there is uncertainty about which assay(s) should be selected and when they should be implemented during drug development. Here, we evaluated both total antibody (TAb) and neutralizing antibody (NAb) screening assays for two AAV serotypes to determine their suitability for patient enrollment in clinical studies. 

Background 

TAb assays detect all binding antibodies generated in response to an AAV vector, regardless of whether they are functionally neutralizing. NAb assays, on the other hand, measure only those antibodies that functionally inhibit AAV transduction, typically via a cell-based in vitro assay that quantifies the ability of serum antibodies to block reporter gene expression in target cells transfected with AAV. Each of these assays has its pros and cons, and different AAV serotypes have varying tropisms, immunogenicity profiles, and capsid antigenicity, all of which can influence the performance and interpretation of these assays. 

To investigate these differences, TAb and NAb assays were compared for suitability as screening assays for clinical trial enrollment for AAV2- and AAV8-based gene therapies. 

Methods 

TAb assays 

Utilizing a sequential bridging ELISA format, AAV capsids were coated on microwell plates. After sample incubation, an HRP-conjugated anti-human antibody was used for detection of binding antibodies. For both AAV2 and AAV8, the resulting signal was proportional to the TAbs in the sample.  

NAb assays 

A cell-based assay was set up using HuH7 cells seeded and pre-incubated with a modified adenovirus serotype 5 (Ad5) vector used as a helper virus for AAV transduction. The AAV vector transgene encoded a luciferase reporter gene (AAV-Luc). AAV-Luc was mixed with serum samples and then added to the cells for transduction, and luciferase expression was detected in the absence of NAbs (see Figure 1). 

Figure 1. NAb assay scheme 

Results 

AAV2 serotype 

461 serum samples were screening using both the TAb and NAb assays: 

• 42% of the samples screened negative in both assays 

• 32% of the samples screened positive in both assays 

• 6% of samples that screened positive in the TAb assay did not demonstrate positivity for NAbs to AAV2 

• 20% of samples that screened positive in the NAb assay were negative in the TAb assay 

The Cohen’s kappa correlation coefficient was 0.49, denoting only moderate agreement between the TAb and NAb assays. 

AAV8 serotype 

474 serum samples were screened using both TAb and NAb assays: 

• 49% of the samples screened negative in both assays 

• 38% of the samples screened positive in both assays 

• 4% of samples that screened positive in the TAb assay did not demonstrate positivity for NAbs to AAV8 

• 9% of samples that screened positive in the NAb assay were negative in the TAb assay 

The Cohen’s kappa correlation coefficient was 0.74, denoting substantial agreement between the TAb and NAb assays. 

Discussion 

The concordance data presented here shows a moderate agreement for TAb and NAb assay results for AAV2 and a substantial agreement for AAV8. Since NAbs are a subset of TAbs, correlation is typically expected to be high, but may vary based on a range of biological, analytical, and methodological factors. Here, the TAb-/NAb+ samples may reflect the presence of non-antibody or other uncharacterized neutralizing serum components, the clinical relevance of which is not currently known. 

Key Takeaway 

These data demonstrate the suitability of either TAb or NAb assays as a screening tool for patient enrollment into AAV2 or AAV8 gene therapy clinical studies. TAb assays offer the advantages of short turnaround time, high throughput and robustness, and ability to detect antibodies that may impact transduction by different mechanisms. NAb assays are assumed to be better predictors of transduction outcomes and to be more clinically relevant but are more labor intensive and demonstrate higher variability with potential false positives due to non-NAb inhibitors.  

Thus, method suitability for improved selection of patients during clinical study enrollment should be determined for each AAV serotype under consideration and is best evaluated on a case-by-case basis. 

To learn more about developing anti-AAV antibody screening assays for gene therapies, contact us.