(W1130-02-11) Comparison of a Microfluidic Generic Total Antibody (TAb) Assay with Independent Neutralizing Antibody (NAb) and TAb Assays for the Detection of Pre-existing Antibodies against AAV Vectors

Purpose: Screening participants in clinical trials involving adeno-associated virus (AAV) vectors is essential to find individuals without pre-existing neutralizing antibodies that can restrict the effectiveness of AAV gene therapies. Various methods have been developed to screen for anti-AAV antibodies. Neutralizing (NAb) assays assess how NAbs and other non-antibody factors hinder AAV transduction as measured by the expression of a reporter gene mediated by the AAV therapy. Meanwhile, TAb assays detect all capsid-binding antibodies, regardless of their ability to neutralize. The TAb assay, utilizing usually direct coat or bridging binding formats, stands out for its simpler design, lower variability, and ease of operational monitoring. However, TAb assays demand the creation of serotype-specific assays or AAV capsid labeling (when bridging format is used), steps that consume substantial time but also introduce result variability due to diverse labeling protocols. We developed a generic anti-AAV TAb immunoassay on the automated Gyrolab^® platform that enables the detection of anti-AAV antibodies in human samples without the need to develop serotype-specific assays or labeling of AAV capsids. To confirm the robustness of the generic assay to screen for binding antibodies against AVV vectors for different serotypes, a comparison was performed with independent NAb and TAb assays.

Methods: Anti-AAV TAb analysis in an automated, CD-based format was performed on the Gyrolab microfluidic platform with commercially available Gyrolab Generic Anti-AAV kit reagents on human serum samples. In the automated microfluidic method, biotinylated capture reagent is automatically introduced into a microstructure in the Gyrolab Bioaffy 1000 CD and captured on streptavidin-coated beads in the flow-through affinity column. The second capture reagent, an AAV capsid of choice, is introduced and interacts with the first capture reagent forming a complex. Subsequently, samples are introduced into the microstructures where anti-AAV antibodies are captured on the capture column. Finally, a detecting reagent labeled with Alexa Fluor®647 is added. The integrated laser-induced fluorescent signal represents the response from the bound anti-AAV antibody to the AAV capsid. To address background variability caused by nonspecific antibodies in the sample binding to the capture reagent, the assay is run twice for each sample, once with capsid as a secondary capture and once with only buffer, allowing the determination of individualized cut-off points based on individual background readings. NAb assays were completed using SVAR Life Science iLite^® AAV Neutralizing Platform for 2 different serotypes (AAV2 and AAV8), whereas SVAR Life Science ELISA TAb assay was performed for serotype AAV2.

Results: The NAb results for 12 human samples are given in % neutralization (transduction inhibition). For this comparison, a cut point was set at 35% for AAV2, with samples reading above qualifying as positive and samples reading below qualifying as negative. For AAV2, 8/12 NAb-samples correlated well with the Gyrolab microfluidic assay. Samples S58, S72 and S74 came in as negative in the NAb assay and positive in the Gyrolab assay while S69 qualified as positive in the NAb and negative in the Gyrolab assay. For AAV8, with a cut point of 38%, the NAb and Gyrolab assays correlated completely. In the comparison of the AAV2 ELISA TAb assay to the Gyrolab assay, samples quantifying above 1 ug/mL in a 1/30 dilution in the TAb are considered positive. There is a clear correlation between samples qualifying as positive in both assays. The TAb data for samples S58, S69, S72 and S74 is consistent with the Gyrolab assay. This speaks to the conclusion that the differences in qualification for these samples are due to the assay formats (NAb vs TAb) rather than an inaccurate read.

Conclusion: We have developed a Gyrolab generic assay that allows for the qualitative assessment/screening of binding antibodies against AVV vectors for different serotypes. When compared to independent NAb and TAb assay results, the results demonstrate a remarkably strong correlation even when considering the limited sample size. This underscores the potential of the Gyrolab generic TAb assay to be employed as a standalone method or as a complementary tool alongside NAb assays, offering a robust means of qualitative assessment and screening of pre-existing anti-AAV antibodies in patient populations.

References: 1. Mendell JR, Connolly AM, Lehman KJ, et al. Mol Ther Methods Clin Dev. 2022 Jun: 25: 74–83. DOI: 10.1016/j.omtm.2022.02.011
2. Gorovits B, Azadeh M, Buchlis G et al. AAPS J 2021 23; 108.

Microfluidic flow-through AAV ADA assay where the immunoassay is built on a streptavidin coated bead within a 15 nL flow-through affinity column and fluid flow (reagents, buffers) controlled with centrifugal (spinning) during the method.