Bioanalytical support for anti-AAV antibody assays

Written by Yanshan Dai (Bristol Myers Squibb)
Antibodies | ADCs Expert opinion

This expert opinion piece from Yanshan Dai, Bristol Myers Squibb (NY, USA), reviews two assay formats for total AAV binding antibody assays, as well as a method to determine Sulfo-tag labeling on AAV5 vectors and its impact on assay sensitivity and antibody binding affinity.

Yanshan Dai, PhD
Senior Principal Scientist, Bioanalysis, Translational Medicine
Bristol Myers Squibb

Yanshan Dai is a Senior Principal Scientist in the Biotherapeutic Bioanalysis Group at Bristol Myers Squibb. He obtained his PhD degree in Molecular and Cell Biology from Columbia University (NY, USA). For the past 9 years at BMS, he has been working on biologics, PK and biomarker analysis using Simoa®, AAV total antibody assays, neutralizing antibody (NAb) immunogenicity assessments, ELISpot assays and whole blood immunity assays. He also performs CAR T-cell PK analysis utilizing ddPCR methods.

Pre-existing anti-AAV antibodies may pose a significant challenge in AAV gene therapy, depending on the delivery routes, and potentially hinder the efficacy of AAV-based treatments‘ efficacy. Accurate detection of these antibodies is required for patient screening to ensure the success of systemic gene therapy, delivering agents throughout the body. This review focuses on two assay formats for total AAV binding antibody assays. We have also developed a method to determine Sulfo-tag labeling on AAV5 vectors and its impact on assay sensitivity and antibody binding affinity, which I will discuss here.

We developed an ELISA-based direct total antibody (TAb) binding assay to detect anti-AAV8 binding antibodies [1]. In this format, AAV8 vectors are coated on the plate, followed by adding samples. The detection antibody is a biotinylated anti-human light chain antibody. Due to the high background noise, a specific diluent is required to block nonspecific binding so that the screening assay cut point can be set.  However, some samples still have nonspecific bindings, causing false positivity. Therefore, a confirmatory assay is needed to identify false-positive samples [1].

Another TAb assay is the step-wise bridging assay format, in which unlabeled AAV5 vectors are coated on a meso scale discovery (MSD) plate, followed by adding samples [2]. This MSD-based bridging assay platform utilizes Sulfo-tag-labeled AAV5 vectors as detection reagents. We developed liquid chromatography-high resolution mass spectrometry (LC-HRMS) methods to determine the degree of Sulfo-Tag-ruthenium labeling on AAV5 vectors, as well as biotin labeling on AAV5 vectors [2]. The increased degree of labeling (DoL) of Sulfo-tag did not compromise the binding affinity of the anti-AAV5 mAb. We further employed the MSD-bridging assay to assess the S/N ratios of the binding between four anti-AAV5 mAbs and various Sulfo-tag-labeled AAV5 vectors. A strong correlation was observed: the higher the degree of Sulfo-tag labeling, the higher the S/N ratios and sensitivity. Similar results were obtained with positive human serum samples [2]. The results suggest the importance of optimizing the labeling of detection reagents to enhance assay sensitivity.

You may also be interested in:

In comparison with the ELISA-based direct binding assay, we found one human serum sample out of 14 positive anti-AAV5 samples was not detected by Sulfo-tag-labeled AAV5 in the MSD-bridging assay but was detected by the ELISA and cell-based NAb assay, suggesting epitope masking on AAV5 due to Sulfo-tag labeling occurring at low frequency (data unpublished) [3]. Further research is needed to address this issue.

We have also developed a cell-based NAb assay for various AAV serotypes (AAV1, 2, 5, 6, 8) using a COS-7 cell line to screen NAbs in serum and plasma samples from pigs, monkeys and humans [4]. To minimize interference from the matrix, we reduced the incubation time of samples, AAV-luciferase reporter and COS-7 cell line, to 30–60 min while maintaining high transduction efficiency. Similar results were obtained for the AAV9 serotype using a CHO Lec2 cell line. Furthermore, we found that an ELISA-based direct binding assay requires a confirmatory assay to reduce the occurrence of false positive samples. The confirmed TAb-positive samples also tested positive for NAbs [1].

In conclusion, our findings show that increased Sulfo-tag labeling enhances the sensitivity of the MSD bridging assay. These results are consistent with those obtained from the ELISA-based direct binding TAb assay and the cell-based NAb assay. These TAb and NAb assays can be used to determine the anti-AAV titers that may inhibit in vivo AAV-mediated gene transduction.

Disclaimer: the opinions expressed are solely those of the author and do not express the views or opinions of Bioanalysis Zone or Taylor & Francis Group.

Source

  1. Dai Y, Dong H, Gleason C, et al. Comparison of pre-existing anti-AAV8 total antibody screening and confirmatory assays with a cell-based neutralizing assay in normal human serum. AAPS J. 25(3), 35 (2023).
  2. Dai Y, Wu X, Sun X, et al. Determining the degree of Sulfo-tag conjugation to AAV5 vectors by LC-HRMS and evaluating the effects on antibody binding affinity and bridging assay sensitivity. AAPS J. 26, 104 (2024).
  3. Dai Y. (2024) Comparison of different platforms for evaluating cellular and humoral immunogenicity. Presentation delivered at AAPS PharmSci 360, Salt Lake City, 2024.
  4. Dai Y, Kavita U, Lampen MH, et al. Prevalence of pre-existing neutralizing antibodies against adeno-associated virus serotypes 1, 2, 5, 6, 8, and 9 in sera of different pig strains. Hum. Gene. Ther. 33, 451-459. (2022).