A sensitive and reproducible cell-based assay via secNanoLuc to detect neutralizing antibody against adeno-associated virus vector capsid

Abstract

Most gene therapy clinical trials that systemically administered adeno-associated virus (AAV) vector enrolled only patients without anti-AAV-neutralizing antibodies. However, laboratory tests to measure neutralizing antibodies varied among clinical trials and have not been standardized. In this study, we attempted to improve the sensitivity and reproducibility of a cell-based assay to detect neutralizing antibodies and to determine the detection threshold to predict treatment efficacy. Application of the secreted type of NanoLuc and AAV receptorexpressing cells reduced the multiplicity of infection (MOI) for AAV transduction and improved the sensitivity to detect neutralizing antibodies with a low coefficient of variation, whereas the detection threshold could not be improved by the reduction of MOI to <100. After human immunoglobulin administration into mice at various doses, treatment with high-dose AAV8 vector enabled evasion of the inhibitory effect of neutralizing antibodies. Conversely, gene transduction was slightly influenced in the mice treated with low-dose AAV8 vector, even when neutralizing antibodies were determined to be negative in the assay. In conclusion, we developed a reliable and sensitive cell-based assay to measure neutralizing antibodies against AAV and found that the appropriate MOI to detect marginal neutralizing antibodies was 100. Other factors, including noninhibitory antibodies, marginally influence in vivo transduction at low vector doses.

Immunogenicity assessment of AAV-based gene therapies: An IQ consortium industry white paper

“…In those cases, the sponsor(s) should consider developing a CDx at least during pivotal studies for its availability upon market approval. A CDx is defined as an in vitro diagnostic test providing information that is essential for the safe and effective use of a corresponding drug or immunoassay (e.g., bridging and sandwich assay formats) 170 anti-capsid TI (i.e., neutralizing antibodies) cell-based neutralization assay [171][172][173][174] Cellular T cell response to capsid by measuring secreted factors (e.g., IFN-g)…”

MSD-based assays facilitate a rapid and quantitative serostatus profiling for the presence of anti-AAV antibodies

“…35 We observed a strong overlap between MSDbased BAb and NAb assays throughout all in-house measurements in NHP and human sera. However, transduction of cells is inefficient in vitro with many AAVs including AAV8 and AAV9 36,37 and might require different cell lines for AAV transduction. 38 The NAb escape effect can be masked by transduction impairment in cellular assays, and even minor changes of the capsid surface residues can negatively affect transduction efficiency.…”

The seroprevalence of neutralizing antibodies against the adeno-associated virus capsids in Japanese hemophiliacs

“…The AAV vector was isolated by CsCl-based ultracentrifugation, as described previously 39 . Finally, the titer of the AAV vector was determined, as described previously 40 .…”