Confirmatory detection of neutralizing antibodies to AAV gene therapy using a cell-based transduction inhibition assay.

Successful treatment with adeno-associated virus (AAV)-based gene therapies can be limited by pre-existing anti-AAV antibodies. Cell-based transduction inhibition (TI) assays are useful to characterize the neutralizing potential of anti-AAV antibodies in patient samples. While these assays are commonly used, they are not specific for neutralizing antibodies (NAbs) against AAV, also detecting non-antibody-based factors that inhibit AAV transduction in vitro but may not substantially decrease efficacy in vivo. This paper describes the development and bioanalytical validation of a confirmatory assay to improve the specificity of detecting anti-AAV5 NAbs in cell-based TI assays. Samples that screen positive for transduction inhibitors are subsequently depleted of all classes of immunoglobulins using agarose resins conjugated with protein A, G, and L (AGL), which restores AAV5 transduction for NAb-containing samples. Unconjugated agarose resin serves as a mock control for non-specific depletion effects and facilitates normalization of the transduction efficiencies between an AGL- and mock-treated sample; the normalized value is termed the AGL/mock ratio. During validation, a confirmatory cut point for the AGL/mock ratio was derived; sensitivity, precision, selectivity, and matrix interference were also assessed. This confirmatory TI assay facilitates a characterization of humoral immunity to AAV gene therapy by reliably distinguishing NAbs from non-antibody-based neutralizing factors.