(M1030-02-09) Assessment of Pre-existing AAV8 Neutralizing Antibody and Total Binding Antibody Assays for Patient Selection

Purpose: Pre-existing AAV neutralizing antibody (NAb) can prevent AAV vectors from transducing target tissues. To study the correlation between AAV8 total binding antibody (TAb) and Nab and to determine which assays should be selected for patient enrollment in AAV8 gene therapy clinical trials, we developed a chemiluminescent-based ELISA assay to analyze pre-existing AAV8 total antibody levels in normal healthy subjects.

Methods: Eighty-four serum samples of normal human subjects with ages of 18 to 21 were purchased from BioIVT. For chemiluminescent AAV8 TAb ELISA analysis, AAV8 vector was coated on 96-well plate. The serum samples were diluted at MRD20 in a diluent containing heterophilic antibody blockers. The detection was with biotin-labeled rabbit anti-human light chain antibody and streptavidin-HRP, followed by chemiluminescent substrate. The assay sensitivity for positive control anti-AAV8 monoclonal antibody was 50 ng/ml in neat serum. To determine the specificity of AAV8 TAb screening test, a confirmatory assay was applied to AAV8 TAb assay in which AAV8 vector was spiked in serum samples to compete for anti-AAV8 antibody binding to the AAV8-coated wells. COS-7 -based AAV8 NAb assay was used to analyze serum NAb levels at MRD 10 with % inhibition >50 % as cut-off point for positive NAb.

Results: AAV8 TAb Screening cut point factor (SCPF) and confirmatory cut point (CCP) calculations were performed for 84 samples based on the relative luminescence units with and without excess AAV8 vector. For calculation of the SCPF and CCP, those samples with % Inhibition >50% in the confirmatory assay were excluded as confirmed samples with pre-existing TAb. The calculations were then performed on the smaller data sets (n=61 for SCPF and CCP). Outliers were assessed using the Tukey boxplot approach and removed.

AAV8 TAb screening cut point factor was determined to be 2.65 Signal/Negative control ratio, assuming 5% false positive rate. The TAb CCP was calculated with 60 subjects at the 99th percentile and was calculated to be 57% of inhibition.

The results showed that the prevalence of AAV8 TAb in 84 normal healthy subjects was 38% at MRD 20, of which 24% was also NAb positive, but the other 14 % was NAb negative. All 24% of NAb positive subjects also tested positive to TAb, which was also confirmed by TAb confirmatory assay (CCP). All 14% of NAb negative subjects was determined to be negative in TAb confirmatory assay with the assay cut point of 57% inhibition.

Conclusion: This study indicated that the confirmatory determination of AAV8 TAb concurred with cell-based NAb assay when 57% of inhibition was determined by statistical analysis as cut point in TAb confirmatory test. The TAb confirmatory test improved the specificity of AAV8 TAb screening test and identified the subjects with true positive TAb. Our data suggest that AAV8 total binding antibody screening test in conjunction with the TAb confirmatory test can be utilized for patient enrolment in clinical AAV8-mediated gene therapy.