Abstract 3249: Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum

Abstract Adeno-associated viruses (AAV) are prevalent gene therapy vectors, delivering therapeutic transgenes to treat various diseases including cancer and rare monogenic diseases. Individuals with pre-existing immunity to AAVs are less likely to benefit from AAV-based therapies due to both, a reduction in cellular AAV uptake and activation of a cytotoxic T-cell response to transduced cells, decreasing efficacy or changing the safety profile. Measurement of serum neutralizing antibodies (NAbs) against AAV is essential to determine treatment eligibility and to identify clinical trial candidates. Here we introduce a cell-based assay using NanoLuc® luciferase to detect NAbs in serum samples. The superior brightness of NanoLuc® luciferase in our AAV reporter system results in the need of low concentrations of AAV and a short 24h assay time. The minimal AAV concentration in our assay aids in detecting low NAb titers in serum samples, reducing false negatives and enhancing decision-making accuracy. Testing 60 human serum samples against a range of AAV serotypes, we found that a large fraction of the population had pre-existing NAbs to at least one serotype, and of the pre-exposed individuals a majority displayed NAbs against multiple AAV serotypes. Interestingly, fewer samples were seropositive for AAV-DJ than for either parental serotype (AAV2, AAV8 or AAV9), highlighting the assays utility in the development of engineered capsids. Furthermore, the broad dynamic range of our assay facilitates serum sample categorization into four NAb tiers: negative, low, medium, and high using a single serum dilution. Neutralizing titers (ND50) which was determined by serial dilution of the serum samples strongly agreed with the assigned NAb tiers. Our study with 40 naïve mice further validated the assay's robustness, showing a clear correlation between NAb levels and injected AAV viral loads. Taken together, this rapid, highly sensitive, and reliable assay enabled by NanoLuc® AAV reporter technology precisely measures NAbs against AAVs in both human and animal serum. Further validation of the assay is required for future use in a clinical setting. Citation Format: Morten Seirup, Kirsten Wingate, Jeff Nelson, Kara Machleidt, Vanessa Ott, Trish Hoang, Marjeta Urh. Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3249.