Abstract 472: A simple, rapid, and robust bioluminescent assay for detecting anti-AAV neutralizing antibodies in serum samples

Abstract Adeno-associated viruses (AAVs) are widely used as vectors in gene therapy, including for the delivery of therapeutic genes in cancer treatments. In the context of cancer therapeutics, AAVs can be engineered to deliver tumor-suppressing genes or immune-modulating factors directly to cancer cells, enhancing the efficacy of immunotherapy and oncolytic strategies. However, these treatments can be compromised by pre-existing neutralizing antibodies (NAbs) in human serum. To address the need for a sensitive and efficient screening method, we present a novel cell-based transduction inhibition (TI) assay using NanoLuc luciferase, an engineered enzyme known for its high brightness and stability. This assay enables rapid and sensitive detection of NAb activity, utilizing low multiplicities of infection (MOI: 100-10, 000) and incubation times as short as 4 hours. Our workflow employs a straightforward add-mix-read format with a luminescent detection reagent, facilitating luminescence measurements with high reproducibility and minimal hands-on time. The assay's ability to tolerate high serum concentrations, allowing dilutions as low as 1:3, significantly enhances sensitivity for detecting neutralizing activity. Comparative analysis with firefly luciferase demonstrated that NanoLuc produces over 700-fold brighter signals, providing superior detection at lower MOIs. We validated the assay using HEK293 cells, achieving consistent intra-assay variability (%CV <18%) and precise neutralizing dose (ND50) determinations in a 3-fold dilution series of 60 human serum samples. Single-dilution screening highlighted the impact of serum dilution on hit rates, while robust 4-parameter logistic (4PL) curve fitting enabled reliable ND50 determinations for 28 of the tested samples. The assay’s simplicity, high sensitivity, and rapid turnaround make it an invaluable tool for the preclinical and clinical evaluation of NAbs against AAV serotypes, thereby supporting patient eligibility assessments and therapeutic development. These findings underscore the NanoLuc-based assay as a transformative solution for advancing AAV-based gene therapy, particularly in the realm of cancer therapeutics, where precise and rapid NAb screening is crucial for successful treatment outcomes. Citation Format: Morten Seirup, Trish Hoang, Jeff Nelson, Brock Binkowski, Marjeta Urh. A simple, rapid, and robust bioluminescent assay for detecting anti-AAV neutralizing antibodies in serum samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 472.