(T1330-06-31) Method Development and Validation of a Cell-Based Assay for the Detection of Anti-AAV Neutralizing Antibodies in Human Serum

Purpose: Adeno-associated virus (AAV) is a small, non-enveloped virus with a single-stranded DNA genome. AAV is known for its low immunogenicity, high safety profile, wide host range, long-term transgenic expression, and the ability to target specific tissue sites for gene delivery. These characteristics make AAV-based vectors highly promising for gene therapy applications in humans. In recent years, many AAV gene therapy products have been approved by regulatory authorities, but more are in different stages of clinical trials. However, the production of neutralizing antibodies against AAV in humans may block the transduction of AAV, thereby impacting the efficacy of therapeutic products. The current published AAV neutralizing antibody analysis methods either lack high sensitivity or miss comprehensive validation data following principles of immunogenicity validation guidance, which limits their applications in drug development or clinical testing. This study established a series of cell-based methods for detecting neutralizing antibodies against different serotypes of AAV (AAV2, AAV8 and AAV9) in human serum.

Methods: In the assays, AAV carrying the luciferase reporter gene infected 2V6.11 cells and expressed luciferase within the cells to produce a luminescence signal. With the presence of neutralizing antibodies (NAb) against AAV in human serum, the luminescence signal will decrease. By measuring the luminescence signal in the presence and absence of neutralizing antibodies, the presence and quantity of neutralizing antibodies against AAV were determined in the tested sample. The assay procedure is as follows: 2V6.11 cells were coated on a cell culture plate overnight at 37℃,5% CO₂ incubator. Test samples were mixed with luciferase-modified AAVs. Then they were transferred to the cell coated plate and incubated overnight at 37℃. After that, the luminescence signal was detected with luciferase detection reagent.

Results: These methods for neutralizing antibody determination of AAV2, AAV8, and AAV9 therapeutic products were established. The host cell lines for AAV infection and AAV infection ratios (MOI) were screened and optimized in method development to achieve high sensitivity. We found that 2V6.11 cells worked best among the screened cell lines, the MOI values for AAV2, AAV8, and AAV9 were determined as 300, 10000, and 30000, and the sensitivity for detecting anti-AAV2, AAV8, and AAV9 neutralizing antibodies reached to 100 ng/mL, 100 ng/mL, and 300 ng/mL respectively. At the same time, methods were validated by assessing the cut point, sensitivity, precision, robustness, drug interference, selectivity, and stability. The cut-point factors of AAV2, AAV8, and AAV9 are determined as 0.70, 0.67, and 0.74. Precision was assessed by analyzing six sets of HPCs, LPCs and NCs six times by two analysts. Titer precision was assessed by analyzing three sets of TPC dilution samples in a plate. The intra and inter precision and titer precision of these three methods were all within 30.0%. Matrix effect/selectivity was assessed using ten lots of healthy human serum as well as hemolysis and lipemic serum; no matrix effect was observed in the above selectivity assessment. Bench top stability, 2-8℃ stability, and freeze-thaw stability were evaluated, with the results showing 72 hours of stability at RT and 2-8℃ and eight cycles of freeze-thaw stability. Other assessed parameters also met the requirement of immunogenicity guidelines.

Conclusion: Based on the above results, we have established a series of cell-based methods for detecting multiple serotypes of AAV neutralizing antibodies in human serum. These methods have high sensitivity and reliability for detecting neutralizing antibodies of AAV2, AAV8 and AAV9, and meet the requirements of health authority guidelines of immunogenicity. This research has the potential to significantly impact the field of gene therapy, as it provides a reliable and sensitive method for detecting neutralizing antibodies, thereby enhancing the safety and efficacy of AAV-based gene therapy products.  These methods could be used for subject screening and clinical sample analysis of AAV therapeutic products, further advancing the development and application of gene therapy.

Figure 1. The diagram of the principle of anti-AAV neutralizing antibodies detection methods. AAV carrying the luciferase reporter gene infected 2V6.11 cells and expressed luciferase within the cells to produce a luminescence signal with luciferase substrate supplied. With the presence of neutralizing antibodies (NAb) against AAV in human serum, the luminescence signal will decrease.

Figure 2. Sensitivity. The positive control antibodies were spiked into pooled human serum at an initial concentration of 1,000 ng/mL. Then, they were serially diluted with pooled human serum. The sensitivity for detecting anti-AAV2, AAV8, and AAV9 neutralizing antibodies reached 100 ng/mL, 100 ng/mL, and 300 ng/ML, respectively.

Table 1. Precision results of AAV NAb assays. Precision was evaluated by testing six sets of PCs and NCs in each un by two analysts in three days. Both intra and inter precision of AAV2, AAV8, and AAV9 assays are within 30%.