(W1130-06-37) Validation of an In Vitro Cell-Based Assay for the Detection of Neutralizing Antibodies against Adeno-Associated Virus 9 in Cynomolgus Monkey

Purpose: Gene therapy due to its wide applicability in cancer and rare diseases. Among the variety of gene therapy delivery systems, adeno-associated virus has been widely recognized as a valuable vector due to its effectiveness and safety (1). There has been increasing enthusiasm for developing AAV-based gene therapies around the world especially following the promising results obtained in clinical trials of AAV vector-mediated gene therapeutics (2-4). Despite the safety profile of AAV, the high prevalence of naturally occurred AAV infections correlates with high incidence of pre-existing neutralizing antibodies against AAV in individuals, which decreases cell transduction efficiency and is thought to be one of the factors in early clinical failures. Besides humans, non-human primates are also natural hosts of AAV. Therefore, it is critical to screen for the presence of AAV-neutralizing antibodies in non-human primates and set an exclusion criterion for participation in pre-clinical trials. Towards this end, the objective of this study is to develop and validate an efficient and reliable flow cytometry-based cell assay for detecting anti-AAV neutralizing antibodies in cynomolgus monkey serum.

Methods: The detection of neutralizing antibodies was carried out by measuring the inhibition of AAV transduction rate. AAV9 carrying a GFP gene was utilized to transduce target cells in vitro in the presence of anti-AAV9 positive control antibody (Anti-AAV9 (intact particle) mouse recombinant IgG 1 (clone ADK9-1R)) or cynomolgus monkey serum samples. Flow cytometry was used to quantify the percentage of GFP-expressing cells as an indication of AAV transduction. The transduction inhibition rate is calculated according to the following formula: inhibition rate (inhibition %) = 100 - [(transduction efficiency of test sample – the mean transduction efficiency of current batch blank sample) / (the mean transduction efficiency of current batch negative controls – the mean transduction efficiency of current batch blank samples)] * 100.

Results: Method development and optimization: During assay development, we tested several cell lines (HeLa, HEK293, HEK293T, and Huh7 cells), a range of cell densities (1×10⁵, 1.5×10⁵, 2×10⁵, 4×10⁵cells/mL), a variety of multiplicity of infections (MOIs, 40000, 20000, 10000 and 5000), and different incubation times to optimize the transduction efficiency for the AAV9 in vitro. In addition, the minimum required dilution factors (MRD, 1:20, 1:10, 1:5) of cynomolgus monkey serum samples were tested in the feasibility runs to balance the sensitivity and specificity. After systemic optimization, we found that HEK293T with the density of 1.5×10⁵ cells/mL, MOI of 20000, and MRD of 1:5 provided the best assay performance for AAV9 neutralizing antibody detection. The method development result is illustrated in Figure 1 A to D. Method validation: A method validation was conducted in line with current guidelines and whitepaper recommendations. Our results showed that in monkey serum, the assay sensitivity reached 250 ng/mL with a cut point of 50% inhibition. No hook effect was observed when evaluating a set of different concentrations of positive control antibody (10000 ng/mL~ 50 ng/mL) in pooled cynomolgus monkey serum. Drug tolerance testing revealed that 500 ng/mL Anti-AAV9 (intact particle) mouse recombinant IgG 1 could tolerate 2x10^10 GC/mL AAV9. The intra/inter-precision and robustness were assessed. All parameter acceptance criteria meet regulatory requirements (5). The method validation result was illustrated in Figure 2 A to D. Sample analysis: Fifty cynomolgus monkey serum samples have been collected and tested for the presence of anti-AAV9 neutralizing antibodies. Twenty-three out of fifty cynomolgus monkeys were tested positive and thus excluded for participation in pre-clinical study. The screening result is illustrated in Figure 3.

Conclusion: Here we reported the development and validation of a cell-based assay to determine the neutralizing antibodies against AAV9, which could be used to prescreen animal subjects with pre-existing neutralizing antibodies in preclinical studies. In this assay, flow cytometry was utilized to measure the inhibition of AAV transduction rate, which streamlined the analytical procedure and improved the efficiency of sample analysis. Moreover, a battery of parameters has been systemically optimized and standardized to ensure the reliability and reproducibility of the assay. Ultimately, a rigorous validation process following current guidelines and whitepaper recommendations has been completed. The resulting method is considered valid for supporting the GLP preclinical toxicology studies.

References: 1. Amine Meliani, Christian Leborgne, et al. Determination of Anti-Adeno-Associated Virus Vector Neutralizing Antibody Titer with an In Vitro Reporter System. Human gene therapy methods 26:45–53 (April 2015)
2. Nathwani AC, Reiss UM, Tuddenham EG, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 2014;371:1994–2004.
3. Nathwani AC, Tuddenham EG, Rangarajan S, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 2011;365:2357–2365.
4. Bainbridge JW, Smith AJ, Barker SS, et al. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N Engl J Med 2008;358:2231–2239.
5. Guidance for industry: immunogenicity testing of therapeutic protein products-developing and validating assays for anti-drug antibody detection. FDA Guidance, January, 2019.

Acknowledgements: Cynomolgus monkeys used in this report were originated from China and maintained in compliance with the Guide for the Care and Use of Laboratory Animals. All animal research was approved by the Institutional Animal Care and Use Committee.

Figure 1. The optimization of assay parameters during method development: A) cell line, B) MRD, C) cell density, and D) MOI.

Figure 2. The assay performance and validation. A) Sensitivity determination. B) Hook effect evaluation. C) Drug tolerance assessment. D) Precision evaluation.

Figure 3. Screening for anti-AAV9 neutralizing antibody in cynomolgus monkey serum samples.