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Comparison of Pre-existing Anti-AAV8 Total Antibody Screening and Confirmatory Assays with a Cell-Based Neutralizing Assay in Normal Human Serum

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Abstract

Pre-existing adeno-associated viruses (AAV) neutralizing antibodies (NAb) can prevent AAV vectors from transducing target tissues. The immune responses can include binding/total antibodies (TAb) and neutralizing antibodies (NAb). This study is aimed at comparing total antibody assay (TAb) and cell-based NAb assay against AAV8 to help inform the best assay format for patient exclusion criteria. We developed a chemiluminescence-based enzyme-linked immunosorbent assay to analyze AAV8 TAb in human serum. The specificity of AAV8 TAb was determined using a confirmatory assay. A COS-7-based assay was used to analyze anti-AAV8 NAbs. The TAb screening cut point factor was determined to be 2.65, and the confirmatory cut point (CCP) was 57.1%. The prevalence of AAV8 TAb in 84 normal subjects was 40%, of which 24% were NAb positive and 16% were NAb negative. All NAb-positive subjects were confirmed to be TAb-positive and also passed the CCP-positive criteria. All 16 NAb-negative subjects did not pass the CCP criterion for the positive specificity test. There was a high concordance between AAV8 TAb confirmatory assay and NAb assay. The confirmatory assay improved the specificity of the TAb screening test and confirmed neutralizing activity. We proposed a tiered assay approach, in which an anti-AAV8 screening assay should be followed by a confirmatory assay during pre-enrollment for patient exclusions for AAV8 gene therapy. This approach can be used in lieu of develo** a NAb assay and can be also implemented as a companion diagnostic assay for post-marketing seroreactivity assessments due to ease of development and use.

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Acknowledgements

We would like to thank uniQure company for providing AAV8 vectors. The geometric shapes of AAV8 and anti-AAV antibody in the graphical abstract were designed with www.biorender.com.

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  1. Clinical Pharmacology, Pharmacometrics, Disposition & Bioanalysis, Bristol Myers Squibb Company, Princeton, New Jersey, 08543, USA

    Yanshan Dai, Hui** Dong, Carol Gleason, Johanna Mora, Gerry Kolaitis, Nanda Balasubramanian, Sekhar Surapaneni, Alexander Kozhich & Vibha Jawa

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Contributions

Study conception and design: Y.D., H.D, C.G., J.M., G.K, N.B., S.S., A.K., and V.J. Manuscript writing: Y.D. Statistical analysis and report: C.G. Manuscript edited by J.M., A.K., and V.J. Data analysis and interpretation and figure preparation: Y.D. and C.G. Final manuscript and figure approval: all authors. Experimental procedures and data acquisition: Y.D., H.D., and C.G.

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Correspondence to Yanshan Dai.

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All authors are employees of Bristol Myers Squibb Company.

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Dai, Y., Dong, H., Gleason, C. et al. Comparison of Pre-existing Anti-AAV8 Total Antibody Screening and Confirmatory Assays with a Cell-Based Neutralizing Assay in Normal Human Serum. AAPS J 25, 35 (2023). https://doi.org/10.1208/s12248-023-00805-6

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