Biacore Analysis of Cross-Reactive Adeno-Associated Virus Antibodies in Monkeys Following Intravenous Administration of AAV5, AAV8, and AAV9 Vectors

In gene therapy using adeno-associated virus (AAV) vectors, treatment-induced anti-AAV antibodies pose barriers for re-administration of the same or different AAV serotype vectors. We aimed to investigate whether the administration of AAV5, AAV8, or AAV9 in Cynomolgus monkeys resulted in the formation of cross-reactive antibodies. To achieve this, we developed a Biacore SPR-based total binding antibody (TAb) assay to identify anti-AAV antibodies in monkey plasma and assess the cross-reactivity of these antibodies against AAV5, AAV8, or AAV9 vectors on a sensor chip. AAV5, AAV8, and AAV9 vectors were immobilized onto the surface of a CM5 sensor chip on Fc2, Fc3, and Fc4 flow cells, respectively, using amine coupling, while Fc1 served as a reference. Plasma samples flowed through four channels, followed by injecting anti-monkey IgG and IgM antibodies to determine the immunoglobulin (Ig) isotypes. We analyzed TAb against the AAV serotypes in the plasma using a Biacore-based TAb assay 29 days after administration to evaluate the anti-AAV antibody responses. The TAb detected by the Biacore-based assay showed cross-reactivity between antibodies against AAV8 and AAV9; however, there was minimal cross-reactivity between antibodies against AAV5 and those against AAV8 or AAV9. Both IgG and IgM TAb were detected at 29 days post-dosing, and the antibody profiles determined by both the Biacore and ELISA platforms were comparable. The Biacore assessment confirmed the absence of cross-reactivity of anti-AAV5 antibodies against AAV8 and AAV9 vectors, and vice versa. This absence of cross-reactive antibodies against a specific AAV serotype indicated the possibility of re-administering a different AAV serotype.