A Total ADA Assay and Pre-Existing Antibodies to AAV9

Recombinant adeno-associated virus serotype 9 (rAAV9) based gene therapies are gaining significant momentum as a novel therapeutic modality. However, AAV9 can stimulate both humoral and cellular responses, which are often exaggerated by preexisting immunity due to previous exposure to wild type AAV9. In this project, we developed and validated a sensitive and drug tolerant total anti-drug antibody (ADA) assay for AAV9 following FDA guidance. The assay format was hard coating affinity capture and elution (HC-ACE) with Ruthenium-AAV9 as detection. Upon evaluation, an equal mixture of three commercial anti-AAV9 monoclonal antibodies (clones HI17, HI35, and HL2374) was chosen as positive control material for assay development and validation. The assay has a sensitivity of ~ 2 ng/mL and drug tolerance of at least 1×10e11 DRP/mL. Using this total ADA assay, significant pre-existing antibodies (PEA) were detected from normal human sera, which was consistent with literature.