Scientist Sangamo Therapeutics Brisbane, California
Recombinant adeno-associated virus (AAV) vectors have been the most frequently used delivery vehicle for clinical stage gene therapy programs. Immunogenicity analysis of anti-AAV antibody levels is critical for the enrollment of patients with no pre-existing immunity to ensure adequate treatment efficacy, and for detection of treatment-induced antibody response to assess safety. A cell-based transduction inhibition anti-AAV6 neutralizing antibody (NAb) assay has been previously developed and used for patient screening. This abstract describes the development of an anti-AAV6 total antibody assay to monitor patients’ antibody responses post-treatment in clinical studies. This direct ELISA uses an anti-human IgG detection antibody that cross-reacts with all five human immunoglobulin isotypes. As commercial human anti-AAV6 antibodies are currently not available, we generated the positive control antibody by purifying IgG from human sera with pre-existing antibodies to AAV6. We compared two different approaches to determine the total antibody assay cut point: (i) using a panel of healthy donors without pre-screening (used in NAb assay for patient enrollment) and (ii) using negative healthy donors pre-screened by the NAb assay having less than 30% transduction inhibition. The first approach inflated the cut point factor (2.6 vs. 1.7) and resulted in more than three-fold increase in the false-negative rate as compared with the second method (10% vs. 3%). The second approach, furthermore, provided better sensitivity and correlation with the anti-AAV6 NAb assay. In conclusion, establishing cut point using prescreened healthy donors who have no pre-existing immunity to AAV6 is the method of choice based on its context of use of detecting anti-AAV6 antibody response induced by gene therapy.
compare two approaches to determine cut point for immunogenicity assay and identify which one fits for their specific purpose.
acquire how to develop and optimize an immunogenicity assay, including preparation of the negative control serum, selection of the detection antibody and generation of the positive control antibody.
verify the correlation between anti-AAV6 total antibody assay and anti-AAV6 neutralizing antibody assay