Bioanalytical Methods & Strategies to Monitor Immunogenicity for AAV Mediated Gene Therapy Brian Long, PhD, Associate Director, Immunogenicity Assessment, BioMarin Pharmaceutical Inc.
This presentation describes bioanalytical methods for detecting pre-existing immunity to AAV vectors, recommendations for pre-clinical evaluation of immunogenicity and monitoring immunogenicity in the clinical trial setting. Clinical immunogenicity results following administration of valoctocogene roxaparvovec, an investigational gene therapy for the treatment of Hemophilia A will be discussed.
Pre-existing antibodies to AAV can impact efficacy by inhibiting transduction efficiency (neutralizing Abs) or by altering biodistribution of vector to phagocytic cells and non-target tissue via formation of immune complexes (binding Abs). Two analytical methods have commonly been employed to screen patients for ant-AAV antibodies prior to enrollment; cell base neutralization assays measuring transduction inhibition (TI), and ELISA-based assays measuring AAV binding antibodies. Non-clinical animal models have been used to evaluate the effect of pre-existing immunity and to inform on the utility of testing methodologies monitoring immunogenicity in the clinic. For clinical studies of valoctocogene roxaparvovec, an AAV5-mediated gene therapy encoding a B-domain deleted human FVIII protein (hFVIII-SQ) for the treatment of hemophilia A, patients were excluded based on the presence of pre-existing total antibodies (TAb) against AAV5. Additionally, patients with a history of FVIII inhibitors (FVIII neutralizing antibodies) were excluded from the study. Valoctocogene roxaparvovec has been administered to greater than 150 hemophilia-A patients in the clinical trial setting, with no patients having developed FVIII inhibitors with a follow up time ranging from 6 months to nearly five years. Dose administration was well tolerated across all doses and was associated with clinically meaningful efficacy. All patients seroconverted to AAV5 TAb positive after dosing and remained positive at all subsequent time points. Emergent AAV5 TAb titers have not been associated with measures of safety (liver transaminase elevations) or efficacy (FVIII activity measures). PBMCs were analyzed by IFN-g ELISpot assay for capsid-specific and hFVIII-SQ-specific cellular immune responses. Though most patients tested positive for cellular immune responses to AAV5, the response was largely transient, self-limiting, and not consistently associated with changes in safety or efficacy parameters. Detection of FVIII-specific cellular immune responses was rare. Cellular immune responses to both AAV5 and FVIII could be detected during and outside periods of glucocorticoid use, though continued use of immune suppressive agents can complicate interpretation of negative ELISpot results. A well characterized and comprehensive immune monitoring program has revealed that the immune response to valoctocogene roxaparvovec is primarily directed toward the AAV5 capsid with only sporadic, transient responses toward hFVIII-SQ. Immune responses detected through clinical monitoring have not been associated with safety or efficacy outcomes.
1. Differentiate between bioanalytical methods used to detect pre-existing immunity to AAV serotypes.
2. Describe the use of non-clinical animal models to inform immunogenicity monitoring strategies in the clinical trial setting.
3. Distinguish between immunogenicity parameters specific to the viral vector and the expressed transgene product.
4. Establish an appropriate immunogenicity monitoring program for clinical trials.
5. Review the clinical trial experience of an immunogenicity monitoring program applied to a case study.