730. T Cell Responses to AAV Vector Capsid in Normal Donors and Subjects Who Have Undergone Liver-Directed AAV-Mediated Gene Transfer

In a Phase I/II study of gene transfer for hemophilia B, an adeno-associated viral vector serotype 2 (AAV-2) was introduced into the liver of human subjects and therapeutic levels of transgene expression (up to 11% of normal) were reached. However, the duration of gene expression was limited; four weeks after infusion, levels of circulating factor IX (F.IX) began to decline, and returned to baseline by week 10. This phenomenon was accompanied by a mild, self-limited, increase in liver enzymes. After a similar phenomenon was observed in another patient, the clinical trial was halted. We hypothesized that T cells directed towards AAV capsid antigens harbored by transduced hepatocytes were activated and these mediated destruction of the transduced hepaotocytes, thereby causing loss of transgene expression and a transient transaminitis. We used IFN|[gamma]| ELISpot analysis of peripheral blood mononuclear cells (PBMCs) from a subject in the clinical study, combined with bioinformatics tools for the prediction of MHC class I binders, to define an MHC Class I-restricted T cell epitope for an HLA type common in the general population (B|[ast]|0702). We then designed pentamers in which five peptide-loaded MHC class I molecules are linked to a fluorochrome to detect and study AAV-specific B|[ast]|0702-restricted CD8+ T cells. Indeed, in the HLA B|[ast]|0702 subject who developed transaminitis after vector infusion, AAV-specific CD8+ T cells were detected by pentamer staining of PBMCs up to two years later. In contrast, we have not detected AAV-specific CD8+ T cells in the peripheral blood of normal donors, though after several rounds of ex vivo stimulation with AAV capsid or peptide epitopes, we have been able to enrich a population of AAV-specific T cells, suggesting that AAV-specific T cells exist in the T cell memory pool. Functional assays on expanded AAV-specific T cells have demonstrated that these T cells specifically produced IFN|[gamma]| upon detection of AAV antigen and specifically lyse target cells presenting antigen in the context of the HLA B|[ast]|0702. These data provide direct evidence that humans mount a cytotoxic immune response to AAV capsid proteins, and that a contracted pool of AAV-specific T cells can remain as memory T cells in normal donors. Thus, re-activation of an AAV-specific T cell response may account for the limited duration of transgene expression that we observed in our clinical study. Moreover, the use of alternate serotypes may not easily avoid this immune response, as T cells from subjects infused with AAV-2 showed functional responses to AAV-1 and AAV-8-derived peptides by both cytokine production and killing assays, and T cells from normal donors that were expanded with an AAV-2 derived epitope also cross-react with the homologous epitope from AAV-5 by pentamer staining. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.