Interim Analysis of Immunogenicity to the Vector Capsid and Transgene-Expressed Human FVIII in a Phase-1/2 Clinical Study of Bmn 270, an AAV5-Mediated Gene Therapy for Hemophilia a

When assessing the immunogenicity of gene therapy drug products, two components need to be considered: immunogenicity toward the vector delivery system and immunogenicity specific for the transgene product. BMN 270 is an AAV5-mediated gene therapy indicated for the treatment of Hemophilia A, and encodes for a codon-optimized B-domain deleted human FVIII protein (hFVIII-SQ) under control of a liver specific promoter. In study BMN 270-201, the first-in-human clinical trial, prospective patients with severe Hemophilia A were screened for both pre-existing antibodies directed against the AAV5 vector and inhibitors of transduction using a cell-based in vitro transduction inhibition (TI) assay. Patients testing positive in either assay were excluded from the trial, as were patients who had a history of FVIII inhibitors. Following infusion of BMN 270, patient plasma was analyzed for total antibody (TAb) responses specific for the AAV5 capsid and TAb responses directed against FVIII, using bridging ECLA immunoassays. Development of FVIII inhibitors was monitored using the Nijmegen-modified Bethesda assay. Additionally, peripheral blood mononuclear cells were collected for analysis by IFN-γ and TNF-α ELISpot assays for detection of capsid-specific and hFVIII-SQ specific cellular immune responses. The available data indicate that, as expected, all patients develop anti-AAV5 TAb by Week 8 post BMN 270 infusion, the first immunogenicity time-point assessed. One patient screened and confirmed positive at a single time point in the anti-FVIII TAb assay. This response was below the minimum required dilution to determine a titer, and was negative at all subsequent time points. No patients have tested positive in the Bethesda assay for FVIII inhibitors.

We investigated the possibility that cell mediated immunity may explain the observed elevation in hepatic enzymes. To date, several patients dosed with BMN 270 experienced asymptomatic elevation of alanine aminotransferase (ALT) laboratory values ranging from 44 to 141 U/L (normal, 6-43 U/L) that resolved without sequelae. Analysis of cellular immune response in these patients by IFN-γ ELISpot assay against peptides spanning AAV5 capsid or the hFVIII-SQ protein was negative, as it was for all other subjects in the study across all time points tested to date. Unlike the IFN-γ assay, intermittent responses were detected across several patients in response to hFVIII-SQ peptide pools in the TNF-α ELISpot assay. In general, these responses were self-limiting and were not temporally associated with increases in ALT or a decline in FVIII activity measures, and did not correlate with results from the IFN-γ assay. Similar TNF-α responses to hFVIII-SQ peptides were also observed in a subset of healthy donors. Overall, immune responses to BMN 270 were characterized by the production of anti-AAV5 antibodies, which is an expected finding following viral vector administration. In conclusion, no consistent association could be made with increases in ALT and cellular immune responses. Our data to date indicate that although antibodies to the AAV5 capsid develop in all patients, concomitant cellular immunity is not readily detectable, and has not been associated with liver function abnormalities nor changes in transgene expression levels.