Early Phase Clinical Immunogenicity of Valoctocogene Roxaparvovec, an AAV5-Mediated Gene Therapy for Hemophilia A

Abstract

Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures.

Keywords: AAV; AAV antibody; adeno-associated virus vector; cellular immunity; clinical immunogenicity; clinical safety; cross-reactivity; gene therapy; hemophilia A; humoral immunity.

Graphical abstract

Figure 1

All Subjects Develop a Sustained Anti-AAV5 Antibody Response following Dose Administration Longitudinal assessment of AAV5 capsid-specific total binding antibody. Plasma was collected from clinical patients at regular intervals after valoctocogene roxaparvovec administration and assayed for AAV5-specific TAbs. For similar titers across dose cohorts, titers peaked at week 40 with a mean of 8.8E6, 4.6E6 at year 2, and 7.6E6 at year 3. Box plots show the interquartile range with whiskers indicating minimum and maximum values. Line indicates median, diamond indicates mean

Figure 2

Correlation Analysis of AAV5 TAb Titer with Plasma ALT and FVIII Activity (A and B) The maximum observed AAV5 TAb titers for each patient (upper plots) and the AAV5 TAb titer at week 8 (lower plots) show no association with the maximum observed plasma ALT value matched by patient (A), or with the median FVIII activity measures from weeks 23 to 26 after dose administration (B). Data in (B) are from cohort 3 and cohort 4 patients only. Mean FVIII activity from weeks 23–26 was not calculable for patients in cohorts 1 and 2.

Figure 3

Cross-Reactive Antibody Response against Divergent Capsids (A and B) Plots of AAV2, AAV5, AAV6, AAV8, and AAVrh10 specific TAb titers over time are displayed for individual subjects in cohort 1 (A) cohort 2 (B). (C and D) Grouped individual plots are shown for n = 7 subjects in cohort 3 (C) and n = 6 subjects in cohort 4 (D). The final available sample time point after infusion ranged from week 76 to week 134.

Figure 4

Heatmap of Cellular Immune Responses Heatmaps show instances of IFN-γ- and TNF-α-positive responses for individual patients at baseline and each post-dose administration time point (weeks 4–52 and by quarter [Q1–Q4] as indicated for years 2 [Y2] and 3 [Y3]). Coloration signifies magnitude of response by SFU/10⁶ PBMCs and the highest SFU value, displayed for positive responses, was used when positive responses were detected to more than one peptide pool. Results too numerous to count were interpolated to a value of 5,000 SFU/10⁶ PBMCs. (A) Results of the IFN-γ FluoroSpot assay following stimulation with AAV5-derived peptide pools and (B) FVIII-derived peptide pools. (C) Results of the TNF-α FluoroSpot assay following stimulation with AAV5-derived peptide pools and (D) FVIII- derived peptide pools. Cells in white (blank) indicate time points where there were too few cells to test. Tested samples with a result <50 SFU/10⁶ PBMCs are left blank. SFU counts are shown for positive responses ≥50.

Figure 5

Distribution of FVIII Activity and ALT Levels by FluoroSpot An IFN-γ and TNF-α FluoroSpot assay was used to measure cell-mediated immunity (CMI) specific for AAV5 or FVIII peptides. (A) Pairwise comparison of instances of IFN-γ-positive and IFN-γ-negative time points with corresponding measures of ALT or FVIII activity. Boxplots show mean of pairwise matched ALT (upper plots) or FVIII activity measures (lower plots) following stimulation with AAV5--derived peptides (left plots) or FVIII derived peptides (right plots). (B) Similar pairwise comparison of instances of TNF-α-positive and TNF-α-negative time points with corresponding mean measures of ALT or FVIII activity. Box plots show the interquartile range with whiskers indicating minimum and maximum values. Line indicates median, diamond indicates mean.