First report of high-titer FVIII inhibitor development following AAV-mediated gene therapy in Hemophilia A

Abstract Over the past decade, adeno-associated virus (AAV) vector–based gene therapy (GT) has emerged as a major advance in hemophilia A (HA). To date, over 280 patients have received HA gene therapy, and no confirmed cases of FVIII inhibitor development following GT have been reported. We report the case of a 47 yo male patient (pt) with severe HA (intron 22 inversion) who developed FVIII inhibitors following a single 3x1013 vg/kg dose of giroctocogene fitelparvovec in the ongoing phase 3 AFFINE study. Prior to enrollment, he was on damoctocog alfa pegol prophylaxis (>150 ED). Medical history included HCV and HIV infection, as well as immune thrombocytopenia (ITP) from 1987 to 2007 that was treated with IV gammaglobulin, RH immune globulin and prednisolone, splenectomy in 1995 and IVIG until 2000, with stable platelet counts since then. The pt has no personal or family history of inhibitors. HLA typing revealed no haplotypes associated with increased inhibitor risk. Following gene therapy, FVIII activity peaked above the normal range within the first 3 months (4.42 IU/mL by chromogenic substrate assay). The pt experienced recurrent episodes of mild transaminitis, triggering 3 tapering courses of corticosteroids (CS) of several months in the first year, initiated on Day (D) 25 post infusion. On these occasions when CS were tapered and discontinued a decline in FVIII:C was observed, partially reversed with the reintroduction of CS. After the 3rd CS course discontinuation (D335), FVIII:C continued to decrease (1.1 to 0.2 IU/mL) leading to the initiation of mycophenolate mofetil (MMF) and CS were reintroduced. During the planned W52 visit (D369), a low-titer FVIII inhibitor of 2.7 BU was detected (FVIII:C: 0.016 IU/mL). Subsequent retrospective testing of stored samples allowed the detection of a first positive measurement at D334 (0.6 BU). The pt then developed a high-titer inhibitor of 5.3 BU on D453 (FVIII:C: 0.062 IU/mL). Although no bleeding episodes were observed since screening, the pt reported new, recurrent joint aches suggestive of possible microbleeds, not confirmed by imaging. In response, MMF was increased to 1 g twice daily on D455 to support inhibitor eradication, and emicizumab prophylaxis was initiated on D460 (225 mg QW). Inhibitor titers continued to rise, peaking at 647.9 BU on D592, then declining to 416.9BU on D690. Throughout this period, FVIII:C and FVIII antigen levels were below limit of detection, and liver enzymes remained within normal limits. FVIII C inhibition kinetic was evaluated using a Nijmegen-modified Bethesda assay. Serial dilutions of plasma were incubated with pooled normal plasma and assayed for residual FVIII activity using a bovine chromogenic substrate assay. The data were plotted and analyzed using Hill slope fitting to characterize the steepness and pattern of FVIII recovery, exhibiting a prolonged low-residual plateau, followed by a gradual increase in FVIII activity with increasing dilution. This was followed by a threshold point beyond which an accelerated recovery phase emerged, modestly approaching the activity levels observed in inhibitor-free control plasma. These results are consistent with a Type 2 inhibitor, exhibiting a characteristic two-phase inhibition profile. Total anti-FVIII antibodies were measured using a modified FVIII IgG-specific enzyme immunoassay (EIA). A positive cutoff was established using the mean and 2 standard deviations above a healthy control population (n=24) for anti-FVIII total IgG, IgG1, IgG2, IgG3, and IgG4. A positive total IgG titer was first detected on D219 (~4 months prior to the first positive inhibitor result), however a first IgG3 titer > positive cutoff was detected on D23. IgG1 and IgG3 were the predominant subclasses. To our knowledge this is the first reported case of high-titer FVIII inhibitor development following gene therapy in a patient with hemophilia A. Although this appears to be a rare event, potentially comparable to or even less frequent than inhibitor formation in previously treated patients receiving FVIII prophylaxis, these results suggest that the possibility of inhibitor development should be included in pre-treatment counseling for patients considering gene therapy.