Immune Responses in 101HEMB01, a Phase 1/2 Open-Label, Single Ascending Dose-Finding Trial of DTX101 (AAVrh10FIX) in Patients with Severe Hemophilia B

Abstract Background: Humoral and cellular immune responses to adeno-associated virus (AAV) serotype rh10 (AAVrh10 vector) and to the human FIX transgene were analyzed in blood and serum samples from hemophilia B subjects enrolled in a phase 1/2 open-label, single-arm, multicenter, dose-finding study (101HEMB01; See also ASH 2017 Abstract: Pipe et al., 101HEMB01 is a phase 1/2 open-label, single ascending dose finding trial of DTX101 (AAVrh10FIX) in patients with moderate/severe hemophilia B that demonstrated meaningful but transient expression of human factor IX (hFIX)). Methods: T cell responses in blood PBMCs were analyzed by IFNγ ELISPOT and polychromatic flow cytometry. In addition, a multi-analyte profiling of secreted cytokines, chemokines, and growth factors in serum was performed. Furthermore, we performed a computational HLA class I affinity prediction analysis for each subject's FIX mutations. Humoral immune response in serum to AAVrh10 capsid was analyzed by IgG ELISA and by an in vitro neutralizing antibody (NAb) assay. Results: A total of 6 subjects were dosed. Subjects in Cohort 1 (n=3) and Cohort 2 (n=3) received a dose of DTX101 of 1.6x1012 GC/kg and 5.0x1012 GC/kg, respectively. Five out of six subjects had asymptomatic elevations in ALT of 40 U/L or higher. Subject 6 had a peak ALT of 914 U/L. IFNγ ELISPOT analysis showed a limited response to the AAVrh10 capsid 6-8 weeks after vector administration in subjects 3, 4, 5, and 6. The magnitude of the response was low and did not persist in subjects 3, 5, and 6. Intracellular cytokine analysis also showed low magnitude T cell responses to both the AAVrh10 capsid and FIX transgene. Subject 6 was the only subject with a strong CD4+ T cell IL-2+ IFNγ+ response to FIX that did not persist. HLA class I affinity prediction analysis revealed that subject 6 had high binding affinity for a single 9-mer peptide spanning the FIX mutation. Multi-analyte analysis in serum showed an increase in TGFa, IL-10, IL-7, and PDGF-AB/BB only observed in subject 6. IgG ELISA and NAb assay analyses showed strong antibody responses to AAVrh10 after vector administration in all six subjects. The antibody response remained stable for at least 52 weeks. Conclusion: Low magnitude and non-persisting T cell responses to AAVrh10 and FIX were detected in the peripheral blood of many patients, although there is no clear etiology for the higher elevation of transaminases observed in subject 6. Several aspects of his immune response do suggest that it differed qualitatively from that of the others, such as an exaggerated humoral response to the AAV capsid, a CD4 IL2 response against FIX, increases in serum growth factors associated with cell proliferation, differentiation and angiogenesis, and an increase of the anti-inflammatory cytokine IL10. Studies in nonhuman primates indicate that the T cell compartment in peripheral blood does not reflect that of the target of AAV, or in this case the liver, which could complicate the analysis of host immune responses to gene therapy in humans. Disclosures Wilson: REGENXBIO: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Pennsylvania: Patents & Royalties; Dimension Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Solid Gene Therapy: Membership on an entity's Board of Directors or advisory committees.