Prophylaxis CTLA4-ig reduces anti-AAV neutralizing antibodies in AAV gene therapy for hemophilia

Abstract Objective: This study aimed to investigate the inhibitory effect of CTLA4-Ig on anti-AAV neutralizing antibody (Nab) development after AAV gene therapy in hemophilia patients, providing an immunomodulatory strategy for potential re-treatment. Methods: Key immune targets were identified through analysis of single-cell RNA sequencing (scRNA) data from a hemophilia B gene therapy clinical trial (NCT04135300; n=7) and flow cytometry data from a hemophilia A gene therapy trial (NCT05454774; n=8). To recapitulate human immune responses to AAV in murine models, an AAV immune-adopted murine model was established. Wild-type (WT) and immune-adopted mice received intravenous infusion of AAV843-Gaussia luciferase (Gluc; 5×10¹² vg/kg). Abatacept (CTLA4-Ig, 5 mg/kg) or an equivalent PBS was administered intraperitoneally 3 days prior to vector infusion, followed by twice-weekly maintenance dosing for 6 weeks. Gluc expression and anti-AAV Nab titers were monitored. Concurrently, an Investigational New Drug (IND) clinical trial (CTR20233400) was initiated. Adult male hemophilia A patients with baseline FVIII:C <2 IU/dL, no history of FVIII inhibitors, and pre-existing anti-AAV Nab titers ≤1:4 were enrolled. Prophylaxis CTLA4-Ig (5 mg/kg) was administered 3 days prior to AAV vector (BBM-H803) infusion. Patients were followed for six months to observe anti-AAV Nab titers dynamics. Results: The scRNA and flow cytometry analysis revealed significant activation of monocyte pathways post-AAV infusion. The proportion of CD86⁺CD16⁺ monocytes showed characteristic fluctuations (D0: 25% → D3: 15% [p<0.05] → W2: 30% [p<0.05]). In the mouse model, anti-AAV Nab titers were significantly lower in the CTLA4-Ig group versus the control group: WT mice at Week 6 (1:16 vs. 1:2,048, p<0.05) and pre-immune mice (1:16 vs. 1:4,096, p<0.05). In the clinical trial, among 5 evaluable patients with a median follow-up of 26 weeks, anti-AAV Nab titers induction during the CTLA4-Ig treatment period (≤Week 12) was significantly attenuated (median log₂[W12/baseline]: 8 vs. 11, p<0.05). Conclusion: Prophylaxis administration of CTLA4-Ig effectively suppresses anti-AAV Nab formation following AAV gene therapy in hemophilia A patients, creating a potential immunological tolerance window for re-treatment.