Purpose: Pompe Disease is an autosomal recessive disease characterized by the loss of the enzyme acid alpha-glucosidase (GAA). Although enzyme replacement with recombinant human GAA (rhGAA) is the first line of therapy, the development of rhGAA-specific anti-drug antibodies (ADA) in 90-100% of patients significantly abrogates the clinical efficacy of this treatment. Once sustained ADA develop, there are no effective clinical options. Therefore, prevention of an antibody response is critical to addressing an unmet clinical need. Our lab has developed a reverse vaccination strategy utilizing phosphatidylserine (PS) nanoparticles to prevent the development of ADA in mouse models of Hemophilia A and Pompe Disease when administered subcutaneously. Here, we identify a key PS species that can promote tolerance induction, and we show that oral administration of Lyso-PS nanoparticles can prevent antibody development in a mouse model of Pompe Disease.
Methods: In vitro cell culture was performed to investigate the mechanistic basis of Lyso-PS tolerance induction. Bone marrow derived cells were collected and cultured to mature dendritic cells (DC). After maturation, DC were exposed to rhGAA with or without Lyso-PS nanoparticles, and expression of cytokines were measured by ELISA. In vivo studies were performed in GAA-/- mice to compare ADA development after pre-treatment with or without Lyso-PS nanoparticles. Mice received weekly oral gavages for nine weeks of 1 µg rhGAA in the presence and absence of PS and Lyso-PS liposomes. Buffer treatment acted as a control. Beginning on week six, mice were also challenged with four weekly intravenous (IV) injections of 1 mg/kg rhGAA 24 hours after oral gavage. Plasma was collected to analyze anti-rhGAA antibodies by ELISA.
Results: DC exposed to Lyso-PS-rhGAA showed an increase in latent TGF-β secretion (521.3±34.45 pg/mL), compared to cells exposed to free rhGAA (255.9±15.12 pg/mL). Dexamethasone-Vitamin D3 (Dex-D3) is used as a tolerogenic DC control, and treated cells showed TGF-β secretion (435.9± 62.28 pg/mL) that is comparable to DC exposed to Lyso-PS-rhGAA. After oral administration, mice treated with Lyso-PS-rhGAA consistently displayed much lower anti-rhGAA antibody levels as compared to treatment with conventional PS-rhGAA and buffer. In the same study, mice that were administered Lyso-PS-rhGAA showed lower mean antibody titers compared to free rhGAA treated animals, but statistical significance could not be established. All mice treated with Lyso-rhGAA showed very little antibody development, while some mice treated with free rhGAA developed antibodies at the end of the study.
Conclusion: Administration of rhGAA complexed with Lyso-PS nanoparticles has the ability to convert rhGAA from an immunogen to a tolerogen. Upon challenge with doses of free rhGAA, a reduction in ADA development was still seen in mice treated with Lyso-PS, suggesting Lyso-PS actively trains the body to tolerate rhGAA through a TGF-β dependent manner. Based on this mechanism, our work identified a novel reverse vaccination strategy utilizing oral administration of Lyso-rhGAA nanoparticles to induce systemic tolerance and prevent antibody development against rhGAA.
Dr. Sathy Balu-Iyer– University At Buffalo School of Pharmacy and Pharmaceutical Sciences