Phosphatidylserine Converts Immunogenic Recombinant Human Acid Alpha-Glucosidase to a Tolerogenic Form in a Mouse Model of Pompe Disease

Abstract

Development of unwanted immune responses against therapeutic proteins is a major clinical complication. Recently, we have shown that exposure of Factor VIII (FVIII) in the presence of phosphatidylserine (PS) induces antigen-specific hypo-responsiveness to FVIII re-challenge, suggesting that PS is not immune suppressive, but rather immune regulatory in that PS converts an immunogen to a tolerogen. Since PS is exposed in the outer leaflet during apoptosis, we hypothesize that PS imparts tolerogenic activity to this natural process. Thus, immunization with PS containing liposomes would mimic this natural process. Here, we investigate the immune regulatory effects of PS in inducing tolerance towards recombinant human acid alpha-glucosidase (rhGAA). rhGAA was found to complex with PS liposomes through hydrophobic interactions and incubation PS-rhGAA with dendritic cells resulted in the increased secretion of TGF-β. Immunization with PS-rhGAA or OPLS-rhGAA led to a reduction in anti-rhGAA antibody response which persisted despite re-challenge with free rhGAA. Importantly, the titer levels in a majority of these animals remained unchanged after rechallenge and can be considered non-responders. These data provide evidence that PS liposomes can be utilized to induce tolerance towards therapeutic proteins, in general.

Liposomes used as a vaccine adjuvant-delivery system: From basics to clinical immunization

“…Moreover, PS-containing liposomes neither directly inhibited DC maturation in vitro in response to different stimuli, nor prevented DC migration to dLNs in vivo, hinting the complicated interactions among various immunocytes involved in the suppressive process sponsored by PS. Recently, Balu-lyer's group prepared Factor VIII-loaded liposomes with PS/PC (3:7 mol ratio), which when incubated with DCs in vitro caused increased secretion of TGF-β, and, when immunized to mice, due to PS incorporation, not only lowered the Ag-specific Ab response but also induced the hypo-responsiveness to the Ag re-challenge [96]. These outcomes suggest that PS-containing liposomes may function to exert the immune regulatory effects capable of converting an immunogen to a tolerogen, opposing the use as a VADS against pathogens.…”

Nanoparticles for the Induction of Antigen-Specific Immunological Tolerance

“…A similar approach was demonstrated with alpha-glucosidase (GAA) in a mouse model of Pompe disease. Administration of GAA-containing PS liposomes provided therapeutically active enzyme while preventing the formation of inhibitory antibodies ( 56 ). In an autoimmune setting, PS liposomes loaded with disease-relevant peptides were protective in the NOD animal model of T1D ( 54 ) and PLGA nanoparticles displaying PS and containing peptide autoantigens (from myelin oligodendrocyte protein, MOG 35–55 ) were also efficacious in an acute model of EAE (in B6 mice) ( 55 ).…”

Immunogenicity of Protein Pharmaceuticals

“…Upon cellular apoptosis, PS flips to the outer membrane and acts as an "eat me" signal to phagocytic cells that engulf and digest cellular debris without the initiation of an immune response. We have shown that coadministration of PS with various therapeutic proteins and antigens (including rhGAA, FVIII and ovalbumin) can induce durable tolerance and prevent the development of antibodies 78,126 (unpublished data). Additionally, we have shown this method to be antigen specific, as rechallenge with an unrelated antigen showed a robust immune response 31 .…”

“…e) ELISA: ELISAs can also be used to detect ADA by coating the plate with the protein and incubating samples to measure ADA bound to the plate. This method has been used in our lab in conjunction with the Frey method to measure anti-rhGAA antibodies 78,79 . The utility of ELISAs can be limited because standards are not available for all ADA.…”

“…In a study by Lu et al, BALC/c mice were used to compare the relative immunogenicity of DNA vaccines by different routes of administration and showed that intramuscular administration was the least immunogenic 125 . Studies from our lab have used mouse models to show that administration of FVIII or rhGAA complexed with phosphatidylserine based liposomes is significantly less immunogenic than administration of the free protein alone 31,78,126 . B6-129 mice with the GAA gene knocked out 127 have been used as a model to assess immunogenicity of rhGAA 78,128 .…”