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 in the presence of phosphatidylserine (PS) induces antigen-specific hyporesponsiveness to Factor VIII rechallenge, 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 toward 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 transforming growth factor-β. Immunization with PS-rhGAA or O-phospho-L-serine-rhGAA led to a reduction in anti-rhGAA antibody response which persisted despite rechallenge with free rhGAA. Importantly, the titer levels in a majority of these animals remained unchanged after rechallenge and can be considered nonresponders. These data provide evidence that PS liposomes can be used to induce tolerance toward therapeutic proteins, in general.

Keywords: biotechnology; immunology; liposomes; phospholipids; protein delivery.

FIGURE 1

A) Fluorescent spectra to examine the tertiary structure of rhGAA in the presence and absence of PS liposomes. The solid black line represents rhGAA and the dashed gray line represents PS-rhGAA. B) Acrylamide quenching to examine location of rhGAA within the PS liposome bilayer. The solid black line represents rhGAA and the dashed gray line represents PS-rhGAA

FIGURE 2

Immunogenicity and tolerance induction study with PS-rhGAA. (A) Immunization schedule where an arrow indicates an injection and an asterisk (*) indicates blood sample. (B) Anti-rhGAA antibody (mean ± SEM) titer response in animals that received 4 weekly injections of pre-treatment with formulation: free rhGAA (circles), PS-rhGAA (squares), or PG-rhGAA (triangles). (C) Anti-rhGAA antibody titer response after re-challenge with free rhGAA. (D) Comparison of anti-rhGAA antibody titer in individual animals before and after re-challenge with free rhGAA.

FIGURE 3

Immunogenicity and tolerance induction study with OPLS-rhGAA given together and sequentially. (A) Anti-rhGAA antibody titer response (mean ± SEM) in animals that received 4 weekly injections of pre-treatment with formulation: free rhGAA (circles), OPLS-rhGAA together (triangles), or OPLS-rhGAA sequential (diamonds). (B) Anti-rhGAA antibody titer response after re-challenges with free rhGAA. (C) Comparison of anti-rhGAA antibody titer in individual animals before and after re-challenge with free rhGAA.