ABSTRACT
The strongest mechanism for adsorption of antigens to aluminum adjuvants is ligand exchange, which involves the replacement of a surface hydroxyl on the adjuvant by a terminal phosphate group of the antigen. A novel phosphonate linker was developed that allows the addition of phosphonate (C-PO3) groups to proteins under controlled and chemically mild conditions. Increasing the number of linkers per protein molecule progressively increased the adsorption strength to aluminum hydroxide adjuvant (AH) as measured by elution in serum. The effect of phosphonate conjugation on the antibody response was determined with hen egg lysozyme (HEL), a protein that has the same charge as AH at neutral pH and does not adsorb to AH. The phosphonylated form of HEL (HEL-P) adsorbed to AH, indicating that the ligand exchange interaction could overcome the electrostatic repulsion. Mice injected with HEL-P/AH had a higher antibody titer to HEL than mice injected with HEL/AH, especially at lower antigen doses, suggesting that adsorption of antigen has a dose-sparing effect. Conjugation of CRM197, an antigen that adsorbs electrostatically to AH, with phosphonate linkers did not enhance the antibody response, indicating that adsorption by either electrostatic or ligand exchange to AH is sufficient to enhance the antibody response.
Subject(s)
Adjuvants, Immunologic/chemistry , Aluminum Hydroxide/chemistry , Antibody Formation/immunology , Muramidase/immunology , Organophosphonates/chemistry , Adjuvants, Immunologic/chemical synthesis , Adsorption , Aluminum Hydroxide/immunology , Animals , Antibodies/blood , Bacterial Proteins/chemistry , Bacterial Proteins/immunology , Female , Humans , Mice , Mice, Inbred BALB C , Muramidase/chemistry , Protein Binding/immunology , RatsABSTRACT
Our laboratory recently reported the development of novel prodrug chemistry for the intracellular delivery of phosphotyrosine mimetics. This chemistry has now been adapted for the synthesis of a prodrug that delivers the nonhydrolyzable difluoromethylphosphonate moiety intracellularly. Activation of the prodrug generates a difluoromethylphosphonamidate anion that undergoes subsequent cyclization and hydrolysis with a t1/2 = 44 min. A highly potent and selective inhibitor of protein tyrosine phosphatase 1B (PTP1B) with a nanomolar Ki has been reported, but this bis(difluoromethylphosphonate) lacks potential utility due to its exceedingly low membrane permeability at physiological pH. A prodrug of this inhibitor has been synthesized and evaluated in a cell-based assay. The prodrug exhibits nanomolar PTP1B inhibitory activity in this assay, confirming the efficacy of intracellular phosphonate delivery using this prodrug approach.