Thermally stabilized immunoconjugates: Conjugation of antibodies to alkaline phosphatase stabilized with polymeric cross-linkers

Abstract

A method of conjugating poly(glutamic acid) poly(phosphorothioate)-cross-linked alkaline phosphatase to maleimide-derivatized immunoglobulin is described. Intramolecular autocatalyzed cross-linking of alkaline phosphatase at 2:1 to 4:1 polymer:enzyme ratios introduced 32-68 thiolates on the surface of the enzyme. Depending on the stoichiometry of polymer to enzyme, the cross-linked alkaline phosphatase retained 75-90% of its native catalytic activity. The cross-linked thiolate-functionalized alkaline phosphatase was conjugated to maleimide-derivatized immunoglobulin. Compared to a control prepared using non-cross-linked alkaline phosphatase, these conjugates were smaller in size and more stable to heat. The enzymatic activity of the cross-linked conjugates after incubation at 45 °C and pH 7.5 for 25 days was 35% higher than those of the highest-activity control conjugates. The conjugation process could be controlled by varying the stoichiometries of poly(glutamic acid) poly(phosphorothioate), alkaline phosphatase, and immunoglobulin.