Thermally stabilized immunoconjugates: conjugation of antibodies to alkaline phosphatase stabilized with polymeric cross-linkers.
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 degrees 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.
Bieniarz
C
C
Advanced Drug Delivery, Abbott Laboratories, Abbott Park, Illinois 60064-3500, USA. bienica@hpd.abbott.com
Young
D F
DF
Cornwell
M J
MJ
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism; D004795:Enzyme Stability / Q000502:physiology; D018796:Immunoconjugates / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D008301:Maleimides / Q000138:chemical synthesis; D015394:Molecular Structure; D011099:Polyglutamic Acid / Q000138:chemical synthesis; D011108:Polymers / Q000138:chemical synthesis; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D013972:Thyrotropin / Q000276:immunology
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D018796:Immunoconjugates; D007074:Immunoglobulin G; D008301:Maleimides; D011108:Polymers; D013438:Sulfhydryl Compounds; D011099:Polyglutamic Acid; D013972:Thyrotropin; D000469:Alkaline Phosphatase