title: Affinity purification and characterization of anti-Tac(Fv)-C3-PE38KDEL: A highly potent cytotoxic agent specific to cells bearing IL-2 receptors.
abstract: A chimeric, single chain antibody fused immunotoxin, denoted anti-Tac(Fv)-C3-PE38KDEL, was engineered and expressed in Escherichia coli. The microbially expressed anti-Tac(Fv)-C3-PE38KDEL was solubilized from inclusion bodies using guanidine hydrochloride, and subsequently refolded in a redox buffer via thiol/disulfide exchange. The recombinant immunotoxin from the crude extract was purified employing receptor-affinity chromatography, which is based upon biological function and involved the immobilized p55 subunit of human IL-2 receptor. The cytotoxic activity of this immunotoxin was measured by the IL-2 dependent phytohemagglutinin (PHA) blast proliferation inhibition and HUT-102 protein synthesis inhibition assays, in which the IC50 values were 41.5 and 0.8 pM, respectively. The biochemical homogeneity and authenticity of the purified material were determined by gel permeation chromatography, amino acid composition and N-terminal sequence analyses, SDS-PAGE, isoelectric focusing, and Western blotting. The receptor-affinity-purified immunotoxin was shown to be highly effective in specifically killing cells bearing IL-2 receptors. Anti-Tac(Fv)-C3-PE38KDEL is a powerful immunosuppressant which may be a potentially useful therapeutic agent in the prevention of allograft rejection and in the treatment of autoimmune diseases. Another anticipated application of this fusion protein is as a chemotoxin in the treatment of some forms of cancer.
authors.affiliation: Protein Biochemistry Department, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110.
authors.forename: C
authors.initials: C
authors.lastname: Spence
authors.forename: M
authors.initials: M
authors.lastname: Nachman
authors.forename: M K
authors.initials: MK
authors.lastname: Gately
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: I
authors.initials: I
authors.lastname: Pastan
authors.forename: P
authors.initials: P
authors.lastname: Bailon
mesh_terms: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001427:Bacterial Toxins; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004247:DNA / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000008:administration & dosage* / Q000737:chemistry; D000922:Immunotoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D007525:Isoelectric Focusing; D010835:Phytohemagglutinins / Q000494:pharmacology; D015375:Receptors, Interleukin-2 / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004247:DNA; D005098:Exotoxins; D000922:Immunotoxins; D010835:Phytohemagglutinins; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A