title: Single-chain immunotoxin fusions between anti-Tac and Pseudomonas exotoxin: relative importance of the two toxin disulfide bonds.
abstract: Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin in which the variable heavy and light domains of the anti-IL2 receptor antibody, anti-Tac, are connected to each other by a peptide linker and then fused to PE40, a truncated form of Pseudomonas exotoxin (PE). This fusion protein has four disulfide bonds: one in each of the two variables domains, one in domain II (Cys 265-287), and one in domain Ib (Cys 372-379) of PE. To study the importance of the disulfide bonds of the toxin to the activity of single-chain immunotoxins, we constructed mutants in which either the cysteines in the toxin were changed to alanines or the amino acids 365-380 of PE were deleted. We began this study with anti-Tac(Fv)-PE40 and a more active variant, anti-Tac(Fv)-PE40KDEL, in which the carbonyl terminus is changed from REDLK to KDEL. From these proteins we made anti-Tac(Fv)-PE40(4)A and anti-Tac(Fv)-PE40KDEL4A, respectively, by converting cysteins at amino acids 265, 287, 372, and 379 of PE to alanines. This change resulted in a 20-100-fold loss of activity toward human target cells, but no significant change in binding affinity to p55. To determine the importance of the second toxin disulfide bond, we removed amino acids 365-380 from anti-Tac(Fv)-PE40, anti-Tac(Fv)-PE40KDEL, and anti-Tac(Fv)-PE40KDEL4A, resulting in anti-Tac(Fv)-PE38, anti-Tac(Fv)-PE38KDEL, and anti-Tac(Fv)-PE38KDEL2A, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: J K
authors.initials: JK
authors.lastname: Batra
authors.forename: S
authors.initials: S
authors.lastname: Seetharam
authors.forename: V K
authors.initials: VK
authors.lastname: Chaudhary
authors.forename: D J
authors.initials: DJ
authors.lastname: FitzGerald
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D001483:Base Sequence; D016923:Cell Death / Q000187:drug effects; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics; D005260:Female; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015375:Receptors, Interleukin-2 / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D013329:Structure-Activity Relationship; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D000922:Immunotoxins; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A