title: Cytotoxicity of chimeric (human-murine) monoclonal antibody BR96 IgG, F(ab')2, and Fab' conjugated to Pseudomonas exotoxin.
abstract: We have made antigen-specific cytotoxic reagents by conjugating the chimeric antibody BR96 (chiBR96) to Pseudomonas exotoxin A (PE), as either native PE or a truncated form (LysPE40) devoid of the cell-recognition region (domain I). PE kills cells by ADP-ribosylation of elongation factor 2, thereby inhibiting protein synthesis. Chimeric BR96 immunotoxins were constructed by chemical conjugation of the toxin to Fab', F(ab')2, and intact IgG and purified by anion-exchange and gel-filtration chromatography. Chimeric BR96 [IgG and F(ab')2] immunotoxins were cytotoxic against tumor cell lines displaying the BR96 antigen, with EC50 values ranging from 0.1 to 110 pM. Immunotoxins constructed with chiBR96 Fab' were 50-100-fold less cytotoxic. Competition analysis showed that the immunotoxins were specifically active through their BR96 antigen-binding ability. The binding of chiBR96-PE and chiBR96-LysPE40 to antigen was equivalent to that of BR96 itself and these immunotoxins were found to internalize very rapidly, displaying 90% of their cytotoxicity within 1 h. Binding assays determined that chiBR96 F(ab')2-LysPE40 bound as well as chiBR96-LysPE40; however, chiBR96 Fab'-LysPE40 bound 20-fold less efficiently. The chiBR96 Fab'-LysPE40 internalized similarly to the F(ab')2 or the IgG immunotoxins. Therefore, the chiBR96 Fab'-LysPE40 immunotoxin is less cytotoxic toward target cells because of reduced antigen binding. This is may be due to the monovalent nature of chiBR96 Fab'-LysPE40. This study shows that the monoclonal antibody chiBR96-Pseudomonas exotoxin A immunotoxins can be effective at inhibiting protein synthesis in target cells.
authors.affiliation: Cellular and Molecular Biology Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492.
authors.forename: C B
authors.initials: CB
authors.lastname: Siegall
authors.forename: S L
authors.initials: SL
authors.lastname: Gawlak
authors.forename: J J
authors.initials: JJ
authors.lastname: Chin
authors.forename: M E
authors.initials: ME
authors.lastname: Zoeckler
authors.forename: K F
authors.initials: KF
authors.lastname: Kadow
authors.forename: J P
authors.initials: JP
authors.lastname: Brown
authors.forename: G R
authors.initials: GR
authors.lastname: Braslawsky
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000912:Antibodies, Neoplasm / Q000737:chemistry*; D000918:Antibody Specificity; D001667:Binding, Competitive / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005098:Exotoxins / Q000737:chemistry / Q000633:toxicity*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D000922:Immunotoxins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D007202:Indicators and Reagents; D051379:Mice; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010957:Plasmids; D011549:Pseudomonas
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D005098:Exotoxins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D000922:Immunotoxins; D007202:Indicators and Reagents; D009363:Neoplasm Proteins