title: Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli.
abstract: Recent advances in the expression of Abs in Escherichia coli have raised the possibility that virtually any specificity can be obtained by either cloning Ab genes from characterized hybridomas or by de novo selection using Ab gene libraries. Bispecific Abs have been more difficult to engineer because of problems inherent in the proper folding and association of VH and VL domains. In this report, a model system for expressing and testing the activity of a single chain bispecific Ab was used. The Ab contained the VH and VL genes from the anti-TCR Ab 1B2 joined by a 25 amino acid residue linker to the VH and VL genes from the anti-fluorescein Ab 4420. The 57-kDa single chain bispecific Ab (scFv2) was purified in a single step by affinity chromatography through a fluorescein column at a yield of 1 mg/L of bacterial culture. Despite the presence of 1B2 V regions at the NH2-terminus and a 10-residue c-myc peptide at the COOH-terminus, the refolded protein had an affinity for fluorescein that was nearly identical with the monospecific single chain Ab. The scFv2 also bound the TCR of the mouse CTL clone 2C and redirected the lysis of human tumor cells that had fluorescein covalently linked to their surface. Lysis was mediated at scFv2 concentrations that were 100-fold lower than the concentrations of Ab that inhibited normal recognition by CTL 2C. These results show that single chain bispecific Abs can mediate CTL lysis of target cells without the immunosuppressive side effects associated with the use of anti-TCR Abs.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana 61801.
authors.forename: M
authors.initials: M
authors.lastname: Gruber
authors.forename: B A
authors.initials: BA
authors.lastname: Schodin
authors.forename: E R
authors.initials: ER
authors.lastname: Wilson
authors.forename: D M
authors.initials: DM
authors.lastname: Kranz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D003602:Cytotoxicity, Immunologic; D004926:Escherichia coli / Q000235:genetics; D019793:Fluorescein; D005452:Fluoresceins; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D005452:Fluoresceins; D011948:Receptors, Antigen, T-Cell; D019793:Fluorescein