title: A single-chain bispecific Fv2 molecule produced in mammalian cells redirects lysis by activated CTL.
abstract: Single-chain Fv (sFv) molecules consist of the two variable domains of an antibody (Ab) connected by a polypeptide spacer and contain the binding activities of their parental antibodies (Abs). In this paper we have attached the C-terminus of 2C11-sFv (anti-mouse CD3 epsilon-chain) to the N-terminus of OKT9-sFv (anti-human transferrin receptor [TfR]) through a 23 amino acid inter-sFv linker consisting primarily of CH1 region residues from 2C11, to form a single-chain bispecific Fv2 [bs(sFv)2] molecule. The bs(sFv)2 was expressed in COS-7 cells, and was secreted at the same rate as the two parental sFvs. The secreted protein had both anti-CD3 and anti-TfR binding activities. Essentially all of the secreted bs(sFv)2 molecules bound TfR and the binding affinity of the bs(sFv)2 was comparable to that of OKT9 sFv and Fab. Thus, the attachment of the inter-sFv linker to the N-terminus of OKT9-sFv did not impair its binding function. The bs(sFv)2 retained both binding specificities after long-term storage at 4 degrees C or overnight incubation at 37 degrees C. It redirected activated mouse CTL to specifically lyse human TfR+ target cells at low (ng/ml) concentrations and was much more active than a chemically cross-linked heteroconjugate prepared from the same parental mAbs. Because bs(sFv)2 molecules secreted by mammalian cells are homogeneous proteins containing two binding sites in a single polypeptide chain, they hold great promise as an easily obtainable, economic source of a bispecific molecule suitable for in vivo use.
authors.affiliation: Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: C R
authors.initials: CR
authors.lastname: Jost
authors.forename: J A
authors.initials: JA
authors.lastname: Titus
authors.forename: I
authors.initials: I
authors.lastname: Kurucz
authors.forename: D M
authors.initials: DM
authors.lastname: Segal
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017252:CD3 Complex / Q000276:immunology; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011990:Receptors, Transferrin / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D018033:Antibodies, Bispecific; D017252:CD3 Complex; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv