title: T-cell-targeted immunofusion proteins from E. coli.
abstract: Antibodies and antibody domains are ideal agents for targeting the surface of cells, and fusion proteins between cell-targeting domains and cytotoxic proteins may be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking the humanized Fab, F(ab')2, or single-chain antibody form of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. To maximize the product yield and simplify the production process, each fusion protein was linked to a bacterial signal sequence for expression in E. coli as a secreted protein. More than 30 fusion genes were assembled with antibody domains and gelonin in various physical orientations. Each immunofusion accumulated in the bacterial culture supernatant in a properly folded, active form. Bacteria transformed with each fusion gene were then grown in a fermentor, and product was recovered from the cell-free fermentation broth by column chromatography. All of the immunofusion proteins were purified by a single process and each was tested for cytotoxicity toward antigen-positive human cells. A compact cGMP fermentation area was built to manufacture these fusion proteins. Our integrated approach to microbial protein production, including molecular genetics, bacterial fermentation, and initial isolation, is described in detail.
authors.affiliation: XOMA Corporation, Santa Monica, California 90404, USA.
authors.forename: M
authors.initials: M
authors.lastname: Better
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology; D001666:Binding Sites, Antibody; D018956:CD5 Antigens / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D004926:Escherichia coli; D005803:Genes, Immunoglobulin; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000276:immunology; D010940:Plant Proteins / Q000096:biosynthesis / Q000276:immunology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D054789:Ribosome Inactivating Proteins, Type 1; D012441:Saccharomyces cerevisiae; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D018956:CD5 Antigens; D007140:Immunoglobulin Fab Fragments; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C025674:GEL protein, Gelonium multiflorum