T-cell-targeted immunofusion proteins from E. coli.
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.
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XOMA Corporation, Santa Monica, California 90404, USA.
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*
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