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.