T cell-targeted immunofusion proteins from Escherichia coli.
Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking humanized Fab, F(ab')2, or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A. H. C., and Carroll, S. F. (1994) J. Biol. Chem. 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found. Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form. All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells. A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates. Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome-inactivating protein.
Better
M
M
XOMA Corporation, Santa Monica, California 90404, USA.
Bernhard
S L
SL
Williams
R E
RE
Leigh
S D
SD
Bauer
R J
RJ
Kung
A H
AH
Carroll
S F
SF
Fishwild
D M
DM
D000328:Adult; D000936:Antigen-Antibody Complex; D015703:Antigens, CD / Q000276:immunology*; D018956:CD5 Antigens; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis*; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis; D010940:Plant Proteins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D054789:Ribosome Inactivating Proteins, Type 1; D013601:T-Lymphocytes / Q000276:immunology*
D000936:Antigen-Antibody Complex; D015703:Antigens, CD; D018956:CD5 Antigens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C025674:GEL protein, Gelonium multiflorum