Gelonin analogs with engineered cysteine residues form antibody immunoconjugates with unique properties
We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired cysteine residue. The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of Gel, or with the positions of endogenous cysteine...
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Published in | The Journal of biological chemistry Vol. 269; no. 13; pp. 9644 - 9650 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
01.04.1994
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Subjects | |
Online Access | Get full text |
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Summary: | We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired
cysteine residue. The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of
Gel, or with the positions of endogenous cysteine residues. In most cases, enzymatic activity in vitro was unaltered by this
modification. The rGel analogs were conjugated via their unpaired cysteine residue to the anti-CD5 antibody H65, or to H65
Fab and F(ab')2. Several rGel analogs formed immunoconjugates that were up to 6-fold more cytotoxic to antigen-bearing cells
than those made with linker-modified rGel, whereas others were less potent. In the rat, the in vivo clearance rates of whole
antibody conjugates correlated with their relative in vitro disulfide bond stability, and deconjugation to intact antibody
and rGel was the predominant clearance mechanism. Fab conjugates to rGel analogs which differed in their in vitro disulfide
bond stability had similar serum clearance rates, suggesting that clearance occurs mainly by removal of intact immunoconjugate
from the serum, and is less dependent on deconjugation. Our results demonstrate that rGel analogs with a single cysteine at
various positions on the solvent exposed surface are produced efficiently in Escherichia coli (>1 g/liter), and that the position
of the cysteine greatly influences the potency and stability of the resulting immunoconjugates. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(17)36930-2 |