Irreversible Engineering of the Multielement-Binding Antibody 2D12.5 and Its Complementary Ligands

Engineering the permanent formation of a receptor−ligand complex has a number of potential applications in chemistry and biology, including targeted medical imaging and therapy. Starting from the crystal structure of the rare-earth-DOTA binding antibody 2D12.5 (Corneillie, T. M., Fisher, A. J., and...

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Bibliographic Details
Published in:Bioconjugate chemistry 2004-11, Vol.15 (6), p.1392-1402
Main Authors: Corneillie, Todd M, Lee, Kelvin C, Whetstone, Paul A, Wong, Jeremy P, Meares, Claude F
Format: Article
Language:English
Subjects:
Animals
Binding Sites, Antibody - physiology
Cell Line
Chemistry
Cysteine - genetics
Cysteine - metabolism
Drosophila
Immunoglobulin Fab Fragments - genetics
Immunoglobulin Fab Fragments - metabolism
Ligands
Mutation
Protein Binding - physiology
Protein Engineering - methods
Transfection - methods
Yttrium - metabolism
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Summary:Engineering the permanent formation of a receptor−ligand complex has a number of potential applications in chemistry and biology, including targeted medical imaging and therapy. Starting from the crystal structure of the rare-earth-DOTA binding antibody 2D12.5 (Corneillie, T. M., Fisher, A. J., and Meares, C. F. (2003) J. Am. Chem. Soc. 125, 15039−15048), we used the site-directed incorporation of cysteine nucleophiles at the periphery of the antibody's binding site, paired with the chemical design of a weakly electrophilic ligand, to produce a receptor−ligand pair that associates efficiently and permanently. Protein residues proximal to the ligand's side chain were identified for engineering cysteine mutants. Fab fragments incorporating a cysteine at position 54, 55, or 56 of the heavy chain (complementarity determining region 2) were designed from the structure and then cloned, expressed in Drosophila S2 cells, and tested for reactivity with mildly electrophilic DOTA−yttrium ligands. All showed permanent binding activity, indicating that there is some tolerance for the location of the reactive mutant on the protein surface near the binding site. The G54C Fab mutant displayed the highest expression levels and permanent binding activity in initial experiments and was produced in high yield for further study. Upon examining the behavior of the G54C mutant with a small set of electrophilic ligands, differences in reactivity were observed which indicated that the substituents near the electrophilic atom can be important determinants of permanent binding. The G54C mutant permanently attaches to Y3+ complexes of (S)-2-(4-acrylamidobenzyl)-DOTA with a half-time of ≈13 min at 37 °C, making it potentially useful for in vivo pretargeting applications.
ISSN:1043-1802
1520-4812
DOI:10.1021/bc049824m