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

Abstract

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 Y(3+) complexes of (S)-2-(4-acrylamidobenzyl)-DOTA with a half-time of approximately 13 min at 37 degrees C, making it potentially useful for in vivo pretargeting applications.

Cysteinylated Protein as Reactive Disulfide: An Alternative Route to Affinity Labeling

“…2 In each case the half-lives for covalent attachment were a few minutes at 37 °C, pH 7.4, and the attached ligands migrated with the protein during denaturing (non-reducing) gel electrophoresis. The results for ligands 1 and 2, shown in Figure 2, are similar to the previous results for electrophiles 3 and 4 reacting with the free sulfhydryl form of this antibody (10). As will be shown below, the protein as expressed contains a small fraction of heavy chain cysteine-54 with a free thiol side chain (~7%), and a larger fraction of cysteinylated cysteine-54.…”

“…We expressed the cysteinylated anti-DOTA 1 antibody Fab fragment 2D12.5 G54C in Drosophila S2 cells as described (10). After DTT reduction, we used amino-acid analysis to confirm the release of cysteine monomer from the Fab.…”

Engineered Antibody Fragments with Infinite Affinity as Reporter Genes for PET Imaging

“…The bifunctional chelating agent AABD was prepared following the method of Corneillie et al (24). 90 Y in 0.05 M hydrochloric acid (specific activity, 18.5 TBq/mg) was purchased from PerkinElmer, and 86 Y in 0.1 M hydrochloric acid (specific activity, 1 TBq/mg) was purchased from Isotrace Technologies (25).…”

Removal of autoantibodies by 4-mercaptoethylpyridine-based adsorbent

“…When the pH of the mobile phase is decreased, desorption of the bound proteins can be readily achieved by electrostatic repulsion via the conversion of ionizable groups to their charged forms. MEP-based HCIC has proven effective in the purification of IgG from various feedstocks [14][15][16][17].…”