title: Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.
abstract: Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.
authors.affiliation: a Department of Pharmacology and Cell Biophysics ; College of Medicine ; University of Cincinnati ; Cincinnati , OH USA.
authors.forename: Terence L
authors.initials: TL
authors.lastname: Kirley
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Norman
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D055598:Chemical Phenomena; D003042:Cocaine / Q000031:analogs & derivatives / Q000276:immunology*; D004220:Disulfides / Q000032:analysis*; D005470:Fluorometry; D005936:Glucans / Q000032:analysis*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D055672:Static Electricity
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D004220:Disulfides; D005936:Glucans; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; C005618:benzoylecgonine; C066444:cocaethylene; D003042:Cocaine
keywords: ABD-F, 7-fluorobenz-2-oxa-1, 3-diazole-4-sulfonamide; ADCC, antibody-dependent cell mediated cytotoxicity; BE, benzoylecgonine; CDR, complementarity determining regions; CE, cocaethylene; CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; Endo H, endoglycosidase H; Endo Lys-C, lysyl endoproteinase; Fab fragment; Fab, fragment; Fc, fragment, crystallizable; HPLC, high performance liquid chromatography; IEF, isoelectric focusing; KD, dissociation constant; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; NEPHGE, non-equilibrium pH gel electrophoresis; PNGase-F, peptide N-glycosidase F; SCX, strong cation exchange; TBP, tributylphosphine; TBS, tris-buffered saline; antigen-binding; cocaine; fluorescence quenching, heterogeneity; h2E2, humanized monoclonal antibody against cocaine; high performance ion exchange chromatography; ligand binding; mAb, monoclonal antibody; monoclonal antibody; non-equilibrium pH gel electrophoresis