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.

Keywords:

• cocaine
• Fab fragment
• fluorescence quenching, heterogeneity
• high performance ion exchange chromatography
• ligand binding
• monoclonal antibody
• non-equilibrium pH gel electrophoresis

Abbreviations:

• mAb, monoclonal antibody
• h2E2, humanized monoclonal antibody against cocaine
• Fab, fragment
• antigen-binding
• Fc, fragment, crystallizable
• ADCC, antibody-dependent cell mediated cytotoxicity
• PNGase-F, peptide N-glycosidase F
• Endo H, endoglycosidase H
• TBP, tributylphosphine
• ABD-F, 7-fluorobenz-2-oxa-1, 3-diazole-4-sulfonamide
• IEF, isoelectric focusing
• NEPHGE, non-equilibrium pH gel electrophoresis
• MOPS, 3-(N-morpholino)propanesulfonic acid
• MES, 2-(N-morpholino)ethanesulfonic acid
• TBS, tris-buffered saline
• Endo Lys-C, lysyl endoproteinase
• BE, benzoylecgonine
• CE, cocaethylene
• HPLC, high performance liquid chromatography
• SCX, strong cation exchange
• CHO, Chinese hamster ovary
• CDR, complementarity determining regions
• ELISA, enzyme-linked immunosorbent assay
• K_D, dissociation constant

Disclosure of Potential Conflicts of Interest

Dr. Norman is named as a co-inventor on a patent application for the use of the humanized anti-cocaine monoclonal antibody that is the subject of this manuscript.

Acknowledgments

We thank Dr. Thomas Thompson for the use of the Akta HPLC needed for the high performance cation exchange chromatography experiments, and Ryan Walker in Dr. Thompson's laboratory for instruction and help in its use. We also thank Dr. Jim Ball and Mike Tabet for their helpful comments and critiques of the manuscript.

Funding

This work was supported by the National Institutes of Health National Institute on Drug Abuse Grant DP1DA031386.