Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

Abstract

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.

Keywords: :

• ADCC
• Glycosylation
• high mannose
• pharmacokinetics
• therapeutic antibody

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Feng Li, Efren Pacis, Amy Shen, and Domingos Ng for initial efforts to generate the Man5 glycoform. We thank Mike Laird and Clarissa Chui for cloning the Aspergillus saitoi a-1,2-mannosidase gene into our in-house vector, and Dr. Roland Contreras for providing the a-1,2-mannosidase from Trichoderma reesei. We also thank Rod Keck and Tomasz Baginski for support with the MALDI-TOF assay. Finally, we thank everyone mentioned above and Reed Harris, Steve Meier, Peter Thiel, and Ashraf Amanullah for valuable discussions, review of this manuscript, and support for this work.