Protocol abstract
Nature Protocols 1, - 2048 - 2060 (2006)
Published online: 30 November 2006 | doi:10.1038/nprot.2006.322
Subject Categories: Immunological techniques | Isolation, purification and separation
Tunable pharmacokinetics: modifying the in vivo half-life of antibodies by directed mutagenesis of the Fc fragment
Tove Olafsen1, Vania E Kenanova1 & Anna M Wu1
Abstract
Immunoglobulins (Igs) are large proteins of 150 kDa with prolonged residence time in blood. Their half-life is controlled by their ability to interact with the protective neonatal Fc receptor (FcRn, Brambell receptor) present on endothelial cells. Here, we describe a protocol using site-specific mutagenesis of individual residues responsible for this interaction, resulting in engineered antibodies with distinct half-lives. The method is a powerful tool that enables manipulation of half-lives and is applicable to all antibodies and Fc fusion proteins for the development of agents with controlled pharmacokinetic properties. Moreover, the protocol is applicable to any situation where the structure and/or function of engineered proteins are to be studied. The protocol begins with the mutagenesis reaction at the DNA level and proceeds to describe mammalian expression and purification of recombinant proteins, radiolabeling and evaluation in vivo. The time frame for completing the procedure is about 6 months, provided that no complications are encountered.
- Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California Los Angeles, 700 Westwood Plaza, Los Angeles, California 90095, USA.
Correspondence to: Tove Olafsen1 e-mail: tolafsen@mednet.ucla.edu
