Methods to Engineer and Identify IgG1 Variants with Improved FcRn Binding or Effector Function
Antibodies as therapeutic agents have gained broad acceptance as shown by the number of antibodies in clinical use and many more in clinical development. This utility is an outcome of the high specificity and affinity of the antigen-binding site comprised of the heavy and light chain variable domains. In addition, the Fc portion of human or humanized IgG1 antibodies promotes long half-life through interaction with the recycling FcRn receptor and effects killing functions through interaction with complement and Fcγ receptors. Engineering the Fc portion to increase half-life through stronger binding to FcRn, or to increase complement or cell-mediated killing may lead to improved therapeutic antibodies. These improvements may benefit the patients through convenience in dosing or increased efficacy. Here we describe protocols for generating Fc-engineered IgG1 antibodies and assays to measure Fc receptor binding, antibody dependent cellular cytotoxicity activity, and complement dependent cytotoxicity activity to identify variants with improved FcRn binding or effector function.
Key wordsAntibody engineering FcRn binding FcγR binding ADCC CDC
We thank Devin Tesar for providing the protocol for surface plasmon resonance measurement of FcRn binding, Gerald Nakamura for advice on ADCC and CDC protocols, and Yanmei Lu for reviewing the Fc receptor binding ELISA protocols.
- 11.Yamane-Ohnuki N, Kinoshita S, Inoue-Urakubo M et al (2004) Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng 87:614–622PubMedCrossRefGoogle Scholar