Introducing antigen-binding sites in structural loops of immunoglobulin constant domains: Fc fragments with engineered HER2/neu-binding sites and antibody properties
- 11 February 2010
- journal article
- Published by Oxford University Press (OUP) in Protein Engineering, Design and Selection
- Vol. 23 (4), 289-297
- https://doi.org/10.1093/protein/gzq005
Abstract
Yeast surface display libraries of human IgG1 Fc regions were prepared in which loop sequences at the C-terminal tip of the CH3 domain were randomized. A high percentage of these library members bound to soluble CD64 and Protein A indicating that the randomization step did not grossly interfere with the overall structure of the displayed Fc. Sorting these libraries by FACS for binders against HER2/neu yielded antigen-specific Fc binders (Fcab; Fc antigen binding) of which one was affinity matured, resulting in Fcab clone H10-03-6 which showed >10-fold improvement in antigen-binding activity versus the parental clone. Pre-equilibrium surface plasmon resonance experiments revealed a KD value of 69 nM. H10-03-6 did not react with other members of the HER family and specifically interacted with HER2-positive but not with HER2-negative cells. Importantly, Fcab H10-03-6 elicited potent antibody-dependent cellular cytotoxicity in vitro. Finally, the in vivo half-life in mice was similar to wild-type Fc indicating that the amino acid changes in the CH3 domain did not affect the pharmacokinetic behavior of the recombinant Fc. Our data demonstrate that the Fcab scaffold combines all features of normal antibodies in a small 50 kD homodimeric protein: antigen binding, effector functions and long half-life in vivo.Keywords
This publication has 22 references indexed in Scilit:
- A large library based on a novel (CH2) scaffold: Identification of HIV-1 inhibitorsBiochemical and Biophysical Research Communications, 2009
- Targeting of HER2-Expressing Tumors with a Site-Specifically 99mTc-Labeled Recombinant Affibody Molecule, ZHER2:2395, with C-Terminally Engineered CysteineJournal of Nuclear Medicine, 2009
- Development trends for therapeutic antibody fragmentsNature Biotechnology, 2009
- IG, TR and IgSF, MHC and MhcSF: what do we learn from the IMGT Colliers de Perles?Briefings in Functional Genomics and Proteomics, 2008
- A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2Journal of Molecular Biology, 2007
- Design, expression, and stability of a diverse protein library based on the human fibronectin type III domainProtein Science, 2007
- High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG methodNature Protocols, 2007
- Strategies to improve plasma half life time of peptide and protein drugsAmino Acids, 2006
- SWISS-MODEL: an automated protein homology-modeling serverNucleic Acids Research, 2003
- The fibronectin type III domain as a scaffold for novel binding proteinsJournal of Molecular Biology, 1998