PEDS Advance Access originally published online on August 8, 2005
Protein Engineering Design and Selection 2005 18(9):435-444; doi:10.1093/protein/gzi050
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© The Author 2005. Published by Oxford University Press. All rights reserved.
Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two
1Phylos, Inc., succeeded by Compound Therapeutics, 100 Beaver Street, Waltham, MA 02453, USA 2Present address: Merrimack Pharmaceuticals, Inc., Cambridge, MA 02142, USA 3Present address: Novartis Institute for Biomedical Research, Cambridge, MA 02139, USA 4Present address: Idenix, Cambridge, MA 02138, USA 5Present address: Harvard Medical School, Boston, MA 02115, USA 6Present address: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA 7Present address: Massachusetts General Hospital, Boston, MA 02114, USA 8Present address: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
9 To whom correspondence should be addressed. E-mail: dlipovsek{at}ml1.net
The tenth human fibronectin type three domain (10Fn3) is a small (10 kDa), extremely stable and soluble protein with an immunoglobulin-like fold, but without cysteine residues. Selections from 10Fn3-based libraries of proteins with randomized loops have yielded high-affinity, target-specific antibody mimics. However, little is known about the biophysical properties of such antibody mimics, which will determine their suitability for in vitro and medical applications. We characterized target binding and biophysical properties of two related 10Fn3-based antibody mimics that bind vascular endothelial growth factor receptor two (VEGF-R2). The first antibody mimic, which has a dissociation constant (Kd) of 13 nM, is highly stable [melting temperature (Tm) = 62°C] and soluble, whereas the second, which binds VEGF-R2 with 40x higher affinity, is less stable (Tm < 40°C) and relatively insoluble. We used our understanding of these two 10Fn3 derivatives and of wild-type 10Fn3 structure to engineer the next generation of antibody mimics, which have an improved combination of high affinity (Kd = 0.59 nM), stability (Tm = 53°C) and solubility. Our findings illustrate that 10Fn3-based antibody mimics can be engineered for favorable biophysical properties even when 20% of the wild-type 10Fn3 sequence is mutated in order to satisfy target-binding requirements.
Keywords: antibody mimic/fibronectin/Fn3/VEGF-R2
Received April 21, 2005; accepted July 4, 2005.
Edited by Elizabeth Meiering
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Shen, M. D. Vil, X. Jimenez, M. Iacolina, H. Zhang, and Z. Zhu Single Variable Domain-IgG Fusion: A NOVEL RECOMBINANT APPROACH TO Fc DOMAIN-CONTAINING BISPECIFIC ANTIBODIES J. Biol. Chem., April 21, 2006; 281(16): 10706 - 10714. [Abstract] [Full Text] [PDF]
|
|