CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an “i-body,” the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor.
antibody engineering
CXC chemokine receptor type 4 (CXCR-4)
G protein-coupled receptor (GPCR)
hematopoietic stem cells
protein structure
single-domain antibody (sdAb, nanobody)
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The atomic coordinates and structure factors (code5AEA) have been deposited in the Protein Data Bank (http://wwpdb.org/).
This work was supported by a CSIRO OCE Science Leader Fellowship (to S. K. N.). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. K. Lim, J. L. Casey, K. Parisi, and M. Foley are shareholders in Adalta. K. Griffiths, T. Murray-Rust, and M. Foley are currently employed by Adalta. A. Pow, K. Viduka, K. Lim, B. G. C. Lu, D. H. C. Chang, K. Parisi, and J. Casey are former employees of Adalta. All authors or institutions received payment or services from a third party to conduct the research reported here. Some authors have non-financial relationships with entities in the biomedical arena that could be perceived to influence, or that give the appearance of potentially influencing, what is written in this work. K. G. and S. D. N. have an issued patent relating to the reported work, and K. G., A. P., K. V., and M. F. have a pending patent relating to the reported work.
Present address: Dept. of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria 3010, Australia.
