i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4

Katherine Griffiths; Olan Dolezal; Benjamin Cao; Susan K Nilsson; Heng B See; Kevin D G Pfleger; Michael Roche; Paul R Gorry; Andrew Pow; Katerina Viduka; Kevin Lim; Bernadine G C Lu; Denison H C Chang; Thomas Murray-Rust; Marc Kvansakul; Matthew A Perugini; Con Dogovski; Marcel Doerflinger; Yuan Zhang; Kathy Parisi; Joanne L Casey; Stewart D Nuttall; Michael Foley

doi:10.1074/jbc.M116.721050

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4

J Biol Chem. 2016 Jun 10;291(24):12641-12657. doi: 10.1074/jbc.M116.721050. Epub 2016 Apr 1.

Authors

Katherine Griffiths¹, Olan Dolezal², Benjamin Cao³, Susan K Nilsson³, Heng B See⁴, Kevin D G Pfleger⁵, Michael Roche⁶, Paul R Gorry⁷, Andrew Pow¹, Katerina Viduka¹, Kevin Lim¹, Bernadine G C Lu¹, Denison H C Chang¹, Thomas Murray-Rust¹, Marc Kvansakul⁸, Matthew A Perugini⁸, Con Dogovski⁸, Marcel Doerflinger¹, Yuan Zhang⁹, Kathy Parisi¹⁰, Joanne L Casey¹, Stewart D Nuttall², Michael Foley¹¹

Affiliations

¹ From AdAlta Pty. Ltd., 15/2 Park Dr., Bundoora, Victoria 3083.
² Biomedical Manufacturing, CSIRO Manufacturing, 343 Royal Parade, Parkville, Victoria 3052.
³ the Australian Regenerative Medicine Institute, Monash University, Wellington Road, Clayton, Victoria 3800,; Biomedical Manufacturing, CSIRO Manufacturing, Bayview Avenue, Clayton, Victoria 3168.
⁴ the Harry Perkins Institute of Medical Research, Nedlands, Western Australia 6009,; the Centre for Medical Research, University of Western Australia, Crawley, Western Australia 6009.
⁵ the Harry Perkins Institute of Medical Research, Nedlands, Western Australia 6009,; the Centre for Medical Research, University of Western Australia, Crawley, Western Australia 6009,; Dimerix Bioscience Ltd., Nedlands, Western Australia 6009.
⁶ the Doherty Institute for Infection and Immunity, University of Melbourne, 792 Elizabeth Street, Melbourne, Victoria 3000,; the Burnet Institute, 85 Commercial Road, Melbourne, Victoria 3004.
⁷ the School of Health and Biomedical Sciences, College of Science, Engineering and Health, RMIT University, Melbourne, Victoria 3001.
⁸ the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, and.
⁹ the Department of Medicine, St. Vincent's Hospital, University of Melbourne, Fitzroy, Victoria 3065, Australia.
¹⁰ From AdAlta Pty. Ltd., 15/2 Park Dr., Bundoora, Victoria 3083,; the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, and.
¹¹ From AdAlta Pty. Ltd., 15/2 Park Dr., Bundoora, Victoria 3083,; the Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, and. Electronic address: m.foley@latrobe.edu.au.

Abstract

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.

Keywords: CXC chemokine receptor type 4 (CXCR-4); G protein-coupled receptor (GPCR); antibody engineering; hematopoietic stem cells; protein structure; single-domain antibody (sdAb, nanobody).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibody Specificity / immunology
Binding Sites / immunology
Cell Line, Tumor
Cell Movement / drug effects
Cell Movement / immunology
Cells, Cultured
Crystallography, X-Ray
Epitope Mapping
HEK293 Cells
HIV Infections / immunology
HIV Infections / prevention & control
HL-60 Cells
Humans
Jurkat Cells
Mice, Inbred BALB C
Mice, Inbred NOD
Mice, Knockout
Mice, SCID
Models, Molecular
Protein Binding / immunology
Protein Domains
Receptors, CXCR4 / antagonists & inhibitors*
Receptors, CXCR4 / immunology*
Receptors, CXCR4 / metabolism
Single-Domain Antibodies / chemistry
Single-Domain Antibodies / immunology*
Single-Domain Antibodies / pharmacology*
Surface Plasmon Resonance

Substances

Receptors, CXCR4
Single-Domain Antibodies

Associated data

PDB/1QZ1
PDB/3ODU
PDB/5AEA