Submitted March 6, 2006
Accepted December 1, 2006
Generation of activation-specific human anti-
M
2 single-chain antibodies as potential diagnostic tools and therapeutic agents
Steffen U Eisenhardt, Meike Schwarz, Nils Schallner, Juliana Soosairajah, Nicole Bassler, Dexing Huang, Christoph Bode, and Karlheinz Peter*
Baker Heart Research Institute, Melbourne, Victoria, Australia
Dept of Cardiology, University of Freiburg, Freiburg, Germany
* Corresponding author; email: karlheinz.peter{at}baker.edu.au.
The leukocyte integrin Mac-1 (
M
2) plays a pivotal role in inflammation and host defense. Upon leukocyte activation, Mac-1 undergoes a conformational change exposing interaction sites for multiple ligands. We aimed to generate single-chain antibodies (scFv) directed against activation-specific Mac-1 ligand binding sites. Using human scFv phage libraries, we developed subtractive strategies with depletion of phages binding to non-activated Mac-1 and selection of phages binding to activated Mac-1, using monocytes as well as CHO cells transfected with native or mutated, activated Mac-1. Three scFv clones demonstrated exclusive binding to activated Mac-1. Mac-1 binding of the ligands fibrinogen, heparin and ICAM-1, but not C3bi, was inhibited. Using alanine substitutions, the paratope was identified within the scFvs' heavy chain HCDR3. The epitope was localized to Lys245-Arg261 of the M I-domain. In a pilot study with septicaemic patients, we provide initial support for these scFvs' use as markers of monocyte activation and as potential diagnostic tools. Potential therapeutic use was tested in adhesion assays under static and flow conditions demonstrating the selective blockade of activated monocytes only. Furthermore, scFv HCDR3-derived peptides retain selectivity for the activated integrin, providing a unique template for the potential development of inhibitors that are specific for the activated Mac-1.
