Generation of activation-specific human anti-alphaMbeta2 single-chain antibodies as potential diagnostic tools and therapeutic agents.
The leukocyte integrin Mac-1 (alpha(M)beta(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's) directed against activation-specific Mac-1 ligand-binding sites. Using human scFv phage libraries, we developed subtractive strategies with depletion of phages binding to nonactivated 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 heavy chain HCDR3s of the scFv's. The epitope was localized to Lys(245)-Arg(261) of the alpha(M) I-domain. In a pilot study with septicemic patients, we provide initial support for the use of these scFv's 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.
Eisenhardt
Steffen U
SU
Baker Heart Research Institute, Melbourne, Victoria, Australia.
Schwarz
Meike
M
Schallner
Nils
N
Soosairajah
Juliana
J
Bassler
Nicole
N
Huang
Dexing
D
Bode
Christoph
C
Peter
Karlheinz
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology / Q000627:therapeutic use; D016466:CHO Cells; D002448:Cell Adhesion / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D007249:Inflammation / Q000175:diagnosis / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D008262:Macrophage Activation / Q000187:drug effects; D016177:Macrophage-1 Antigen / Q000276:immunology*; D008264:Macrophages / Q000276:immunology* / Q000473:pathology
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D016177:Macrophage-1 Antigen