title: Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.
abstract: The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.
authors.affiliation: Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., P.O. Box 368, Ridgefield, CT 06877, USA. jwoska@rdg.boehringer-ingelheim.com
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Woska
authors.forename: Kathleen
authors.initials: K
authors.lastname: Last-Barney
authors.forename: Robert
authors.initials: R
authors.lastname: Rothlein
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Kroe
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Reilly
authors.forename: Deborah D
authors.initials: DD
authors.lastname: Jeanfavre
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Mainolfi
authors.forename: Terence A
authors.initials: TA
authors.lastname: Kelly
authors.forename: Gary O
authors.initials: GO
authors.lastname: Caviness
authors.forename: Steven E
authors.initials: SE
authors.lastname: Fogal
authors.forename: Maret J
authors.initials: MJ
authors.lastname: Panzenbeck
authors.forename: Takashi K
authors.initials: TK
authors.lastname: Kishimoto
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Giblin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity / Q000276:immunology; D001667:Binding, Competitive; D040881:CD11a Antigen / Q000276:immunology / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007093:Imidazoles / Q000378:metabolism* / Q000494:pharmacology; D048289:Imidazolidines; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology* / Q000378:metabolism; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism; D002679:Pan troglodytes; D016028:Receptors, Leukocyte-Adhesion / Q000276:immunology / Q000502:physiology; D012453:Saimiri
chemical_list: D000911:Antibodies, Monoclonal; C400461:BIRT 377; D040881:CD11a Antigen; D015815:Cell Adhesion Molecules; D007093:Imidazoles; D048289:Imidazolidines; D016169:Lymphocyte Function-Associated Antigen-1; D016028:Receptors, Leukocyte-Adhesion