title: A novel multi-parameter assay to dissect the pharmacological effects of different modes of integrin αLβ2 inhibition in whole blood.
abstract: OBJECTIVE
  The integrin αLβ2 plays central roles in leukocyte adhesion and T cell activation, rendering αLβ2 an attractive therapeutic target. Compounds with different modes of αLβ2 inhibition are in development, currently. Consequently, there is a foreseeable need for bedside assays, which allow assessment of the different effects of diverse types of αLβ2 inhibitors in the peripheral blood of treated patients.
  
  
  METHODS
  Here, we describe a flow cytometry-based technology that simultaneously quantitates αLβ2 conformational change upon inhibitor binding, αLβ2 expression and T cell activation at the single-cell level in human blood. Two classes of allosteric low MW inhibitors, designated α I and α/β I allosteric αLβ2 inhibitors, were investigated. The first application revealed intriguing inhibitor class-specific profiles.
  
  
  RESULTS
  Half-maximal inhibition of T cell activation was associated with 80% epitope loss induced by α I allosteric inhibitors and with 40% epitope gain induced by α/β I allosteric inhibitors. This differential establishes that inhibitor-induced αLβ2 epitope changes do not directly predict the effect on T cell activation. Moreover, we show here for the first time that α/β I allosteric inhibitors, in contrast to α I allosteric inhibitors, provoked partial downmodulation of αLβ2, revealing a novel property of this inhibitor class.
  
  
  CONCLUSIONS
  The multi-parameter whole blood αLβ2 assay described here may enable therapeutic monitoring of αLβ2 inhibitors in patients' blood. The assay dissects differential effect profiles of different classes of αLβ2 inhibitors.
authors.affiliation: Novartis Pharma AG, Novartis Institutes of Biomedical Research, Basel, Switzerland.
authors.forename: Karl
authors.initials: K
authors.lastname: Welzenbach
authors.affiliation: Division of Clinical Pharmacology and Toxicology, University Hospital, Basel, Switzerland.
authors.forename: Riccardo V
authors.initials: RV
authors.lastname: Mancuso
authors.affiliation: Division of Clinical Pharmacology and Toxicology, University Hospital, Basel, Switzerland.
authors.forename: Stephan
authors.initials: S
authors.lastname: Krähenbühl
authors.affiliation: Novartis Pharma AG, Novartis Institutes of Biomedical Research, Basel, Switzerland.|AlloCyte Pharmaceuticals AG, Basel, Switzerland.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Weitz-Schmidt
mesh_terms: D001681:Biological Assay; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D008213:Lymphocyte Activation; D016169:Lymphocyte Function-Associated Antigen-1 / Q000097:blood*; D009281:Naphthalenes / Q000494:pharmacology*; D010078:Oxazines / Q000494:pharmacology*; D010795:Phthalic Acids / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000276:immunology; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000939:Epitopes; C494439:LFA 878; D016169:Lymphocyte Function-Associated Antigen-1; D009281:Naphthalenes; D010078:Oxazines; D010795:Phthalic Acids; C546917:XVA 143; D015091:beta-Alanine