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A novel multi-parameter whole blood assay to dissect the pharmacologic effects of different modes of LFA-1 receptor occupancy

Welzenbach, Karl, Mancuso, Riccardo, Krähenbühl, Stephan and Weitz-Schmidt, Gabriele (2015) A novel multi-parameter whole blood assay to dissect the pharmacologic effects of different modes of LFA-1 receptor occupancy. British Journal of Pharmacology.


The integrin lymphocyte function associated antigen-1 (LFA-1) plays central roles in leukocyte adhesion as well as T cell activation and proliferation, rendering LFA-1 an attractive therapeutic target. Different modes of LFA-1 inhibition have been identified and are reported in preclinical and clinical development, respectively. An important question which remains to be answered is whether these different modes of inhibition will translate into different pharmacodynamic effect profiles. Here we describe a multi-parameter flow cytometry-based methodology which allowed, for the first time, to relate different modes and levels of LFA-1 occupancy to distinct pharmacologic effects at the single cell level in human whole blood cultures. Two classes of allosteric small molecule inhibitors, designated αI and α/β I allosteric LFA-1 inhibitors, were investigated. Interestingly, more than 80% LFA-1 occupancy (as measured by distinct compound-induced LFA-1 epitope changes) was required with αI allosteric inhibitors to achieve half-maximal inhibition of T cell activation and proliferation while less than 40 % receptor occupancy by α/β I allosteric inhibitors was sufficient for the same effect size. Further, α/β I allosteric inhibitors induced partial downregulation of LFA-1 from the cell surface revealing a novel property of this inhibitor class distinct from αI allosteric inhibitors. The assay described here opens the possibility to dissect the molecular mechanisms of LFA-1 inhibitors in a physiological environment. This will be essential to identify the best-suited inhibitors for clinical development and to direct them to the most appropriate target indications. The assay may also be used for therapeutic monitoring of LFA-1 inhibitors and possibly other immunosuppressants.

Item Type: Article
Keywords: beta2 integrins, allosteric inhibition, receptor epitope monitoring, LFA-1, whole blood , pharmacodynamic assay, flow cytometry, allosteric inhibitor, T cell activation,
Date Deposited: 07 Jun 2016 23:45
Last Modified: 07 Jun 2016 23:45


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