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A novel multi-parameter assay to dissect the pharmacological effects of different modes of integrin ?L?2 inhibition in whole blood.

ABSTRACT: BACKGROUND AND PURPOSE:The integrin ?L?2 plays central roles in leukocyte adhesion and T cell activation, rendering ?L?2 an attractive therapeutic target. Compounds with different modes of ?L?2 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 ?L?2 inhibitors in the peripheral blood of treated patients. EXPERIMENTAL APPROACH:Here, we describe a flow cytometry-based technology that simultaneously quantitates ?L?2 conformational change upon inhibitor binding, ?L?2 expression and T cell activation at the single-cell level in human blood. Two classes of allosteric low MW inhibitors, designated ? I and ?/? I allosteric ?L?2 inhibitors, were investigated. The first application revealed intriguing inhibitor class-specific profiles. KEY RESULTS:Half-maximal inhibition of T cell activation was associated with 80% epitope loss induced by ? I allosteric inhibitors and with 40% epitope gain induced by ?/? I allosteric inhibitors. This differential establishes that inhibitor-induced ?L?2 epitope changes do not directly predict the effect on T cell activation. Moreover, we show here for the first time that ?/? I allosteric inhibitors, in contrast to ? I allosteric inhibitors, provoked partial downmodulation of ?L?2, revealing a novel property of this inhibitor class. CONCLUSIONS AND IMPLICATIONS:The multi-parameter whole blood ?L?2 assay described here may enable therapeutic monitoring of ?L?2 inhibitors in patients' blood. The assay dissects differential effect profiles of different classes of ?L?2 inhibitors.

SUBMITTER: Welzenbach K

PROVIDER: S-EPMC4621979 | BioStudies | 2015-01-01

REPOSITORIES: biostudies

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Project description:Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

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A novel multi-parameter assay to dissect the pharmacological effects of different modes of integrin ?L?2 inhibition in whole blood.

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