Differential effects of low molecular weight inhibitors on the conformation and the immunologic function of the adhesion receptor LFA-1 (2004)

Previous studies of our group 1,2 and by others 3 on the isolated ligand binding&#13;\ndomain of LFA-1 (αL I domain) have suggested that some LFA-1 inhibitors act&#13;\nallosterically while other inhibitors were proposed to competitively block the LFA-&#13;\n1/ligand interaction 4. We postulated that LMW LFA-1 inhibitors allosterically alter the&#13;\nLFA-1 receptor conformation, resulting in shielding or neo-expression of epitopes&#13;\nrecognized by monoclonal antibodies (mAbs) mapping to regulatory domains of the&#13;\naL or b2 chains. Our data revealed that LFA-1 inhibitors can be differentiated according to their mode&#13;\nof action on the receptor level.&#13;\nThe first group of lovastatin-derived LFA-1 inhibitors strongly induced conformational&#13;\nchanges within the aL I domain. This was detected by the potent inhibition of the&#13;\nbinding of the mAb R7.1 (anti CD11a, aL I domain specific) to either purified LFA-1 or&#13;\nLFA-1 expressed on Jurkat T-cells. The degree of epitope reduction by the LFA-1&#13;\nantagonists tested, correlated well with the potency in inhibition of the LFA-1/ICAM-1&#13;\ninteraction. These LFA-1 inhibitors had no effect on the binding of mAbs directed to&#13;\nother domains within LFA-1.&#13;\nIn contrast, one lovastatin-derived inhibitor (LFA703) induced epitope changes in the&#13;\naL I domain and also in the b2 I-like domain, a regulatory domain located on the b2&#13;\nchain of LFA-1. This effect became evident by the reduced binding of mAb IB4 (anti&#13;\nCD18; b2 I-like domain specific) to cation-activated LFA-1 in the presence of&#13;\nLFA703. These results demonstrated that amongst lovastatin-derived inhibitors&#13;\nsubclasses exit, which exert differential effects on the LFA-1 receptor conformation.&#13;\nMoreover, the antibody binding patterns observed on native LFA-1 receptors in the&#13;\npresence of various inhibitors demonstrated that upon receptor activation a&#13;\nconformational interaction between the aL I domain and the b2 I-like domain is formed. These findings have meanwhile been confirmed by others in more&#13;\ncomprehensive biochemical studies 5.&#13;\nFor the first time our results provided strong evidence that the b2 I-like domain&#13;\nembodies a target for allosteric LFA-1 inhibition similar to the well established&#13;\nregulatory L-site in the aL I domain. XVA143, a suggested ICAM-1 mimetic, which&#13;\nwas proposed by the inventors to be a competitive aL I domain inhibitor 4, blocked&#13;\nthe binding of the β2 I-like domain specific mAb IB4 with nM potency. XVA143 had no&#13;\neffect on the binding of mAb R7.1 or other anti CD11a mAbs under all experimental&#13;\nconditions, and did not bind to the αL L-site as determined by NMR studies.&#13;\nFurthermore, we showed that the target of XVA143 is most probably located on the&#13;\nβ2 chain, as the compound also blocked the binding of mAb IB4 to purified Mac-1&#13;\n(αMβ2) and inhibited the interaction of purified Mac-1 with ICAM-1. The compound&#13;\ntypifies therefore a novel class of LFA-1 inhibitors with a distinct, probably allosteric&#13;\nmode of action. These findings provided evidence that the β2 I-like domain could&#13;\nrepresent a new target for potent inhibition of adhesion receptors of the β2 integrin&#13;\nsubgroup. Potent LMW inhibitors like XVA143 may open new opportunities for&#13;\nspecific intervention with the function of β2 integrins. These inhibitors could be&#13;\ntherapeutically useful in transplantation, autoimmune diseases and inflammatory&#13;\ndisorders.&#13;\nCompellingly, the combined use of various LFA-1 inhibitors and selected monitoring&#13;\nmAbs contributed to the understanding of the mode of action of LFA-1 inhibitors and&#13;\nthe function of β2 integrins on a molecular level. In addition, our findings show that&#13;\ncurrently available LFA-1 inhibitors can be differentiated into two major groups&#13;\naccording to their mode of action on the receptor level: the αL L-site inhibitors and the&#13;\nputative β2 I-like domain inhibitors. LMW LFA-1 inhibitors may soon enter clinical trials. For their pharmacological and&#13;\nsafety evaluation in clinical studies, it will be mandatory to provide, in addition to&#13;\npharmacokinetic (PK) measurements, insights in the pharmacodynamic (PD)&#13;\nproperties of these potentially immunosuppressive and anti-inflammatory&#13;\ncompounds. The aim of the studies described here was to develop the methodology&#13;\nfor studying the effect of LFA-1 inhibitors on receptor occupancy, receptor expression&#13;\nand T-cell function in whole blood. These studies are intended as a basis for the&#13;\npharmacodynamic characterization of LFA-1 inhibitors in clinical trials. Furthermore,&#13;\nthe effect of LFA-1 inhibitors on T-cell function was compared to the&#13;\nimmunosuppressants cyclosporine A and everolimus. LFA-1 inhibitors of different chemical classes were tested in novel whole blood&#13;\nreceptor epitope monitoring assays (REMAs). We designate here REMAs as&#13;\ncytometric methods which use target-specific mAbs to detect receptor occupancy by&#13;\nLMW compounds in whole blood. The lovastatin-derived LFA-1 inhibitor LFA878 and&#13;\nthe experimental COMPOUND X, a non lovastatin-derived LFA-1 inhibitor, blocked&#13;\nthe binding of mAb R7.1 to leukocytes in undiluted blood with nM potencies. As&#13;\nexpected, the putative β2 I-like domain inhibitor XVA143 was unable to alter the&#13;\nbinding of mAb R7.1 to leukocytes in whole blood. In contrast, we found that LFA-1&#13;\nreceptor occupancy by XVA143 led to a significantly increased binding of the β2&#13;\nchain, stalk region specific mAb MEM48 to whole blood leukocytes. These results&#13;\ndemonstrated for the first time that LFA-1 inhibitors with different modes of action can&#13;\ninteract with LFA-1 in undiluted human blood and that target occupancy can be&#13;\nmonitored by selected mAbs.&#13;\nThe REMA principle was validated ex vivo by measuring LFA-1 receptor occupancy&#13;\nin blood of rabbits after i.v. administration of LFA878. LFA878 blocked the binding of&#13;\nthe mAb R7.1 with transient duration of action. Dependent on the dose administered the pharmacodynamic half-life was 0.6 h (11.5 mg/kg i.v.) or 3.3 h (50mg/kg i.v.).&#13;\nThese data showed for the first time that the REMA can be applied to study&#13;\npharmacodynamic effects of αL L-site inhibitors in rabbits ex vivo. Our results&#13;\nfurthermore suggested that the αL L-site and the mAb R7.1 epitope are conserved&#13;\nbetween man and rabbit. The pharmacodynamic effects of XVA143 could not be&#13;\ninvestigated because the mAb MEM48 did not cross-react with LFA-1 of other&#13;\nspecies.&#13;\nTo allow the assessment of the effect of LFA-1 inhibitors on several T-cell&#13;\nparameters, we developed an anti CD3 (OKT3) mAb stimulated T-cell activation&#13;\nassay (CD69 readout) and combined it with the REMAs described above. The socalled&#13;\nEA-REMAs allowed us to quantify simultaneously receptor occupancy by LFA-&#13;\n1 inhibitors (REMA), the cell surface LFA-1 expression (E) and the upregulation of&#13;\nthe activation marker CD69 (A) on individual T-lymphocytes after in vitro stimulation&#13;\nof 1:1 diluted blood with immobilized mAb OKT3.&#13;\nLFA878, COMPOUND X and XVA143 completely blocked mAb OKT3 stimulated&#13;\nCD69 upregulation with IC50s of 2 μM, 1 μM and 0.05 μM respectively, while&#13;\npravastatin, a statin that does not bind to LFA-1, was completely inactive at 50 μM.&#13;\nThe LFA-1 inhibitors tested were completely inactive in blood cultures stimulated with&#13;\na combination of mAbs OKT3 and anti CD28, demonstrating the specific inhibition of&#13;\nLFA-1 dependent T-cell responses by the compounds tested.&#13;\nAn additional pharmacodynamic property of XVA143 was revealed by the EA-REMA.&#13;\n22 h incubation of whole blood with XVA143 led to a partial (35-55%) downregulation&#13;\nof LFA-1 cell surface receptors on T-cells, a phenomenon not observed for the αL Lsite &#13;\ninhibitors tested. The compounds were then assessed on their effect on mAb OKT3 stimulated T-cell&#13;\nproliferation in 1:10 diluted blood. All LFA-1 inhibitors blocked mAb OKT3 stimulated T-lymphocyte proliferation with nearly equal potencies than observed in the mAb&#13;\nOKT3 stimulated T-cell activation assay.&#13;\nApplying these protocols, experimental evidence was obtained for the first time that&#13;\nLFA-1 receptor occupancy by LFA-1 inhibitors can translate into efficient blockade of&#13;\nin vitro stimulated T-cell activation and proliferation in whole blood. The correlation&#13;\nbetween receptor occupancy and blockade of T-cell activation and proliferation&#13;\n(response) revealed that a &gt;85% receptor occupancy in whole blood is required by&#13;\nthe αL L-site inhibitors tested for the suppression of T-cell responses in whole blood&#13;\ncultures by 50%. In contrast, an almost 1:1 correlation between receptor occupancy&#13;\nand the resulting suppression of T-cell responses was observed for the β2 I-like&#13;\ndomain inhibitor XVA143. A comparison of LFA-1 inhibitors with cyclosporin A (CsA) and everolimus in the&#13;\nwhole blood assays suggested that the structurally different LFA-1 inhibitors could be&#13;\nuseful as immunosuppressants. XVA143 blocked T-cell activation (0.05 μM) and&#13;\nproliferation (0.02 μM) with higher potency than CsA (0.8 μM; 0.15 μM respectively)&#13;\nand was nearly equipotent to everolimus (0.01 μM) in the whole blood proliferation&#13;\nassay. In contrast, αL L-site inhibitors were nearly as potent as CsA in the CD69 Tcell&#13;\nactivation assay, but significantly less active in whole blood proliferation assays&#13;\n(1-2 μM). As expected, CsA or everolimus did not interfere with LFA-1 expression or&#13;\nthe binding of the monitoring mAbs R7.1 or MEM48.&#13;\nDuring the development of the EA-REMA we found that supplemental MgCl2 strongly&#13;\nsynergi