Distinct Conformations Of The Chemokine Receptor CCR4 With Implications For Biased Agonist Activity And Therapeutic Targeting In Allergy

J Pease1, J Viney1, D Andrew3, R Phillips1, A Meiser1, P Patel1, M Lennartz-Walker2, D Cousins2, N Barton3, D Hall3. 1Imperial College London, London, UK, 2King’s College London, London, UK, 3Glaxo-Smith Kline, Stevenage, UK

The chemokine receptor CCR4 is expressed by both structural cells and Th2 lymphocytes implicated in the allergic response and induces cell migration and activation in response to the chemokines CCL17 and CCL22. Expression of both chemokines is elevated in allergic diseases such as asthma and atopic dermatitis, making CCR4 a therapeutic target for the treatment of allergy. As a GPCR, CCR4 is inherently druggable and several small molecule antagonists have been described in the literature. One possible caveat with such therapies is that CCR4 also plays a role in the recruitment of T regulatory cells, which play a role in immunosuppression. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4 (1), which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function using assays of calcium release, chemotaxis, receptor endocytosis and ligand binding (2). Data are described as mean ± SEM (n) with analysis performed using one-way ANOVA or unpaired Student’s T-test, as applicable.

Both ligands had comparable potency and efficacy in Ca2+ assays, with human Th2 cells (EC50 values of 1.6 nM (CCL17) and 2.5 nM (CCL22; n=3) and induced comparable bell shaped responses in chemotaxis assays with the human T cell line Hut-78 (n=3). However, as previously reported, CCL22 showed dominance in receptor endocytosis assays and competitive ligand binding assays. Using the 1G1 mAb to measure CCR4 endocytosis, 100nM CCL22 induced significant internalization of CCR4 (p<0.001) whilst CCL17 was without effect (n=3). Similarly, in ligand binding assays, whilst 125I-CCL17 was fully displaced by a thousand-fold excess of unlabelled CCL17 or CCL22, a remnant of 125I-CCL22 was unable to be displaced by a similar excess of CCL17 (p<0.001; n=3). Using the 1G1 mAb and an alternative mAb 10E4, we found CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. 10E4 stained a major population of CCR4 on Hut-78 cells (10,749 ± 417 binding sites) whilst 1G1 stained a minor population 590 ± 356 binding sites (n=3). Similar staining profiles for the two mAbs were also observed on human Th2 cells and CCR4 transfectants. 10E4 mAb blockade ablated chemotactic responses of CCR4 transfectants to CCL17 (p<0.05), whilst responses to CCL22 were not significantly affected (n=3). Mutation of a single C-terminal residue (K310) within a putative CCR4 antagonist binding site, had no significant effect upon receptor expression or ligand binding, but ablated the activity of CCL17 in chemotaxis assays (p<0.001) whilst CCL22 responses were largely intact (n=3).

We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms (2). This supports our previous finding that the ligands are biased agonists of CCR4 on lung epithelial cells, with CCL17, but not CCL22, inducing transcription of α-CGRP (3). Moreover, our data suggest that selective blockade of CCR4 in allergy may be feasible where one CCR4 ligand dominates, allowing the inhibition of Th2 signalling via one ligand whilst sparing T regulatory cell recruitment via another.

(1) Mariani et al, Eur. J. Immunol. 34:231, 2004.

(2) Viney et al, J. Immunol. In press, 2014

(3) Bonner et al, J. Allergy. Clin. Immunol. 132:942, 2013.