Characterizing the interaction of CCL17 with CCR4: Elucidating requirements to elicit cellular function

Abstract

Using two unique antibodies that selectively inhibit the function of CCL17 we have revealed that in order for this chemokine to function productively through CCR4 it requires concurrent interaction with two different binding sites on CCL17 [1] . CCR4 is known to interact with only one other ligand, CCL22, and the consequences of CCL17 vs CCL22 interaction with CCR4 are quite different. We have generated two antibodies, B202 and B225, which inhibit CCL17 function yet CCL22 inhibition can be mediated only by B202. Both antibodies perform comparably to block CCL17 function in vitro but differences become apparent in vivo. Methacholineinduced airway hyperresponsiveness (AHR) is effectively reversed by both antibodies but B225 was more effective at lowering IL-4 levels in the lung in the A. fumigatus model of chronic fungal asthma. Mice treated with B225 exhibited a more profound impact on inflammation in the lung accompanied by a decrease in mucus production compared to B202 treated mice as evident in histological analysis. One possible explanation for these disparities could be attributed to binding affinity for CCL17 as the difference in KD between the two antibodies is ~7-fold. However, this cannot fully account for the similarity in vitro. Further investigation of binding characteristics of B225 and B202 using competition binding studies revealed that B202 and B225 bind to non-competing epitopes on CCL17 and treatment with either one of these antibodies blocks CCL17 function through CCR4. This was somewhat surprising in that the size differential between CCL17 and the antibodies is > 15-fold so that the expectation was that any antibody binding to CCL17 would be capable of neutralizing function due to steric hindrance. This is the first report showing that CCL17 is required to engage two different binding sites to mediate CCR4 function.