Engagement of Two Distinct Binding Domains on CCL17 Is Required for Signaling through CCR4 and Establishment of Localized Inflammatory Conditions in the Lung

Abstract

CCL17 (TARC) function can be completely abolished by mAbs that block either one of two distinct sites required for CCR4 signaling. This chemokine is elevated in sera of asthma patients and is responsible for establishing inflammatory sites through CCR4-mediated recruitment of immune cells. CCL17 shares the GPCR CCR4, with CCL22 (MDC) but these two chemokines differentially affect the immune response. To better understand chemokine mediated effects through CCR4, we have generated chimeric anti-mouse CCL17 surrogate antibodies that inhibit function of this ligand in vitro and in vivo. The affinities of the surrogate antibodies for CCL17 range from 685 pM for B225 to 4.9 nM for B202. One antibody, B202, also exhibits weak binding to CCL22 (KD∼2 µM) and no binding to CCL22 is detectable with the second antibody, B225. In vitro, both antibodies inhibit CCL17-mediated calcium mobilization, β-arrestin recruitment and chemotaxis; B202 can also partially inhibit CCL22-mediated β-arrestin recruitment. Both B202 and B225 antibodies neutralize CCL17 in vivo as demonstrated by reduction of methacholine-induced airway hyperreactivity in the A. fumigatus model of asthma. That both antibodies block CCL17 function but only B202 shows any inhibition of CCL22 function suggests that they bind CCL17 at different sites. Competition binding studies confirm that these two antibodies recognize unique epitopes that are non-overlapping despite the small size of CCL17. Taking into consideration the data from both the functional and binding studies, we propose that effective engagement of CCR4 by CCL17 involves two distinct binding domains and interaction with both is required for signaling.

Biased agonism at chemokine receptors: obstacles or opportunities for drug discovery?

“…Data from our own group have provided further insight into the modes of ligand binding to CCR4 (63). In support of the data by Santulli-Marotto et al (62), there appears to be a distinct difference in the molecular mechanisms by which CCL17 and -22 activate CCR4. Whereas mutation of the C-terminal residue K310 had little effect on migration in response to CCL22, it abolished the chemotactic activity of CCL17.…”

“…However, recent work has suggested that this simplistic view of CCR4 activation does not explain experimental observations. Using 2 distinct recombinant monoclonal antibodies against CCL17, each composed of a chimeric molecule with rat V L and V H domains fused with mouse IgG1 Fc, Santulli-Marotto et al (62) found that, whereas both antibodies inhibited CCL17 function, only 1 of the 2 was effective in blocking CCL22 activity. Furthermore, in competitive binding assays, CCL17, but not -22, competed for CCL17 binding to either antibody, indicating that the 2 antibodies bind to nonoverlapping sites on CCL17.…”

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

“…Tested in the A. fumigatus model of chronic asthma, only B225 was able to ameliorate methacholine-induced AHR at the lower dose of antibody (200μg/mL) whereas both antibodies were able to do this at a 5-fold higher dose [1] . In addition B202 was less effective at modulating goblet cell metaplasia and mucus production in the lung; however, the difference is less obvious with respect to cytokine production.…”

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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.

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CCL22-specific Antibodies Reveal That Engagement of Two Distinct Binding Domains on CCL22 Is Required for CCR4-mediated Function

“…(26) This is in contrast to what was observed following neutralization of CCL17 in the same model system. (26,27) Following four rounds of phage library panning, a panel of eight CCL22 specific antibodies was identified from which three unique clones were chosen for in vitro characterization. Based on the ability to block CCL22-mediated calcium flux and b-arrestin recruitment comparably to a commercially available antibody, MAB4391, we characterized the CCL22 binding characteristics of two of these antibodies and established that MC2B7 recognizes a distinct epitope from MAB4391.…”