Surrogate Antibodies That Specifically Bind and Neutralize CCL17 But Not CCL22

Abstract

The chemokines CCL17 (TARC) and CCL22 (MDC) function through the same receptor, CCR4, but have been proposed to differentially affect the immune response. To better understand the role of the individual ligands, a panel of rat anti-mouse CCL17 surrogate antibodies was generated that can be used to differentiate CCL17 and CCL22 function in vitro and in vivo. We have successfully identified a panel of neutralizing antibodies by screening hybridomas for the ability to inhibit CCL17-mediated calcium mobilization. Chemotaxis in response to CCL17 is also inhibited, providing further evidence that the antibodies in this panel are antagonistic. Using a recombinant cell line expressing human CCR4, we show that the antibodies block β-arrestin recruitment as evidence that the antibodies are specifically blocking CCL17 signaling through CCR4. The antibodies within this panel inhibit calcium mobilization with varying potency in the calcium flux assay, having apparent IC50 ranging from approximately 1 to >400 ng/mL. Although both CCL17 and CCL22 function through CCR4, only a single antibody was identified as having detectable binding to CCL22. This panel of CCL17-specific antibodies provides tools that can be used to differentiate CCL17 and CCL22 function through CCR4 interaction in vitro and in vivo.

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

“…CCL17 protein was expressed in E. coli then isolated from inclusion bodies and refolded to generate functional chemokine as previously described [30]. Briefly, inclusion bodies were collected in solubilization buffer consisting of 8 M urea, 5 mM EDTA, 20 mM Tris HCl, pH 7, 10 mM DTT.…”

“…Studies blocking CCL17 in vivo are reported in the literature and these studies have been conducted using commercially available polyclonal antibodies or monoclonal rat anti-CCL17 antibody, as in the murine A. fumigatus model of invasive lung disease [12]. To study the effects of inhibiting CCL17 function in vivo , we have generated rat:mouse (IgG1) chimeric mAbs and demonstrated that upon binding CCL17 they inhibit signaling mediated through CCR4 interaction [30]. These antibodies were generated using the antigen-binding region of hybridomas from rats immunized with CCL17 and fused to mouse IgG1 Fc.…”

“…These antibodies were generated using the antigen-binding region of hybridomas from rats immunized with CCL17 and fused to mouse IgG1 Fc. Chimeric mAbs were characterized for binding specificity and ability to inhibit CCL17 function as previously described [30]. Two antibodies were chosen for further study based on their unique characteristics compared to the rest of the antibodies in this panel.…”

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

“…Since both chemokines are known to interact exclusively with CCR4, both compete for CCR4 binding and the most conserved regions are predicted to reside within the receptor binding site of each chemokine, one would expect to obtain a set of antibodies which crossreact with CCL17 and CCL22 [8,17] . Despite this, only a single dual-reactive antibody, B202, was identified in our screen, representing <1% of the entire panel [15] . In addition to binding, B202 was also shown to block CCL22 function, although this effect was weak in that only partial inhibition was evident using conditions that were sufficient to completely inhibit CCL17 function.…”

“…The commercially available monoclonal anti-CCL17 antibody, MAB529, is a rat IgG2a antibody that inhibits CCL17 but not CCL22 function and although useful in vitro, may complicate interpretation of in vivo results due to Fcmediated effector function. In contrast, B225 and B202 are mouse: rat chimeric antibodies such that the Fv regions are derived from rat antibodies but the Fc regions have been converted to mouse IgG1which lacks effector function [15] . This eliminates any ambiguity surrounding contribution of the rat Fc in the interpretation of in vivo findings.…”