Pauline Bonvin

Targeting chemokines: Pathogens can, why can't we?

Chemoattractant cytokines, or chemokines, are the largest sub-family of cytokines. About 50 distinct chemokines have been identified in humans. Their principal role is to stimulate the directional migration of leukocytes, which they achieve through activation of their receptors, following immobilization on cell surface glycosaminoglycans (GAGs). Chemokine receptors belong to the G protein-coupled 7-transmembrane receptor family, and hence their identification brought great promise to the pharmaceutical industry, since this receptor class is the target for a large percentage of marketed drugs. Unfortunately, the development of potent and efficacious inhibitors of chemokine receptors has not lived up to the early expectations. Several approaches to targeting this system will be described here, which have been instrumental in establishing paradigms in chemokine biology. Whilst drug discovery programs have not yet elucidated how to make successful drugs targeting the chemokine system, it is now known that certain parasites have evolved anti-chemokine strategies in order to remain undetected by their hosts. What can we learn from them?

Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models

Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine‐binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin‐4. Evasin‐4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase‐type plasminogen activator signal peptide. Using SPR, Evasin‐4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine‐induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin‐4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin‐4 did not inhibit CCL25‐induced chemotaxis. Size‐exclusion chromatography confirmed that Evasin‐4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin‐4 was rapidly cleared following in vivo administration. To enhance the in vivo half‐life and optimize its potential as a therapeutic agent, Fc fusions of Evasin‐4 were created. Both the N‐ and C‐terminal fusions were shown to retain binding activity, with the C‐terminal fusion showing a modest reduction in potency.

De novo isolation of antibodies with pH-dependent binding properties

pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.

Evasins: Therapeutic Potential of a New Family of Chemokine-Binding Proteins from Ticks

Blood-sucking parasites, such as ticks, remain attached to their hosts for relatively long periods of time in order to obtain their blood meal without eliciting an immune response. One mechanism used to avoid rejection is the inhibition of the recruitment of immune cells, which can be achieved by a class of chemokine-binding proteins (CKBPs) known as Evasins. We have identified three distinct Evasins produced by the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. They display different selectivities for chemokines, the first two identified show a narrow selectivity profile, while the third has a broader binding spectrum. The Evasins showed efficacy in animal models of inflammatory disease. Here, we will discuss the potential of their development for therapeutic use, addressing both the advantages and disadvantages that this entails.

Glycosaminoglycan Interactions with Chemokines Add Complexity to a Complex System

Chemokines have two types of interactions that function cooperatively to control cell migration. Chemokine receptors on migrating cells integrate signals initiated upon chemokine binding to promote cell movement. Interactions with glycosaminoglycans (GAGs) localize chemokines on and near cell surfaces and the extracellular matrix to provide direction to the cell movement. The matrix of interacting chemokine–receptor partners has been known for some time, precise signaling and trafficking properties of many chemokine–receptor pairs have been characterized, and recent structural information has revealed atomic level detail on chemokine–receptor recognition and activation. However, precise knowledge of the interactions of chemokines with GAGs has lagged far behind such that a single paradigm of GAG presentation on surfaces is generally applied to all chemokines. This review summarizes accumulating evidence which suggests that there is a great deal of diversity and specificity in these interactions, that GAG interactions help fine-tune the function of chemokines, and that GAGs have other roles in chemokine biology beyond localization and surface presentation. This suggests that chemokine–GAG interactions add complexity to the already complex functions of the receptors and ligands.

Identification of the Pharmacophore of the CC Chemokine-binding Proteins Evasin-1 and -4 Using Phage Display

Background: The selectivity profiles of the closely related chemokine-binding proteins Evasin-1 and -4 differ. Results: Using phage display, we identified the N-terminal region of Evasin-4 as key for the interaction with CC chemokines. Conclusion: Evasin-1 and -4 use different domains for target binding. Significance: Phage display allowed rapid insight into their different selectivities, which could aid rational design of inhibitory proteins. To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.

CCL18 Exhibits a Regulatory Role through Inhibition of Receptor and Glycosaminoglycan Binding

CCL18 has been reported to be present constitutively at high levels in the circulation, and is further elevated during inflammatory diseases. Since it is a rather poor chemoattractant, we wondered if it may have a regulatory role. CCL18 has been reported to inhibit cellular recruitment mediated by CCR3, and we have shown that whilst it is a competitive functional antagonist as assessed by Schild plot analysis, it only binds to a subset of CCR3 receptor populations. We have extended this inhibitory activity to other receptors and have shown that CCL18 is able to inhibit CCR1, CCR2, CCR4 and CCR5 mediated chemotaxis, but has no effect on CCR7 and CCR9, nor the CXC receptors that we have tested. Whilst CCL18 is able to bind to CCR3, it does not bind to the other receptors that it inhibits. We therefore tested the hypothesis that it may displace glycosaminoglycan (GAG) chemokines bound either in cis- on the leukocyte, or in trans-presentation on the endothelial surface, thereby inhibiting the recruitment of leukocytes into the site of inflammation. We show that CCL18 selectivity displaces heparin bound chemokines, and that chemokines from all four chemokine sub-classes displace cell bound CCL18. We propose that CCL18 has regulatory properties inhibiting chemokine function when GAG-mediated presentation plays a role in receptor activation.

Antibody neutralization of CXCL10 in vivo is dependent on binding free and not endothelial bound chemokine: implications for the design of a new generation of anti-chemokine therapeutic antibodies

To improve our understanding of properties that confer successful inhibition of chemokines in vivo, we analyzed anti-murine CXCL10 monoclonal antibodies (mAb) having different characteristics. 1B6 displayed potent inhibition of cell recruitment in vitro with an IC50 of 0.5 nm but demonstrated little efficacy in various animal models of human disease. On the contrary, 1F11 showed efficacy in several models of inflammation yet was less potent at inhibiting chemotaxis in vitro with an IC50 of 21 nm. Furthermore, we observed that 1B6 displayed a rapid dose-dependent clearance (t½ 10–60 h) in contrast to 1F11, which presented a dose-proportional pharmacokinetic profile and a half-life of 12 days. Moreover, 1B6 recognized glycosaminoglycan (GAG)-bound CXCL10, resulting in target-mediated clearance, which was corroborated using CXCL10-deficient mice. In contrast to 1B6, 1F11 inhibited the interaction of CXCL10 with GAGs, did not recognize GAG-bound CXCL10, and did not display target-mediated drug disposition. Confirming previous animal studies, 1B6 was poor at reversing glycemia in a model of type 1 diabetes, whereas 1F11 induced early and prolonged control of diabetes. Furthermore, when using 1A4, a subsequently generated anti-mCXCL10 mAb that shares the property with 1F11 of being unable to recognize CXCL10 immobilized on GAG, we observed a similar superior control of diabetes as compared with 1B6. We therefore concluded that targeting chemokines with antibodies such as 1B6 that recognize the more abundant GAG-bound form of the chemokine may not be the optimal strategy to achieve disease control.

Methods for the Recognition of GAG-Bound Chemokines.

Chemokines play a pivotal role in the multistep cascade of cellular recruitment, where they provide the directional signal. They activate cells through a high-affinity interaction with their receptors, members of the large family of heptahelical G protein-coupled receptors. In order to provide the directional signal, they bind to cell surface proteoglycans through a low-affinity interaction with the glycosaminoglycan (GAG) moiety. While several methods have been described to measure the chemokine-GAG interaction, this chapter describes methods to identify whether anti-chemokine antibodies or chemokine-binding proteins recognize the GAG-bound chemokine.

Mutagenesis by Phage Display

Chemokines are small chemoattractant proteins involved in the recruitment of leukocytes to the site of inflammation. Due to their prominent role in the inflammatory process, chemokine inhibitors have been developed by parasites to remain undetected not only by the host immune system but also by various laboratories to develop anti-inflammatory compounds. Taking advantage of the small size of natural chemokine-binding proteins, we report here several methods to facilitate their characterization using phage display to identify the chemokine-binding site and to modulate the selectivity of such inhibitors. Interestingly, these methods could be adapted to display the natural inhibitors of other cytokines or even cytokines on phage surface.