Targeting of CCL2-CCR2-Glycosaminoglycan Axis Using a CCL2 Decoy Protein Attenuates Metastasis through Inhibition of Tumor Cell Seeding.

The CCL2-CCR2 chemokine axis has an important role in cancer progression where it contributes to metastatic dissemination of several cancer types (e.g., colon, breast, prostate). Tumor cell-derived CCL2 was shown to promote the recruitment of CCR2(+)/Ly6C(hi) monocytes and to induce vascular permeability of CCR2(+) endothelial cells in the lungs. Here we describe a novel decoy protein consisting of a CCL2 mutant protein fused to human serum albumin (dnCCL2-HSA chimera) with enhanced binding affinity to glycosaminoglycans that was tested in vivo. The monocyte-mediated tumor cell transendothelial migration was strongly reduced upon unfused dnCCL2 mutant treatment in vitro. dnCCL2-HSA chimera had an extended serum half-life and thus a prolonged exposure in vivo compared with the dnCCL2 mutant. dnCCL2-HSA chimera bound to the lung vasculature but caused minimal alterations in the leukocyte recruitment to the lungs. However, dnCCL2-HSA chimera treatment strongly reduced both lung vascular permeability and tumor cell seeding. Metastasis of MC-38GFP, 3LL, and LLC1 cells was significantly attenuated upon dnCCL2-HSA chimera treatment. Tumor cell seeding to the lungs resulted in enhanced expression of a proteoglycan syndecan-4 by endothelial cells that correlated with accumulation of the dnCCL2-HSA chimera in the vicinity of tumor cells. These findings demonstrate that the CCL2-based decoy protein effectively binds to the activated endothelium in lungs and blocks tumor cell extravasation through inhibition of vascular permeability.

PA401, a novel CXCL8-based biologic therapeutic with increased glycosaminoglycan binding, reduces bronchoalveolar lavage neutrophils and systemic inflammatory markers in a murine model of LPS-induced lung inflammation.

RATIONALE: Neutrophils play a fundamental role in a number of chronic lung diseases. Among the mediators of their recruitment to the lung, CXCL8 (IL-8) is considered to be one of the major players. CXCL8 exerts its chemotactic activity by binding to its GPCR receptors (CXCR1/R2) located on neutrophils, as well as through interactions with glycosaminoglycans (GAGs) on cell surfaces including those of the microvascular endothelium. Binding to GAG co-receptors is required to generate a solid-phase haptotactic gradient and to present IL-8/CXCL8 in a proper conformation to its receptors on circulating neutrophils. METHODS: We have engineered increased GAG-binding affinity into human CXCL8, thereby obtaining a competitive inhibitor that displaces wild-type IL-8/CXCL8 from GAGs. By additionally knocking-out the GPCR binding domain of the chemokine, we generated a dominant negative protein (dnCXCL8; PA401) with potent anti-inflammatory characteristics proven in vivo in a murine model of LPS-induced lung inflammation (Adage et al., 2015). Here we have further investigated PA401 activity in this pulmonary model by evaluating plasma changes induced by LPS on white blood cells (WBC) and a broad range of inflammatory markers, especially chemokines, by addressing immediate effects of PA401 on these parameters in healthy and LPS exposed mice. RESULTS: Aerosolized LPS induced a significant increase in bronchoalveolar lavage (BAL) neutrophils after 3 and 7h, as well as an increase in total WBC and changes in 21 of the 59 measured plasma markers, mostly belonging to the chemokine family. PA401 treatment in saline exposed mice didn't induce major changes in any of the measured parameters. When administered to LPS aerosolized mice, PA401 caused a significant normalization of KC/mCXCL1 and other inflammatory markers, as well as of blood WBC count. In addition, BAL neutrophils were significantly reduced, confirming the previously observed lung anti-inflammatory activity of PA401 in this experiment. CONCLUSIONS: PA401 is a new promising biologic therapeutic with a novel and unique mechanism of action for interfering with neutrophilic lung inflammation, that also normalizes plasma inflammatory markers.

Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity.

Chemokines like CCL2 mediate leukocyte migration to inflammatory sites by binding to G-protein coupled receptors on the target cell as well as to glycosaminoglycans (GAGs) on the endothelium of the inflamed tissue. We have recently shown that the dominant-negative Met-CCL2 mutant Y13A/S21K/Q23R with improved GAG binding affinity is highly bio-active in several animal models of inflammatory diseases. For chronic indications, we have performed here a fusion to human serum albumin (HSA) in order to extend the serum half-life of the chemokine mutant. To compensate a potential drop in GAG-binding affinity due to steric hindrance by HSA, a series of novel CCL2 mutants was generated with additional basic amino acids which were genetically introduced at sites oriented towards the GAG ligand. From this set of mutants, the Met-CCL2 variant Y13A/N17K/S21K/Q23K/S34K exhibited high GAG-binding affinity and a similar selectivity as wild type (wt) CCL2. From a set of different HSA-chemokine chimeric constructs, the linked HSA(C34A)(Gly)4Ser-Met-CCL2(Y13A/N17K/S21K/Q23K/S34K) fusion protein was found to show the best overall GAG-binding characteristics. Molecular modeling demonstrated an energetically beneficial fold of this novel protein chimera. This was experimentally supported by GdmCl-induced unfolding studies, in which the fusion construct exhibited a well-defined secondary structure and a transition point significantly higher than both the wt and the unfused CCL2 mutant protein. Unlike the wt chemokine, the quaternary structure of the HSA-fusion protein is monomeric according to size-exclusion chromatography experiments. In competition experiments, the HSA-fusion construct displaced only two of seven unrelated chemokines from heparan sulfate, whereas the unfused CCL2 mutant protein displaced five other chemokines. The most effective concentration of the HSA-fusion protein in inhibiting CCL2-mediated monocyte attachment to endothelial cells, as detected in the flow chamber, was 8.6 µg/ml. This novel HSA-fusion protein exhibits not only high affinity but also selective displacement of chemokines from GAGs binding. HSA is therefore proposed to be a highly promising scaffold candidate for therapeutic, GAG-targeting chemokine mutants.

Targeting glycosaminoglycans in the lung by an engineered CXCL8 as a novel therapeutic approach to lung inflammation.

It is broadly recognized that chemokine-activated neutrophils play a crucial role in the inflammation and disruption of lung tissue observed in several acute and chronic lung diseases. Since glycosaminoglycan side chains of proteoglycans act as chemokine co-receptors in inflammation, we have used a CXCL8-based dominant-negative mutant, dnCXCL8, to displace neutrophil-related chemokines in murine lungs using models of lung inflammation. Treatment with dnCXCL8 resulted in a dose-dependent reduction of neutrophil counts in bronchoalveolar lavage (BAL) of mice exposed to lipopolysaccharide after intravenous, subcutaneous and intratracheal administration. A strong and significant therapeutic effect was achieved already at a dose of 40 µg/kg of dnCXCL8. A similar dose response, but showing a broader spectrum of reduced inflammatory cells and soluble inflammatory markers, was observed in a murine model of tobacco smoke (TS)-induced lung inflammation. The broad spectrum of reduced inflammatory cells and markers can be due to the strong inhibition of neutrophil extravasation into the lung parenchyma, and/or to a relatively broad protein displacement profile of dnCXCL8 which may compete not only with wtCXCL8 for glycosaminoglycan-binding but possibly also with other related glycosaminoglycan-binding pro-inflammatory chemokines. Overall our results demonstrate that antagonizing CXCL8/glycosaminoglycan binding reduces lung inflammation as well as associated lung tissue damage due to LPS and TS and may therefore be a new therapeutic approach for lung pathologies characterized by a neutrophilic inflammatory phenotype.

Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells.

The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a "public" mechanism of aging.