Development and characterization of humanized and human forms of ELR-CXC chemokine antagonist, bovine CXCL8(3-74)K11R/G31P
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Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5’ half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia. As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. In vitro, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. In vivo, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorGordon, John R.
CommitteeSingh, Baljit; Misra, Vikram; Liu, Lixin; Babiuk, Lorne A.; Yang, Xi