Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-03-17
2002-04-02
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C435S325000, C435S326000, C435S334000, C435S358000, C435S361000, C435S366000, C435S365100, C435S365000
Reexamination Certificate
active
06365356
ABSTRACT:
Chemotactic cytokines, also called chemokines, induce cell migration and activation by binding to surface receptors on a large number of target cells. Chemokines are involved in a variety of diseases. For a review, see Luster, New England Journal of Medicine 338, 436 (1998).
The chemokines are peptides that contain four highly conserved Cys residues that form two disulfide bonds, Baggiolini, et al., The New England Journal of Medicine, 338:436-445 (1998). There are several classes of chemokines. The two largest classes are CXC chemokines, in which two highly conserved Cys residues at the peptide amino terminus are separated by any amino acid, and CC chemokines, in which the Cys residues are proximate to one another.
Chemokines act via G protein-coupled receptors (GPCRs). GPCRs represent the largest family of signal-transducing molecules known, and convey signals for a diverse array of extracellular regulatory molecules. By coupling to G proteins and then activating effectors, GPCRs initiate a series of intracellular signal transduction cascades. In a major pathway, chemokine receptors activate phospholipase C to form second messengers, such as inositol 1,4,5-trisphosphate, which mobilizes calcium ion, and 1,2-diacylglycerol, which activates protein kinase C. Other protein kinases are activated by GPCRs and lead to cell proliferation.
A number of GPCRs for chemokines have been cloned. Epstein, The New England Journal of Medicine, 338:436-445. CXC receptors are designated CXCR1, CXCR2, etc. CC receptors are designated CCR1, CCR2, etc.
The CXC chemokines can be subdivided into peptides that contain the sequence Glu-Leu-Arg (ELR) at their amino termini (ELR+) and those that do not (ELR−). In general, CXCRs are activated by ELR+ or ELR− CXC chemokines, but usually not both. In fact, CXC/ELR− chemokines can act as antagonists at receptors for CXC/ELR+ chemokines. For example, interleukin-8 (IL-8) activation of CXCR2 can be antagonized by an analog of PF-4. Jones, et al., Journal of Biological Chemistry 272:16166-16169 (1997).
Chemokine receptors are found on a variety of cells, such as on hematopoietic cells, neurons, astrocytes, epithelial cells, and endothelial cells. The presence of chemokine receptors on these different types of cells suggests various roles for chemokine receptors, such as leukocyte chemotaxis and stimulation of endothelial cell proliferation. Luster, New England Journal of Medicine 338, 436 (1998).
The Duffy antigen receptor for chemokines (DARC) is found on various types of cells, such as endothelial cells and blood cells, including erythrocytes and leukocytes. DARC binds both CXC/ELR+ and CXC/ELR− chemokines. Hadley, et al., J. Amer. Soc. Hematol. 89: 3077-3091 (1997); J. Clin. Invest. 94:985-991 (1994).
DARC does not, however, transduce a signal in activation assays, at least when expressed in transfected cells alone. In fact, DARC is missing some of the highly conserved residues that are known to be important for signaling of the rhodopsin/&bgr;-adrenergic subfamily of GPCRs, such as the Asp-Arg-Tyr (DRY) sequence at the intracellular aspect of putative transmembrane helix-3. In a recent review article, Hadley and Peiper stated in a section entitled “The Riddle of DARC Function” that the role of DARC in normal and pathologic physiology remains uncertain. See Blood 89 3077 (1997).
As is apparent from the above, chemokines exhibit a large number of important physiological and pathophysiological functions, such as in angiogenesis. For example, angiogenesis is the proliferation of new blood vessels from preexisting vasculature. Net neovascularization is a balance between the effects of angiogenesis-stimulatory (angiogenic) factors and angiogenesis-inhibitory (angiostatic) factors.
During some pathological conditions, it is desirable to bias the balance between angiogenic factors and angiostatic factors. For example, in order for tumors to grow and metastasize, net angiogenesis must be persistent. Therefore, it is desirable to bias the balance in favor of angiostatic factors in order to inhibit tumor growth.
On the other hand, atherosclerosis is a condition caused by a compromised blood supply. Therefore, it is desirable to bias the balance in favor of angiogenic factors in order to treat atherosclerosis.
There are a number of peptide factors that regulate angiogenesis. For example, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are angiogenic factors. Angiostatin and endostatin are angiostatic factors.
Chemokines constitute another class of peptides that appear to regulate angiogenesis, Keane, et al., Proc Assoc Amer Phys 110:288-296 (1998). Chemokines have not been studied as intensively as other angiogenic factors, and the receptor(s) that mediates chemokine regulation of angiogenesis and angiostasis has not been identified.
In general, putative angiogenic chemokines are CXC/ELR+, e.g., growth-related protein-&agr; (Gro-&agr;) and IL-8, and putative angiostatic chemokines are CXC/ELR−, e.g., interferon &ggr;-inducible protein-10 (IP-10) and platelet factor-4 (PF-4). Moore, et al., J. Invest. Med. 46:113-120 (1998).
It is likely that stimulation of these pathways by CXC/ELR+ chemokines leads to proliferation of endothelial cells and angiogenesis. The receptor(s) that mediates these angiogenic effects, however, is not known.
Similarly, several CXC/ELR− chemokines are known to be angiostatic factors. The receptor(s) that mediates angiostasis is also not known.
There is considerable uncertainty regarding how chemokine receptors activate angiogenesis. It was suggested by Keane, et al., (Proceeding of the Association of American Physicians, 110:288-296 (1998)), that CXCR2 may mediate the angiogenic effects of IL-8, Watson, et al., Science 268:447-448. However, it is not clear that CXCR2 is expressed on endothelial cells. Moreover, mice that lack CXCR2 appear to have a normal vasculature.
By contrast, CXCR4, the receptor for the CXC/ELR− chemokine stromal cell-derived factor-1 (SDF-1), is found on endothelial cells. Mice lacking CXCR4 have defective vasculogenesis. Tachibana, et al., Nature, 393:591-594 (1998); Zou, et al., Nature, 393:595-599 (1998). Similarly, mice lacking SDF-1 have defective vasculogenesis. Nagasawa, et al., Nature, 382:635-638 (1996).
However, neither of the putative angiogenic chemokines Gro-&agr; and IL-8 (see above) activates CXCR4. Therefore, if CXCR4 is involved in regulating angiogenesis, it cannot be acting alone, or is mediated by an unknown ligand.
Additional uncertainties exist. For example, it is not known whether any known CXC receptor mediates the angiostatic effects of the CXC/ELR− chemokines PF-4 and IP-10. The receptor for IP-10 is CXCR3; however, CXCR3 is not expressed on endothelial cells. Moreover, there is no known GPCR for PF-4.
It is of potential interest in this regard that, as mentioned above, the Duffy antigen receptor for chemokines (DARC) is found on endothelial cells, and binds both CXC/ELR+ and CXC/ELR− chemokines. Hadley, et al., J. Amer. Soc. Hematol. 89: 3077-3091 (1997); J Clin. Invest. 94:985-991 (1994).
The identification of the chemokine receptor that regulates angiogenesis is of potentially enormous value because receptors that regulate angiogenesis and angiostasis are potential targets for drug therapy of many human diseases, including diseases in which angiostasis is desirable, (e.g., cancers) and diseases in which angiogenesis is desirable (e.g., atherosclerosis). The value of such receptors as targets in drug screening, however, cannot be realized, until the receptors have been identified.
It is an object of the present invention to enable the screening for drugs that regulate the activities of chemokine receptors, especially of angiogenesis-mediating chemokine receptors, as well as the receptors that can be used as targets for the drug in such screens.
SUMMARY OF THE INVENTION
These and other objectives as will be apparent to those having skill in the art have been achieved by p
Cornell Research Foundation Inc.
Feit Irving N.
Hamud Fozia
Hoffmann & Baron , LLP
Kunz Gary L.
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