UPAR mimicking peptide

Details UPAR mimicking peptide UPAR mimicking peptide

1999-10-20

2002-10-08

Carlson, Karen Cochrane (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C435S004000, C435S069700, C435S320100, C435S325000, C435S334000, C530S326000, C530S327000, C530S328000, C530S330000, C536S023100, C536S023400

Reexamination Certificate

active

06462170

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for preventing or activating the migration of cells in a mammal, in particular a human. The method comprises the use of synthetic peptides, or of defined recombinant soluble forms of the urokinase receptor to activate the recruitment of inflammatory cells in a mammal, by stimulating a cellular adaptor that mediates the chemotactic activity of uPA. This mechanism does not require the protease activity of urokinase. Actually it by-passes it by acting on a downstream step, namely the interaction of such a peptide or recombinant soluble form of urokinase receptor with a cellular adaptor. Additionally, the reagents produced are also useful to identify and isolate drugs that can inhibit said processes of cell recruitment, and hence may be employed in blocking the malignant phenotype of cancer and of aggressive hyper-or auto-inflammatory diseases.
GENERAL BACKGROUND
Cell Migration and Disease
The response of the human body to noxic stimuli is dependent on the local production of inflammatory molecules as well as on the recruitment of specialized cells that migrate from the blood and the neighbouring tissue into the damaged site. These cells, neutrophils, monocyte-macrophages, lymphocytes and endothelial cells, respond to specific migratory stimuli and build up a defense response that results in the destruction and elimination of the nocive agent or organism. While essential and beneficial for the host defense, this type of response may also prove dangerous for the host or may hamper the success of some therapeutic or preventive approaches like transplantation and vaccination. In fact, a series of severe pathological entities exist which can be ascribed to excess or deficient migratory cell response. For example, immunodeficient patients fail to respond to infective agents and this may be due to inability of the cells to rush to the damaged or infected site. Excess of recruitment, on the other hand, may be responsive of destructive pathologies in which defense cells attack cells and functions of the host, as it happens for example in autoimmune diseases. Failure to properly respond to vaccination, moreover, may also depend on unwanted recruitment of host inflammatory cells that destroy the antigenic cells too quickly to promote an immunological response. The availability of a specific “adjuvant” to natural immunity cells, would be advantageous as it would produce a stronger antibody response.
The malignancy of cancer cells mostly consists in the ability to invade and metastasize at a distance; in this process, however, non-malignant stromal host cells actively participate in establishing the malignant phenotype and hence the destructive and invasive phenotype. While the mechanisms involved are largely unknown, it is clear that stromal cells not only respond to stimuli arriving from the cancer cells but also signal to cancer cells through mechanisms of their own. In order to block the invasive phenotype of cancer, it is thus essential to act both on cancer and on stromal cells.
The Urokinase/urokinase Receptor System
The activity of urokinase (uPA) can be confined to the cell surface by the presence of a specific receptor (uPAR, CD87). uPA is a serine protease important in maintaining the fibrinolytic state of the body as it generates plasmin from plasminogen and hence prevents fibrin deposition. Plasmin is a broad spectrum protease that can destroy many proteins of the extracellular matrix and hence the inter-cellular and cell-to-extracellular matrix connections. UPA and uPAR have long been recognised as regulators of cell migration and hence to be important in inflammation and cancer invasion. In addition to functions connected to its proteolytic activity, uPA has other properties that do not require proteolytic activity, but simply the binding to the receptor. In fact, the proteolytic and the receptor binding activities are separated on the uPA molecule and can be assayed individually (receptor binding in the amino terminal fragment, proteolysis in the carboxy-terminal fragment). Among the functions that do not require the proteolytic moiety of uPA, are the stimulation of mitogenesis, cell migration, adhesion and, in particular, chemotaxis (Gudewicz and Bilboa, 1987; Gyetko et al., 1994; Resnati et al., 1996; Besser et al., 1996).
The specific uPA receptor (uPAR) is a GPI-anchored plasma membrane protein endowed with a very high affinity (Kd of 0.1-1 nM) for uPA, pro-uPA and inhibited forms of uPA, like the uPA-PAI-1 complex (Fazioli and Blasi, 1994). In addition to uPA, uPAR also binds vitronectin with an about 10-20 nM affinity as well as integrins. Structurally, uPAR is formed by three repeats of about 90 amino acid residues connected by two linker regions (Danø, Blasi et al., 1990; Behrendt et al., 1991) which define functionally and structurally different domains: the amino terminal domain (D1) contains the uPA binding site, while the carboxyterminal region containing domains D2 and D3 binds vitronectin. However, the integrity of the three-domains structure is necessary for high affinity binding to uPA at least with the purified, soluble protein (Danø et al., 1994). To date, no information has been provided as to the function of the linker regions.
uPAR is expressed in circulating blood cells, in particular monocytes, neutrophils and T-lymphocytes but not in erythrocytes or B-lymphocytes., In addition, uPAR is a target gene in lymphocytes and macrophage activation (CD87). Indeed, monocytes and monocyte-like cells (like HL60, U937) express uPAR, or are induced to overexpress uPAR by a variety of cytokines and other agents, like phorbol ester PMA, phytohemagglutinin, bacterial liposaccharide, TGFb1/vitamin D3, GM-CSF, IFNg, TNFa and others. In human T-lymphocytes, stimulation of the TCR/CD3 complex, lymphokines IL2, IL4, IL7 or the concomitant activation of the T cell receptor and integrins engagement, all induce uPAR expression (Nykjmr et al., 1994; Bianchi et al., 1996). It is also noteworthy, that tumor infiltrating T-lymphocytes heavily express UPA-R and some of the properties of the activated T-lymphocytes, in particular their migration through reconstituted basement membranes, appear to be at least in part uPA- and uPAR-dependent (Bianchi et al., 1996). In view of the important contribution of stromal cells to the invasiveness of cancer, it is also important to stress that macrophages present in human breast cancer and other cancers, where the level of uPAR production by the overall tumor importantly contributes to its malignancy, express high levels of uPAR (Brunner et al., 1996). The cooperation between stromal and cancer cells in cancer invasiveness, implies that high expression of uPAR by cancer or stromal cells, can affect cell prognosis possibly through different mechanisms depending on the overexpressing cell-type.
uPA/uPAR and Chemotaxis
Cooperation between cancer and stromal cells poses the problem of how uPAR expression on stromal cells influences the malignancy of cancer cells. Since the uPA/uPAR system has chemotactic activity, the production of uPAR by stromal cells can attract cancer cells through a chemokine like action. Indeed, chemokines produced by certain cells are anchored to a presentation molecule in the extracellular matrix and attract other cells having specific receptors (Schall and Bacon, 1996). The information available on the uPA/uPAR system supports this possibility.
DISCLOSURE OF THE INVENTION
uPA/uPAR and Chemotaxis
Stimulation of chemotaxis in monocyte-like cells, fibroblasts and some cancer cells requires the specific cell surface uPAR (CD87) which mediates the chemoattractant activity of uPA (Resnati et al., 199.6). Chemotaxis by uPA does not require its protease activity, but only the occupancy of its receptor, and can be reproduced with the enzymatically inactive receptor binding moiety ATF (amino terminal fragment), pro-uPA and by chymotrypsin-cleaved soluble uPAR (Resnati et al., 1996). In mouse, the importance of this system in cell recruitment in vivo is shown by the fa

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