Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Utility Patent
1998-03-25
2001-01-02
Mertz, Prema (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S388100, C530S388230, C530S388240, C530S389100, C530S389200
Utility Patent
active
06169167
ABSTRACT:
The invention relates to polypeptides which bind to one or more EPH-like receptors. More particularly, the invention relates to polypeptides which bind to the HEK4 receptor, to nucleic acids encoding same and to expression vectors and host cells for the production of the polypeptides.
BACKGROUND OF THE INVENTION
The response of cells to their environment is often mediated by soluble protein growth and differentiation factors. These factors exert their effects by binding to and activating transmembrane receptors. This interaction is the initial event in a cascade which culminates in a biological response by the cell. An important class of transmembrane receptors is the receptor protein tyrosine kinases (receptor PTKs, reviewed in van der Geer et al. Ann. Rev. Cell. Biol. 10, 251-337 (1994). PTKs consist of an extracellular domain which interacts specifically with the receptor's cognate ligand, a membrane spanning domain, and an intracellular domain which harbors the tyrosine kinase activity. Receptor PTKs are activated by ligand-mediated dimerization followed by autophosphorylation of tyrosine residues in the cytoplasmic domain. The receptor PTK can then in turn phosphorylate substrate molecules in the signal transduction pathway, leading to a cellular response.
The family of receptor PTKs can be divided into a number of sub-families based on the general structure of the extracellular domain and on amino acid sequence relationships within the catalytic domain. Currently, the largest known sub-family of receptor protein tyrosine kinases is the EPH-like receptors, consisting of at least 13 members. Members of this sub-family include the following: EPH (Hirai et al., Science 238, 1717-1725 (1987)), ECK (Lindberg et al., Mol. Cell. Biol. 10, 6316-6324 (1990)), Cek4, Cek5, Cek6, Cek7, Cek8, Cek9, Cek10 (Pasquale, Cell Regulation 2, 523-534 (1991); Sajjadi et al., The New Biologist 3, 769-778 (1991); Sajjadi and Pasquale, Oncogene 8, 1807-1813 (1993)), Eek, Erk (Chan and Watt, Oncogene 6, 1057-1061 (1991)), Ehk1, Ehk2 (Maisonpierre et al., Oncogene 8, 3277-3288 (1993)), HEK (PCT Application No. WO93/00425; Wicks et al., PNAS 89, 1611-1615 (1992)), HEK2 (Bohme et al., Oncogene 8, 2857-2862 (1993)), HEK5, HEK7, HEK8, HEK11 (U.S. Ser. No. 08/229,509) and HTK (Bennett et al. J. Biol. Chem. 269, 14211-14218 (1994)).
Until recently, no ligands for any member of the EPH sub-family had been identified. A ligand for the Eck receptor was described in PCT Application No. WO 94/11020 and Bartley et al. (Nature 368, 558-560 (1994)) and identified earlier as B61, a polypeptide encoded by a cDNA of unknown function (Holzman et al., Mol. Cell Biol. 10, 5830-5838 (1990)). Ligands for Elk and Ehk1 receptors have also been reported (PCT Application No. WO94/11384; Davis et al., Science 266, 816-819 (1994)). Most recently, a polypeptide (ELF-1) identified from a mouse embryo midbrain and hindbrain cDNA library has been reported to be a ligand for Mek4 and Sek (Cheng and Flanagan, Cell 79, 157-168 (1994).
Most attempts to purify soluble factors from complex biological fluids have depended on cell-based bioassays of the response to stimulation by the factor. These include increased cell growth or survival, increased DNA synthesis, a chemotactic response, or some other downstream consequence of receptor activation. Receptor autophosphorylation has also been used as an assay to detect stimulation of the cell. We have recently described a method for the isolation of ligands based on direct detection of receptor/ligand binding and the use of receptor affinity chromatography for purification (Bartley et al., supra). Here we report the application of this method to purify, sequence, and molecularly clone one of a family of ligands corresponding to the EPH sub-family of receptor tyrosine kinases.
Although the EPH sub-family is the largest known sub-family of receptor PTKs, few ligands have been identified which bind to and activate an EPH sub-family receptor. It is therefore an objective to identify additional ligands for EPH sub-family receptor PTKs. These ligands will be useful for modulating responses of EPH sub-family receptor bearing cells.
SUMMARY OF THE INVENTION
The present invention relates to polypeptides capable of binding to one or more EPH-like receptor PTKs. More particularly, the invention provides polypeptides which bind to the HEK4 receptor, but may also bind to other members of the sub-family of EPH-like receptor PTKs. These polypeptides are referred to as HEK4 binding proteins (HEK4 BPs). In one embodiment, the polypeptide binds to and activates HEK4 and ECK receptors. Also encompassed by the invention are nucleic acids encoding HEK4 BPs and nucleic acids which hybridize to HEK4 BP nucleic acids and encode polypeptides having at least one of the biological properties of a HEK4 BP. Biologically active HEK4 BP fragments and analogs and nucleic acids encoding same as well as fusion proteins comprising HEK4 BP are also encompassed by the invention.
Expression vectors and host cells for the production of biologically active HEK4 BP and processes for the production of HEK4 BP using the expression vectors and host cells are also within the scope of the invention. Antibodies specifically binding HEK4 BP are also provided for.
Polypeptides of the invention are useful for modulating (i.e., increasing or decreasing) the growth and/or differentiation of EPH sub-family receptor-bearing cells, particularly cells expressing HEK4 or ECK receptors. Based on levels of expression of HEK4, ECK, and HEK4 BP in a variety of tissues, it is expected that HEK4 BP will be useful for modulating the growth and/or differentiation, for example, liver, kidney, lung, skin or neural tissues. Administration of HEK4 BP to mammals is useful in the treatment of nervous system disorders and in the regeneration of damaged or depleted tissues. HEK4 BP antagonists are also useful for the treatment of cancers.
REFERENCES:
patent: 5457048 (1995-10-01), Pasquale et al.
patent: 5512457 (1996-04-01), Lyman et al.
Ullrich et al. (1990) Cell. vol. 61, pp. 203-212.*
Bartley Timothy D.
Fox Gary M.
Amgen Inc.
Levy Ron K.
Mertz Prema
Odre Steven M.
Winter Robert B.
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