EPH receptor ligands, and uses related thereto

Abstract

The present invention relates to the discovery of a novel EPH receptor ligand, referred to hereinafter as “Elf-1”, which protein has apparently broad involvement in the formation and maintenance of ordered spatial arrangements of differentiated tissues in vertebrates, and can be used to generate and/or maintain an array of different vertebrate tissue both in vitro and in vivo.

1 . A substantially pure preparation of an Elf-1 polypeptide having a sequence identical or homologous to an amino acid sequence represented in SEQ ID No. 2.  
     
     
         2 . The Elf-1 polypeptide of  claim 1 , which polypeptide modulates at least one of proliferation, differentiation or survival of a cell which expresses an EPH-type receptor.  
     
     
         3 . The Elf-1 polypeptide of  claim 2 , which polypeptide stimulates intracellular signal transduction pathways mediated by an EPH-type receptor.  
     
     
         4 . The Elf-1 polypeptide of  claim 2 , which polypeptide antagonizes intracellular signal transduction pathways mediated by an EPH-type receptor.  
     
     
         5 . The Elf-1 polypeptide of  claim 1 , which polypeptide comprises Cys-69 through Cys-159 of the amino acid sequence represented by SEQ ID No. 2, or a sequence homologous thereto.  
     
     
         6 . The Elf-1 polypeptide of  claim 1 , which polypeptide is post-translationally modified to include a covalently added moiety selected from the group consisting of a phosphatidylinositol and a carbohydrate.  
     
     
         7 . The Elf-1 polypeptide of  claim 1 , which polypeptide specfically binds to an EPH-type receptor.  
     
     
         8 . An immunogen comprising the Elf-1 polypeptide of  claim 1 , in an immunogenic preparation, said immunogen being capable of eliciting an immune response specific for said Elf-1 polypeptide.  
     
     
         9 . An antibody preparation specifically reactive with an epitope of the Elf-1 polypeptide of  claim 1 .  
     
     
         10 . A recombinant polypeptide comprising an Elf-1 polypeptide sequence identical or homologous with an amino acid sequence represented by SEQ ID No. 2.  
     
     
         11 . The polypeptide of  claim 10 , which protein is a fusion protein further comprising in addition to said Elf-1 polypeptide sequence, a second polypeptide sequence having an amino acid sequence unrelated to the amino acid sequence represented by SEQ ID No. 2.  
     
     
         12 . The polypeptide of  claim 11 , wherein said fusion protein includes, as a second polypeptide sequence, a polypeptide which functions as a detectable label for detecting the presence of said fusion protein or as a matrix-binding domain for immobilizing said fusion protein.  
     
     
         13 . The polypeptide of  claim 12 , wherein said second polypeptide sequence has an enzymatic activity.  
     
     
         14 . An isolated or recombinant Elf-1 polypeptide having an amino acid sequence crossreactive with an antibody which specifically binds the Elf-1 protein represented by SEQ ID No. 2.  
     
     
         15 . An isolated or recombinant Elf-1 polypeptide encoded by a nucleic acid which hybridizes under stringent conditions to a nucleic acid sequence represented in SEQ ID No.2.  
     
     
         16 . A substantially pure nucleic acid encoding a recombinant polypeptide comprising an Elf-1 polypeptide sequence homologous to an amino acid sequence represented in SEQ ID No. 2.  
     
     
         17 . The nucleic acid of  claim 16 , wherein said polypeptide modulates at least one of proliferation, differentiation or survival of a cell which expresses an EPH-type receptor.  
     
     
         18 . The nucleic acid of  claim 16 , wherein said nucleotide sequence hybridizes under stringent conditions to a nucleic acid probe having a sequence represented by at least consecutive nucleotides of SEQ ID No. 1.  
     
     
         19 . The nucleic acid of  claim 16 , further comprising a transcriptional regulatory sequence operably linked to said nucleotide sequence so as to render said nucleic acid suitable for use as an expression vector.  
     
     
         20 . An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the nucleic acid of  claim 16 .  
     
     
         21 . A host cell transfected with the expression vector of  claim 20  and expressing said recombinant polypeptide.  
     
     
         22 . A method of producing a recombinant Elf-1 polypeptide comprising culturing the cell of  claim 21  in a cell culture medium to express said Elf-1 polypeptide and isolating said Elf-1 polypeptide from said cell culture.  
     
     
         23 . A transgenic animal having cells which harbor a transgene comprising the nucleic acid of  claim 16 .  
     
     
         24 . A recombinant transfection system, comprising 
 (i) a gene construct including the nucleic acid of  claim 16  and operably linked to a transcriptional regulatory sequence for causing expression of said Elf-1 polypeptide in eukaryotic cells, the Elf-1 polypeptide being characterized by an ability to bind to an EPH-type receptor, and    (ii) a gene delivery composition for delivering said gene construct to a cell and causing the cell to be transfected with said gene construct.    
     
     
         25 . The recombinant transfection system of  claim 24 , wherein the gene delivery composition is selected from a group consisting of a recombinant viral particle, a liposome, and a poly-cationic nucleic acid binding agent,  
     
     
         26 . A transgenic animal in which Elf-1 stimulated signal transduction pathways are inhibited in one or more tissue of said animal by one of either expression of an antagonistic Elf-1 polypeptide or disruption of an Elf-1 gene.  
     
     
         27 . A recombinant gene comprising a nucleotide sequence homologous with SEQ ID No.  1 , or a fragment thereof, said nucleotide sequence operably linked to a transcriptional regulatory sequence in an open reading frame and translatable to a polpeptide capable of specifically binding to an EPH-type receptor.  
     
     
         28 . A probe/primer comprising a substantially purified oligonucleotide. said oligonucleotide containing a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence of SEQ ID No. 1, or naturally occuring mutants thereof.  
     
     
         29 . The probe/primer of  claim 28 , which probe/primer further comprises a label group attached theretc and able to be detected.  
     
     
         30 . A test kit for detecting cells which contain an Elf-1 mRNA transcript, comprising a probe/primer of  claim 29  for measuring, in a sample of cells, a level of nucleic acid encoding an ELf-1 protein.  
     
     
         31 . A method for modulating, in an animal, cell growth, differentiation or survival, comprising administering a therapeutically effective amount of an Elf-1 polypeptide which binds to an EPH-type receptor.  
     
     
         32 . The method of  claim 31 , which polypeptide stimulates intracellular signal transduction pathways mediated by an EPH-type receptor.  
     
     
         33 . The method of  claim 31 , which polypeptide antagonizes intracellular signal transduction pathways mediated by an EPH-type receptor.  
     
     
         34 . An assay for screening test compounds that modulate the binding of an Elf-1 polypeptide with an EPH-type receptor comprising: 
 i. combining an Elf-1 polypeptide, an EPH receptor protein, and a test compound; and    ii. detecting the formation of a complex comprising said Elf- polypeptide and said EPH receptor protein,    wherein a change in the formation of said complex in the presence of said test compound is indicative of a modulator of the interaction between Elf-1 and an EPH receptor.    
     
     
         35 . A peptidomimetic of a portion of an Elf-1 protein which specifically binds an EPH-type receptor.  
     
     
         36 . An assay for detecting a receptor ligand or its cognate receptor, comprising 
 (i) providing an affinity probe including a binding agent selected from the group consisting of a receptor, and a receptor ligand, which binding agent is covalently linked to an enzymatically active tag for which a chromogenic substrate exist,    (ii) contacting a tissue or an organism with the affinity probe to form complexes between the probe and a cognate receptor or ligand in the sample, and removing any unbound affinity probe after such time as the probe complexes have formed, and    (iii) detecting the affinity complex using a chromogenic substrate for the enzymatic tag of the affinity probe.    
     
     
         37 . The assay of  claim 36 , wherein the enzymatic tag is selected from a group consisting of alkaline phosphatase, horseradish peroxidase, β-galactosidase, malate dehydrogenase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, and urease.  
     
     
         38 . The assay of  claim 36 , wherein the binding agent is selected from the group consisting of an EPH receptor and an EPH ligand.  
     
     
         39 . The assay of  claim 36 , provided in a kit for detecting expression of a receptor or receptor ligand in a non-dispersed tissue biopsy.