Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-05-21
2004-02-03
Kunz, Gary (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S351000, C514S012200, C424S085100
Reexamination Certificate
active
06686448
ABSTRACT:
BACKGROUND OF THE INVENTION
Cytokines are soluble proteins that influence the growth and differentiation of many cell types. Their receptors are composed of one or more integral membrane proteins that bind the cytokine with high affinity and transduce this binding event to the cell through the cytoplasmic portions of the certain receptor subunits. Cytokine receptors have been grouped into several classes on the basis of similarities in their extracellular ligand binding domains. For example, the receptor chains responsible for binding and/or transducing the effect of interferons (IFNs) are members of the type IIcytokine receptor family (CRF2), based upon a characteristic 200 residue extracellular domain. The demonstrated in vivo activities of these interferons illustrate the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists.
SUMMARY OF THE INVENTION
The present invention fills this need by providing novel cytokine receptors and related compositions and methods. In particular, the present invention provides for an extracellular ligand-binding region of a mammalian Zcytor7 receptor, alternatively also containing either a transmembrane domain or both an intracellular domain and a transmembrane domain.
Within one aspect, the present invention provides an isolated polynucleotide encoding a ligand-binding receptor polypeptide. The polypeptide comprises a sequence of amino acids selected from the group consisting of (a) residues 30 through 250 of SEQ ID NO:2; (b) allelic variants of (a); and (c) sequences that are at least 80% identical to (a) or (b). Within one embodiment, the polypeptide comprises residues 30 through 250 of SEQ ID NO:2. Within another embodiment, the polypeptide encoded by the isolated polynucleotide further comprises a transmembrane domain. The transmembrane domain may comprise residues 251 through 274 of SEQ ID NO:2, or an allelic variant thereof. Within another embodiment, the polypeptide encoded by the isolated polynucleotide further comprises an intracellular domain, such as an intracellular domain comprising residues 275 through 553 of SEQ ID NO:2, or an allelic variant thereof. Within further embodiments, the polynucleotide encodes a polypeptide that comprises residues 1 through 553, 1 through 274, 1 through 250, 30 through 274 or 30 through 553 of SEQ ID NO:2. Within an additional embodiment, the polypeptide further comprises an affinity tag. Within a further embodiment, the polynucleotide is DNA. Also claimed are the isolated polypeptides encoded by these polynucleotides.
Within a second aspect of the invention there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding a ligand-binding receptor polypeptide, wherein the ligand-binding receptor polypeptide comprises a sequence of amino acids selected from the group consisting of: (i) residues 30 through 250 of SEQ ID NO:2; (ii) allelic variants of (i); and (iii) sequences that are at least 80% identical to (i) or (ii); and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked. The ligand-binding receptor polypeptide may further comprise a secretory peptide, a transmembrane domain, a transmembrane domain and an intracellular domain, or a secretory peptide, a transmembrane domain and an intracellular domain.
Within a third aspect of the invention there is provided a cultured eukaryotic cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses a receptor polypeptide encoded by the DNA segment. Within one embodiment, the cell further expresses a necessary receptor subunit which forms a functional receptor complex. Within another embodiment, the cell is dependent upon an exogenously supplied hematopoietic growth factor for proliferation.
Within a fourth aspect of the invention there is provided an isolated polypeptide comprising a sequence selected from the group consisting of (a) residues 30, a valine, through residue 250, a lysine, of SEQ ID NO:2; (b) allelic variants of (a); and (c) sequences that are at least 80% identical to (a) or (b), wherein said polypeptide is substantially free of transmembrane and intracellular domains ordinarily associated with hematopoietic receptors. Also claimed are polypeptides comprised of a sequence defined by residues 30, a valine, through residue 274, a tyrosine; and a polypeptide comprised of a sequence defined by residues 30, a valine, through residue 553 an asparagine. Also claimed are the polypeptides and polynucleotides defined by the sequences of SEQ ID NOs: 13-60.
Within a further aspect of the invention there is provided a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond. The first portion of the chimeric polypeptide consists essentially of a ligand binding domain of a receptor polypeptide selected from the group consisting of (a) a receptor polypeptide as shown in SEQ ID NO:2; (b) allelic variants of SEQ ID NO:2; and (c) receptor polypeptides that are at least 80% identical to (a) or (b). The second portion of the chimeric polypeptide consists essentially of an affinity tag. Within one embodiment the affinity tag is an immunoglobulin F
c
polypeptide. The invention also provides expression vectors encoding the chimeric polypeptides and host cells transfected to produce the chimeric polypeptides.
The present invention also provides for an isolated polynucleotide encoding a polypeptide selected from a group defined SEQ ID NO:2 consisting of residues 1 through 250, residues 1 through 274, residues 1 through 553, residues 2 through 250, residues 2 through 274, residues 2 through 553, residues 251 through 274, residues 251 through 553 and residues 275 through 553. Also claimed are the isolated polypeptide expressed by these polynucleotides.
The invention also provides a method for detecting a ligand within a test sample, comprising contacting a test sample with a polypeptide as disclosed above, and detecting binding of the polypeptide to ligand in the sample. Within one embodiment the polypeptide further comprises transmembrane and intracellular domains. The polypeptide can be membrane bound within a cultured cell, wherein the detecting step comprises measuring a biological response in the cultured cell. Within another embodiment, the polypeptide is immobilized on a solid support.
Within an additional aspect of the invention there is provided an antibody that specifically binds to a polypeptide as disclosed above, as well as an anti-idiotypic antibody which binds to the antigen-binding region of an antibody to Zcytor7.
In still another aspect of the present invention, polynucleotide primers and probes are provided which can detect mutations in the Zcytor7 gene. The polynucleotide probe should at least be 20-25 bases in length, preferably at least 50 bases in length and most preferably about 80 to 100 bases in length. In addition to the detection of mutations, these probes can be used to discover the Zcytor7 gene in other mammalian species. The probes can either be positive strand or anti-sense strands, and they can be comprised of DNA or RNA.
These and other aspects of the invention will become evident upon reference to the following detailed description and the attached drawing.
DETAILED DESCRIPTION OF THE INVENTION
The term “allelic variant” is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
The term “expression vector” is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such a
Adams Robyn L.
Farrah Theresa M.
Jelmberg Anna C.
Kho Choon J.
Lok Si
Kunz Gary
Li Ruixiang
Walker Shelby J.
ZymoGenetics Inc.
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