WO2001000664A2 - Secreted alpha-helical protein-36 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for mammalian secreted alpha helical protein-36 (Zalpha36). The polypeptides, and polynucleotides encoding them, are hormonal and may be used to regulate the functioning of the immune system. The present invention also includes antibodies to the Zalpha36 polypeptides. The Zalpha36 protein can be used to stimulate the production of leukocytes and to inhibit the production of platelets.

Description

PATENT APPLICATION DOCKET 99-42PC

SECRETED ALPHA-HELICAL PROTEIN - 36

BACKGROUND OF THE INVENTION

Proliferation, maintenance, survival and differentiation of cells of multi cellular organisms are controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other and act in concert to form cells and organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones (e.g. estrogen, testosterone), parathyroid hormone, follicle stimulating hormone, the interleukins, platelet derived growth factor (PDGF), epidermal growth factor (EGF), granulocyte- macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding to proteins. Proteins may be integral membrane proteins that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of proteins are soluble molecules, such as the transcription factors.

Of particular interest are cytokines, molecules that promote the proliferation, maintenance, survival or differentiation of cells. Examples of cytokines include erythropoietin (EPO), which stimulates the development of red blood cells; thrombopoietin (TPO), which stimulates development of cells of the megakaryocyte lineage; and granulocyte-colony stimulating factor (G-CSF), which stimulates development of neutrophils. These cytokines are useful in restoring normal blood cell levels in patients suffering from anemia or receiving chemotherapy for cancer. The demonstrated in vivo activities of these cytokines illustrates the enormous clinical potential of, and need for, other cytokines, cytokine agonists, and cytokine antagonists. DESCRIPTION OF THE INVENTION

The present invention addresses this need by providing novel polypeptides and related compositions and methods useful to promote proliferation of leukocytes. Within one aspect, the present invention provides an isolated polynucleotide encoding a mammalian cytokine termed 'Secreted alpha helical protein- 36', hereinafter referred to as "Zalpha36". Human Zalpha36, SEQ ID NOs 1 and 2, has four alpha helices A, B, C and D. Amino acid residues 1-25 of SEQ ID NO: 2 define a signal sequence. Thus, the mature sequence extends from amino acid residue 26, a glutamine, to and including amino acid residue 199, a lysine of SEQ ID NO: 2. The mature sequence, which is also defined by SEQ ID NO: 3, has an unglycosylated molecular weight of about 19,917 Daltons (D).

The present invention further provides for murine Zalpha36. Murine Zalpha36, SEQ ID NOs: 4 and 5, also has four alpha helices, A, B, C and D. Amino acid residues 1-25 of SEQ ID NO: 5 define a signal sequence. Thus, the mature sequence extends from amino acid residue 26, a glutamine, to and including amino acid residue 199, a lysine of SEQ ID NO: 5. The mature sequence, which is also represented by SEQ Id NO: 6, has an unglycosylated molecular weight of about 20,019 D.

Within a second aspect of the invention there is provided an expression vector comprising (a) a transcription promoter; (b) a DNA segment encoding Zalpha36 polypeptide, and (c) a transcription terminator, wherein the promoter, DNA segment, and terminator are operably linked.

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 protein polypeptide encoded by the DNA segment.

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) a Zalpha36 polypeptide as shown in SEQ ID NO: 2 (b) allelic variants of SEQ ID NO: 2; and (c) protein polypeptides that are at least 80% identical to (a) or (b). The second portion of the chimeric polypeptide consists essentially of another polypeptide such as 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.

Within an additional aspect of the invention there is provided an antibody that specifically binds to a Zalpha36 polypeptide as disclosed above, and also an anti-idiotypic antibody that neutralizes the antibody to a Zalpha36 polypeptide.

An additional embodiment of the present invention relates to a peptide or polypeptide that has the amino acid sequence of an epitope-bearing portion of a Zalpha36 polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zalpha36 polypeptide of the present invention include portions of such polypeptides with at least nine, preferably at least 15 and more preferably at least 30 to 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Examples of such epitope-bearing polypeptides are SEQ ID NOs: 16-20 for human Zalpha36 and SEQ ID NOs: 21-24 for mouse Zalpha36. Also claimed are any of these polypeptides that are fused to another polypeptide or carrier molecule.

The present invention is also comprised of a method for inducing proliferation of peripheral blood leukocytes in a mammal comprising administering a therapeutically effective amount of Zalpha36 to said mammal. The present invention further comprises a method for inhibiting the production of platelets in a mammal comprising administering a therapeutically effective amount of Zalpha36 to said mammal.

The present invention further comprises a method for inducing the production of platelets in a mammal comprising administering a therapeutically effective amount of an antagonist to Zalpa36 to said mammal.

Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:

The term "affinity tag" is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly- histidine tract, protein A, Nilsson et al, EMBOJ. 4:1075 (1985); Nilsson et al., Methods Enzymol. 198:3 (1991), glutathione S transferase, Smith and Johnson, Gene (57:31 (1988), Glu-Glu affinity tag, Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 52:7952-4 (1985), substance P, Flag™ peptide, Hopp et al, Biotechnology r5:1204-1210 (1988), streptavidin binding peptide, or other antigenic epitope or binding domain. See, in general, Ford et al., Protein Expression and Purification 2: 95-107 (1991). DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia Biotech, Piscataway, NJ).

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 terms "amino-terminal" and "carboxyl-terminal" are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

"Angiogenic" denotes the ability of a compound to stimulate the formation of new blood vessels from existing vessels, acting alone or in concert with one or more additional compounds. Angiogenic activity is measurable as endothelial cell activation, stimulation of protease secretion by endothelial cells, endothelial cell migration, capillary sprout formation, and endothelial cell proliferation.

The term "complement/anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair. Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/anti sense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <10⁹ M^-1.

The term "complements of a polynucleotide molecule" is a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.

The term "contig" denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-ATGGCTTAGCTT-3' are 5'- TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5'.

The term "degenerate nucleotide sequence" denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp). 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 additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.

The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:114-1% (1985).

An "isolated" polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

A leukocyte is a type of cell formed in the myelopoietic, lymphoid, and reticular portions of the reticuloendothelial system in various part of the body. The cells include granulocytes, monocytes and lymphocytes.

The term "operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator. The term "ortholog" denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.

"Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a- globin, b-globin, and myoglobin are paralogs of each other.

A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules it is used to denote overall length and will be understood to be equivalent to the term "base pairs". It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.

A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides".

The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes.

A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell. Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).

The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.

The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "about" X or "approximately" X, the stated value of X will be understood to be accurate to ±10%.

The present invention provides novel cytokine polypeptides/proteins.

The novel cytokine, termed "alpha helical protein-36" hereinafter referred to as "Zalpha36" was discovered and identified to be a cytokine by the presence of polypeptide and polynucleotide features characteristic of four-helix-bundle cytokines (e.g., erythropoietin, thrombopoietin, G-CSF, IL-2, IL-4, leptin and growth hormone). Analysis of the amino acid sequence shown in SEQ ID NO: 2 indicates a signal sequence which extends from the methionine at position 1 to and including amino acid residue 25. Thus the mature sequence extends from amino acid residue 26, a glutamine, to an including amino acid residue 199, a lysine. The mature Zalpha36 polypeptide is also represented by the amino acid sequence of SEQ ID NO: 3, which has an unglycosylated molecular weight of approximately 19,917 D.

Further analysis of SEQ ID NO: 2, human Zalpha36 indicates the presence of four amphipathic, alpha-helical regions, namely helices A, B, C and D. Each helix contains an external region having amino acid residues that are generally hydrophilic, and an internally located region that generally contains hydrophobic amino acid residues. The amino acid residues that are positioned on the exterior of the helices are considered crucial for receptor binding and should not be changed to another amino acid residue except to one that is almost identical in charge. The amino acid residues that are positioned on the interior of the helix may be changed to any hydrophobic amino acid residue.

Helix A contains at least amino acid residue 54, a serine, to and including amino acid residue 68, a leucine of SEQ ID NO: 2. Helix A is also represented by SEQ ID NO: 7. Amino acid residues 54, 57, 58, 61, 64, 65 and 68 of SEQ ID NO: 2 are positioned towards the interior of helix A, while amino acid residues 55, 56, 59, 60, 62, 63, 66 and 67 of SEQ ID NO: 2 are positioned towards the exterior of helix A. Helix B contains at least amino acid residue 88, a valine, to and including amino acid residue 102, a leucine of SEQ ID NO: 2. Helix B is also represented by SEQ ID NO: 8. Amino acid residues 88, 91, 92, 95, 98, 99 and 102 of SEQ ID NO: 2 are positioned towards the interior of helix B, while amino acid residues 89, 90, 93, 94, 96, 97, 100 and 101 of SEQ ID NO: 2 are positioned towards the exterior of helix B.

Helix C contains at least amino acid residue 110, an isoleucine, to and including amino acid residue 124, a tyrosine of SEQ ID NO: 2. Helix C is also represented by SEQ ID NO: 9. Amino acid residues 110, 113, 114, 117, 120, 121 and 124 of SEQ ID NO: 2 are positioned towards the interior of helix C, while amino acid residues 111, 112, 115, 116, 118, 119, 122 and 123 of SEQ ID NO: 2 are positioned towards the exterior of helix C.

Helix D contains at least amino acid residue 172, a leucine, to and including amino acid residue 186, a methionine of SEQ ID NO: 2. Helix D is also represented by SEQ ID NO: 10. Amino acid residues 172, 175, 176, 179, 182, 183 and 186 of SEQ ID NO: 2 are positioned towards the interior of helix D, while amino acid residues 173, 174, 177, 178, 180, 181, 184 and 185 of SEQ ID NO: 2 are positioned towards the exterior of helix D.

Further analysis of SEQ ID NO: 5, mouse Zalpha36, indicates the presence of four amphipathic, alpha-helical regions, namely helices A, B, C and D. Each helix contains an external region having amino acid residues that are generally hydrophilic, and an internally located region that generally contains hydrophobic amino acid residues. The amino acid residues that are positioned on the exterior of the helices are considered crucial for receptor binding and should not be changed to another amino acid residue except to one that is almost identical in charge. The amino acid residues that are positioned on the interior of the helix may be changed to any hydrophobic amino acid residue.

Helix A contains at least amino acid residue 54, a serine, to and including amino acid residue 68, a leucine of SEQ ID NO: 5. Helix A is also represented by SEQ ID NO: 11. Amino acid residues 54, 57, 58, 61 , 64, 65 and 68 of SEQ ID NO: 5 are positioned towards the internal portion of helix A, while amino acid residues 55, 56, 59, 60, 62, 63, 66 and 67 of SEQ ID NO: 5 are positioned on the external portion of helix A.

Helix B contains at least amino acid residue 88, a valine, to and including amino acid residue 102, a leucine of SEQ ID NO: 5. Helix B is also represented by SEQ ID NO: 12. Amino acid residues 88, 91, 92, 95, 98, 99 and 102 of SEQ ID NO: 5 are positioned towards the interior of helix B while amino acid residues 89, 90, 93, 94, 96, 97, 100 and 101 of SEQ ID NO: 5 are positioned towards the exterior of helix B.

Helix C contains at least amino acid residue 110, a isoleucine, to and including amino acid residue 124, a tyrosine of SEQ ID NO: 5. Helix C is also represented by SEQ ID NO: 13. Amino acid residues 110, 113, 1 14, 1 17, 120, 121 and 124 of SEQ ID NO: 5 are positioned towards the interior of helix C, while amino acid residues 111, 112, 115, 116, 118, 119, 122 and 123 of SEQ ID NO: 5 are positioned towards the exterior of helix C.

Helix D contains at least amino acid residue 172, a leucine, to and including amino acid residue 186, a methionine of SEQ ID NO: 5. Helix D is also represented by SEQ ID NO: 14. Amino acid residues 172, 175, 176, 179, 182, 183, and 186 of SEQ ID NO: 5 are positioned towards the interior of helix D, while amino acid residues 173, 174, 177, 178, 180, 181, 184 and 185 of SEQ ID NO: 5 are positioned towards the exterior of helix D.

POLYNUCLEOTIDES:

The present invention also provides polynucleotide molecules, including DNA and RNA molecules, which encode the Zalpha36 polypeptides disclosed herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules.

Polynucleotides, generally a cDNA sequence, of the present invention encode the described polypeptides herein. A cDNA sequence that encodes a polypeptide of the present invention is comprised of a series of codons, each amino acid residue of the polypeptide being encoded by a codon and each codon being comprised of three nucleotides. The amino acid residues are encoded by their respective codons as follows. Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;

Cysteine (Cys) is encoded by TGC or TGT;

Aspartic acid (Asp) is encoded by GAC or GAT; Glutamic acid (Glu) is encoded by GAA or GAG;

Phenylalanine (Phe) is encoded by TTC or TTT;

Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;

Histidine (His) is encoded by CAC or CAT;

Isoleucine (He) is encoded by ATA, ATC or ATT; Lysine (Lys) is encoded by AAA, or AAG;

Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;

Methionine (Met) is encoded by ATG;

Asparagine (Asn) is encoded by AAC or AAT;

Proline (Pro) is encoded by CCA, CCC, CCG or CCT; Glutamine (Gin) is encoded by CAA or CAG;

Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;

Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;

Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;

Valine (Val) is encoded by GTA, GTC, GTG or GTT; Tryptophan (Tip) is encoded by TGG; and

Tyrosine (Tyr) is encoded by TAC or TAT.

It is to be recognized that according to the present invention, when a polynucleotide is claimed as described herein, it is understood that what is claimed are both the sense strand, the anti-sense strand, and the DNA as double-stranded having both the sense and anti-sense strand annealed together by their respective hydrogen bonds. Also claimed is the messenger RNA (mRNA) that encodes the polypeptides of the president invention, and which mRNA is encoded by the cDNA described herein. Messenger RNA (mRNA) will encode a polypeptide using the same codons as those defined herein, with the exception that each thymine nucleotide (T) is replaced by a uracil nucleotide (U).

One of ordinary skill in the art will also appreciate that different species can exhibit "preferential codon usage." In general, see, Grantham, et al., Nuc. Acids Res. 5:1893-1912 (1980); Haas, et al. Curr. Biol. (5:315-324 (1996); Wain-Hobson, et al., Gene 73:355-364 (1981); Grosjean and Fiers, Gene 75:199-209 (1982); Holm, Nuc. Acids Res. 14:3015-3081 (1986); Ikemura, J. Mol. Biol. 158:513-591 (1982). As used herein, the term "preferential codon usage" or "preferential codons" is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid. For example, the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.

Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.

As previously noted, the isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of Zalpha36 RNA. Such tissues and cells are identified by Northern blotting, Thomas, Proc. Natl. Acad. Sci. USA 77:5201 (1980) and are discussed below. Total RNA can be prepared using guanidine HC1 extraction followed by isolation by centrifugation in a CsCl gradient, Chirgwin et al, Biochemistry 18:52-94 (1979). Poly (A)⁺ RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA 69:1408-1412 (1972). Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding Zalpha36 polypeptides are then identified and isolated by, for example, hybridization or PCR. A full-length clone encoding Zalpha36 can be obtained by conventional cloning procedures. Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library. Expression libraries can be probed with antibodies to Zalpha36, receptor fragments, or other specific binding partners.

The polynucleotides of the present invention can also be synthesized using DNA synthesizers. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. See Glick and Pasternak, Molecular Biotechnology, Principles & Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem. 53: 323-356 (1984) and Climie et al, Proc. Natl. Acad. Sci. USA 87:633-631 (1990).

The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are Zalpha36 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human Zalpha36 can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses Zalpha36 as disclosed herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A Zalpha36-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human Zalpha36 sequence disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to Zalpha36 polypeptide. Similar techniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequence disclosed in SEQ ID NOs: 1 and 4 represent single alleles of the human and mouse Zalpha36 respectively, and that allelic variation and alternative splicing are expected to occur. One can clone allelic variants of this sequence by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: 1, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the Zalpha36 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.

The present invention also provides isolated Zalpha36 polypeptides that are substantially identical to the polypeptides of SEQ ID NOs: 2, 3, 5 and 6 and their orthologs. The term "substantially identical" is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO: 2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or its orthologs.) Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48: 603-616 (1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 59:10915-10919 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter codes).

The percent identity is then calculated as:

Those skilled in the art appreciate that there are many established algorithms to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence and the amino acid sequence of a putative variant. The FASTA algorithm is described by Pearson and Lipman, Proc. Nat'lAcad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions or deletions. The ten regions with the highest density of identities are then re-scored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAMJ. Appl. Math. 26:181 (1974), which allows for amino acid insertions and deletions. Illustrative parameters for FASTA analysis are: ktup=l, gap opening penalty=10, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 753:63 (1990).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one and six, preferably from four to six.

The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID NO: 3. The BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins [Henikoff and Henikoff, Proc. Nat 'I Acad. Sci. USA 59:10915 (1992)]. Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the language "conservative amino acid substitution" refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0,1,2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1,2 or 3), while more preferred conservative substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3). Accordingly the present invention claims those polypeptides which are at least 90%, preferably 95% and most preferably 99% identical to SEQ ID NO: 3 and which are able to stimulate antibody production in a mammal, and said antibodies are able to bind the native sequence of SEQ ID NO: 3.

Variant Zalpha36 polypeptides or substantially homologous Zalpha36 polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides of from

20 to 30 amino acid residues that comprise a sequence that is at least 90%, preferably at least 95%, and more preferably 99% or more identical to the corresponding region of SEQ ID NO: 4. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the Zalpha36 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites. Table 2

Conservative amino acid substitutions

Basic: arginine lysine histidine

Acidic: glutamic acid aspartic acid

Polar: glutamine asparagine

Hydrophobic: leucine isoleucine valine

Table 2 cont. Aromatic: phenylalanine tryptophan tyrosine

Small: glycine alanine serine threonine methionine

The present invention further provides a variety of other polypeptide fusions [and related multimeric proteins comprising one or more polypeptide fusions]. For example, a Zalpha36 polypeptide can be prepared as a fusion to a dimerizing protein as disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include immunoglobulin constant region domains. Immunoglobulin-Zalpha36 polypeptide fusions can be expressed in genetically engineered cells [to produce a variety of multimeric Zalpha36 analogs]. Auxiliary domains can be fused to Zalpha36 polypeptides to target them to specific cells, tissues, or macromolecules (e.g., collagen). For example, a Zalpha36 polypeptide or protein could be targeted to a predetermined cell type by fusing a Zalpha36 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A Zalpha36 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See, Tuan et al., Connective Tissue Research 34:1-9 (1996).

The proteins of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans -3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, allo-threonine, mefhylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,

3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incoφorating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRΝAs.

Methods for synthesizing amino acids and aminoacylating tRΝA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2122 (1991); Ellman et al., Methods Enzymol. 202:301 (1991; Chung et al, Science 259:806- 809 (1993); and Chung et al, Proc. Natl. Acad. Sci. USA 90:10145-1019 (1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRΝA and chemically aminoacylated suppressor tRΝAs, Turcatti et al., J.

Biol. Chem. 277:19991-19998 (1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al, Biochem. 33:7470-7476 (1994). Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site- directed mutagenesis to further expand the range of substitutions, Wynn and Richards, Protein Sci. 2:395-403 (1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for Zalpha36 amino acid residues.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis, Cunningham and Wells, Science 244: 1081-1085 (1989); Bass et al, Proc. Natl. Acad. Sci. USA 55:4498-502 (1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, J. Biol. Chem. 277:4699-708, 1996. Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-312 (1992); Smith et al., J. Mol. Biol. 224:899-904 (1992); Wlodaver et al, FEBS Lett. 309:59-64 (1992).

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer, Science 241:53-51 (1988) or Bowie and Sauer, Proc. Natl Acad. Sci. USA 5(5:2152-2156 (1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display, e.g., Lowman et al, Biochem. 30:10832-10837 (1991); Ladner et al, U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region- directed mutagenesis, Derbyshire et al, Gene 46:145 (1986); Ner et al, DNA 7:127 (1988).

Variants of the disclosed Zalpha36 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389-391, (1994), Stemmer, Proc. Natl. Acad. Sci. USA 97:10747-10751 (1994) and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. One can modify this technique by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modem equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID NOs:2, 4 or 6 or that retain the properties of the wild-type Zalpha36 protein. For any

Zalpha36 polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant.

PROTEIN PRODUCTION

The Zalpha36 polypeptides of the present invention, including full- length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multi cellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., (Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY, 1989), and Ausubel et al., eds., Current Protocols in Molecular Biology (John Wiley and Sons, Inc., NY, 1987). In general, a DNA sequence encoding a Zalpha36 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.

To direct a Zalpha36 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of Zalpha36, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the Zalpha36 DNA sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al, U.S. Patent No. 5,037,743; Holland et al, U.S. Patent No. 5,143,830).

Alternatively, the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway. The present invention provides for such fusion polypeptides. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Such constructs have numerous applications known in the art. For example, these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. Such fusions may be used in vivo or in vitro to direct peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, Wigler et al, Cell 14:125 (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb, Virology 52:456 (1973), electroporation, Neumann et al, EMBO J. 7:841-845 (1982), DEAE- dextran mediated transfection (Ausubel et al, ibid., and liposome-mediated transfection, Hawley-Nelson et al, Focus 15:13 (1993); Ciccarone et al, Focus 75:80 (1993), and viral vectors, Miller and Rosman, BioTechniques 7:980(1989); Wang and Finer, Nature Med. 2:714 (1996). The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al, U.S. Patent No. 4,713,339; Hagen et al, U.S. Patent No. 4,784,950; Palmiter et al, U.S. Patent No. 4,579,821 ; and Ringold, U.S. Patent No. 4,656, 134. Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al, J. Gen. Virol 36:59 (1977) and Chinese hamster ovary (e.g. CHO- Kl ; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification." One can carry out amplification by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4,

CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells. Sinkar et al, J. Biosci. (Bangalore) 77:47 (1987) have reviewed the use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al, U.S. Patent No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). DNA encoding the Zalpha36 polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods. The first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the Zalpha36 flanked by AcNPV sequences. Suitable insect cells, e.g. SF9 cells, are infected with wild-type AcNPV and transfected with a transfer vector comprising a Zalpha36 polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences. See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, (Chapman & Hall, London); O'Reilly, D.R. et al, Baculovirus Expression Vectors: A Laboratory Manual (Oxford University Press, New York, New York, 1994); and, Richardson, C.

D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, (Humana

Press, Totowa, NJ 1995). Natural recombination within an insect cell will result in a recombinant baculovirus that contains Zalpha36 driven by the polyhedrin promoter.

Recombinant viral stocks are made by methods commonly used in the art.

The second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow, V.A, et al, J Virol 67:4566 (1993).

This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the Zalpha36 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid." The pFastBacl™ transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case Zalpha36. However, pFastBacl™ can be modified to a considerable degree. The polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter), which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R.D., J Gen Virol 77:971 (1990); Bonning, B.C. et al, J Gen Virol 75:1551 (1994); and, Chazenbalk, G.D., and Rapoport, B., J Biol Chem 270:1543 (1995). In such transfer vector constructs, a short or long version of the basic protein promoter can be used. One can construct transfer vectors that replace the native Zalpha36 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin (Invitrogen, Carlsbad, CA), or baculovirus gρ67 (PharMingen, San Diego, CA) can be used in constructs to replace the native Zalpha36 secretory signal sequence. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed Zalpha36 polypeptide, for example, a Glu-Glu epitope tag, Grussenmeyer, T. et al, Proc Natl Acad Sci. 52:7952 (1985). Using a technique known in the art, a transfer vector containing Zalpha36 is transformed into E. coli, and screened for bacmids that contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses Zalpha36 is subsequently produced. Recombinant viral stocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA (ASM Press, Washington, D.C., 1994). Another suitable cell line is the High

FiveO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent #5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 10⁵ cells to a density of 1-2 x 10⁶ cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3. The recombinant virus-infected cells typically produce the recombinant Zalpha36 polypeptide at 12-72 hours post-infection and secrete it with varying efficiency into the medium. The culture is usually harvested 48 hours post- infection. Centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the Zalpha36 polypeptide is filtered through micropore filters, usually 0.45 μm pore size. Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid.; O'Reilly, D.R. et al, ibid.; Richardson, C. D., ibid.). Subsequent purification of the Zalpha36 polypeptide from the supernatant can be achieved using methods described herein.

Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichiapastoris, and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,31 1 ; Kawasaki et al, U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al, U.S. Patent No. 5,037,743; and Murray et al, U.S. Patent No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly dmg resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311 ; Kingsman et al, U.S.

Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol 732:3459 (1986) and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al, U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.

The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (A UG1 and A UG2) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.

Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art, see, e.g., Sambrook et al, ibid.). When expressing a Zalpha36 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient, which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto™ Peptone (Difco Laboratories, Detroit, MI), 1 % Bacto™ yeast extract (Difco

Laboratories), 0.004% adenine and 0.006% L-leucine).

Another embodiment of the present invention provides for a peptide or polypeptide comprising an epitope-bearing portion of a Zalpha36 polypeptide of the invention. The epitope of the polypeptide portion is an immunogenic or anti genie epitope of a polypeptide of the invention. A region of a protein to which an antibody can bind is defined as an "antigenic epitope". See for instance, Geysen, H.M. et al, Proc. Natl. Acad Sci. USA 57:3998-4002 (1984). As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in the art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See Sutcliffe, J.G. et al. Science 279:660-666 (1983). Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical mles, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer soluble peptides, especially those containing proline residues, usually are effective.

Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, which bind specifically to a polypeptide of the invention. Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, preferably between 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that react with the protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred. All of the polypeptides shown in the sequence listing contain antigenic epitopes to be used according to the present invention, however, specifically designed antigenic epitopes include the peptides defined by SEQ ID NOs: 16-20 for human Zalpha36 and SEQ ID NOs: 21-24 for murine Zalpha36. The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a Zalphal polypeptide described herein. Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen. Immunogenic epitope-bearing peptides can be identified using standard methods [see, for example, Geysen et al, supra. See also U.S. Patent No. 4,708,781

(1987) further describes how to identify a peptide bearing an immunogenic epitope of a desired protein.

Examples of human Zalpha36 epitope-bearing polypeptides are the following from SEQ ID NO: 2: amino acid residue 36, an arginine, to and including amino acid residue 60, a histidine, also represented by SEQ ID NO: 16; amino acid residue 177, a glutamine, to and including amino acid residue 199, a lysine, also represented by SEQ ID NO: 17; amino acid residue 155, an asparagine, to and including amino acid residue 199, a lysine, also represented by SEQ ID NO: 18; amino acid residue 70, a threonine, to and including amino acid residue 116, a glutamate, also represented by SEQ ID NO: 19; amino acid residue 70, a threonine, to and including amino acid residue 126, an asparagine, also represented by SEQ ID NO: 20.

Examples of mouse Zalpha36 epitope-bearing polypeptides are the following subsequences from SEQ ID NO: 5: amino acid residue 36 and arginine to and including amino acid residue 60, a histidine, also represented by SEQ ID NO: 21 ; amino acid residue 184, a glutamate, up to and including amino acid residue 199, a lysine, also represented by SEQ ID NO: 22; amino acid residue 70, a threonine, up to and including amino acid residue 126, a serine, also represented by SEQ ID NO: 23; amino acid residue 137, a serine, up to and including amino acid residue 163, a histidine, also represented by SEQ ID NO:24.

Protein Isolation

It is preferred to purify the polypeptides of the present invention to >80% purity, more preferably to >90% purity, even more preferably >95% purity, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.

Expressed recombinant Zalpha36 polypeptides (or chimeric Zalpha36 polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred. Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross- linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art. Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988).

The polypeptides of the present invention can be isolated by exploitation of their properties. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3:1 (1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography. Methods in Enzymol, Vol. 752, "Guide to Protein Purification", M. Deutscher, (ed.),page 529-539 (Acad. Press, San Diego, 1990). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.

Moreover, using methods described in the art, polypeptide fusions, or hybrid Zalpha36 proteins, are constructed using regions or domains of the inventive

Zalpha36, Sambrook et al, ibid., Altschul et al, ibid., Picard, Cur. Opin. Biology, 5:511 (1994). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure. Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domain(s) conferring a biological function may be swapped between Zalpha36 of the present invention with the functionally equivalent domain(s) from another family member. Such domains include, but are not limited to, the secretory signal sequence, conserved, and significant domains or regions in this family. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known family proteins, depending on the fusion constmcted. Moreover, such fusion proteins may exhibit other properties as disclosed herein.

Zalpha36 polypeptides or fragments thereof may also be prepared through chemical synthesis. Zalpha36 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.

Chemical Synthesis of Polypeptides

Polypeptides, especially polypeptides of the present invention can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by solid phase peptide synthesis, for example as described by Meπϊfield, J. Am. Chem. Soc. 55:2149 (1963).

ASSAYS

The activity of molecules of the present invention can be measured using a variety of assays.

An in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovims, herpesvirus, vaccinia vims and adeno-associated vims (AAV). Adenovims, a double- stranded DNA vims, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see T.C. Becker et al, Meth. Cell Biol. 43:161 (1994); and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44 (1997). The adenovims system offers several advantages: adenovims can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenovimses are stable in the bloodstream, they can be administered by intravenous injection.

By deleting portions of the adenovims genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co- transfected plasmid. In an exemplary system, the essential El gene has been deleted from the viral vector, and the vims will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary). When intravenously administered to intact animals, adenovims primarily targets the liver. If the adeno viral delivery system has an El gene deletion, the vims cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.

The adenovims system can also be used for protein production in vitro. By culturing adenovims-infected cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum- free conditions, which allows infected cells to survive for several weeks without significant cell division. Alternatively, adenovims vector infected 293 S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al, Cytotechnol 75:145 (1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293 S cell production protocol, non- secreted proteins may also be effectively obtained.

Antagonists

Antagonists are also useful as research reagents for characterizing sites of ligand-receptor interaction. Inhibitors of Zalpha36 activity (Zalpha36 antagonists) include anti-Zalpha36 antibodies and soluble Zalpha36 receptors, as well as other peptidic and non-peptidic agents (including ribozymes).

Zalpha36 fusion proteins

A Zalpha36 polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an F_c fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to affinity purify the ligand, such as a Protein A column. For use in assays, the chimeras are bound to a support via the F_c region and used in an ELISA format.

A Zalpha36 ligand-binding polypeptide can also be used for purification of ligand. The polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross- linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine HC1), or pH to dismpt ligand-receptor binding.

Zalpha36 polypeptides can also be used to prepare antibodies that specifically bind to Zalpha36 epitopes, peptides or polypeptides. The Zalpha36 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response. Suitable antigens would be the Zalpha36 polypeptides encoded by SEQ ID NOs: 2-14, and 16-24. Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, (John Wiley and Sons, Inc., 1995); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, NY, 1989); and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Inc., Boca Raton, FL, 1982).

As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a Zalpha36 polypeptide or a fragment thereof. The immunogenicity of a Zalpha36 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of Zalpha36 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine semm albumin (BSA) or tetanus toxoid) for immunization.

As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included. Non-human antibodies may be humanized by grafting non-human

CDRs onto human framework and constant regions, or by incoφorating the entire non- human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered" antibody). In some instances, humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.

Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to Zalpha36 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled Zalpha36 protein or peptide). Genes encoding polypeptides having potential Zalpha36 polypeptide-binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides that interact with a known target, which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances. Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al, US Patent NO. 5,223,409; Ladner et al, US Patent NO. 4,946,778; Ladner et al, US Patent NO. 5,403,484 and Ladner et al, US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the Zalpha36 sequences disclosed herein to identify proteins that bind to Zalpha36. These "binding proteins" which interact with Zalpha36 polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to d gs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as Zalpha36 "antagonists" to block Zalpha36 binding and signal transduction in vitro and in vivo.

Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and 2) they do not significantly cross-react with related polypeptide molecules. First, antibodies herein specifically bind if they bind to a Zalpha36 polypeptide, peptide or epitope with a binding affinity (K_a) of 10⁶ M^{- 1} or

greater, preferably 10⁷ M^{- 1} or greater, more preferably 10⁸ M^{- 1} or greater, and most preferably 10 9 M -1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis.

Second, antibodies are determined to specifically bind if they do not significantly cross-react with related polypeptides. Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect Zalpha36 but not known related polypeptides using a standard Western blot analysis (Ausubel et al, ibid.). Examples of known related polypeptides are orthologs, proteins from the same species that are members of a protein family (e.g. IL-16), Zalpha36 polypeptides, and non-human Zalpha36. Moreover, antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides. For example, antibodies raised to Zalpha36 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to Zalpha36 will flow through the matrix under the proper buffer conditions. Such screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to closely related polypeptides, Antibodies: A Laboratory Manual, Harlow and Lane (eds.) (Cold Spring Harbor Laboratory Press, 1988); Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health (John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.) (Raven Press, 1993); Getzoff et al, Adv. in Immunol. 43: 1-98 (1988); Monoclonal Antibodies: Principles and Practice, Goding, J.W. (eds.), (Academic Press Ltd., 1996); Benjamin et al., Ann. Rev. Immunol. 2: 67-101 (1984).

A variety of assays known to those skilled in the art can be utilized to detect antibodies that specifically bind to Zalpha36 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.) (Cold Spring Harbor Laboratory Press, 1988). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno- precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild type versus mutant Zalρha36 protein or polypeptide.

Antibodies to Zalpha36 may be used for tagging cells that express Zalpha36; for isolating Zalpha36 by affinity purification; for diagnostic assays for determining circulating levels of Zalpha36 polypeptides; for detecting or quantitating soluble Zalpha36 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block Zalpha36 in vitro and in vivo. Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates. Antibodies herein may also be directly or indirectly conjugated to dmgs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. Moreover, antibodies to Zalpha36 or fragments thereof may be used in vitro to detect denatured Zalpha36 or fragments thereof in assays, for example, Western Blots or other assays known in the art.

BIOACTIVE CONJUGATES:

Antibodies or polypeptides herein can also be directly or indirectly conjugated to dmgs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for instance). More specifically, Zalpha36 polypeptides or anti-Zalpha36 antibodies, or bioactive fragments or portions thereof, can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.

Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like. Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Polypeptides or antibodies may also be conjugated to cytotoxic dmgs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these puφoses, biotin/streptavidin is an exemplary complementary/ anticomplementary pair.

In another embodiment, polypeptide-toxin fusion proteins or antibody- toxin fusion proteins can be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues). Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a targeting domain), a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest. In instances where the domain only fusion protein includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue- specific delivery of generic anti-complementary-detectable/ cytotoxic molecule conjugates.

In another embodiment, Zalpha36-cytokine fusion proteins or antibody- cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, blood and bone marrow cancers), if the Zalpha36 polypeptide or anti- Zalpha36 antibody targets the hypeφroliferative blood or bone marrow cell. See, generally, Homick et al., Blood 59:4437 (1997). They described fusion proteins enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine. Suitable Zalpha36 polypeptides or anti-Zalpha36 antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediated improved target cell lysis by effector cells. Suitable cytokines for this puφose include interleukin 2 and granulocyte-macrophage colony-stimulating factor (GM- CSF), for instance.

In yet another embodiment, if the Zalpha36 polypeptide or anti- Zalpha36 antibody targets vascular cells or tissues, such polypeptide or antibody may be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy. For instance, iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a bioactive conjugate containing a radionuclide, as described herein.

The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.

USE OF ZALPHA36 TO PROMOTE THE PROLIFERATION OF

LEUKOCYTES

The present invention is also comprised of a method for inducing proliferation of peripheral blood leukocytes in a mammal comprising administering a therapeutically effective amount of Zalpha36 to said mammal.

For pharmaceutical use, the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a Zalpha36 protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed.,(Mack Publishing Co., Easton, PA, 19th ed., 1995). Therapeutic doses will generally be in the range of 0.1 to 100 μg/kg of patient weight per day, preferably 0.5-20 mg/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years.

An example of a composition containing Zalpha36 is the following: 300 μg of Zalpha36 (SEQ ID NO: 3), 0.59 mg acetate, 50.0 mg of mannitol, 0.004%

TWEEN , and 0.035 mg sodium in 1 mL of water for injection. An optimized dose to stimulate the production of peripheral blood leukocytes would range from 4 to 18 μg/kg/day for 4 to 19 days administered either subcutaneously or intravenously. This could be used to stimulate the production of leukocytes depleted through chemotherapy, radiation, or immunodeficiency diseases caused by such agents as the human immunodeficiency vims (HIV) or by aging in general. Thus, Zalpha36 can be administered in conjunction with a vaccine in immunodeficient patients to promote greater and quicker efficacy of the vaccine.

USE OF ZALPHA36 TO INHIBIT PLATELET PROLIFERATION

The present invention further comprises a method for inhibiting the production of platelets in a mammal comprising administering a therapeutically effective amount of Zalpha36 to said mammal. Zalpha36 can be administered to inhibit the production of platelets in amounts similar to those administered to an individual to promote the production of leukocytes. Zalpha36 can be administered to a patient several days prior to undergoing percutaneous transluminal coronary angioplasty (PCTA). The amount administered should be about 0.25 mg/kg 3-5 days prior to PCTA and about lOμg/min continuous intravenous administration for 12 hours after PCTA. Zalpha36 can be administered to a patient during and after a coronary infarct. An ideal dose would be 0.25 mg/kg and then a continuous administration of 10 μg/min administered intravenously for 2-3 days. Likewise, a person who has undergone an ischemic cerebral stroke would also benefit from the administration of Zalpha36 to prevent platelet aggregation. An ideal dose would range from 0.25 mg/kg to 0.50 mg/kg and then continuous intravenous infusion of 10 μg/kg for 3-4 days or until the attending physician concludes that the patient is stable.

USE OF ANTAGONISTS TO ZALPHA36 TO PROMOTE THE PRODUCTION OF PLATELETS

The present invention further comprises a method for inducing the production of platelets in a mammal comprising administering a therapeutically effective amount of an antagonist to Zalpa36 to said mammal. To promote the production platelets antagonists of Zalpha36 can be administered to a patient. For example, antibodies can be administered, preferable the Fab fragment, which is obtained by papain digestion and purification, typically by using a Protein A column which binds the remaining Fc portion of the antibody. Typically, antagonists to Zalpha36 can be administered if the platelet count in the peripheral blood goes below 100,000/μL. The Fab fragments of an antibody to Zalpha36 should be administered at a dose of 0.25 mg to 0.50 mg/kg of body weight/day for 5 to 10 days or until the platelet count reaches about 100,000/μL.

GENE THERAPY:

Polynucleotides encoding Zalpha36 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit Zalpha36 activity. If a mammal has a mutated or absent Zalpha36 gene, one can introduce the Zalpha36 gene into the cells of the mammal. In one embodiment, a gene encoding a Zalpha36 polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA vims, such as, but not limited to, heφes simplex vims (HSV), papillomavirus, Epstein Barr vims (EBV), adenovims, adeno-associated vims (AAV), and the like. Defective vimses, which entirely or almost entirely lack viral genes, are preferred. A defective vims is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective heφes simplex vims 1 (HSV1) vector, Kaplitt et al, Molec. Cell. Neurosci. 2:320 (1991); an attenuated adenovims vector, such as the vector described by Stratford-Peπϊcaudet et al, J. Clin. Invest. 90:626 (1992); and a defective adeno-associated vims vector, Samulski et al, J. Virol. 61:3096 (1987); Samulski et α/., J. Virol. 63:3822 (1989).

In another embodiment, a Zalpha36 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al, U.S. Patent No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al, U.S. Patent No. 4,650,764; Temin et al, U.S. Patent No. 4,980,289; Markowitz et al, J. Virol. (52:1120 (1988); Temin et al, U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al; and Kuo et al, Blood 52:845 (1993). Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker, Feigner et al, Proc. Natl. Acad. Sci. USA 84:1413 (1987); Mackey et al, Proc. Natl. Acad. Sci. USA 55:8027 ( 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the puφose of targeting. Targeted peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically. It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al, J. Biol. Chem. 267:963 (1992); Wu et al, J. Biol. Chem. 2(53:14621-4, 1988. Antisense methodology can be used to inhibit Zalpha36 gene transcription, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a Zalpha36-encoding polynucleotide (e.g., a polynucleotide as set froth in SEQ ID NO: 1) are designed to bind to Zalpha36- encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of Zalpha36 polypeptide-encoding genes in cell culture or in a subject.

The present invention also provides reagents that will find use in diagnostic applications. For example, the Zalpha36 gene, a probe comprising Zalpha36 DNA or RNA or a subsequence thereof can be used to determine if the Zalpha36 gene is present on chromosome 3q25-3q26 or if a mutation has occurred. Detectable chromosomal aberrations at the Zalpha36 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymoφhism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et αl, ibid.; Ausubel et. al., ibid.; Marian, Chest 705:255 (1995).

Transgenic mice, engineered to express the Zalpha36 gene, and mice that exhibit a complete absence of Zalpha36 gene function, referred to as "knockout mice", Snouwaert et αl., Science 257:1083 (1992), may also be generated, Lowell et αl., Nature 366:140-42 (1993). These mice may be employed to study the Zalpha36 gene and the protein encoded thereby in an in vivo system.

CHROMOSOMAL LOCALIZATION:

Radiation hybrid mapping is a somatic cell genetic technique developed for constmcting high-resolution, contiguous maps of mammalian chromosomes (Cox et al, Science 250:245 (1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL). These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymoφhic and polymoφhic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers. The precise knowledge of a gene's position can be useful for a number of puφoses, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.

Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example, Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences. Tissue Expression and Use

Zalpha36 represents a novel polypeptide with a putative signal peptide leader sequence and alpha helical stmcture. Therefore this gene may encode a secreted polypeptide with secondary stmcture indicating it is a member of the four-helix bundle cytokine family. Northern blot analysis reveals that Zalpha36 is expressed in many tissues including pancreas, heart, skeletal muscle, kidney, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, thyroid, spinal cord, lymph node and adrenal gland.

Most four-helix bundle cytokines as well as other proteins produced by cells of the immune system play an important biological role in cell differentiation, activation, recmitment and homeostatsis of cells throughout the body. Antagonists to Zalpha36 may be important in the regulation of inflammation and therefore would be useful in treating rheumatoid arthritis, asthma and sepsis.

EDUCATIONAL KIT UTILITY OF ZALPHA36 POLYPEPTIDES, POLYNUCLEOTIDES AND ANTIBODIES

Polynucleotides and polypeptides of the present invention will additionally find use as educational tools as a laboratoryφracticum kits for courses related to genetics and molecular biology, protein chemistry and antibody production and analysis. Due to its unique polynucleotide and polypeptide sequence molecules of Zalpha36 can be used as standards or as "unknowns" for testing puφoses. For example, Zalpha36 polynucleotides can be used as an aid, such as, for example, to teach a student how to prepare expression constmcts for bacterial, viral, and/or mammalian expression, including fusion constmcts, wherein Zalpha36 is the gene to be expressed; for determining the restriction endonuclease cleavage sites of the polynucleotides; determining mRNA and DNA localization of Zalpha36 polynucleotides in tissues (i.e., by Northern and Southern blotting as well as polymerase chain reaction); and for identifying related polynucleotides and polypeptides by nucleic acid hybridization. Zalpha36 polypeptides can be used educationally as an aid to teach preparation of antibodies; identifying proteins by Western blotting; protein purification; determining the weight of expressed Zalpha36 polypeptides as a ratio to total protein expressed; identifying peptide cleavage sites; coupling amino and carboxyl terminal tags; amino acid sequence analysis, as well as, but not limited to monitoring biological activities of both the native and tagged protein (i.e., receptor binding, signal transduction, proliferation, and differentiation) in vitro and in vivo. Zalpha36 polypeptides can also be used to teach analytical skills such as mass spectrometry, circular dichroism to determine conformation, in particular the locations of the disulfide bonds, x-ray crystallography to determine the three-dimensional stmcture in atomic detail, nuclear magnetic resonance spectroscopy to reveal the stmcture of proteins in solution. For example, a kit containing the Zalpha36 can be given to the student to analyze. Since the amino acid sequence would be known by the professor, the protein can be given to the student as a test to determine the skills or develop the skills of the student, the teacher would then know whether or not the student has correctly analyzed the polypeptide. Since every polypeptide is unique, the educational utility of Zalpha36 would be unique unto itself.

The antibodies which bind specifically to Zalpha36 can be used as a teaching aid to instmct students how to prepare affinity chromatography columns to purify Zalpha36, cloning and sequencing the polynucleotide that encodes an antibody and thus as a practicum for teaching a student how to design humanized antibodies. The Zalpha36 gene, polypeptide or antibody would then be packaged by reagent companies and sold to universities so that the students gain skill in art of molecular biology. Because each gene and protein is unique, each gene and protein creates unique challenges and learning experiences for students in a lab practicum. Such educational kits containing the Zalpha36 gene, polypeptide, or antibody, are considered within the scope of the present invention.

The invention is further illustrated by the following non-limiting examples. Example 1 Cloning of Human Zalpha36

Zalpha36 was discovered by using the Expressed Sequence Tag of SEQ ID NO: 15 as a probe. A clone containing the EST was discovered in a pancreatic islet cell cDNA library produced according to the procedure described below in Example 2 below. The clone was isolated and sequenced resulting in the sequences of SEQ ID NOs: 1 and 2.

Example 2

Production a Pancreatic Islet Cell cDNA Library

RNA extracted from pancreatic islet cells was reversed transcribed in the following manner. The first strand cDNA reaction contained 10 μl of human pancreatic islet cell poly d(T)-selected poly (A)⁺ mRNA (Clontech, Palo Alto, C A) at a concentration of 1.0 mg/ml, and 2 μl of 20 pmole/μl first strand primer SEQ ID NO:25 (GTC TGG GTT CGC TAC TCG AGG CGG CCG CTA TTT TTT TTT TTT TTT TTT) containing an Xho I restriction site. The mixture was heated at 70°C for 2.5 minutes and cooled by chilling on ice. First strand cDNA synthesis was initiated by the addition of 8 μl of first strand buffer (5x SUPERSCRIPT™ buffer; Life Technologies, Gaithersburg, MD), 4 μl of 100 mM dithiothreitol, and 3 μl of a deoxynucleotide triphosphate (dNTP) solution containing 10 mM each of dTTP, dATP, dGTP and 5- methyl-dCTP (Pharmacia LKB Biotechnology, Piscataway, NJ) to the RNA-primer mixture. The reaction mixture was incubated at 40° C for 2 minutes, followed by the addition of 10 μl of 200 U/μl RNase H^" reverse transcriptase (SUPERSCRIPT II®; Life

Technologies). The efficiency of the first strand synthesis was analyzed in a parallel reaction by the addition of 10 μCi of 32p._αdCTP to a 5 μl aliquot from one of the reaction mixtures to label the reaction for analysis. The reactions were incubated at 40°C for 5 minutes, 45°C for 50 minutes then incubated at 50°C for 10 minutes. Unincoφorated ^?- dCT_? in the labeled reaction was removed by chromatography on a 400-pore size gel filtration column (Clontech Laboratories, Palo Alto, CA). The unincoφorated nucleotides and primers in the unlabeled first strand reactions were removed by chromatography on 400-pore size gel filtration column (Clontech Laboratories, Palo Alto, CA). The length of labeled first strand cDNA was determined by agarose gel electrophoresis. The second strand reaction contained 102 μl of the unlabeled first strand cDNA, 30 μl of 5x polymerase I buffer (125 mM Tris: HC1, pH 7.5, 500 mM KC1, 25 mM MgCl , 50mM [(NH ) 2SO4], 2.0 μl of 100 mM dithiothreitol, 3.0 μl of a solution containing 10 mM of each deoxynucleotide triphosphate, 7 μl of 5 mM β-NAD, 2.0 μl of 10 U/μl E. coli DNA ligase (New England Biolabs; Beverly, MA), 5 μl of 10 U/μl E. coli DNA polymerase I (New England Biolabs, Beverly, MA), and 1.5 μl of 2 U/μl RNase H (Life Technologies, Gaithersburg, MD). A 10 μl aliquot from one of the second strand synthesis reactions was labeled by the addition of 10 μCi ³²p-αdCTP to monitor the efficiency of second strand synthesis. The reactions were incubated at 16° C for two hours, followed by the addition of 1 μl of a 10 mM dNTP solution and 6.0 μl T4 DNA polymerase (10 U/μl, Boehringer Mannheim, Indianapolis, IN) and incubated for an additional 10 minutes at 16°C. Unincoφorated ³²p-αd TP in the labeled reaction was removed by chromatography through a 400-pore size gel filtration column (Clontech Laboratories, Palo Alto, CA) before analysis by agarose gel electrophoresis. The reaction was terminated by the addition of 10.0 μl 0.5 M EDTA and extraction with phenol/chloroform and chloroform followed by ethanol precipitation in the presence of 3.0 M Na acetate and 2 μl of Pellet Paint carrier (Novagen, Madison, WI). The yield of cDNA was estimated to be approximately 2 μg from starting mRNA template of 10 μg.

Eco RI adapters were ligated onto the 5' ends of the cDNA described above to enable cloning into an expression vector. A 12.5 μl aliquot of cDNA (~2.0 μg) and 3 μl of 69 pmole/μl of Eco RI adapter (Pharmacia LKB Biotechnology Inc.,

Piscataway, NJ) were mixed with 2.5 μl lOx ligase buffer (660 mM Tris-HCl pH 7.5, 100 mM MgCl₂), 2.5 μl of l0 mM ATP, 3.5 μl 0.1 M DTT and 1 μl of l5 U/μl T4

DNA ligase (Promega Coφ., Madison, WI). The reaction was incubated 1 hour at 5°C, 2 hours at 7.5°C, 2 hours at 10°C, 2 hours at 12.5°C and 16 hours at 10° C. The reaction was terminated by the addition of 65 μl H₂O and 10 μl 10X H buffer (Boehringer Mannheim, Indianapolis, IN) and incubation at 70°C for 20 minutes.

To facilitate the directional cloning of the cDNA into an expression vector, the cDNA was digested with Xho I, resulting in a cDNA having a 5' Eco RI cohesive end and a 3' Xho I cohesive end. The Xho I restriction site at the 3' end of the cDNA had been previously introduced. Restriction enzyme digestion was carried out in a reaction mixture by the addition of 1.0 μl of 40 U/μl Xho I (Boehringer Mannheim, Indianapolis, IN). Digestion was carried out at 37°C for 45 minutes. The reaction was terminated by incubation at 70°C for 20 minutes and chromatography through a 400- pore size gel filtration column (Clontech Laboratories, Palo Alto, CA).

The cDNA was ethanol precipitated, washed with 70% ethanol, air-dried and resuspended in 10.0 μl water, 2 μl of 10X kinase buffer (660 mM Tris-HCl, pH 7.5, 100 mM MgCl₂), 0.5 μl 0.1 M DTT, 2 μl 10 mM ATP, 2 μl T4 polynucleotide kinase (10 U/μl, Life Technologies, Gaithersburg, MD). Following incubation at 37° C for 30 minutes, the cDNA was ethanol precipitated in the presence of 2.5 M Ammonium Acetate, and electrophoresed on a 0.8% low melt agarose gel. The contaminating adapters and cDNA below 0.6 kb in length were excised from the gel. The electrodes were reversed, and the cDNA was electrophoresed until concentrated near the lane origin. The area of the gel containing the concentrated cDNA was excised and placed in a microfuge tube, and the approximate volume of the gel slice was determined. An aliquot of water approximately three times the volume of the gel slice (300 μl) and 35 μl 1 Ox β-agarose I buffer (New England Biolabs) was added to the tube, and the agarose was melted by heating to 65°C for 15 minutes. Following equilibration of the sample to 45°C, 3 μl of 1 U/μl β-agarose I (New England Biolabs, Beverly, MA) was added, and the mixture was incubated for 60 minutes at 45 °C to digest the agarose. After incubation, 40 μl of 3 M Na acetate was added to the sample, and the mixture was incubated on ice for 15 minutes. The sample was centrifuged at 14,000 x g for 15 minutes at room temperature to remove undigested agarose. The cDNA was ethanol precipitated, washed in 70% ethanol, air-dried and resuspended in 40 μl water.

Following recovery from low-melt agarose gel, the cDNA was cloned into the Eco RI and Xho I sites of pBLUESCRIPT SK+ vector (Gibco/BRL,

Gaithersburg, MD) and electroporated into DH10B cells. Bacterial colonies containing ESTs of known genes were identified and eliminated from sequence analysis by reiterative cycles of probe hybridization to hi-density colony filter arrays (Genome Systems, St. Louis, MI). cDNAs of known genes were pooled in groups of 50 - 100 inserts and were labeled with ³²P-αdCTP using a MEGAPRIME labeling kit

(Amersham, Arlington Heights, IL). Colonies that did not hybridize to the probe mixture were selected for sequencing. Sequencing was done using an ABI 377 sequencer using either the T3 or the reverse primer. The resulting data were analyzed which resulted in the identification of the novel ESTs of SEQ ID NOs:l-38. Each polynucleotide corresponds to a clone in the pancreatic islet cell library. Analyzing the ESTs, a bioinformaticist can identify one of interest and the clone or clones identified which contain the EST. If the clone did not contain the full-length cDNA, the EST of the present invention can be radiolabeled by standard techniques to screen cDNA libraries by nucleic hybridization to isolate a full-length clone. The labeled EST may further be used to probe Northern blots or by other hybridization techniques known in the art to determine mRNA localization and transcription levels. EST sequence information may also be used to design primers to enable the isolation of full-length cDNA sequencing using 5' or 3' rapid amplification of DNA (RACE) polymerase chain reaction. The radiolabeled EST can further be used in the technique of in situ hybridization to locate the specific nucleic acid sequences in cells or chromosomes. Furthermore, the EST can facilitate the diagnosis of genetic abnormalities using standard hybridization techniques.

Example 3 Mouse Zalpha36

Using the human sequence the public database was scanned to identify a mouse EST that would correspond with the human nucleotide sequence. SEQ ID NO: 26 was discovered and the clone, which was from a cDNA library from 13.5-14 day old whole mouse, was pulled and sequenced. This resulted in the mouse sequence of SEQ ID NOs: 4 and 5.

Example 4

Adenovims Administration of Zalpha36 to Normal Mice

Zalpha36 was administered using adenovims containing the Zalpha36 gene. There were three groups of mice, one group receiving adenovims containing the Zalpha36 gene, a second group receiving only the adenovims, and a third group receiving no treatment. The adenovims was injected in the tail veins of the mice. Each mouse receiving the adenovims was given one dose containing 1 x 10^u in 0.1ml of phosphate buffered saline (PBS). Blood was drawn from the tail vein at day eleven after the injections and leukocyte count and platelet count were determined.

Experimental Design Group 1 Zalpha36 (SEQ ID NO: l)/pAdV/Zalpha36

1 x 10^{1 1} particles/dose

There were 8 females and 8 males. Blood was drawn on day 11 from the tale vein. In this group of mice zalpha36 was administered by inserting the Zalpha36 gene into adenovims. The adenovims was injected into the tail vein and localized in the liver. The transfected liver cells would then express the Zalpha36 gene and produce Zalpha36, which is secreted by the liver cells.

Group 2 null /AdV control 1 x 10" particles/dose

There were 8 females and 8 males. Blood was drawn from the tail vein on day 11. This group of mice only received adenovims without the Zalpha36 gene.

Group 3 No treatment was given to this group of mice.

There were 8 females and 8 males. Blood was drawn on day 11 from the tale vein.

Results The most striking effect was a significant increase in white blood cell count in Zalpha36 treated animals compared with the controls that received no treatment and the control group that received adenovims that did not have the Zalpha36 gene. At the end of 11 days from the date of injection, the average white blood cell count for the mice treated with adenovims containing the Zalpha36 gene was 6,735/μL, 5,615/μL for the mice treated with adenovims not containing the Zalpha36 gene, and

3,761/μL of blood for the untreated mice.

From the data it can also be concluded that Zalpha36 decreases platelet count. The platelet count at day 11 was 800,000/μL of blood for the mice treated with adenovims containing the Zalpha36 gene, 910,000/μL for the mice treated with adenovims that did not contain the Zalpha36 gene, and 940,000/μL for the mice that were untreated. This shows that Zalpha36 inhibits the production of platelets. Thus, Zalpha36 can be administered to inhibit the production of platelets, which may be necessary after a coronary infarction, ischemic stroke or deep-vein thrombosis are involved and clotting needs to be prevented. This also indicates that one can induce the production of platelets by administering antagonists such as antibodies to Zalpha36 to an individual.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for puφoses of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMSWhat is claimed is:

1 . A polypeptide comprised of a sequence selected from the group consisting of SEQ ID NOs.2, 3, 5 and 6.

2 . An isolated polynucleotide that encodes a polypeptide selected from the group of SEQ ID NOs. 2,3,5 and 6.

3. An antibody that specifically binds to a polypeptide selected from the group of SEQ ID NOs. 2, 3, 5 and 6.

4. A method for inducing the proliferation of leukocytes comprising administering a therapeutically effective amount of Zalpha36.

5. A method for inducing the proliferation of platelets comprising administering a therapeutically effective amount of an antagonist to Zalpha36.

6. The method of claim 5 wherein the antagonist is an antibody to Zalpha36.

7. A method for inhibiting the production of platelets comprising administering a therapeutically effective amount of Zalpha36.

8. An educational kit comprised of a polynucleotide that encodes Zalpha36.

9. The educational kit of claim 8 further comprising a Zalpha36 protein.

10. The educational kit of claim 8 further comprising an antibody that specifically binds to Zalpha36.

11. The use of a Zalpha36 polypeptide in the manufacture of a medicament to stimulate the production of leukocytes.

12. The use of a Zalpha36 polypeptide in the manufacture of a medicament to inhibit the production of platelets.

13. The use of an antagonist to a Zalpha36 polypeptide in the manufacture of a medicament to stimulate the production of platelets.

14. The use of claim 13 wherein the antagonist is an antibody to a Zalpha36 polypeptide.

SEQUENCE LISTING

<110> ZymoGenetics, Inc..

<120> Mammalian alpha helical protein - 36

<130> 99-42

<150> 09/343,152 <151> 1999-06-28

<160> 26

<170> FastSEQ for Windows Version 3.0

<210> 1 <211> 1098 <212> DNA <213> Homo sapiens

<220>

<221> CDS

<222> (33)... (629)

<400> 1 gcgctgccgg gtgaaatcgt aggacagtga ag atg ctg ctg gaa ttg tec gag 53

Met Leu Leu Glu Leu Ser Glu 1 5

gag cat aag gaa cac ctg gcc ttc ctg cct caa gtg gac age gcg gtg 101 Glu His Lys Glu His Leu Ala Phe Leu Pro Gin Val Asp Ser Ala Val 10 15 20

gtc gcc gag ttt ggg egg att get gtg gaa ttc ctg aga cgc ggc gca 149 Val Ala Glu Phe Gly Arg He Ala Val Glu Phe Leu Arg Arg Gly Ala 25 30 35

aac cca aaa ate tac gaa ggc gcc gcc aga aaa etc aat gtg agt agt 197

Asn Pro Lys He Tyr Glu Gly Ala Ala Arg Lys Leu Asn Val Ser Ser 40 45 50 55

gac act gtc cag cat ggt gtg gaa gga tta acg tat etc etc act gag 245 Asp Thr Val Gin His Gly Val Glu Gly Leu Thr Tyr Leu Leu Thr Glu 60 65 70 age tea aag etc atg att tct gaa ctg gat ttc caa gac tct gtt ttt 293 Ser Ser Lys Leu Met He Ser Glu Leu Asp Phe Gin Asp Ser Val Phe 75 80 85

gtt ctg gga ttc tct gaa gaa tta aac aaa ttg ttg ctt cag ctt tat 341 Val Leu Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu Gin Leu Tyr 90 95 100

ctg gac aac aga aaa gag ate aga acg ctt ctg agt gaa ttg gca cca 389 Leu Asp Asn Arg Lys Glu He Arg Thr Leu Leu Ser Glu Leu Ala Pro 105 110 115

age ctt ccc agt tat cat aac ctt gaa tgg cga eta gat gta cag ctt 437 Ser Leu Pro Ser Tyr His Asn Leu Glu Trp Arg Leu Asp Val Gin Leu 120 125 130 135

gca agt aga agt etc agg caa cag att aaa cca gca gtg act ata aag 485 Ala Ser Arg Ser Leu Arg Gin Gin He Lys Pro Ala Val Thr He Lys 140 145 150

eta cac ctt aat caa aat gga gat cac aac ace aaa gtt ctg cag aca 533 Leu His Leu Asn Gin Asn Gly Asp His Asn Thr Lys Val Leu Gin Thr 155 160 165

gac cca gcc ace ctg etc cat ttg gtt caa caa ctg gaa caa gca ttg 581 Asp Pro Ala Thr Leu Leu His Leu Val Gin Gin Leu Glu Gin Ala Leu 170 175 180

gaa gag atg aag aca aac cac tgt agg aga gtt gtt cgc aac ate aag 629 Glu Glu Met Lys Thr Asn His Cys Arg Arg Val Val Arg Asn He Lys 185 190 195

tagtaccagt tttaaggttt taattcattt gaatcactta tgaattgatg atatacagca 689 attacttttc aaaattaatt ttttattaat tcatgatgat aaatacatag tattcctcag 749 tatctattcc aagatactga ggtcataate agaagctaag ctgggtgcag tggctcatge 809 cagttatccc agcactttgg gaggccgagg tgggcaaatc atgaggtcag gagattgaga 869 ccttcctggc taacatggtg aaaccccatc tctactaaaa atataaaaaa ttagccaggt 929 gtggtggcac gcatctatea gagtcccagc taetcaggag gctgaggcag gagaatcgct 989 tgaacctggg aggtggaggt tgcagtgagc tgagattgtg ccactgcact ecagcctggg 1049 tgacagagtg agactccatc tcaaaaataa taataataat aataaagta 1098

<210> 2 <211> 199 <212> PRT <213> Homo sapiens

<400> 2

Met Leu Leu Glu Leu Ser Glu Glu His Lys Glu His Leu Ala Phe Leu

1 5 10 15

Pro Gln Val Asp Ser Ala Val Val Ala Glu Phe Gly Arg He Ala Val 20 25 30

Glu Phe Leu Arg Arg Gly Ala Asn Pro Lys He Tyr Glu Gly Ala Ala

35 40 45

Arg Lys Leu Asn Val Ser Ser Asp Thr Val Gln His Gly Val Glu Gly

50 55 60

Leu Thr Tyr Leu Leu Thr Glu Ser Ser Lys Leu Met He Ser Glu Leu

65 70 75 80

Asp Phe Gln Asp Ser Val Phe Val Leu Gly Phe Ser Glu Glu Leu Asn 85 90 95

Lys Leu Leu Leu Gln Leu Tyr Leu Asp Asn Arg Lys Glu He Arg Thr 100 105 110

Leu Leu Ser Glu Leu Ala Pro Ser Leu Pro Ser Tyr His Asn Leu Glu

115 120 125

Trp Arg Leu Asp Val Gln Leu Ala Ser Arg Ser Leu Arg Gin Gin He

130 135 140

Lys Pro Ala Val Thr He Lys Leu His Leu Asn Gin Asn Gly Asp His

145 150 155 160

Asn Thr Lys Val Leu Gin Thr Asp Pro Ala Thr Leu Leu His Leu Val 165 170 175

Gin Gin Leu Glu Gin Ala Leu Glu Glu Met Lys Thr Asn His Cys Arg

180 185 190

Arg Val Val Arg Asn He Lys

195

<210> 3

<211> 174

<212> PRT

<213> Homo sapiens

<400> 3 Glu Phe Gly Arg He Ala Val Glu Phe Leu Arg Arg Gly Ala Asn Pro 1 5 10 15

Lys He Tyr Glu Gly Ala Ala Arg Lys Leu Asn Val Ser Ser Asp Thr

20 25 30

Val Gin His Gly Val Glu Gly Leu Thr Tyr Leu Leu Thr Glu Ser Ser

35 40 45

Lys Leu Met He Ser Glu Leu Asp Phe Gin Asp Ser Val Phe Val Leu

50 55 60

Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu Gin Leu Tyr Leu Asp 65 70 75 80

Asn Arg Lys Glu He Arg Thr Leu Leu Ser Glu Leu Ala Pro Ser Leu

85 90 95

Pro Ser Tyr His Asn Leu Glu Trp Arg Leu Asp Val Gin Leu Ala Ser

100 105 110

Arg Ser Leu Arg Gln Gln He Lys Pro Ala Val Thr He Lys Leu His

115 120 125

Leu Asn Gin Asn Gly Asp His Asn Thr Lys Val Leu Gin Thr Asp Pro

130 135 140

Ala Thr Leu Leu His Leu Val Gin Gin Leu Glu Gin Ala Leu Glu Glu 145 150 155 160

Met Lys Thr Asn His Cys Arg Arg Val Val Arg Asn He Lys 165 170

<210> 4 <211> 965 <212> DNA <213> mus musculus

<220>

<221> CDS

<222> (44)... (640)

<400> 4 ggcggggccg gcgccgggtg gcaaagtctg aagagccgct aaa atg ctg ctg gat 55

Met Leu Leu Asp 1

ttg tec gag gag cac aag gag cac ttg gcc ttc ctg ccg caa gtg gac 103 Leu Ser Glu Glu His Lys Glu His Leu Ala Phe Leu Pro Gin Val Asp 5 10 15 20

act gca gtg gtc gcc gag ttc ggg agg ate gcc gtg gag ttc etc cga 151 Thr Ala Val Val Ala Glu Phe Gly Arg He Ala Val Glu Phe Leu Arg 25 30 35

cgg ggc tea aac ccg aag ate tac gaa ggc gcc gcc aga aaa ctg aac 199 Arg Gly Ser Asn Pro Lys He Tyr Glu Gly Ala Ala Arg Lys Leu Asn 40 45 50

gtg agt agt gac act ate cag cat ggt gtg gaa gga tta acg tat etc 247 Val Ser Ser Asp Thr He Gin His Gly Val Glu Gly Leu Thr Tyr Leu 55 60 65

etc ace gag age tea aag ctt atg att tct gaa ctg gat ttc caa gac 295 Leu Thr Glu Ser Ser Lys Leu Met He Ser Glu Leu Asp Phe Gin Asp 70 75 80

tct gtc ttt gtt ctg gga ttc tct gaa gaa eta aac aaa tta ttg ctt 343 Ser Val Phe Val Leu Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu 85 90 95 100

cag etc tac ctg gac aac aga aag gag ate aga act att ctg aac gag 391 Gin Leu Tyr Leu Asp Asn Arg Lys Glu He Arg Thr He Leu Asn Glu 105 110 115

tta gca cct cgt ctt ccc agt tac cat agt ctt gag tgg egg eta gat 439 Leu Ala Pro Arg Leu Pro Ser Tyr His Ser Leu Glu Trp Arg Leu Asp 120 125 130

gta cag ctt gca age aga agt etc egg caa cag att aag cca gca gtg 487 Val Gin Leu Ala Ser Arg Ser Leu Arg Gin Gin He Lys Pro Ala Val 135 140 145

ace ata aag ctg cac etc gat cag aat ggc gac cac age act cat ttc 535 Thr He Lys Leu His Leu Asp Gin Asn Gly Asp His Ser Thr His Phe 150 155 160

ttg cag aca gac cca get ace ctg ctt cat ttg gtt cag cag ctg gag 583 Leu Gin Thr Asp Pro Ala Thr Leu Leu His Leu Val Gin Gin Leu Glu 165 170 175 180

cag gcg tta gag gaa atg aaa aca aac cac tgc agg aga gta gtc cgc 631 Gin Ala Leu Glu Glu Met Lys Thr Asn His Cys Arg Arg Val Val Arg 185 190 195

age ate aag tagctctgag gtttccttcc ttccagttgg agacactggc 680

Ser He Lys

gtggcagtgg tacgtctgga ttcgtggagt ttctttccct gctcaggatg ttgaaatctt 740 gatcagaage tgagccgeag etecgttttg gtgetgaatg cggtcagctt cttgeaggtt 800 ggaaagcatt ttttacttea agtgtgtaaa gaggatgcae caactaagat ggateaatae 860 tgetgatttc tgtggactet tcggacttgg atattttttt tatctacttt atttectttg 920 ctcttgatca aaatttgaaa ataaaattca ctgttactac ttaga 965

<210> 5 <211> 199 <212> PRT <213> mus musculus <400> 5 Met Leu Leu Asp Leu Ser Glu Glu His Lys Glu His Leu Ala Phe Leu 1 5 10 15

Pro Gin Val Asp Thr Ala Val Val Ala Glu Phe Gly Arg He Ala Val

20 25 30

Glu Phe Leu Arg Arg Gly Ser Asn Pro Lys He Tyr Glu Gly Ala Ala

35 40 45

Arg Lys Leu Asn Val Ser Ser Asp Thr He Gin His Gly Val Glu Gly

50 55 60

Leu Thr Tyr Leu Leu Thr Glu Ser Ser Lys Leu Met He Ser Glu Leu 65 70 75 80

Asp Phe Gln Asp Ser Val Phe Val Leu Gly Phe Ser Glu Glu Leu Asn

85 90 95

Lys Leu Leu Leu Gln Leu Tyr Leu Asp Asn Arg Lys Glu He Arg Thr

100 105 110

He Leu Asn Glu Leu Ala Pro Arg Leu Pro Ser Tyr His Ser Leu Glu

115 120 125

Trp Arg Leu Asp Val Gln Leu Ala Ser Arg Ser Leu Arg Gin Gin He

130 135 140

Lys Pro Ala Val Thr He Lys Leu His Leu Asp Gin Asn Gly Asp His 145 150 155 160

Ser Thr His Phe Leu Gin Thr Asp Pro Ala Thr Leu Leu His Leu Val

165 170 175

Gin Gin Leu Glu Gin Ala Leu Glu Glu Met Lys Thr Asn His Cys Arg

180 185 190

Arg Val Val Arg Ser He Lys 195

<210> 6 <211> 174 <212> PRT <213> Mus musculus

<400> 6 Glu Phe Gly Arg He Ala Val Glu Phe Leu Arg Arg Gly Ser Asn Pro 1 5 10 15

Lys He Tyr Glu Gly Ala Ala Arg Lys Leu Asn Val Ser Ser Asp Thr

20 25 30

He Gin His Gly Val Glu Gly Leu Thr Tyr Leu Leu Thr Glu Ser Ser

35 40 45

Lys Leu Met He Ser Glu Leu Asp Phe Gin Asp Ser Val Phe Val Leu

50 55 60

Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu Gin Leu Tyr Leu Asp 65 70 75 80

Asn Arg Lys Glu He Arg Thr He Leu Asn Glu Leu Ala Pro Arg Leu 85 90 95

Pro Ser Tyr His Ser Leu Glu Trp Arg Leu Asp Val Gin Leu Ala Ser

100 105 110

Arg Ser Leu Arg Gln Gln He Lys Pro Ala Val Thr He Lys Leu His

115 120 125

Leu Asp Gin Asn Gly Asp His Ser Thr His Phe Leu Gin Thr Asp Pro

130 135 140

Ala Thr Leu Leu His Leu Val Gln Gln Leu Glu Gln Ala Leu Glu Glu 145 150 155 160

Met Lys Thr Asn His Cys Arg Arg Val Val Arg Ser He Lys 165 170

<210> 7

<211> 15

<212> PRT

<213> Homo sapiens

<400> 7 Ser Ser Asp Thr Val Gin His Gly Val Glu Gly Leu Thr Tyr Leu 1 5 10 15

<210> 8

<211> 15

<212> PRT

<213> Homo sapiens

<400> 8 Val Leu Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu Gin Leu 1 5 10 15

<210> 9

<211> 15

<212> PRT

<213> Homo sapiens

<400> 9 He Arg Thr Leu Leu Ser Gl u Leu Al a Pro Ser Leu Pro Ser Tyr 1 5 10 15

<210> 10

<211> 15

<212> PRT

<213> Homo sapiens

<400> 10 Leu Leu His Leu Val Gin Gin Leu Glu Gin Ala Leu Glu Glu Met 1 5 10 15

<210> 11

<211> 15

<212> PRT

<213> Mus musculus

<400> 11 Ser Ser Asp Thr He Gin His Gly Val Glu Gly Leu Thr Tyr Leu 1 5 10 15

<210> 12

<211> 15

<212> PRT

<213> Mus musculus

<400> 12 Val Leu Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu Gin Leu 1 5 10 15

<210> 13

<211> 15

<212> PRT

<213> Mus musculus

<400> 13 He Arg Thr He Leu Asn Glu Leu Ala Pro Arg Leu Pro Ser Tyr 1 5 10 15

<210> 14

<211> 15

<212> PRT

<213> Mus musculus

<400> 14 Leu Leu His Leu Val Gln Gln Leu Glu Gln Ala Leu Glu Glu Met 1 5 10 15

<210> 15 <211> 599 <212> DNA <213> Homo sapiens

<220> <221> mιsc_feature <222> (1)...(599) <223> n = A.T.C or G

<400> 15 ggacagtgaa gatgctgctg gaattgtccg aggagcataa ggaacacctg gccttcctgc 60 ctcaagtgga cagcgcggtg gtcgccgagt ttgggcggat tgctgtggaa ttcctgagac 120 gcggcgcaaa cccaaaaatc tacgaaggcg ccgcagaaaa ctcaatgtga gtagtgacac 180 tgtccagcat ggtgtggaag gattaacgta tctcctcact gagagctcaa agctcatgat 240 ttctgaactg gatttccaag actctgtttt tgttctggga ttctctgaag aattaaacaa 300 attgttgctt cagctttatc tggacaacag aaaagagatc agaacgattc tgagtgaatt 360 ggcaccaagc cttcccagtt atcataacct tgaatggcga ctagatgtac agcttgcaag 420 tagaagtctc aggcaaeaga ttaaccagea gtgactataa agctacacct taatcaaaat 480 ggagatcaca acaccaagtt ctgcagacag acccagccac cctgctccat ttggntcaca 540 atngaacaag cattggaaga gatgaagnca acccctgttg gagaattgtt gcaacatca 599

<210> 16

<211> 29

<212> PRT

<213> Homo sapiens

<400> 16

Arg Arg Gly Al a Asn Pro Lys H e Tyr Gl u Gly Al a Al a Arg Lys Leu 1 5 10 15

Asn Val Ser Ser Asp Thr Val Gi n Hi s Gly Val Gl u Gly 20 25

<210> 17

<211> 23

<212> PRT

<213> Homo sapiens

<400> 17 Gi n Gi n Leu Gl u Gi n Al a Leu Gl u Gl u Met Lys Thr Asn Hi s Cys Arg 1 5 10 15

Arg Val Val Arg Asn H e Lys 20

<210> 18

<211> 45

<212> PRT

<213> Homo spaiens

<400> 18 Asn Gin Asn Gly Asp His Asn Thr Lys Val Leu Gin Thr Asp Pro Ala

1 5 10 15

Thr Leu Leu His Leu Val Gin Gin Leu Glu Gin Ala Leu Glu Glu Met

20 25 30

Lys Thr Asn His Cys Arg Arg Val Val Arg Asn He Lys 35 40 45

<210> 19

<211> 47

<212> PRT

<213> Homo sapiens

<400> 19 Thr Glu Ser Ser Lys Leu Met He Ser Glu Leu Asp Phe Gin Asp Ser 1 5 10 15

Val Phe Val Leu Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu Gin

20 25 30

Leu Tyr Leu Asp Asn Arg Lys Glu He Arg Thr Leu Leu Ser Glu 35 40 45

<210> 20

<211> 57

<212> PRT

<213> Homo sapiens

<400> 20 Thr Glu Ser Ser Lys Leu Met He Ser Glu Leu Asp Phe Gin Asp Ser 1 5 10 15

Val Phe Val Leu Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu Gin

20 25 30

Leu Tyr Leu Asp Asn Arg Lys Glu He Arg Thr Leu Leu Ser Glu Leu

35 40 45

Ala Pro Ser Leu Pro Ser Tyr His Asn 50 55

<210> 21

<211> 25

<212> PRT

<213> Mus musculus

<400> 21 Arg Arg Gly Ser Asn Pro Lys He Tyr Glu Gly Ala Ala Arg Lys Leu

1 5 10 15

Asn Val Ser Ser Asp Thr He Gln His 20 25 <210> 22

<211> 16

<212> PRT

<213> Mus musculus

<400> 22 Glu Glu Met Lys Thr Asn His Cys Arg Arg Val Val Arg Ser He Lys 1 5 10 15

<210> 23

<211> 57

<212> PRT

<213> Mus musculus

<400> 23 Thr Glu Ser Ser Lys Leu Met He Ser Glu Leu Asp Phe Gin Asp Ser 1 5 10 15

Val Phe Val Leu Gly Phe Ser Glu Glu Leu Asn Lys Leu Leu Leu Gin

20 25 30

Leu Tyr Leu Asp Asn Arg Lys Glu He Arg Thr He Leu Asn Glu Leu

35 40 45

Ala Pro Arg Leu Pro Ser Tyr His Ser 50 55

<210> 24

<211> 27

<212> PRT

<213> Homo sapiens

<400> 24 Ser Arg Ser Leu Arg Gin Gin He Lys Pro Ala Val Thr He Lys Leu 1 5 10 15

His Leu Asp Gin Asn Gly Asp His Ser Thr His 20 25

<210> 25

<211> 48

<212> DNA

<213> Homo sapiens

<400> 25 gtctgggttc gctactcgag gcggccgcta tttttttttt tttttttt 48 <210> 26 <211> 448 <212> DNA <213> Mus musculus

<400> 26 ggcggggccg gcgccgggtg gcaaagtctg aagagccgct aaaatgctgc tggatttgtc 60 cgaggagcac aaggagcact tggccttcct gccgcaagtg gacactgcag tggtcgccga 120 gttcgggagg atcgccgtgg agttcctccg acggggctca aacccgaaga tctacgaagg 180 cgccgccaga aaactgaacg tgagtagtga cactatccag catggtgtgg aaggattaac 240 gtatctccte acegagagct caaagcttat gatttctgaa ctggatttcc aagactctgt 300 ctttgttctg ggattctctg aagaactaaa caaatgattg cttcagctct acctggacaa 360 cagaaaggag atcagaacta ttctgaacga gttagcacct cgtcttccca gttaccatag 420 tcttgagtgg cggctagatg tacagctt 448