MXPA01007161A - Mammalian alpha-helical protein - 12 - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for mammalian alpha helix-12 (Zalpha12). 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 Zalpha12 polypeptides. Antagonists may be used to treat inflammation and inflammation-related diseases.

Description

PROTEIN ALPHA HELICOIDAL-12 MAMMALS BACKGROUND OF THE INVENTION Inflammation is normally a localized and protective response to trauma or microbial invasion that destroys, dilutes or removes the damaging agent and damaged tissue from the walls. It is characterized in the acute form by the classic signs of pain, heat, inflammation, swelling and loss of function. Microscopically it involves a complex series of events, including dilation of arterioles, capillaries and venules, with increased permeability and blood flow, fluid exudation, including plasma proteins, and migration of leukocytes to the area of inflammation. Diseases characterized by inflammation are significant causes of morbidity and mortality in humans. Commonly, inflammation occurs as a defensive response to host invasion by foreign material, particularly microbial. The responses to mechanical trauma, toxins and neoplasia can also result in inflammatory reactions. The accumulation and subsequent activation of leukocytes are central events Ref: 131311 on the pathogenesis of most forms of inflammation. Deficiencies of inflammation compromise the host. Excessive inflammation caused by normal recognition of host tissue as foreign or prolongation of the inflammatory process can lead to inflammatory diseases as diverse as diabetes, arteriosclerosis, cataracts, reperfusion injury and cancer, to post-infectious syndromes such as infectious meningitis, fever rheumatic, and rheumatic diseases such as systemic lupus erythematosus and rheumatoid arthritis. The centrality of the inflammatory response in these varied disease processes makes its regulation an important element in the control of the prevention or cure of the human disease. Thus, there is a need to discover cytokines that contribute to inflammation and inflammation-related diseases in such a way that antagonists such as antibodies can be administered to down-regulate the cytokine so that inflammation is decreased.

BRIEF DESCRIPTION OF THE INVENTION The present invention satisfies this need by providing novel polypeptides and related compositions, and methods and their antagonists. In one aspect, the present invention provides an isolated polynucleotide that encodes a mammalian cytokine called Zalfal2. The data show that the cytokine is involved in the inflammation response. In this manner, Zalfal2 antagonists can be used to decrease inflammation especially inflammation associated with coronary heart disease, arteriosclerosis, Crohn's disease and inflammatory bowel disease. Three variants have been discovered as shown in SEQ ID NOs: 1, 2, 3, 4, 5 and 6. Each Zalfal2 polypeptide has a signal sequence extending from amino acid residue 1, a methionine, up to and including the amino acid residue 34, a serine of SEQ ID NOs: 2, 4 and 6. In this manner, the Zalfal2 polypeptide represented by SEQ ID NO: 2 has a mature sequence extending from amino acid residue 35, an alanine, up to and including amino acid residue 202, an asparagine, also represented by SEQ ID NO: 8.

The Zalfal2 polypeptide represented by SEQ ID NO: 4 has a mature sequence extending from amino acid residue 35, an alanine, up to and including an amino acid residue 288, an asparagine, also represented by SEQ ID NO: 9. The polypeptide Zalfal2 represented by SEQ ID NO: 6 has a mature sequence extending from amino acid residue 35, an alanine, up to and including amino acid residue 158, an asparagine, also represented by SEQ ID NO: 10. In a second aspect of the invention provides an expression vector comprising a) a transcription promoter; b) a segment of DNA encoding the Zalafal2 polypeptide, and c) a transcription terminator, wherein the promoter, DNA segment and terminator are operably linked. In a third aspect of the invention there is provided a cultured eukaryotic or prokaryotic cell in which an expression vector as described above has been introduced, wherein said cell expresses a protein polypeptide encoded by the .DNA segment. In 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 Zalfal2 polypeptide, b) allelic variants of Zalfal2; 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. In one embodiment the affinity tag is an immunoglobulin Fc polypeptide. The invention also provides expression vectors that encode the chimeric polypeptides and transfected host cells to produce the chimeric polypeptides. In a further aspect of the invention there is provided an antibody that specifically binds a Zalfal2 polypeptide as described above, and also an anti-idiotypic antibody that neutralizes the antibody to a Zalfal2 polypeptide. A further embodiment of the present invention relates to a peptide or polypeptide having the amino acid sequence of an epitope-bearing portion of a Zalfal2 polypeptide having an amino acid sequence described above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a Zalfal2 polypeptide of the present invention include portions of those polypeptides with at least nine, preferably at least 15 and most preferably at least 30 a 50 amino acids, although epitope-bearing polypeptides of any length up to and including the complete amino acid sequence of a polypeptide of the present invention described above are also included in the present invention. Examples of such epitope binding regions are SEQ ID NOs: 18, 19, 20, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44 Any of these polypeptides that have fused to another polypeptide or carrier molecule are also claimed. These and other aspects of the invention will become apparent from the reference of the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION The teachings of all references cited herein are incorporated herein in their entirety by way of reference. Before describing the invention in detail, it could be useful for the understanding of it to define the following terms: The term "affinity tag" is used herein to denote a polypeptide segment that can be linked to a second polypeptide to provide for the purification or detection of the second polypeptide or provide sites for the binding of the second polypeptide to a substrate. Primarily, 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. , EMBO J. 4: 1075 (1985); Nilsson et al. , Methods Enzymol. 198: 3 (1991), glutathione S transferase, Smith and Johnson, Gene 67:31 (1988), affinity tag Glu-Glu, Grussenmeyer et al. , Proc. Nati Acad. Sci. USA 82: 7952-4 (1985), substance P, Flag ™ peptide, Hopp et al. , Biotechnology 6: 1204-1210 (1988), streptavidin-binding peptides, or other antigenic epitope or binding domain. See, in general, Ford et al. , Protein Expression and Purification 2: 95-107 (1991). The .ADNs that encode affinity tags are available from commercial providers (eg Pharmacia Biotech, Piscataway, NJ). The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene that occupies the same chromosomal locus.

Allelic variation originates naturally through mutation, and can result in phenotypic polymorphism in populations. Mutations of genes can be silent (without changes in the encoded polypeptide) or can encode polypeptides having altered amino acid sequences. 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 in the polypeptides. Where the context permits, 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 placed 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. The term "complement pair / anticomplement" denotes non-identical portions that form an associated stable pair non-covalently under suitable conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anticomplement pair. Other exemplary complement / anticomment pairs include the receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs and the like. When the subsequent dissociation of the complement / anticomplement 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 5 'sequence ATGCACGGG 3' is complementary to 5 '• CCCGTGCAT 3'. The term "contig" denotes a polynucleotide having a contiguous length of sequence identical or complementary to another polynucleotide. The contiguous sequences are said to "overlap" a given stretch of the polynucleotide sequence either in its entirety or along a partial stretch of the polynucleotide. For example, the contiguous representatives for the sequence of polynucleotides 5 '-ATGGCTTAGCTT-3' are 5 'TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5 '.

The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that codes for a polypeptide). Degenerate codons contain different triplets of nucleotides, but code for the same amino acid residue (ie, the triplets GAU and GAC encode each for Asp). The term "expression vector" is used to denote a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to additional segments that provide 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 environment and is thus free of other foreign or unwanted coding sequences, and is in a form suitable for use. in genetically engineered protein production systems. These isolated molecules are those that are separated from their natural environment and include .DNA and genomic clones. The isolated DNA molecules of the present invention are free of other genes with which they are normally associated, but can include natural 5 'and 3' untranslated regions such as promoters and terminators. The identification of the associated regions will be apparent to one skilled in the art (see, for example, Dynan and Tijan, Nature 316: 774-78 (1985).) An "isolated" polypeptide or protein is a polypeptide or protein found in a condition that is not its natural environment, such as apart from animal blood and 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, that is, more than 95% pure, most preferably more 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 glycosylated forms or derived alternatively.

The term "operably linked", when referring to DNA segments, indicates that the segments are arranged in such a way that they function in concert for their intended purposes, for example, the transcription starts at the promoter and proceeds through the coding segment until the terminator. The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The sequence differences between orthologs are the result of speciation. "Pars" are different but structurally related proteins made by an organism. It is believed that paralogs originate through the duplication of genes. For example, a-globin, b-globin and myoglobin are paralogs with each other. A "polynucleotide" is a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'end to the 3' end. The polynucleotides include .RNA and AD ?, and can be isolated from natural sources, synthesized in vi tro or prepared from a combination of synthetic and natural molecules. The sizes of the polynucleotides are expressed as base pairs (abbreviated as "pb"), nucleotides ("nt") or kilobases ("kb"). When the context permits, the last 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 full length and will be understood to be equivalent to the term "base pairs". The experts. in the art they will recognize that the two strands of a double-stranded polynucleotide can have a slightly different length and that the ends thereof can be staggered as a result of the enzymatic cut; in this manner, all nucleotides within a double-stranded polynucleotide molecule can not be aligned in pairs. Those ends not aligned in pairs do not generally exceed 20 nt in length. A "polypeptide" is a polymer of amino acid residues linked 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 by its recognized meaning in the art to denote a portion of a gene that contains .DNA sequences that provide for the binding of RNA polymerase and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'non-coding regions of the genes. A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents can 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 nevertheless may be present. 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. The binding of a ligand to a receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecules in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are related to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, cellular calcium mobilization, membrane lipid mobilization, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane, cytosolic or nuclear; monomeric (eg, thyroid hormone receptor, beta-adrenergic receptor) or multimeric receptor (eg, 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 sequence of .DNA that codes for 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 cut to remove the secretory peptide during its transit through the secretory path. The term "splice variant" is used herein to denote alternative forms of .RNA transcribed from a gene. The splicing variation naturally results from the use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several .RNAM molecules transcribed from the same gene. . The splice variants can encode polypeptides having an altered amino acid sequence. The term "splice variant" is also used herein to denote a protein encoded by a splice variant of an .RNA. transcribed from a gene. The molecular weights and lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis) will be understood as approximate values. When such value is expressed as "approximately" X or "around" X, it will be understood that the indicated value of X will be accurate to ± 10%.

Introduction The Zalfal2 protein is an alpha helical protein. SEQ ID NO: 2 has four alpha helices, A, B, C and D. Propeller A of SEQ ID NO: 2 extends from amino acid residue 56, a leucine, up to and including amino acid residue 70, an isoleucine also defined by SEQ ID NO: 21. Propeller B of SEQ ID NO: 2 extends from amino acid residue 96, a leucine, up to and including amino acid residue 110, a tyrosine, also defined by SEQ ID NO: 22 The helix C of SEQ ID NO: 2 extends from amino acid residue 130, a leucine, up to and including amino acid residue 144, a leucine, also defined by SEQ ID NO: 23. Propeller D of SEQ ID NO : 2 extends from amino acid residue 162, a methionine, up to and including amino acid residue 176, a serine, also defined by SEQ ID NO: 24. SEQ ID NO: 25 contains helices A + B; SEQ ID NO: 26 contains the helices A + B + C; SEQ ID NO: 27 contains the helices A + B + C + D; SEQ ID NO: 28 contains the helices B + C + D; SEQ ID NO: 29 contains the helices B + C and SEQ ID NO: 30 contains the helices C + D of SEQ ID NO: 2. The polypeptide of SEQ ID NO: 4 also contains four helices A, B, C and D. The helix of SEQ ID NO: 4 extends from amino acid residue 45, a histidine, up to and including amino acid residue 59, a leucine, also defined by SEQ ID NO: 31. Propellent B of SEQ ID NO: 4 extends from amino acid residue 116, a valine, up to and including amino acid residue 130, a lysine, also defined by SEQ ID NO: 32. Propellent C of SEQ ID NO: 4 extends from amino acid residue 142 , a leucine, up to and including amino acid residue 156, an isoleucine, also defined by SEQ ID NO: 33. Propeller D of SEQ ID NO: 4 extends from amino acid residue 182, a leucine, up to and including the amino acid residue 196, a tyrosine, also defined by SEQ ID NO: 34. SEQ ID NO: 35 contains the helices A + B of SEQ ID N OR: 4. SEQ ID NO: 36 contains the helices A + B + C of SEQ ID NO: 4. SEQ ID NO: 37 contains the helices A + B + C + D of SEQ ID NO: 4. SEQ ID NO: 38 contains the helices B + C + D of SEQ ID NO: 4. SEQ ID NO: 39 contains the helices B + C of SEQ ID NO: 4. SEQ ID NO: 40 contains the helices C + D of SEQ ID NO: 4. The expression of the Zalfal2 gene is observed in a number of different tissues including the spleen, thymus, testis, small intestine, colon, peripheral blood lymphocytes (PBL), stomach, trachea, T cells that include CD4 + and CD8 + cells and bone marrow that is. This pattern of expression suggests that Zalfal2 could play a general role in the development and exercise significant regulatory control of testicular differentiation, the hypothalamic-pituitary-gonadal axis and steroidogenesis and gonadal spermatogenesis. The development of the production of testicular hormones can be divided into initial and terminal stages, with the latter depending on the activation of Leydig cell precursors functionally determined by luteinizing hormone (LH). However, the factors that control the initial stages in this process remain unknown, Huhtaniemi, Reprod. Fertile. Dev. 7: 1025-1035 (1995) suggesting that Zalfal2 could be responsible for the activation of a precursor cell not responsive to LH and non-steroidogenic. Once the differentiation of Leydig cells has occurred, the production of steroid hormones in the testes depends on the secretion of the gonadotro inas, hormone LH and follicle stimulator (FSH), by the pituitary. LH stimulates testosterone production by Leydig cells, whereas spermatogenesis depends on both FSH and high intratesticular concentrations of testosterone. The secretion of LH and FSH is in turn under the control of the gonadotropin releasing hormone (GnRH) produced in the hypothalamus, Kaufman, The neuro endocrine regulation of male reproduction. in: Male Infertility. Clinical Investigation, Cause Evaluation and Treatment., FH Comhaire, ed., Pp-29-54 (Chapman and Hall, London, 1996). Since the testicular products have been shown to control the production of LH and FSH, this suggests that Zalfal2 could regulate the production of hormones by the hypothalamus. It is well known that steroidogenesis and sparmatogenesis take place within two different cell compartments of the testes, with cells from Leydig and Sertoli responsible for the first and the last, respectively, Saez, EndoCrin. Rev. 15: 574-626 (1994). The activity of each of these cell types seems to be regulated by the secretory products of the other. Sertoli cell-derived tumor necrosis factor, fibroblast growth factor, interleukin-1, transforming growth factor-B, epidermal growth factor / transforming growth factor-a, activin, inhibin, factor of Growth type insulin-1, platelet-derived growth factor, endothelin and arginine-vasopressin have all been shown to regulate the cell function of Leydig, Saez, Endocrin. Rev. 15: 574-626 (1994). In this way, Zalfal2 could control or modulate the activities of one or more of these genes. In men, aging is associated with a progressive decline in testicular function. These changes are manifested clinically by diminished virility, vigor, and libido that point to a relative testicular deficiency, Vermeulen, Ann. Med. 25: 531-534 (1993); Pugeat et al., Horm. Res. 43: 104-110 (1995). Hormone replacement therapy is not currently recommended in elderly men, which suggests that a new therapy for the male climacteric is of great value.

Polynucleotides The present invention also provides polynucleotide molecules, including .DNA and RNA molecules, which encode the Zalfal2 polypeptides described herein. Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation between these polynucleotide molecules is possible.

The polynucleotides, generally a cDNA sequence, of the present invention encode the polypeptides described herein. A cDNA sequence encoding a polypeptide of the present invention comprises a series of codons, each amino acid residue of the polypeptide being encoded by a codon, and each codon comprising 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 (lie) 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 (Gln) is encoded by CA 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 GTT; Tryptophan (Trp) is encoded by TGG and Tyrosine (Tyr) is encoded by TAC or TAT. It should be recognized that in accordance with the present invention, when a polynucleotide is claimed as described herein, it is understood that what is claimed are both the sense strand, as the antisense strand, and the .DNA as a double strand. having both the sense and antisense strand bound together by their respective hydrogen bonds. The .RNA messenger (mRNA) encoding the polypeptides of the present invention, .RNA, which is encoded by the cDNA described herein, is also claimed. The messenger RNA (mRNA) will code for a polypeptide using the same codons as defined herein, except that each thymine nucleotide (T) is replaced by a uracil nucleotide (U). One skilled in the art will also appreciate that different species may exhibit "preferential codon usage". In general, see Grantham, et al. , Nuc. Acids Res. 8: 1893-1912 (1980); Haas, et al. Curr. Biol. 6: 315-324 (1996); Wain-Hobson, et al. . Gene 13: 355-364 (1981); Grosjean and Fliers, Gene 18: 199-209 (1982); Holm, Nuc. Acids Res. 14: 3075-3087 (1986); Ike ura, J. Mol. Biol. 158: 573-597 (1982). As used herein, the term "preferential codon usage" or "preferential codons" is a term of the art that refers to translation codons of proteins that are very frequently used in cells of a certain species, thus favoring one or few representatives of the possible codons that code for each amino acid (see Table 2). For example, the amino acid Threonine (Thr) can be encoded by ACA, ACC, ACG or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, e.g., insect cells, yeast, virus or bacteria, different Thr codons may be preferred. The codons that are preferred for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferred codon sequences in recombinant .DNA can, for example, increase the production of the protein making the translation of the protein more efficient within a particular cell type or species. Sequences containing preferential codons can be tested and optimized for expression in several species, and tested to verify functionality as described herein. In preferred embodiments of the invention the isolated polynucleotides will hybridize to regions of similar size of SEQ ID NOs: 1, 3 or 5, 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 (Tm) for the specific sequence at the defined ionic strength and pH. The Tm 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 indicated, 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 .RNA Zalfal2. These tissues and cells are identified by Northern blot analysis, Thomas, Proc. Nati Acad. Sci. USA 77: 5201 (1980) and are described below. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a gradient of CsCl, Chirgwin et al. , Biochemistry 18: 52-94 (1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder, Proc. Nati Acad. Sci. USA 55: 1408-1412 (1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. Alternatively, genomic DNA can be isolated. The polynucleotides encoding Zalfal2 polypeptides are then identified and isolated by, for example, hybridization or PCR.

A full-length clone encoding Zalfal2 can be obtained by conventional cloning procedures. Complementary DNA clones (cDNAs) are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferred to use a genomic clone, or modify a clone of .ADNc to include at least one genomic intron. Methods for preparing cDNAs and genomic clones are well known and are at the level of ordinary skill in the art, and include the use of the sequence described herein, or portions thereof, to probe or prime a library. Expression libraries can be probed with antibodies to Zalfal2, 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 double-stranded DNA synthesized chemically 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 direct and can be achieved by synthesizing the complementary strands and then fixing 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 rarely 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from fragments of a single strand that are from 20 to 100 nucleotides in length. See Glick and Pasternak, Molecular Biotechnology, Principies & Aplications of Recombinant DNA, (ASM Press, Washington, D.C., 1984); Itakura et al. , Annu. Rev. Biochem. 53: 323-356 (1984) and Cumie et al. , Proc. Nati Acad. Sci. USA 87: 633-637 (1990). The present invention also provides polypeptides and counterpart polynucleotides from other species (orthologs). These species include, but are not limited to, species of mammals, birds, amphibians, reptiles, fish, insects and other vertebrates and invertebrates. Of particular interest are Zalfal2 polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine and other primate polypeptides. Human Zalfal2 orthologs can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, an .DNA can be cloned using mRNA obtained from a cell or tissue type expressing Zalfal2 as described herein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences described herein. A mRNA library of a positive tissue or cell is then prepared. The cDNA encoding Zalfal2 can then be isolated by a variety of methods, such as by probing with a complete or partial human .DNA, or with one or more sets of degenerate probes based on the described sequences. An .DNA 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 Zalfal2 sequence described herein. In an additional method, the cDNA library can be used to transform or transfect host cells, and the expression of the -DNAc of interest can be detected with an antibody to Zalfal2 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequence described in SEQ ID NOs: 1, 3 or 5 represents specific alleles of human Zalfal2 and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries of different individuals according to standard procedures. Allelic variants of the cDNA 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 that the proteins that are allelic variants of SEQ ID NOs: 4 or 6. The .ADNc molecules generated from alternatively spliced mRNA molecules, which retain the properties of the Zalfal2 polypeptide are included within the scope of the present invention, same as the polypeptides encoded by such .ADNc and mRNA molecules. The allelic variants and splice variants of these sequences can be cloned by probing AD? C or genomic libraries of different individuals or tissues according to standard procedures known in the art. The present invention also provides polypeptides Zalfal2 isolates that are substantially identical to the polypeptides of SEQ ID Os: 2, 4, 6, 8, 9 or 10 and their orthologs. The term "substantially identical" is used herein to indicate polypeptides having 50%, 60%, 70%, 80% and most preferably at least 90%, 95% or 99% sequence identity to the sequences shown in SEQ ID NOs: 2, 4, 6, 8, 9 or 10 or their orthologs. The percentage of sequence identity is determined by conventional methods. See, for example, Altschul et al. , Bull. Match Bio. 48: 603-616 (1996) and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 85: 10915-10919 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a space opening penalty of 10, a space extension penalty of 1, and the "BLOSUM 62" score matrix of Henikoff and Henikoff. { ibid. ) as shown in Table 1 (amino acids are indicated by standard letter codes). The identity percentage is then calculated as: Total number of identical matings x 100 [length of the longest sequence plus the number of spaces entered in the longest sequence to align the two sequences] . Those skilled in the art appreciate that there are many established algorithms for aligning two amino acid sequences. The "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method to examine 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 r 1 Acad. Sci. USA 85: 2444 (1988), and by Pearson, Meth. Enzymol. 183: 63 (1990). Briefly, FASTA first characterizes the sequence similarity by identifying regions shared by the query sequence (eg, SEQ ID NO: 2) and a 'test sequence that has either the highest identity density (if the variable ktup is 1 ) or pairs of identities (if ktup = 2), without considering substitutions, insertions or deletions of conservative amino acids. The ten regions with the highest density of identities are then reclassified by comparing the similarity of all amino acids aligned in pairs using an amino acid substitution matrix, and the ends of the regions are "cut" to include only those residues that contribute to the highest score If there are several regions with scores greater than the "cut" value (calculated by a predetermined formula based on the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine if the regions can be joined to form an approximate alignment with spaces. Finally, the highest scoring regions of two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wusch, J. Mol. Biol. 48: 444 (1970); Sellers, SIAM J. Appl. Math 26: 181 (1974), which allows amino acid insertions and deletions The illustrative parameters of the FASTA analysis are: ktup = l, space opening penalty = 10, space extension penalty = ly substitution matrix BLOSUM62 These parameters can be entered into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol, 183: 63 (1990). determining the sequence identity of nucleic acid molecules using a ratio as described above For comparisons between nucleotide sequences, the ktup value may vary between one to six, preferably four to six.

The present invention includes nucleic acid molecules, which code for a polypeptide having one or more conservative amino acid changes, as compared to the amino acid sequence of SEQ ID Nos: 2, 4, 6, 8, 9 or 10. Table BLOSUM62 is an amino acid substitution matrix derived from approximately 2000, local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 related protein groups [Henikoff and Henikoff, Proc. Nat 'l Acad. Sci. USA 85: 10915 (1992)]. Accordingly, BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that could 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. The conservative amino acid substitutions that are preferred are characterized by a BLOSUM62 value of at least 1 (eg, 1, 2 or 3), while conservative substitutions that are most preferred are characterized by a BLOSUM62 value of at least 2 (for example, 2 or 3). The sequence identity of polynucleotide molecules is determined by similar methods using a ratio as described above. Zalfal2 variant polypeptides or substantially homologous Zalfal2 polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is, conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the replication or activity of the polypeptide.; small deletions, typically from 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 then includes polypeptides of about 20 to 30 amino acid residues comprising a sequence that is at least 90%, preferably at least 95% and most preferably 99% or more identical to the corresponding region of SEQ ID NOs: 2, 4, 6, 8, 9 or 10. Polypeptides comprising affinity tags may further comprise a proteolytic cleavage site between the Zalfal2 polypeptide and the affinity tag. Preferred sites include thrombin cleavage sites and factor Xa cleavage sites.

Table 2 Conservative amino acid substitutions Basic: arginine lysine histidine Acid: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine septen threonine methionine The present invention also provides a variety of other functions of polypeptides [and related multimeric proteins] comprise one or more polypeptide fusions). For example, a Zalfal2 polypeptide can be prepared as a fusion to a dimerization protein as described in the U.S. Patents. Nos. 5,155,027 and 5,567,584. Preferred dimerization proteins in this regard include the immunoglobulin constant region domains. Zalfal2 immunoglobulin-polypeptide fusions can be expressed in genetically engineered cells [to produce a variety of multimeric Zalfal2 analogs]. The helper domains can be fused to Zalfal2 polypeptides or targeted to specific cells, tissues or macromolecules (e.g., collagen). For example, a Zalfal2 polypeptide or protein can be targeted to a predetermined cell type by fusing a Zalfal2 polypeptide to a ligand that specifically binds a receptor on the surface of the target cell. In this way, the polypeptides and proteins can be targeted for therapeutic or diagnostic purposes. A Zalfal2 polypeptide can be fused to two or more portions, such as an affinity tag for purification and a targeting domain. Polypeptide fusions may 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 amino acid residues that occur non-naturally. The non-naturally occurring amino acid residues include, without limitation, trans-3-methylproline, 2,4-methanoproline, c-s-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyltreonin, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidinocarboxylic acid, dehydroproline, 3- and 4-ethylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine . Various methods are known in the art for incorporating amino acid residues that occur non-naturally in proteins. For example, an in vi tro system in which non-sense mutations can be suppressed using chemically aminoacylated .ARNt molecules can be employed. Methods for synthesizing amino acids and inactivating tRNA are known in the art. Transcription and translation of plasmids containing non-sense mutations is carried out in a cell-free system comprising an S30 extract of E. coli and commercially available enzymes and other reagents. The 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 255: 806-809 (1993); and Chung et al., Proc. Nati. Acad. Sci. USA 50: 10145-1019 (1993) .In a second method, transduction is carried out in Xenopus oocytes by microinjection of mutated .RNA and chemically aminoacylated aminoacid-suppressor .ARNt molecules, Turcatti et al., J. Biol., Chem. 271: 1991-1998 (1996). method, E. coli cells are cultured in the absence of a natural amino acid to be replaced (eg, phenylalanine) and in the presence of the non-naturally occurring amino acid (s) (eg, 2-azaphenylalanine, 3-azaphenylalanine , 4-azaphenylalanine or 4-fluorophenylalanine.) The amino acid that occurs is not naturally incorporated in the protein in place of its natural counterpart, see, Koide et al., Biochem 33: 1410-1416 (1994). that occur naturally can become species that occur not naturally by chemical modification in vi tro Chemical modification can be combined with site-directed mutagenesis to further expand the scale 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 non-natural amino acids can be substituted for Zalfal2 amino acid residues. The essential amino acids in the polypeptides of the present invention can be identified according to methods 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, Nati. Acad. Sci. USA 88: 4498-502 (1991). In this latter technique, individual alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested to verify their biological activity as described below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al. , J. Biol. Chem. 271: 4699-108, 1996. The ligand-receptor interaction sites can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity marking, in set with the 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 analysis, such as those described by Reidhaar-Olson and Sauer, Science 241: 53-51 (1988) or Bowie and Sauer, Proc. Nati Acad. Sci. USA 86 '; 2152-2156 (1989). Briefly, these authors describe methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, 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, for example, Lowman et al. , Biochem. 30: 10832-10837 (1991); Ladner et al. , patent of E.U.A. 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). The Zalfal2 .DNA variants and described polypeptide sequences can be generated by DNA intermixing as described by Stemmer, Nature 370: 389-391, (1994), Stemmer, Proc. Na ti. Acad. Sci. USA 51: 10747-10751 (1994) and the WIPO publication WO 97/20078. Briefly, variants of .ADN molecules can be generated by homologous recombination in vi tro by randomly fragmenting a .ADN progenitor followed by reassembly using PCR, resulting in randomly introduced dot mutations. This technique can be modified using a family of progenitor DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. The selection or analysis of the desired activity, followed by additional iterations of mutagenesis and assay provide rapid "evolution" of the sequences by selecting desirable mutations while simultaneously selecting against damaging changes.

Mutagenesis methods such as those described herein can be combined with automated and high emission analysis methods to detect the activity of cloned and mutagenized polypeptides in host cells. The mutagenized .DNA molecules that code for active polypeptides can be recovered from host cells and rapidly sequenced using modern 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 described herein, a person skilled in the art can identify and / or prepare a variety of fragments of polypeptides or variants of SEQ ID NOs: 2, 4 or 6 or which retain the properties of wild-type Zalfal2 protein. For any Zalfal2 polypeptide, including variants and fusion proteins, one skilled in the art can easily generate a completely degenerate polynucleotide sequence that codes for that variant using the information described in Tables 1 and 2 above.

Production of proteins Zalfal2 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 the types of cells that can be transformed or transfected with exogenous DNA and grow in culture, and include bacteria, fungal cells and cultured higher eukaryotic cells. Eukaryotic cells are preferred, particularly cultured cells of multicellular organisms. Techniques for manipulating cloned cDNA molecules and introducing exogenous DNA into a variety of host cells are described 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, an .DNA sequence encoding a Zalfal2 polypeptide is operably linked to other genetic elements that are required for 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 can be provided on separate vectors, and replication of the exogenous DNA can be provided by integration. in the genome of the host cell. The selection of promoters, terminators, selectable markers, vectors and other elements is a routine aspect within the skill level of one skilled in the art. Many of these elements are described in the literature and are available through several commercial providers. To target a Zalfal2 polypeptide in 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 Zalfal2, or it may be derived from another secreted protein (eg, t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the Zalfal2 DNA sequence, ie, the two sequences are linked in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. The secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences can be placed anywhere in the .ADN sequence of interest (see, eg, Welch et al. al., US Patent No. 5,037,743; Holland et al. , patent of E.U.A. 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 such fusion polypeptides. The secretory signal sequence contained in the fusion polypeptides of the present invention is preferably amino-terminally fused to an additional peptide to direct the additional peptide into the secretory pathway. Such constructions have numerous applications known in the art. For example, these novel fusion constructs of secretory signal sequences can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. These fusions can be used in vivo or in vi tro to direct peptides through the secretory pathway.

Cultured mammalian cells are suitable hosts in 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. 1: 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 15:80 (1993) and vectors Virals, Miller and Rosan, BioTecniques 7: 980 (1989), Wang and Finer, Nature Med. 2: 714 (1996) The production of recombinant polypeptides in cells of cultured mammals is described, for example, by Levinson et al. , U.S. Patent No. 4,713,339, Hagn 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 Lines of COS-1 cells (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 (eg, CHO-Kl; ATCC No. CCL 61). Adequate and additional cell lines are known in the technique and they are available from public deposits such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as SV-40 or cytomegalovirus promoters. See, for example, US patent. No. 4,956,288. Other suitable promoters include those of metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major terminal promoter. Drug selection is generally used to select cells from cultured mammals 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 that are able to pass the gene of interest to their progeny. they are referred to as "stable transfectants". A selectable marker that is preferred is a gene that codes for resistance to the antibiotic neomycin. The 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 level of expression of the gene of interest, a process known as "amplification." The amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select cells that produce high levels of the products of the introduced genes. A selectable and amplifiable marker that is preferred is dihydrofolate reductase, which confers resistance to metrotrexate. Other drug resistance genes (eg, hygromycin resistance, multi-drug resistance, puromycin acetyl transferase) can also be used. Alternate markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, MHC class I, placental alkaline phosphatase, can be used to separate transfected cells from non-transfected cells by means such as separation FACS or separation technology with magnetic spheres. Other higher eukaryotic cells also be used as hosts, including plant cells, insect cells and bird cells. The use of Agrobacterium rhizogenes as a vector to express genes in plant cells has been reviewed by Sinkar et al. , J. Biosci.

(Bangalore) 11: 41 (1987). The transformation of insect cells and the production of foreign polypeptides therein is described by Guarino et al. , patent of E.U.A. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells can be infected with recombinant baculoviruses, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). The DNA encoding the Zalfal2 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 recombination of homologous DNA between wild-type AcNPV and a transfer vector containing Zalfal2 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 Zalfal2 polynucleotide operably linked to a promoter, terminator and flanking sequences of the AcNPV polyhedrin gene. See, King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, (Chapman & amp;; 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 Proctocols. Methods in Molecular Biology, (Humana Press, Totowa, NJ 1995). The natural recombination in an insect cell will result in a recombinant baculovirus that will contain Zalfal2 driven by the polyhedrin promoter. Recombinant viral supplies can be made by methods commonly used in the art. The second method for making recombinant baculovirus uses 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 equipment (Life Technologies, Rockville, MD). This system uses a pFastBacl ™ transfer vector (Life Technologies) which contains a Tn7 transposon to move the DNA encoding the Zalfal2 polypeptide in a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid". The pFastBacl ™ transfer vector uses the AcNPV polyhedrin promoter to drive expression of the gene of interest, in this case Zalfal2. However, pFastBacl ™ can be modified to a considerable degree. The polyhedrin promoter can be removed and replaced with the baculovirus basic protein promoter (also known as the Peor promoter, p6.9 or MP) which has been previously expressed in baculovirus infection, and has been shown to be suitable for expressing secreted proteins . See, Hill-Perkins, M.S. and Possee, R.D., J. Gen Virol 71: 911 (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 those transfer vector constructs, a short or long version of the basic protein promoter can be used. In addition, transfer vectors can be constructed that replace the native Zalfal2 secretory signal sequences with secretory signal sequences derived from insect proteins. For example, a secretory signal sequence of ecdysteroid glucosyltransferase (EGT), honey melitin (Invitrogen, Carisbad, CA) or gp67 baculovirus (PharMigen, San Diego, CA) can be used in constructions to replace the native Zalfal2 secretory signal sequence. In addition, the transfer vectors can include a frame fusion with .ADN coding for an epitope tag in the C- or N- term of the expressed Zalfal2 polypeptide, eg, a Glu-Glu epitope tag, Grussenmeyer, T. et al. , Proc Nati Acad Sci. 82: 7952 (1985). Using a known technique, a transfer vector containing Zalfal2 is transformed into E. coli and analyzed to find bacmids containing an interrupted lacZ gene indicating a recombinant baculovirus. The bacmidic DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, for example, Sf9 cells. A recombinant virus expressing Zalfal2 is subsequently produced. Recombinant viral supplies are made by methods commonly used in the art. The recombinant virus is used to infect host cells, typically a cell line derived from the devastating autumn worm, 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 Tri chopl usia ni (U.S. Patent No. 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 Sf9 cells; and Ex-cell 0405 ™ (JRH Biosciences, Lenexa, KS) or Express FiveO ™ (Life Technologies) for the T. ni cells. The cells are cultured from an inoculation density of about 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time a recombinant viral supply is added at a multiplicity of infection (MOI) of 0.1. at 10, very typically close to 3. Cells infected with recombinant virus typically produce the recombinant Zalfal2 polypeptide at 12-72 hours after infection and secrete it with varying efficiency in the medium. The crop is harvested normally 48 hours after infection. Centrifugation is used to separate the cells from the medium (supernatant). The supernatant containing the Zaifal2 polypeptide is filtered through micropore filters, typically a pore size of 0.45 μm. The procedures used are generally described in available laboratory manuals (King, L.A. and Possee, R.D., ibid., O'Relly, D.R., et al., Ibid., Richardson, C.D., ibid.). Subsequent purification of the Zalfal2 polypeptide from the supernatant can be achieved using methods described herein. Fungal cells, including yeast cells, can also be used in the present invention. Yeast species of particular interest in this regard include Sachar omy ees cerevisiae, Pichia pastoris and Pichia methanolica. Methods for transforming S. cerevisiae cells with exogenous DNA and for producing recombinant polypeptides thereof are described, for example, in Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al. , patent of E.U.A. No. 4,931,373; Brake, patent of E.U.A. No.4, 870, 008; Welch et al. , patent of E.U.A. No. 5,037,743 and Murray et al. , patent of E.U.A. No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (eg, leucine). A vector system that is preferred to be used in Saccharomyces cerevisiae is the POTl vector system described by Kawasaki et al. , (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in media containing glucose. Promoters and terminators suitable for use in yeast include those of glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al. , patent of E.U.A. No. 4,615,974 and Bitter, patent of E.U.A. No. 4,977,092) and alcohol dehydrogenase genes. See also patents of E.U.A. Nos. 4,990,974; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts are known in the art, including Hansenula polymorpha, Schizosaccharomyces pombe, Kl uyveromyces lactis, Kl uyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida mal tosa. See, for example, Gleeson et al. , J. Gen. Microbiol. 132: 3459 (1986) and Cregg, patent of E.U.A. No. 4,882,279. Aspergillus cells can be used according to the methods of McNight et al. , patent of E.U.A. No. 4,935,349. The methods for transforming Acremoni um chrysogenum are described by Sumino et al. , patent of E.U.A. No. 5,162,228. Methods for transforming Neurospora are described by Lambowitz, U.S. Pat. No. 4,486,533. The use of Pichia methanoli ca as a host for the production of recombinant proteins is described in the WIPO publications WO 97/17450, WO 97/17451, WO 98/02536 and WO 98/02565. DNA molecules for use in the transformation of P. methanolica will commonly be prepared as double-stranded circular plasmids, which are preferably linearized prior to transformation. For the production of polypeptides in P. methanolica, it is preferred that the promoter and the terminator in the plasmid be those of a P. methanolica gene, such as an alcohol utilization gene of P. methanolica (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 selectable marker that is preferred to be used in Pichia methanolica is an ADE2 gene from P. methanolica, 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 (AUG1 and AUG2) are removed. For the production of secreted proteins, host cells deficient in vacuolar protease genes are preferred. { PEP4 and PRB1). 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 a pulsed electric field which decays exponentially and which has a field strength of 2.5 to 4.5 kV / cm, preferably around 3.75 kV / cm, and a time constant (t) of 1 to 40 milliseconds, most preferably around 20 milliseconds. Prokaryotic host cells, including strains of the bacterium Escherichia coli, Bacillus and other genera are also useful host cells in the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned thereon are well known in the art, (see, for example, Sambrook et al., Ibid.). When a Zalfal2 polypeptide is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed to the periplasmic space by a bacterial secretion sequence. In the first case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can be refolded and dimerized after dilution to the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a saline solution of regulated pH. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by interrupting the cells (by, for example, sonification or osmotic shock) to release the contents of the periplasmic space and recover the protein, thus obviating the need of denaturation and withdrawal. The transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components necessary for the growth of the chosen host cells. A variety of suitable means, including defined means and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The media may also contain components such as growth factors or serum, as required. The growth medium will generally select cells containing the .DNA added exogenously by, for example, drug selection or deficiency in an essential nutrient that is supplemented by the selectable marker carried on the expression vector or cotransfected in the host cell. P. methanolica cells are grown in a medium comprising suitable 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 small flasks or bubbling fermenters. 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 a peptide or polypeptide comprising an epitope-bearing portion of a Zalfal2 polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. A region of a protein to which an antibody can be attached is defined as an "antigenic epitope". See, for example, Geysen, H.M. et al. , Proc. Na ti. Acad. Sci. USA 81: 3998-4002 (1984). As for the selection of peptides or polypeptides that carry an antigenic epitope (ie, which 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 emit part of a protein sequence are routinely capable of developing an antiserum that reacts with the partially imitated protein. See Sutcl, J.G. et al. Science 219: 660-666 (1983). Peptides capable of developing protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules and are not confined to immunodominant regions of intact proteins (i.e., immunogenic epitopes) or to amino or carboxyl terminals. Peptides that are extremely hydrophobic and those with six or less residues are generally not effective in inducing antibodies that bind to the mimicked protein; Longer soluble peptides, especially those containing proline residues, are usually effective. The peptides and polypeptides carrying the antigenic epitope of the invention are therefore useful for developing antibodies, including monoclonal antibodies, that specifically bind to a polypeptide of the invention. The peptides and polypeptides carrying the antigenic epitope 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 complete amino acid sequence of a polypeptide of the invention, also they 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 (ie, the sequence includes relatively hydrophilic residues, and hydrophobic residues are preferably avoided); and sequences containing proline residues are particularly preferred. All polypeptides shown in the sequence listing contain antigenic epitopes that will be used in accordance with the present invention. The present invention also provides fragments of polypeptides or peptides comprising an epitope-bearing portion of a Zalfal2 polypeptide described herein. These fragments or peptides may comprise an "immunogenic epitope," which is part of a protein that develops an antibody response when the entire protein is used as an immunogen. Immunogenic epitope-bearing peptides can be identified using standard means [see, for example, Geysen et al. , supra. See also the patent of E.U.A. No. 4,708,781 (1987) which further describes how to identify a peptide carrying an immunogenic epitope of a desired protein.

Isolation of Proteins It is preferred to purify the polypeptides of the present invention at = 80% purity, most preferably at = 90% purity, still most preferably = 95% purity, and a pharmaceutically pure state is particularly preferred, ie more than 99.9% pure with regard 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. The expressed recombinant Zalfal2 polypeptides (or chimeric Zalfal2 polypeptides) can be purified using conventional methods and means of fractionation and / or purification. Precipitation in ammonium sulfate and extraction with acid or chaotrope can be used for the fractionation of the samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse phase high performance liquid chromatography. Suitable chromatographic media include dextrans derivatives, agarose, cellulose, polyacrylamide, specialty silicas and the like. Preferred are derivatives of PEI, DE.AE, QAE, and Q. Exemplary chromatographic media include means derived 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 spheres, silica-based resins, cellulosic resins, agarose spheres, interlaced agarose spheres, polystyrene spheres, entangled polyacrylamide resins and the like which are insoluble under the conditions in which they are to be used . These supports can be modified with reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. Examples of chemical coupling reactions include activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, activation with hydrazide and carboxyl and amino derivatives for chemical reactions of coupling with carbodiimide. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for attaching receptor polypeptides to support media are well known in the art. The selection of a particular method is a routine design aspect and is determined in part by the properties of the chosen medium. See, for example, Affini and Chromatography: Principi is & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988). The polypeptides of the present invention can be isolated by exploiting their properties. For example, absorption chromatography of immobilized metal ions (IMAC) can be used to purify histidine rich proteins, including those comprising polyhistidine markers. Briefly, a gel is first charged with divalent metal ions to form a chelate, Sulkowski, Trends in Biochem. 3: 1 (1985). The proteins rich in histidine will be adsorbed to this matrix with different affinities, depending on the metal ion used, and will be eluted by competitive elution, lowering the pH or by using strong chelating agents. Other purification methods include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography. Methods in Enzymol. , Vol. 182, "Guide to Protein Purification", M. Deutscher, '(ed.) Pp. 529-539 (Acad. Press, San Diego, 1990). In additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose binding protein, an immunoglobulin domain) can be constructed to facilitate purification. further, using methods described in the art, polypeptide fusions or hybrid Zalfal2 proteins, are constructed using regions or domains of the inventive Zalfal2, 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. Those hybrids can alter the reaction kinetics, bind, restrict or expand the substrate specificity, or alter the tissue and cell 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 conjugating them chemically. Alternatively, a polynucleotide that encodes 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 conferring a biological function can be exchanged between the Zalfal2 of the present invention with the functionally equivalent domains of another family member. These domains include, but are not limited to, the secretory, conserved signal sequence, and significant domains or regions in this family. It would be expected that said fusion proteins would have a biological functional profile that was the same or similar to that of the polypeptides of the present invention or another known family of proteins, depending on the constructed fusion. In addition, said fusion proteins may exhibit other properties as described herein. Zalfal2 polypeptides or fragments thereof can also be prepared by chemical synthesis. The Zalfal2 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 may 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 Merrifield, J. Am. Chem. 85: 2149 (1963).

Assays The activity of the molecules of the present invention can be measured using a variety of assays. Of particular interest are the changes in steroidogenesis, spermatogenesis, in the testes, production of LH and FSH and GnRH in the hypothalamus. Such assays are well known in the art. The proteins of the present invention are useful for increasing sperm production. Zalfal2 can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. For example, expression host cells transfected (or co-transfected) with Zalfal2 can be embedded in an alginate environment and injected (implanted) into recipient animals. Microencapsulation of alginate-poly-L-lysine, permselective membrane encapsulation and diffusion chambers have been described as a means to trap transfected mammalian cells or primary mammalian cells. These types of non-immunogenic "encapsulations" or microenvironments allow the transfer of nutrients into the microenvironment, and also allow the diffusion of proteins and other macromolecules secreted or released by the cells captured through the environmental barrier to the recipient animal. Most importantly, the capsules or microenvironments mask and protect the encrusted and foreign cells from the immune response of the recipient animal. Said microabsorbent can extend the life of the injected cells from a few hours or days (bare cells) to several weeks (embedded cells). Alginate strands provide a simple and fast means to generate embedded cells. The materials that are required to generate the alginate strands are readily available and relatively inexpensive. Once made, the alginate strands are relatively strong and durable, both in vi tro and, based on data obtained using the strands, in vivo. The alginate strands are easily handled and the methodology can be scaled for the preparation of numerous strands. In an exemplary procedure, 3% alginate is prepared in sterile H20, and sterile filtered. Just before the preparation of the alginate strands, the alginate solution is filtered again. A cell suspension of about 50% (containing about 5 x 10 5 to about 5 x 10 7 cells / ml) is mixed with the 3% alginate solution. One milliliter of the alginate / cell suspension is extruded in a 100 mM sterile filtered CaCl 2 solution for a period of about 15 minutes, forming a "strand". The extruded strand is then transferred to a 50 mM CaCl 2 solution, and then into a 25 mM CaCl 2 solution. The strand is then rinsed with deionized water before coating the strand by incubating it in a 0.01% solution of poly-L-lysine. Finally, the strand is rinsed with lactated Ringer's solution and extracted from the solution in a syringe barrel (without the needle attached). After fixing a large-bore needle to the syringe, the strand is injected intraperitoneally into a recipient at a minimum volume of lactated Ringer's solution.

An alternative in vivo approach to testing the proteins of the present invention includes viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpes virus, vaccinia virus and adeno-associated virus (.AAV). Adenovirus, a double-stranded DNA virus, is currently the best gene transfer vector studied for the delivery of heterologous nucleic acid (for a review, see TC Becker et al., Meth. Cell Biol. 43: 161 (1994 ) and JT Douglas and DT Curiel, Science &; Medicine 4: 44 (1997). The adenovirus system offers several advantages: the adenovirus can i) receive relatively large .DNA inserts; ii) grow up to high titles; iii) infect a wide range of mammalian cell types; and iv) be used with a large number of available vectors containing different promoters. Similarly, since adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By removing portions of the adenovirus genome, larger inserts (up to 70 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 gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary). When administered intravenously to intact animals, the adenovirus is directed primarily to the liver. If the adenoviral delivery system has a deletion of the El gene, the virus can not replicate in the host cells. However, host tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. The secreted proteins will enter the highly vascularized liver circulation, and the effects on the infected animal can be determined. The adenovirus system can also for the production of proteins in vi tro. By culturing non-293 cells infected with adenovirus under conditions in which cells do not divide rapidly, cells can produce proteins for extended periods of time. For example, BHK cells are grown to confluence in cell factories and then exposed to the adenoviral vector encoding the protein of secreted interest. The cells are then cultured under serum free conditions, which allows the infected cells to survive for several weeks without significant cell division. Alternatively, 293S cells infected with adenovirus vector can be cultured in suspension culture at a relatively high cell density to produce significant amounts of protein (see, Garnier et al., Cytotechnol 15: 145 (1994).) With any protocol, A heterologous secreted and expressed protein can be repeatedly isolated from a cell culture supernatant In the production protocol of infected 293S cells, non-secreted proteins can also be obtained effectively.

Agonists and Antagonists In view of the tissue distribution observed for Zalfal2, agonists (including the natural ligand / substrate / cofactor / etc.) And antagonists have enormous potential both in vitro and in vivo applications. The compounds identified as agonists of Zalfal2 are useful to stimulate the immune system or spermatogenesis. For example, Zalfal2 and agonist compounds are useful as components of defined cell culture media, and can be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell cultures.

Antagonists Antagonists are also useful as research reagents for characterizing ligand-receptor interaction sites. Zalfal2 antagonists can also be used to sub-regulate inflammation as described in more detail below. Inhibitors of Zalfal2 activity (Zalfal2 antagonists) include anti-Zalfal2 antibodies and soluble Zalfal2 receptors, as well as other peptide and non-peptide agents (including ribozymes). Zalfal2 can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays described herein to identify compounds that inhibit the activity of Zalfal2. In addition to those assays described herein, samples can be tested to verify the inhibition of Zalfal2 activity in a variety of assays designed to measure receptor binding or stimulation / inhibition of Zalfal2-dependent cellular responses. For example, cell lines responsive to Zalfal2 can be transfected with a reporter gene construct that responds to a cellular pathway stimulated with Zalfal2. Reporter gene constructs of this type are known in the art, and will generally comprise a Zalfal2 DNA response element operably linked to a gene that codes for a protein that can be tested, such as luciferase. Response elements of .DNA may include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE), insulin response element (IRE), Nasrin et al. , Proc. Nati Acad. Sci. USA 87: 5213 (1990) and serum response elements (SRE) (Shaw et al., Cell 56: 563 (1989).) Responses to cyclic AMP are reviewed in Roestler et al., J. Biol. Chem. 263 (19); 9063 (1988) and Habener, Molec. Endocrinol., 4 (8): 1087 (1990). The hormone response elements are reviewed in Beato, Celi 56: 335 (1989). The candidate compounds, solutions, mixtures or extracts are tested to verify their ability to inhibit Zalfal2 activity in target cells as evidenced by a decrease in Zalfal2 stimulation of reporter gene expression. Tests of this type will detect compounds that directly block the binding of Zalfal2 to receptors on the surface of the cell, as well as compounds that block processes in the cell pathway after receptor-ligand binding. Alternatively, compounds or other samples may be tested to verify direct blockade of the Zalfal2 binding to the receptor using Zalfal2 labeled with a detectable label (eg, 125 I, biotin, horseradish peroxidase, FITC and the like). In assays of this type, the ability of a test sample to inhibit the binding of labeled Zalfal2 to the receptor is indicative of the inhibitory activity, which can be confirmed by secondary assays. The receptors used in binding assays can be cellular receptors or isolated and immobilized receptors. A Zalfal2 polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an Fc fragment, which contains two constant region domains and lacks the variable region. The methods for preparing such mergers are described in the U.S. Patents. Nos. 5, 155,027 and 5,567,584. These fusions are typically secreted as multimeric molecules in which the Fc portions are disulfide linked to each other and two non-Ig polypeptides are arranged in close proximity to one another. Mergers of this type can be used to purify the ligand by affinity. For use in assays, the chimeras are bound to a support via the Fc region and used in an ELISA format. A ligand binding polypeptide Zalfal2 can also be used for the purification of a ligand. The polypeptide is immobilized on a solid support, such as spheres of agarose, crosslinked agarose, glass, cellulosic resins, silica-based resins, polystyrene, crosslinked polyacrylamide, or similar materials that are stable under the conditions of use. Methods for attaching polypeptides to solid supports are well known in the art, and include amine chemistry, activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl and activation with hydrazide. 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 the ligand to bind to the receptor polypeptide. The ligand is then eluted using changes in salt concentration, chaotropic agents (guanidine hydrochloride) or pH to interrupt ligand-receptor binding.

A test system 'using a ligand binding receptor (or an antibody, a member of a complement / anti-complement pair) or a binding fragment thereof, and a commercially available biosensing instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ). Such a receptor, antibody, member of a complement / anti-complement pair or fragment is immobilized on the surface of a receptor fragment. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229 (1991) and Cunningham and Wells, J. Mol. Biol. 234: 554 (1993). A receptor, antibody, member or fragment is covalently linked, using amine or sulfhydryl chemistry, to dextran fibers that are attached to a gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope or opposite member of the complement / anticomplement pair is present in the sample, it will bind to the receptor, antibody or immobilized member, respectively, causing a change in the refractive index of the medium, which is detected as a change in the plasmonic resonance of the surface of the gold film. This system allows the determination of active and inactive speeds, from which the binding affinity can be calculated, and the determination of the stoichiometry of the junction. Ligand binding receptor polypeptides can also be used in other assay systems known in the art. Said systems include the Scatchard analysis for the determination of binding affinity, Scatchard, .Ann. NY Acad. Sci. 51: 660 (1949) and calorimetric tests, Cunningham et al. , Science 253: 545 (1991); Cunningham et al. , Science 245: 821 (1991). Zalfal2 polypeptides can also be used to prepare antibodies that specifically bind to Zalfal2 epitopes, peptides or polypeptides. The Zalfal2 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and develop an immune response. Suitable antigens would be the Zalfal2 polypeptides encoded by SEQ ID NOs. 2, 4, 6, 8, 10, 18, 20, 35-30 and 35-44. The antibodies generated from this immune response can be isolated 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, Coligan, et al. , (eds), National Institute of Health, (John Wiley and Sons, Inc., 1995); Sambrook et al. , Molecular Cloning: A Laboratory Manual, second edition (Cold Spring Harbor, NY, 1989); and Hurrel, 'J. G. R., ed., Monoclonal Hybridoma Antibodies: Techniques and Appli cations (CRC Press, Inc., Boca Raton, FL, 1982). As will be apparent to one skilled in the art, polyclonal antibodies can be generated by inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats with a Zalfal2 polypeptide or a fragment thereof. . The immunogenicity of a Zalfal2 polypeptide can be increased by the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as Zalfal2 fusions or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", said portion may be bound or suitably linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum 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 ') 2r 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. Non-human antibodies can be humanized by grafting non-human CDRs into human regions of structure and constants, or by incorporating whole non-human variable domains (optionally "encapsulating" them with a human-like surface by replacing exposed residues, where the result is an antibody "disguised"). In some cases, humanized antibodies may retain non-human residues within the human variable region structure domains to increase appropriate binding characteristics. Through humanizing antibodies, the biological half-life can be increased, and the potential for adverse immune reactions after administration to humans is reduced. Alternative techniques for generating or screening antibodies useful herein include in vitro exposure of lymphocytes to Zalfal2 protein or peptide and selection of antibody display libraries in phage vectors or the like (eg, by using peptide or Zalfal2 protein immobilized or labeled). Genes coding for polypeptides having potential Zalfal2 polypeptide binding domains can be obtained by screening libraries of random peptides displayed on phages (phage display) or on bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as by random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to select polypeptides 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 selecting such random peptide display libraries are known in the art (Ladner et al., U.S. Patent No. 5,223,409; Ladner et al. , patent of E.U.A. No. 4,946,778; Ladner et al. , patent of E.U.A. No. 5,403,484 and Ladner et al. , patent of E.U.A. No. 5,571,698) and random peptide display libraries, and kits for selecting such libraries are commercially available, for example 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 selected using the Zalfal2 sequences described herein to identify proteins that bind Zalfal2. These "binding proteins" that interact with Zalfal2 polypeptides can be used to label cells; for isolating homologous polypeptides by affinity purification; they can be conjugated directly or indirectly to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as to select libraries of expression and neutralizing activity. The binding proteins can also be used for diagnostic tests to determine circulating levels of polypeptides; to detect or quantify soluble polypeptides as markers of underlying pathology or disease. These binding proteins can also act as "antagonists" of Zalfal2 to block Zalfal2 binding and signal transduction in vi tro and in vivo.

Antibodies are determined to be specific binding if: 1) they exhibit a threshold level of binding activity and 2) they do not cross-react significantly with related polypeptide molecules. First, antibodies of the present specifically bind if they bind to a Zalfal2 polypeptide, peptide or epitope with a binding affinity (Ka) of 106 M_1 or more, preferably 107 M ~ 1 or more, most preferably 108 M 1 or more , and more preferably 109 M "1 or more The binding affinity of an antibody can be readily determined by one skilled in the art, for example, by Scatchard analysis .. Second, antibodies are determined to be specific binding if they do not react in Significantly cross-linked with related polypeptides Antibodies do not cross-react significantly with related polypeptide molecules, for example, if they detect Zalfal2 but not known related polypeptides using standard Western blot analysis (Ausubel et al., ibid.) Examples of polypeptides related ones that are known are orthologs, proteins of the same species that are members of a protein family (for example, IL-16), Zalfal2 and Zalfal2 non-human polypeptides. Further, . the antibodies can be "screened against" known related polypeptides to isolate a population that specifically binds to the polypeptides of the invention. For example, antibodies against Zalfal2 are adsorbed to related polypeptides adhered to an insoluble matrix; Zalfal2 specific antibodies will flow through the matrix under the proper pH regulation conditions. Such selection allows the isolation of polyclonal and monoclonal antibodies that do not cross-react to closely related polypeptides, Antibodies: A Laboratory Manual, Harlow and Lane (eds.) (Cold Spring Harbor Laboratory Press, 1988); Current Proctocols in Immunology, Coligan, et al. (eds), National Institutes of Health (John Wiley and Sons, Inc., 1995). The selection 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: Principies and Practice, Goding, J.W. (eds.), (Academic Press Ltd., 1996); Benjamín et al. , Ann. Rev. Inmunol. 2: 67-101 (1984). A variety of assays known to those skilled in the art can be used to detect antibodies that specifically bind Zalfal2 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, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot, inhibition or competition assay, and sandwich assay. In addition, the antibodies can be selected to bind wild-type versus mutant Zalfal2 protein or polypeptide. Antibodies to Zalfal2 can be used to label cells that express Zalfal2; to isolate Zalfal2 by affinity purification; for diagnostic assays to determine circulating levels of Zalfal2 polypeptides; to detect or quantify soluble Zalfal2 as a marker of underlying pathology or disease; in analytical methods using FACS; to analyze expression libraries; to generate anti-idiotypic antibodies and as neutralizing antibodies or as antagonists to block Zalfal2 in vi tro and in vivo. Suitable labels or direct markers include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect labels or markers may include the use of biotin-avidin or other complement / anticomplement pairs as intermediates. The antibodies herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates can be used for diagnostic or therapeutic in vivo applications. Moreover, antibodies to Zalfal2 or fragments thereof can be used in vi tro to detect denatured Zalfal2 or fragments thereof in assays, eg, Western blots or other assays known in the art.

Bioactive conjugates The antibodies or polypeptides herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates can be used for diagnostic or therapeutic applications in vivo. For example, the polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anticomplementary molecule (receptor or antigen, respectively, for example). More specifically, the Zalfal2- polypeptides or anti-Zalfal2 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 anticomplementary molecule. Suitable detectable molecules can be linked directly or indirectly to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. Suitable cytotoxic molecules can bind directly or indirectly to the polypeptide or antibody, and include bacterial or plant toxins (e.g., 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 bound to the polypeptide or antibody, or indirectly linked through means of a chelating portion, for example). The polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect binding of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary / anticomplementary pair, wherein the other member is attached to the polypeptide or antibody portion. For these purposes, biotin / streptavidin is a complementary / anticomplementary pair ej emplar. In another embodiment, polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for the inhibition or ablation of selected cells or tissues (e.g., to treat cancer cells or tissues). Alternatively, if the polypeptide has several functional domains (i.e., a de-activation domain or a ligand binding domain, plus an address domain), a fusion protein that includes only the address domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a molecule complementary to a type of cell or tissue of interest. In cases where the domain-only fusion protein includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. Said domain-complementary molecule fusion proteins then represent a generic targeting vehicle for the cell / tissue specific delivery of conjugates of cytotoxic / detectable anti-complementary molecules. In another embodiment, the Zalfal2-cytoxin fusion proteins or antibody-cytokine fusion proteins can be used to increase the in vivo clearance of target tissues (e.g., blood and bone marrow cancers), if the Zalfal2 polypeptide or anti-Zalfal2 antibody it targets the hyperproliferative blood cell or bone marrow. See, generally, Hornick et al. , Blood 89: 4431 (1997). The fusion proteins described make it possible to direct a cytokine to a desired site of action, thereby providing a high local concentration of cytokine. Zalfal2 polypeptides or suitable anti-Zalfal2 antibodies are directed to an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediates improved target cell lysis by effector cells. Cytoxins suitable for this purpose include interleukin 2 and granulocyte-macrophage colony stimulation factor (GM-CSF), for example. In another modality more, if the Zalfal2 polypeptide or anti-Zalfal2 antibody is targeted to vascular cells or tissues, said polypeptide or antibody can be conjugated with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. This therapeutic approach presents less danger to the medical personnel who administer the radioactive therapy. For example, tapes impregnated with iridium-192 placed in vessels dilated by restenosis (with stent) of patients until the required dose of radiation was delivered showed decreased tissue growth in the vessel and a larger luminal diameter than the control group, which received placebo tapes. In addition, revascularization and artificial graft thrombosis for restenosis (stent) were significantly lower in the treatment group. Similar results are predicted with the direction of a bioactive conjugate containing a radionuclide, as described herein. The bioactive polypeptide or antibody conjugates described herein may be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the desired site of action.

Uses of the polynucleotide / polypeptide The molecules of the present invention can be used to identify and isolate receptors involved in spermatogenesis, steroidogenesis, testicular differentiation and regulatory control of the hypothalamic-pituitary-gonadal axis or receptors of the immune system. For example, the proteins and peptides of the present invention can be immobilized on a column and run membrane preparations on the column, Immobilized Affini and Ligand Techniques, Hermanson et al. , eds., pp. 195-202 (Academic Press, San Diego, CA, 1992). Proteins and peptides can also be radiolabelled, Methods in Enzymol. , vol. 182. "Guide to Protein Purification", M. Deutscher, ed., Pp 721-737 (Acad. Press, San Diego, 1990) or marked by photoaffinity, Brunner et al. , Ann. Rev. Biochem. 62: 483-514 (1993) and Fedan et al. , Biochem. Pharmacol. 33: 1161 (1984) and the specific cell surface proteins can be identified. The molecules of the present invention will be useful for testing disorders of the reproductive system and immune systems.

Gene therapy The polynucleotides encoding Zalfal2 polypeptides are useful in gene therapy applications where it is desired to increase or inhibit the activity of Zalfal2. If a mammal has a mutated or absent Zalfal2 gene, the Zalfal2 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a Zalfal2 polypeptide is introduced in vivo into a viral vector. Those vectors include an attenuated or defective .DNA virus, such as, but not limited to, herpes simplex virus (HSV), papilloma virus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV) and Similar. Defective viruses are preferred, which completely or almost completely lack viral genes. A defective virus is not infectious after its introduction into a cell. The use of defective viral vectors allows administration to cells in a specific and localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, defective herpes simplex virus 1 (HSV1), Kaplitt et al. , Molec. Cell. Neurosci. 2: 320 (1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. , J. Clin.

Invest. 90: 626 (1992); and a defective adeno-associated virus vector, Samulski et al., J. Virol. 61: 3096 (1987); Samulski et al., J. Virol. 63: 3822 (1989). In another embodiment, a Zalfal2 gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Pat. 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. 62: 1120 (1988); Temin et al., U.S. Patent. No. 5,124,263; International Patent Publication No. WO 95/07358, published March 26, 1995 by Dougherty et al., and Kuo et al., Blood 82: 845 (1993). Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for the in vivo transfection of a gene encoding a marker, Felgner et al., Proc. Nati Acad. Sci. USA 84: 7413 (1987); Mackey et al., Proc. Nati Acad. Sci. USA 85: 8021 (1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. The molecular direction of liposomes to specific cells represents an area of benefit. More particularly, directing the transfection to particular cells represents an area of benefit. For example, directing transfection to particular cell types would be particularly suitable in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney and brain. The lipids can be copulated chemically to other molecules for the purpose of direction. 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; introduce the vector as a naked .DNA plasmid; and then reimplant the transformed cells in the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a Gene gun or use of a DNA vector transporter. See, for example, Wu et al. , J. Biol. . Chem. 267: 963 (1992); Wu et al. , J. Biol. Chem. 263: 14621-4, 1988.

Antisense methodology can be used to inhibit transcription of the Zalfal2 gene, such as to inhibit. cell proliferation in vivo. Polynucleotides that are complementary to a segment of a Z-alfal2-encoding polynucleotide (eg, a polynucleotide as described in SEQ ID NO: 1) are designed to bind to .alpha.RNA encoding Zalfal2 and to inhibit the translation of that .ARNm .. Said antisense polynucleotides are used to inhibit the expression of Zalfal2 polypeptide coding genes in cell cultures or in a subject. The present invention also provides reagents that will be useful in diagnostic applications. For example, the Zalfal2 gene, a probe comprising Zalfal2 DNA or RNA or a substance thereof, can be used to determine whether the Zalfal2 gene is present on chromosome 22ql3.1 or whether a mutation has occurred. Chromosomal aberrations detectable at the locus of the Zalfal2 gene include, but are not limited to, aneuploidy, changes in the number of gene copies, insertions, deletions, changes and rearrangements at the restriction site. These aberrations can be detected using polynucleotides of the present invention employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other assay techniques. genetic junction known in the art (Sambrook et al., ibid., Ausubel et al., ibid., Marian, Chest 108: 255 (1995) .Transgenic mice, designed to express the Zalfal2 gene, and mice exhibiting a complete absence of the function of the Zalfal2 gene, known as "mice with suppressed gene", Snouwaert et al., Science 257: 1083 (1992), can also be generated, Loweil et al., Nature 366: 140-42 (1993). they can be used to study the Zalfal2 gene and the protein encoded in this way in a single system.

Chromosomal localization Zalfal2 has been mapped to chromosome 22ql3.1. Hybrid radiation mapping is a somatic cell genetic technique developed to construct contiguous and high resolution maps of mammalian chromosomes (Cox et al., Science 250: 245 (1990).) Partial or complete knowledge of the sequence of a Gene allows the design of PCR primers suitable for use with chromosomal panels of hybrid radiation hybrids. Hybrid radiation mapping panels covering the entire human genome, such as the G3 RH Stanford panel and the GeneBridge 4 RH panel, are commercially available. (Research Genetics, Inc., Huntsville, AL) These panels enable rapid PCR-based chromosomal localizations and gene sorting, site-labeled sequences (STSs), and other non-polymorphic and polymorphic markers in a region of interest. This includes directly establishing proportional physical distances between newly discovered genes of interest and previously mapped markers 2. Accurate knowledge of the position of a gene can be useful for a number of purposes, including: 1) determining whether a sequence is part of an existing "contig" and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA es; 2) provide a possible candidate gene for an inheritable disease that shows binding to the same chromosomal region and 3) cross-reference model organisms, such as mice, which can help determine what function a particular gene might have. Scripted sites (STSs) can also be used independently for chromosomal localization. An STS is a sequence of .DNA that is unique in the human genome and can be used as a reference point for a chromosome or region of a particular 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 rely solely on DNA sequences, they can be fully described in an electronic database, for example, Datábase of Seqúense Tagged Sites (DbSTS), GenBank, (National Center for Biological Information, National Institute of Health, Bethesda, MD, http: /www.nebí.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained in these short genomic STS STS sequences. For pharmaceutical use, the proteins of the present invention are formulated for parenteral administration, particularly intravenous or subcutaneous, according to conventional methods. Intravenous administration will be by bolus injection or infusion during a typical period of one to several hours. In general, the pharmaceutical formulations will include a Zalfal2 protein in combination with a pharmaceutically acceptable carrier, such as saline, pH regulated saline, 5% dextrose in water or the like. The formulations may also include one or more excipients, preservatives, solubilizers, pH regulating agents, albumin to prevent the loss of proteins on flask surfaces, etc. Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., (Mack Publishing Co., Easton, PA, 19th ed., 1985). The 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 doctor according to accepted standards, taking into account the nature and severity of the condition that will be treated, patient characteristics, etc. The determination of the dose is within the level of ordinary skill in the art. The proteins can be administered for acute treatment, for a week or less, commonly for a period of one to three days, or they can be used in chronic treatment, for several months or years. The administration of the protein can be subcutaneous, intraperitoneal or rectal depending on the disease to be treated.

Expression and use of tissues Zalfal2 represents a novel polypeptide with a leader sequence of putative signal peptides and alpha helical structure. Several putative isoforms have been identified. Therefore, this gene can code for a secreted polypeptide with secondary structure indicating that it is a member of the family of cytokines of the group of four helices. Alternatively, this polypeptide may have other activities associated with other biological functions including: enzymatic activity, association with the cell membrane or function as a carrier protein. The Northern blot analysis detects transcripts for Zalfal2 in spleen, thymus, testes, small intestine, colon, PBL, stomach, lymph nodes, trachea and bone marrow. Many of these organs have important immune function or contain cells that play a role in the immune system. Zalfal2 is also expressed in peripheral T cells enriched with CD4 + and to a lesser extent in CD8 + T cells. There is a very weak expression in CD19 + B cells suggesting that Zalfal2 has expression in T-lymphoid lineage cells and very little if any in B-lineage lymphoid cells. There is high expression of Zalfal2 in RNA derived from a mixed lymphocyte reaction 7-day human This suggests that the expression of Zalfal2 is regulated and increases after the activation of T cells.

Use of Zalfal2 Zalfal2 can be administered to an immunocompromised mammal, preferably a human, such as cancer patients who have undergone chemotherapy, AIDS patients and the elderly. This will stimulate your immune systems. Zalfal2 can also be used as a vaccine adjuvant to be administered before, with or after the administration of a vaccine. Zalfal2 can also be used to stimulate the immune system to attack tumors.

Use of Zalfal2 antagonists An antagonist to Zalfal2, such as an antibody, soluble receptor or small molecule antagonist can be administered to a mammal, preferably a human, to alleviate an inflammatory response. Anatagonists, such as antibodies, to Zalfal2 can be used to treat patients having related inflammatory diseases such as arteriosclerotic heart disease [see Paulsson, G. et al. , Arterioscler Thromb. Vasc. Bi ol. , 20: 10-11 (2000)], inflammatory bowel disease, Crohn's disease, rheumatoid arthritis and pancreatitis. The invention is further illustrated by means of the following non-limiting examples.

EXAMPLE 1 Cloning of Zalfal2 Zalfal2 was discovered using the Sequence Tag Expressed of SEQ ID NO: 7 as a probe in a spleen .DNA library. The library was constructed using 1 microgram (μg) of polyA RNA isolated from the spleen tissue of a 2-year-old Hispanic male who died of cerebral anoxia. Syntheses of .AD? C were initiated using a? Otl-oligo (dT) primer. Double-stranded AD? C was shaved, ligated to EcoRI adapters, digested with? Otl, selected by size and cloned into the? Otl and EcoRI sites of a vector. The sequenced clone resulted in AD? full length and polypeptide sequences of SEQ ID Os: 1 and 2.

The EST of SEQ ID NO: 17 resulted in the discovery of a second clone. The clone was sequenced and found to have an unconnected intron. Oligonucleotide primers SEQ ID NOS: 11 and 12 were designed to amplify the full length sequence without the introns sequence. PCR was carried out using MARATON READY® .ADNc lymphatic node cDNA (Cloteach, Palo Alto, CA) as a template. An 823 bp fragment was cut and gel purified using the QIAQUICK® gel extraction equipment (Qiagen, Santa Clarita, CA). The excised PCR fragment was sequenced and did not contain the introns sequence. The next step was to see if the clone of SEQ ID NO: 17 had another initiation codon towards the 5 'end. A RACE® reaction using lymph node cDNA was carried out to obtain a 5 'sequence. Oligonucleotide primers SEQ ID NOs: 13 and 14 were designed for 5 'RACE® and nested RACE® reactions. The RACE® reactions revealed an additional initiation codon towards the 5 'end. The actual full length was obtained by PCR using primers SEQ ID NOs: 15 and 16 flanking the coding sequence. Six bands were amplified and each was gel purified using QIAQUICK® gel extraction equipment and subcloned into plasmid pCR2.1TOPO vector using the TOPO TA® cloning kit (Invitrogen, Carisbad, CA). The clones were 'sequenced and yielded the full length sequences of SEQ ID NOs: 3, 4, 5 and 6. In this way, three variants were discovered, namely SEQ ID NO: 1 and 2; SEQ ID NO: 3 and 4, and SEQ ID NOs: 5 and 6.

EXAMPLE 2 Northern blot analysis of Zalfa.12 A Northern blot analysis was carried out by standard techniques using the polynucleotides of SEQ ID NOs: 1 and 3. The results indicate that Zalfal2 is expressed in lymph nodes, activated mixed lymphocytes, spleen, thymus, testis, small intestine, aortic endothelial cells human, smooth muscle, kidney, mast cells, eosinophils, tonsils, pancreas, colon, peripheral blood lymphocytes (PBL), stomach, trachea, T cells including CD4 + and CD8 + cells and bone marrow. Zalfal2 is also expressed by the following cell lines: HL60 (ATCC 45500), a cell line of acute promyelocytic leukemia; Jurkat cells (ATCC TIB-152), a T-cell lymphocyte of acute T-cell leukemia; MOLT-4 cells (ATCC CRL-1582) a T lymphoblast of acute lymphoblastic leukemia, and HuT 78 cells (ATCC TIB-161), a cutaneous T lymphocyte of a lymphoma. From these data, it can be concluded that Zalfal2 is a cytokine involved in the inflammation cascade. Zalfal2 antagonists can be used to alleviate inflammation related to a number of diseases such as arteriosclerotic heart disease, cardiovascular disease, rheumatoid arthritis, inflammatory bowel disease and Crohn's disease.

EXAMPLE 3 Anti-peptide antibodies Zalfal2 Polyclonal anti-peptide antibodies were prepared by immunizing two female New Zealand albino mice with the peptide huzalfa2XI-AQQHKGSLQKDPLLSQACVGCLEALLDYLDAR (SEQ ID NO: 41) or the peptide of SEQ ID NO: 4 PLPATKDTVLAPLRMSQVRSLVIGLQNLLVC (SEQ ID NO: 42) or the peptide of SEQ ID NO: 4 CEGLPPSTSSQPPLQDMLCLGGVASLSHIRN (SEQ ID NO: 43) or the peptide of SEQ ID NO: 4 FMRYRSSSVLSHEEC (SEQ ID NO: 44). Peptides were synthesized using an Applied Biosystems Model 431A peptide synthesizer (Applied Biosystems, Inc., Foster City, CA); according to the manufacturer's instructions. The peptides were then conjugated to the keyhole limpet hemocyanin carrier protein (KLH) activated with maleimide via cysteine residues according to the manufacturer's instructions. (Pierce, Rockford, IL). Each rabbit was given an initial intraperitoneal (IP) injection of 200 μg of the conjugate peptide in complete Freund's adjuvant (Pierce, Rockford, IL) followed by booster PI injections of 100 μg of peptide penguins in incomplete Freund's adjuvant every three weeks . Seven to ten days after the administration of the third booster injection, the animals were bled and the serum was collected. The rabbits were reinforced and then bled every three weeks. Rabbit sera specific for Zalfal2 peptides were characterized by an ELISA titer verification using 1 μg / ml of the peptide used to make the antibody as an antibody target. The two rabbit sera for the peptide of SEQ ID NO: 41 have their specific peptide titer at a dilution of 1: 5E5 (1: 100000).

The sera of the two rabbits to the peptide of SEQ ID NO: 42 had their specific peptide titer at a dilution of 1: 5E4 (1: 10,000). The two rabbit sera to the peptide of SEQ ID NO: 43 had a titer to their specific peptide at a dilution of 1: 5E4. Sera from the two rabbits to the peptide of SEQ ID NO: 44 had a titer to their specific peptide at a dilution of 1: 5E5. From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is limited except by the accompanying claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

LIST OF SEQUENCES < 110 > ZymoGeneti s. Inc. < 120 > Alpha helical protein - 12 mammals < 130 > 99-01 '< 150 > 09 / 232,427 < 151 > 1999-01-15 < 160 > 44 < 170 > .FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 1117. < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (354) ... (959) < 400 > 1 catcagacca cacccagcag tcagaaaaga ggtgcagggg cccgggctgg gacagtgaag 60 agtgctgggc agtctgtggt cctctgtatc tcaacttttt catcttaaaa aaacaaatag 120 ggttgtgtgt gtggctggtg gtcataaggt cctttctggc tctaataacc tgagcttctg 180 ttatgaagct gggaccctta gagcctcagg atgatcctct gtttgtttgt gaagccccaa 240 tcaggtgcta agcaccatag tggcacttag ctgaagctcc tctgtaactc ctgtgggccc 300 tgccttgccc acccccgaca gctgctgcag tgctcctgag cagcacaggc ctg atg 356 Met 1 gag ctt ctg gag aag atg ctg gcc etc acc ttg gca aag gca gat tet 404 Glu Leu Leu Glu Lys Met Leu Ala Leu Thr Leu Ala Lys Wing Asp Ser 5 10 15 ecc agg act gca etc etc tgc tet gcc tgg ctg etc act gcc tec ttc 452 Pro Arg Thr Wing Leu Leu Cys Ser Wing Trp Leu Leu Thr Wing Being Phe 20 - 25 30 tet gcc cag cag falls aag ggc agt ttg cag aag gac ect cta ttg tec 500 Ser Ala Gln Gln His Lys Gly Ser Leu Gln Lys Asp Pro Leu Leu Ser 35 40 45 cag gcc tgt gtt ggc tgc ctg gag gcc ttg ctt gac tac cg gat gcc 548 Gln Ala Cys Val Gly Cys Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala 50 55 60 65 cgg age cea gac att gct etc falls gtg gcc tec cag ect tgg aat cgg 596 Arg Ser Pro Asp He 'Ala Leu His Val Ala Ser Gln Pro Trp Asn Arg 70 75 80 ttt ttg ctg ttt acc etc. ttg gat gct gag aat tec ttc etc aga 644 Phe Leu Leu Phe Thr Leu Leu Asp Wing Gly Glu Asn Ser Phe Leu Arg 85 90 95 cct. gag 'att ttg agg etc atg acc ctg ttt atg cgg tac cgg agt age 692 Pro Glu He Leu Arg Leu Met Thr Leu Phe Met Arg Tyr Arg Ser Ser 100 105 110 agt gtc etc tet cat gaa gag gtg ggt gat gtt ctg ca ggt gtg gct 740 Ser Val Leu Ser His Glu Glu Val Gly Asp Val Leu Gln Gly Val Wing 115 120 125 ttg gct gac ctg tet acc etc tcg aac acc here etc cag gcc ctg cat 788 Leu Wing Asp Leu Ser Thr Leu Ser Asn Thr Thr Leu Gln Ala Leu His 130 135 140 145 ggc ttc ttc cag cag etc cag age atg gga falls ctg gct gac falls age 836 Gly Phe Gl Gln Le Gln Met Met Gly His Leu Ala Asp His Ser 150 155 160 atg gcc cag acc ctg cag gcc tec ttg gag ggc ctt ecc cct age acc 884 Met Wing Gln Thr Leu Gln Wing Being Leu Glu Gly Leu Pro Pro Being Thr 165 170 175 tec tea ggc cag cea ecc ctg cag gac atg etc tgc ctg gga ggg gtg 932 Being Gly Gln Pro Pro Leu Gln Asp Met Leu Cys Leu Gly Gly Val 180 185 190 gct gta tec ctg tec falls at aga aac tgatcctcag gacttgaagg 979 Wing Val Ser Leu Ser His He Arg Asn 195 200 eccagaagtg gagagagaat g agacctgga gacaaagggc ataattgttg gggaaatgga 1039 tgacagetga agetatteat atggagccat atactctatt gttgaaatag aataaggaaa 1099 taaaatgata cactcaca 1117 < 210 > 2 < 211 > 202 < 212 > PRT < 213 > Homo sapiens < 400 > 2 Met Glu Leu Leu Glu Lys Met Leu Ala Leu Thr Leu Ala Lys Ala Asp 1 5 10 15 Ser Pro Arg Thr Ala Leu Leu Cys Ser Ala Trp Leu Leu Thr Ala Ser 20 25 30 Phe Ser Ala Gln Gln His Lys Gly Ser Leu Gln Lys Asp Pro Leu Leu 35 40 45 Ser Gln Ala Cys Val Gly Cys Leu Glu Ala Leu Leu Asp Tyr Leu Asp 50 55 60 Wing Arg Ser Pro Asp He Wing Leu His Val Wing Being Gln Pro Trp Asn 65 70 75 80 Arg Phe Leu Leu Phe Thr Leu Leu Asp Wing Gly Glu Asn Ser Phe Leu 85 90 95 Arg Pro Glu He Leu Arg Leu Met Thr Leu Phe Met Arg Tyr Arg Ser 100 105 '110 Ser Ser Val Leu Ser His Glu Glu Val Gly Asp Val Leu Gln Gly Val 115 120 125 Wing Leu Wing Asp Leu Ser Thr Leu Ser Asn Thr Thu Leu Gln Wing Leu 130 135 140 His Gly Phe Phe Gln Gln Leu Gln Ser Met Gly His Leu Wing Asp His 145 150 155 160 Ser Met Ala Gln Thr Leu Gln Ala Ser Leu Glu Gly Leu Pro Pro Ser 165 170 175 Thr Ser Gly Gln Pro Pro Leu Gln Asp Met Leu Cys Leu Gly Gly 180 185 190 Val Ala Val Ser Leu Ser His He Arg Asn 195 200 <; 210 > 3 < 211 > 899 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (31) ... (894) 400 > 3 tgtgcagtgc tcctgagcag cacaggcctg atg gag ctt cg gag aag atg ctg 54 Met Glu Leu Leu Glu Lys Met Leu 1 5 gcc etc acc ttg gca aag gca gat tet ecc agg act gca etc etc tgc 102 Ala Leu Thr Leu Ala Lys Ala Asp Ser Pro Arg Thr Ala Leu Leu Cys 10 15 20 tet gcc tgg ctg etc 'act gcc tec ttc tet gcc cag cag cae aag ggc 150 Being Wing Trp Leu Leu Thr Wing Being Phe Being Wing Gln Gln His Lys Gly 25 30 35 40 agt ttg cag gtt falls cag here etc tet gtg gaa atg gac cata gta ttg 198, Ser Leu Gln Val His Gln Thr Leu Ser Val Glu Met Asp Gln Val Leu 45 50 55 aag gct etc age ttt cea aag aaa aag gct gca cta etc tea gct gcc 246 Lys Ala Leu Ser Phe Pro Lys Lys Ala Ala Ala Leu Leu Ser Ala Ala 60 65 70 ate tta tgc ttc ctg cgg here gcc ctg cga ca g age ttt tec tet gcc 294 He Leu Cys Phe Leu Arg Thr Wing Leu Arg Gln Ser Phe Ser Wing 75 80 85 ctg gta gcc ctg gtg ecc tea ggg gcc cag cea ctg cea gcc acc aag 342 Leu Val Ala Leu Val Pro Ser Gly Wing Gln Pro Leu Pro Wing Thr Lys 90 95 100 gac act gtc cta gct cea ctg cga atg tcg ca gtc cgg tec ctg gtc 390 Asp Thr Val Leu Ala Pro Leu Arg Met Ser Gln Val Arg Ser Leu Val 105 110 115 120 att ggg ctg cag aac etc ctg gtg cag aag gac cct cta ttg tec cag 438 He Gly Leu Gln Asn Leu Leu Val Gln Lys Asp Pro Leu Leu Ser Gln 125 130 135 gcc tgt gtt ggc tgc ctg gag gcc ttg ctt gac tac ctg gat gcc cgg 486 Wing Cys Val Gly Cys Leu Glu Wing Leu Leu Asp Tyr Leu Asp Wing Arg 140 145 150 age cea gac att gct etc falls gtg gcc tec cag cct tgg aat cgg ttt 534 Ser Pro Asp He Ala Leu His Val Ala Ser Gln Pro Trp Asn Arg Phe 155 160 165 ttg ctg ttt acc etc. ttg gat gct gag aat tec ttc etc aga cct 582 Leu Leu Phe Thr Leu Leu Asp Wing Gly Glu Asn Ser Phe Leu Arg Pro 170 175 180 gag att ttg agg etc atg acc ctg ttt atg cgg tac cgg agt ge agt 630 Glu He Leu Arg Leu Met Thr Leu Phe Met Arg Tyr Arg Ser Being Ser 185 190 195 200 gtc etc tet cat gaa gag gtg ggt gat gtt ctg ca ggt gtg gtg gt ttg 678 Val Leu Ser His Glu Glu Val Gly Asp Val Leu Gln Gly Val Ala Leu 205 210 215 gct gac ctg tet acc etc. tcg aac acc here etc cag gcc ctg cat ggc 726 Wing Asp Leu Ser Thr Leu Ser Asn Thr Thr Leu Gln Ala Leu His Gly 220 225 230 ttc ttc cag cag etc cag age atg gga falls ctg gct gac falls age atg 774 phe phe Gln Gln Leu Gln Ser Met Gly His Leu Ala Asp His Ser Met 235 240 245 gcc cag acc ctg cag gcc tec ttg g ag ggc ctt ecc cct age acc tec 822 Wing Gln Thr Leu Gln Wing Ser Leu Glu Gly Leu Pro Pro Ser Thr Ser 250 255 260 tea ggc cag cea ecc ctg cag gac atg etc tgc ctg gga ggg gtg gct 870 Ser Gly Gln Pro Pro Leu Gln Asp Met Leu Cys Leu Gly Gly Val Ala 265 270 275 280 gta tec ctg tec falls aga aac tgatc • 899 Val Ser Leu Ser His He Arg Asn 285 < 210 > 4 < 211 > 288 < 212 > PRT < 213 > Homo sapiens < 400 > 4 Met Glu Leu Leu Glu Lys Met Leu Wing Leu Thr Leu Wing Lys Wing Asp 1 5 10 15 Ser Pro Arg Thr Wing Leu Leu Cys Ser Wing Trp Leu 'Leu Thr Wing Ser 20 25 30 Phe Ser Wing Gln Gln His Lys Gly Ser Leu Gln Val His Gln Thr Leu 35 40 v 45 Ser Val Glu Met Asp Gln Val Leu Lys Ala Leu Ser Phe Pro Lys Lys 50 55 60 Lys Ala Ala Leu Leu Ser Ala Ala He Leu Cys Phe Leu Arg Thr Ala 65 70 75 80 Leu Arg Gln Ser Phe Ser Be Ala Leu Val Ala Leu Val Pro Ser Gly 85 90 95 Wing Gln Pro Leu Pro Wing Thr Lys Asp Thr Val Leu Wing Pro Leu Arg 100 105 110 Met Ser Gln Val Arg Ser Leu Val He Gly Leu Gln Asn Leu Leu Val 115 120 125 Gln Lys Asp Pro Leu Leu Ser Gln Wing Cys Val Gly Cys Leu Glu Wing 130 135 140 Leu Leu Asp Tyr Leu Asp Wing Arg Ser Pro Asp He Wing Leu His Val 145 150 155 160 Wing Ser Gln Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu Leu Asp Wing 165 170 175 Gly Glu Asn Ser Phe Leu Arg Pro Glu He Leu Arg Leu Met Thr Leu 180 185 190 Phe Met Arg Tyr Arg Ser Ser Val Leu Ser His Glu Glu Val Gly 195 200 2 05 Asp Val Leu Gln Gly Val Wing Leu Wing Asp Leu Ser Thr Leu Ser Asn 210 215 220 Thr Thr Leu Gln Wing Leu His Gly Phe Gl Gln Gln Ser Met 225 230 235 240 Gly His Leu Wing Asp His Ser Met Wing Gln Thr Leu Gln Ala Ser Leu 245 250 255 Glu Gly Leu Pro Pro Ser Thr Ser Gly Gln Pro Pro Leu Gln Asp 260 265 270 Met Leu Cys Leu Gly Val Val Val Ser Leu Ser His He Arg Asn 275 280 285 < 210 > 5 < 211 > 510 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (32) ... (505) < 400 > 5 tgctgcagtg ctcctgagca gcacaggcct g atg gag ctt cg gag aag atg 52 Met Glu Leu Glu Lys Met 1 5 ctg gcc etc acc ttg gca aag gca gat tet ecc agg act gca etc etc 100 Leu Ala Leu Thr Leu Ala Lys Ala Asp Ser Pro Arg Thr Ala Leu Leu 10 15 20 tgc tet gcc tgg ctg etc act gcc tec ttc tet gcc cag cag falls aag 148 Cys Ser Wing Trp Leu Leu Thr Wing Being Phe Ser Wing Gln Gln His Lys 25 30 35 ggc agt ttg cag aag gac cct cta ttg tec cag gcc tgt gtt ggc tgc 196 Gly Ser Leu Gln Lys' Asp Pro Leu Leu Ser Gln Wing Cys Val Gly Cys 40 45 50 55 ctg gag gcc ttg ctt gac tac ctg gat gcc cgg age cea gac att gct 244 Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg Ser Pro Asp He Wing 60 65 70 etc falls gtg gcc tec cag cct tgg aat cgg ttt ttg ctg ttt acc etc. 292 Leu His Val Wing Pro Gln Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu 75 80 85 ttg gat gct gga gag aat tec ttc etc aga cg cg gag att ttg agg etc 340 Leu Asp Wing Gly Glu Asn Being Phe Leu Arg Pro Glu He Leu Arg Leu 90 95 100 atg acc ctg etc cag age atg gga drops ctg gct gac drops age atg gcc 388 Met Thr Leu Leu Glp Ser Met Gly His Leu Wing Asp His Ser Met Wing 105 110 115 cag acc ctg cag gcc tec ttg gag ggc ctt ecc cct age acc tec te 436 Gln Thr Leu Gln Ala Ser Leu Glu Gly Leu Pro Pro Ser Thr Ser Ser 120 125 130 135 ggc cag cea ecc ctg cag gac atg etc tgc ctg gga ggg gtg gct gta 484 Gly Gln Pro Pro Leu Gln Asp Met Leu Cys Leu Gly Gly Val Wing Val 140 145 150 tec ctg tec falls aga aac a tgatc 510 Ser Leu Ser His He Ar g Asn 155 < 210 > 6 < 211 > 158 < 212 > PRT < 213 > Homo sapiens < 400 > 6 Met Glu Leu Leu Glu Lys Met Leu Wing Leu Thr Leu Wing Lys Wing Asp 1 5 10 15 Ser Pro Arg Thr Wing Leu Leu Cys Ser Wing Trp Leu Leu Thr Wing Ser 20 25 30 Phe Ser Wing Gln Gln His Lys Gly Ser Leu Gln Lys Asp Pro Leu Leu 35 40 45 Be Gln Ala Cys Val Gly Cys Leu Glu Ala Leu Leu Asp Tyr Leu Asp 50 55 60 Wing Arg Ser Pro Asp He Wing Leu His Val Wing Being Gln Pro Trp Asn 65 70 75 80 Arg Phe Leu Leu Phe Thr Leu Leu Asp Wing Gly Glu Asn Being Phe Leu 85 90 95 Arg Pro Glu He Leu Arg Leu Met Thr Leu Leu Gln Ser Met Gly His 100 105 110 Leu Ala Asp His Ser Met Ala Gln Thr Leu Gln Ala Ser Leu Glu Gly 115 120 125 Leu Pro Pro Ser Thr Ser Gly Gln Pro Pro Leu Gln Asp Met Leu 130 135 140 Cys Leu Gly Gly Val Wing Val Ser Leu Ser His He Arg Asn 145 150 155 < 210 > 7 < 211 > 522 < 212 > DNA < 213 > Homo sapiens < 400 > 7 catcagacca cacccagcag tcagaaaaga ggtgcagggg cccgggctgg gacagtgaag 60 agtgctgggc agtctgtggt cctctgtatc tcaacttttt catcttaaaa aaacaaatag 120 ggttgtgtgt gtggctggtg gtcataaggt cctttctggc tctaataacc tgagcttctg 180 ttatgaagct gggaccctta gagcctcagg atgatcctct gtttgtttgt gaaccccaat 240 caggtgctaa gcacatagtg gcacttagct gaagctcctc tgtaactcct gtgggccctg 300 cattgcccac ccccgacagc tgctgcagtg ctcctgagca gcacaggcct gatggagctt 360 tgctggccct ctggagaaga caccttggca aaggcagatt ctcccaggac tgcactcctc 420 tgctctgcct ggctgctcac tgcctccttc tctgcccagc agcacaaggg cagtttgcag 480 aaggaccctc tattgtccca ggcctgtgtt ggctgcctgg ag 522 < 210 > 8 < 211 > 168 < 212 > PRT < 213 > Homo sapiens < 400 > 8 318 Wing Gln Gln His Lys Gly Ser Leu 61n Lys Asp Pro Leu Leu Ser 61n 1 5 10 15 Wing Cys Val Gly Cys Leu Glu Wing Leu Leu Asp Tyr Leu Asp Wing Arg 25 30 Ser Pro Asp He Ala Leu His Val Ala Ser Gln Pro Trp Asn Arg Phe 40 45 Leu Leu Phe Thr Leu Leu Asp Wing Gly Glu Asn Ser Phe Leu Arg Pro 50 55 60 Glu He Leu Arg Leu Met Thr Leu Phe Met Arg Tyr Arg Be Ser 65 '70 75 80 Val Leu Ser His Glu Glu Val Gly Asp Val Leu Gln Gly Val Ala Leu 85 90 95 Wing Asp Leu Being Thr Leu Being Asn Thr Thr Leu Gln Wing Leu His Gly 100 105 110 Phe Phe Gln Gln Leu Gln Ser Met Gly His Leu Ala Asp His Ser Met 115 120 125 Wing Gln Thr Leu Gln Wing Ser Leu Glu Gly Leu Pro Pro Ser Thr Ser 130 135 140 Ser Gly Gln Pro Pro Leu Gln Asp Met Leu Cys Leu Gly Gly Val Ala 145 150 155 160 Val Ser Leu Ser His He Arg Asn 165 < 210 > 9 < 211 > 254 < 212 > PRT < 213 > Homo sapiens < 400 > 9 Wing Gln Gln His Lys Gly Ser Leu Gln Val His Gln Thr Leu Ser Val 1 5 10 15 Glu Met Asp Gln Val Leu Lys Ala Leu Ser Phe Pro Lys Lys Lys Wing 25 30 Ala Leu Leu Ser Ala Ala He Leu Cys Phe Leu Arg Thr Ala Leu Arg 40 45 Gln Ser Phe Ser Be Ala Leu Val Ala Leu Val Pro Ser Gly Ala Gln 50 55 60 Pro Leu Pro Wing Thr Lys Asp Thr Val Leu Wing Pro Leu Arg Met Ser 65 70 75 80 Gln Val Arg Ser Leu Val He Gly Leu Gln Asn Leu Leu Val Gln Lys 85 90 95 Asp Pro Leu Leu Ser Gln Ala Cys Val Gly Cys Leu Glu Ala Leu Leu 100 105 110 Asp Tyr Leu Asp Wing Arg Ser Pro Asp He Wing Leu His Val Wing Being 115 120 125 Gln Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu Leu Asp Wing Gly Glu 130 135 140 Asn Ser Phe Leu Arg Pro Glu He Leu Arg Leu Met Thr Leu Phe Met 145 150 155 160 Arg Tyr Arg Ser Ser Ser Val Leu Ser His Glu Glu Val Gly Asp Val 165 170 175 Leu Gln Gly Val Wing Leu Wing Asp Leu Being Thr Leu Being Asn Thr Thr 180 185 190 Leu Gln Wing Leu His Gly Phe Phe Gln Gln Leu Gln Being Met Gly His 195 200 205 Leu Ala Asp His Ser Met Ala Gln Thr Leu Gln Ala Ser Leu Glu Gly 210 215 220 Leu Pro Pro Ser Thr Ser Gly Gln Pro Pro Leu Gln Asp Met Leu 225 230 235 240 Cys Leu Gly Gly Val Wing Val Ser Leu Ser His He Arg Asn 245 250 < 210 > 10 < 211 > 124 < 212 > PRT < 213 > Homo sapiens < 400 > 10 Ala Gln Gln His Lys Gly Ser Leu Gln Lys • Asp Pro Leu Leu Ser Gln 1 5 10 15 Ala Cys Val Gly Cys Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg 20 25 30 Ser Pro Asp He Ala Leu His Val Ala Ser Gln Pro Trp Asn Arg Phe 35 40 45 Leu Leu Phe Thr Leu Leu Asp Ala Gly Glu Asn Ser Phe Leu Arg Pro 50 55 60 Glu He Leu Arg Leu Met Thr Leu Leu Gln Ser Met Gly His Leu Wing 65 70 75 80 Asp His Ser Met Wing Gln Thr Leu Gln Wing Ser Leu Glu Gly Leu Pro 85 90 95 Pro Ser Thr Be Ser Gly Gln Pro Pro Leu Gln Asp Met Leu Cys Leu 100 105 110 Gly Gly Val Wing Val Ser Leu Ser His He Arg Asn 115 120 < 210 > 11 < 211 > 19 < 212 > DNA < 213 > Homo sapiens < 400 > 11 agtttgcagg ttcaccaga 19 < 210 > 12 '< 211 > 20 < 212 > DNA < 213 = »Homo sapiens < 400 > 12 caattatgcc ctttgtctcc 20 < 210 > 13 < 211 > 24 < 212 > DNA < 213 > Homo sapiens < 400 > 13 ccagggctac cagggcagag gaaa 24 < 210 > 14 < 211 > 24 < 212 > DNA < 213 > Homo sapiens < 400 > 14 ccgcaggaag cataagatgg cage 24 < 210 > 15 < 211 > 23 < 212 > DNA < 213 > Homo sapiens < 400 > 15 cttctgggcc ttcaagtcct gag 23 < 210 > 16 < 211 > 20 < 212 > DNA < 213 > Homo sapiens < 400 > 16 ccttgcccac ccccgacagc 20 < 210 > 17 < 211 > 197 < 212 > DNA - < 213 > Homo sapiens < 400 > 17 geagtttgea. ggttcaccag gcactctctg tggaaatgga ccaagtattg aággctctca 60 gctttccaaa gaaaaaggct gcactactct cagctgccat ettatgette ctgcggacag 120 cccttcgaca aagettttec tctgccctgg tagccctggt gccctcaggg gcccagccac 180 tgccagccac caaggac 197 < 210 > 18 < 211 > 71 < 212 > PRT < 213 > Homo sapiens < 400 > 18 Gln Gln His Lys Gly Ser Leu Gln Val His Gln Thr Leu Ser Val Glu 1 5 10 15 Met Asp Gln Val Leu Lys Ala Leu Ser Phe Pro Lys Lys Wing Ala 20 25 30 Leu Leu Ser Ala Ala He Leu Cys Phe Leu Arg Thr Ala Leu Arg Gln 35 40 45 Be Phe Be Be Wing Leu Val Wing Leu Val Pro Be Gly Wing Gln Pro 50 55 60 Leu Pro Wing Thr Lys Asp Thr 65 70 < 210 > 19 < 211 > 69 < 212 > PRT < 213 > Homo sapiens < 400 > 19 Gln Lys Asp Pro Leu Leu Ser Gln Wing Cys Val Gly Cys Leu Glu Wing 1 5 10 15 Leu Leu Asp Tyr Leu Asp Wing Arg Ser Pro Asp Wing Wing Leu His Val 20 25 30 Wing Being Gln Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu Leu Asp Wing 35 40 45 Gly Glu Asn Ser Phe Leu Arg Pro Glu He Leu Arg Leu Met Thr Leu 50 55 60 Phe Met Arg Tyr Arg 65 < 210 > 20 < 211 > 76 < 212 > PRT < 213 > Homo sapiens < 400 > 20 Arg Being Ser Val Leu Being His Glu Glu Val Gly Asp Val Leu Gln 1 5 10 15 Gly Val Ala Leu Ala Asp Leu Ser Thr Leu Ser Asn Thr Thr Leu Gln 25 30 Ala Leu His Gly Phe Phe Gln Gln Leu Gln Ser Met Gly His Leu Ala 40 45 Asp His Ser Met Ala Gln Thr Leu Gln Ala Ser Leu Glu Gly Leu Pro 50 55 60 Pro Ser Thr Ser Gly Gln Pro Pro Leu Gln Asp 65 70 75 < 210 > 21 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 21 Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg Ser Pro Asp He 1 5 10 15 < 210 > 22 < 211 > 15 < 212 > PRT < 213 > Homo sapiens 400 > 22 Leu Arg Pro 61u He Leu Arg Leu Met Thr Leu Phe Met Arg Tyr 1 5 10 15 < 210 > 23 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 23 Leu Ala Asp Leu Ser Thr Leu Ser Asn Thr Thr Leu Gln Ala Leu 1 5 10 15 < 210 > 24 < 211 > 15 < 212 > PRT < 213 > Hoo sapiens < 400 > 24 Met Wing Gln Thr Leu 61n Wing Being Leu Glu Gly Leu Pro Pro Being 1 5 10 15 • < 210 > 25 < 211 > 55 < 212 > PRT < 213 > Hoo sapiens < 400 > 25 Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg Ser Pro Asp He Ala 1 5 10 15 Leu His Val Wing Ser 61n Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu 25 30 Leu Asp Wing Gly Glu Asn Ser Phe Leu Arg Pro Glu He Leu Arg Leu 40 45 Met Thr Leu Phe Met Arg Tyr 50 55 < 210 > 26 < 211 > 89 < 212 > PRT < 213 > Homo sapiens < 400 > 26 Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg Ser Pro Asp He Ala 1 5 10 15 Leu His Val Ala Ser Gln Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu 25 30 Leu Asp Wing 61 and Glu Asn Ser Phe Leu Arg Pro Glu He Leu Arg Leu 40 45 Met Thr Leu Phe Met Arg Tyr Arg Ser Ser Ser Val Leu Ser His Glu 50 55 60 Glu Val Gly Asp Val Leu Gln Gly Val Ala Leu Ala Asp Leu Ser Thr 65 70 75 80 Leu Ser Asn Thr Thr Leu Gln Ala Leu 85 < 210 > 27 < 211 > 121 < 212 > PRT < 213 > Homo sapiens < 400 > 27 Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg Ser Pro Asp He Ala .1 5 10 15 Leu His Val Ala Ser Gln Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu 25 30 Leu Asp Wing Gly Glu Asn Ser Phe Leu Arg Pro Glu He Leu Arg Leu 40 45 Met Thr Leu Phe Met Arg Tyr Arg Ser Ser Ser Val Leu Ser His Glu 50 55 60 Glu Val Gly Asp Val teu 61n Gly Val Ala Leu Ala Asp Leu Ser Thr 65 70 75 80 Leu Ser Asn Thr Thr Leu Gln Wing Leu His Gly Phe Phe Gln Gln Leu 85 90 95 Gln Ser Met Gly His Leu Wing Asp His Ser Met Wing Gln Thr Leu Gln 100 105 110 Wing Ser Leu Glu Gly Leu Pro Pro Ser 115 120 < 210 > 28 < 211 > 81. < 212 > PRT < 213 > Homo sapiens < 400 > 28 Leu Arg Pro Glu He Leu Arg Leu Met Thr Leu Phe Met Arg Tyr Arg 1 5 10 15 Being Ser Val Leu Being His Glu Glu Val Gly Asp Val Leu Gln Gly 25 30 Val Ala Leu Ala Asp Leu Ser Thr Leu Ser Asn Thr Thr Leu Gln Ala 40 45 Leu His Gly Phe Phe-Gln Gln Leu Gln Ser Met Gly His Leu Ala Asp 50 55 60 His Ser Met Wing Gln Thr Leu Gln Wing Ser Leu Glu Gly Leu Pro Pro 65 70 75 80 Be < 210 > 29 < 211 > 49 < 212 > PRT < 213 > Homo sapiens < 400 > 29 Leu Arg Pro Glu He Leu Arg Leu Met Thr Leu Phe Met Arg Tyr Arg 1 5 10 15 Being Ser Val Leu Ser His Glu Glu Val Gly Asp Val Leu Gln Gly 25 30 Val Ala Leu Ala Asp Leu Ser Thr Leu Ser Asn Thr Thr Leu Gln Ala 40 45 Leu < 210 > 30 < 211 > 47 < 212 > PRT < 213 > Homo sapiens < 400 > 30 Leu Ala Asp Leu Ser Thr Leu Ser Asn Thr Thr Leu Gln Ala Leu His 1 5 10 15 Gly Phe Phe Gln Gln Leu Gln Ser Met Gly His Leu Ala Asp His Ser 25 30 Met Ala Gln Thr Leu Gln Ala Ser Leu Glu Gly Leu Pro Pro Ser 35 40 45 < 210 > 31 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 31 His Gln Thr Leu Ser Val Glu Met Asp Gln Val Leu Lys Ala Leu 1 5 10 15 < 210 > 32 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 32 Val Arg Ser Leu Val He Gly Leu Gln Asn Leu Leu Val Gln Lys 1 5 10 15 < 210 > 33 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 33 Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg Ser Pro Asp He 10 15 < 210 > 34 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 34 Leu Arg Pro Glu He Leu Arg Leu Met Thr Leu Phe Met Arg Tyr i 5 '10 15 < 210 > 35 < 211 > 86 < 212 > PRT < 213 > Homo sapiens 400 > 35 His Gln Thr Leu Ser Val Glu Met Asp 61n Val Leu Lys Ala Leu Ser 1 5 10 15 Phe Pro Lys Lys Lys Wing Wing Leu Leu Wing Wing He Leu Cys Phe 25 30 Leu Arg Thr Ala Leu Arg Gln Ser Phe Ser Be Ala Leu Val Ala Leu 40 45 Val Pro Ser Gly Wing Gln Pro Leu Pro Wing Thr Lys Asp Thr Val Leu 50 55 60 Ala Pro Leu Arg Met Ser Gln Val Arg Ser Leu Val He Gly Leu Gln 65 70 75 80 Asn Leu Leu Val Gln Lys 85 < 210 > 36 < 211 > 112 < 212 > PRT < 213 > Homo sapiens 400 > 36 His Gln Thr Leu Ser Val Glu Met Asp Gln Val Leu Lys Ala Leu Ser 1 5 10 15 Phe Pro Lys Lys Lys Wing Wing Leu Leu Wing Wing He Leu Cys Phe 25 30 Leu Arg Thr Ala Leu Arg Gln Ser Phe Ser Be Ala Leu Val Ala Leu 40 45 Val Pro Ser Gly Wing Gln Pro Leu Pro Wing Thr Lys Asp Thr Val Leu 50 55 60 Wing Pro Leu Arg Met Ser Gln Val Arg Ser Leu Val He Gly Leu Gln 65 70 75 80 Asn-Leu Leu Val Gln Lys Asp Pro Leu Leu Ser Gln Ala Cys Val 61y 85 90 95 Cys Leu 61u Wing Leu Leu Asp Tyr Leu Asp Wing Arg Ser Pro Asp He 100 105 110 < 210 > 37 < 211 > 152 < 212 > PRT < 213 > Homo sapiens < 400 > 37 His 61n Thr Leu Ser Val 61u Met Asp 61n Val Leu Lys Ala Leu Ser 1 5 10 15 Phe Pro Lys Lys Lys Wing Wing Leu Leu Wing Wing He Leu Cys Phe 25 30 Leu Arg Thr Ala Leu Arg Gln Ser Phe Ser Be Ala Leu Val Ala Leu 40 45 Val Pro Ser 61y Wing 61n Pro Leu Pro Wing Thr Lys Asp Thr Val Leu 50 55 60 Ala Pro Leu Arg Met Ser Gln Val Arg Ser Leu Val He Gly Leu 61n 65 70 75 80 Asn Leu Leu Val Gln Lys Asp Pro Leu Leu Ser Gln Ala Cys Val 61y 85 90 95 Cys Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg Ser Pro Asp He 100 105 110 Ala Leu His Val Ala Ser Gln Pro Trp Asn Arg Phe Leu Leu Phe Thr 115 120 125 Leu Leu Asp Wing Gly Glu Asn Being Phe Leu Arg Pro Glu He Leu Arg 130 135 140 Leu Met Thr Leu Phe Met Arg Tyr 145 150 < 210 > 38 < 211 > 81 < 212 > PRT < 213 > Homo sapiens < 400 > 38 Val Arg Ser Leu Val He Gly Leu Gln Asn Leu Leu Val Gln Lys Asp 1 5 10 15 Pro Leu Leu Ser 61n Ala Cys Val Gly Cys Leu Glu Ala Leu Leu Asp 25 30 Tyr Leu Asp Ala Arg Ser Pro Asp He Ala Leu His Val Ala Ser Gln 40 45 Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu Leu Asp Wing 61y 61u Asn 50 55 60 Ser Phe Leu Arg Pro Glu He Leu Arg Leu Met Thr Leu Phe Met Arg 65 70 75 80 Tyr < 210 > 39 < 211 > 41 < 212 > PRT < 213 > Homo sapiens < 400 > 39 Val Arg Ser Leu Val He 61y Leu 61n Asn Leu Leu Val 61n Lys Asp 1 5 10 15 Pro Leu Leu Ser Gln Ala Cys Val Gly Cys Leu Glu Ala Leu Leu Asp 25 30 Tyr Leu Asp Ala Arg Ser Pro Asp He 35 40 < 210 > 40 < 211 > 55 < 212 > PRT < 213 > Homo sapiens < 400 > 40 Leu Glu Ala Leu Leu Asp Tyr Leu Asp Ala Arg Ser Pro Asp He Ala 1 5 10 15 Leu His Val Ala Ser Gln Pro Trp Asn Arg Phe Leu Leu Phe Thr Leu 25 30 Leu Asp Wing Gly Glu Asn Ser Phe Leu Arg Pro Glu He Leu Arg Leu 40 45 Met Thr Leu Phe Met Arg Tyr 50 55 < 210 > 41 < 211 > 32 < 212 > PRT < 213 > Homo sapiens < 400 > 41 Wing Gln 61n His Lys Gly Ser Leu Gln Lys Asp Pro Leu Leu Ser Gln 1 5 10 15 Wing Cys Val 61y Cys Leu 61u Wing Leu Leu Asp Tyr Leu Asp Wing Arg 25 30 < 210 > 42 < 211 > 31 < 212 > PRT < 213 > Homo sapiens < 400 > 42 Pro Leu Pro Wing Thr Lys Asp Thr Val Leu Wing Pro Leu Arg Met Ser 1 5 10 15 61n Val Arg Ser Leu Val He 61and Leu 61n Asn Leu Leu Val Cys 20 25 30 < 210 > 43 < 211 > 33 < 212 > PRT < 213 > Homo sapiens < 400 > 43 Cys 61u 61y Leu Pro Pro Ser Thr Ser Gly 61n Pro Pro Leu Gln 1 5 10 15 Asp Met Leu Cys Leu Gly Gly Val Wing Val Ser Leu Ser His He Arg 25 30 Asn < 210 > 44 < 211 > 15 < 212 > PRT < 213 > Homo sapiens < 400 > 44 Phe Met Arg Tyr Arg Ser Ser Ser Val Leu Ser His Glu 61u Cys 1 5 10 15

Claims (12)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An isolated polypeptide, characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9, 10, 18, 19, 20, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44 or a polypeptide that is at least 80% identical to said polypeptide.

2. An isolated polypeptide, characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9, 10 or a polypeptide that is at least 70% identical to said polypeptide.

3. An isolated polynucleotide encoding a polypeptide, characterized in that said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9, 10, 18, __. L9, 20, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39 and 40 or a polypeptide that is less 80% identical to said polypeptide.

4. An isolated polynucleotide encoding a polypeptide, characterized in that said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9 and 10, or a polypeptide that is at least 70% identical to said polypeptide.

5. An antibody that specifically binds to a polypeptide, characterized in that said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9, 10, 18, 19, 20, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39 and 40.

6. An antibody according to claim 5 that specifically binds to a polypeptide, characterized in that said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9 and 10.

7. An anti-idiotypic antibody that specifically binds to an antibody that specifically binds to a polypeptide, characterized in that said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9, 10, 18, 19, 20, 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39 and 40.

8. An anti-idiotypic antibody according to claim 7, characterized in that the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9 and 10.

9. The use of an antagonist to a polypeptide to treat an inflammation, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9 and 10.

10. The use according to claim 9, wherein the antagonist is an antibody that binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9 and 10.

11. The use of an antagonist to a polypeptide in the preparation of a medicament for treating inflammation, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9 and 10.

12. The use in accordance with the claim 11, wherein the antagonist is an antibody that binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, 4, 6, 8, 9 and 10.