MXPA99002973A - NEUTROKINE&agr; - Google Patents

Abstract

The present invention relates to a novel Neutrokine&agr;protein which is a member of the TNF protein family. In particular, isolated nucleic acid molecules are provided encoding the human Neutrokine&agr;protein including soluble forms of the extracellular domain. Neutrokine&agr;polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of Neutrokine&agr;activity. Also provided are diagnostic methods for detecting immune system-related disorders and therapeutic methods for treating immune system-related disorders.

Description

NEUTROCINA ALFA FIELD OF THE INVENTION * The present invention relates to a novel cytokine expressed by neutrophils, which has been designated as Neutrocin oc ("Neutrocin oc") protein. In particular, isolated nucleic acid molecules encoding the protein Neutrocin oc. Neutrocin oc polypeptides are also provided, such as vectors, host cells and recombinant methods to produce the same. BACKGROUND OF THE INVENTION He The necrosis factors of human tumors (TNF-oc) and (TNF-β, or lymphotoxin) are related members of a broad class of polypeptide mediators, which includes the int er f erones, int erucin s and growth factors / collectively called cytokines (Beutler, B. and Cerami, A., Annu. Re t,.

Im an ol. , 7: 625-655 (1989)). The analysis of REF .: 29576 sequence of cytokine receptors has defined several subfamilies of membrane proteins (1) the Ig superfamily, (2) the superfamily of the hematopoietin receptor (cytokine) and (3) the receptor superfamily of the tumor necrosis factor (TNF) / nerve growth factor (NGF) (for a review of the superfamily of the TNF see, Gruss and Dower, Blood 85 (12): 3318-3404 (1995) and Aggarwal and Natarajan, Eur. Citokine Netw. , 7 (2); 93-124 (1996)). The TNF / NGF receptor superfamily contains at least 10 difference proteins. Gruss and Dower, supra. Ligands have been identified for these receptors and belong to at least two super fi cia of cytokines. Gruss and Dower, supra. The tumor necrosis factor (a mixture of TNF-oc and TNF-β) was originally discovered as a result of its activity against tumors, however, it is now recognized as a pleiotropic cytokine capable of numerous biological activities including the apoptosis of some transformed cell lines, the mediation of cell activation and proliferation and also because it plays important roles in immunological regulation and inflammation. To date the known members of the TNF-superfamily of ligands include TNF-oc, TNF-β (lymphoxoxin-x), LT-β, OX40L, Fas ligand, CD30L, CD27L, CD40L and 4-IBBL. The ligands of the TNF ligand superfamily are acid molecules similar to TNF that have approximately 20% sequence homology in the ex-cellular domains. (range, 12% - 36%) and exist mainly as membrane-bound forms wherein the biologically active form is a complex t / m / m / t / t. Soluble forms of the TNF superfamily of ligands have only been identified so far for the ligand TNF, LT ß, and Fas (for a general review, see Gruss and Dower, SK, Bl ood, 85 (1 2) - .3 3 1 8 - 3 4 0 4 (1995)), which is incorporated herein by reference in its entirety. These proteins are involved in the regulation of cell proliferation, activation, and differentiation, including the control of cell death or survival by apoptosis or cytotoxicity (Armitage, RJ Curr, Opin, Immunol 6: 407 (1994) and Smith, CA CelJ 75: 959 (1994)). The tumor necrosis factor alpha (TNF or, also called cachectin, hereinafter referred to as "TNF") is secreted primarily by monocytes and macrophages in response to endotoxin or other stimuli, such as a soluble first of 17 kD protein subunits (Smith, RA et al., J. Biol. Chem. 262: 6951-6954 (1987)). A precursor form of TNF, 26 Kd, bound to the membrane has also been described (Kriegler, M. et al., Cell 53: 45-53 (1988)). Cumulative evidence indicates that TNF is a regulatory cytokine with pleo tropic biological activities. These activities include: the inhibition of lipoprotein lipase synthesis ("cachectin" activity) (Beutler, B. et al., Nature 316: 552 (1985)), the activation of leukocyte po 1 i or fonucl ear is (Klebanoff, SJ et al., J. Immunol., 136: 4220 (1986); Perussia, B., et al. , J. Immunol. 238: 765 (1987)), inhibition of cell growth or stimulation of cell growth (Vilcek, J. et al., J. Exp. Med. 163: 632 (1986); Sugarman, BJ et al., Science 230 : 943 (1985), Lachman, LB et al., J. Immunol. 138: 2913 (1987)), the cytotoxic action on certain types of transformed cells (Lachman, L. B et al., Supra, Dar zynki ewi cz, Z. et al., Canc. Res. 44: 83 (1984)), antiviral activity (Kohase, M. et al., Cell 45: 659 (1986); Wong, GHW et al., Nature 323: 819 (1986)), the stimulation of bone resorption (Bertolini, DR et al., Nature 319: 516 (1986), Saklatvala, J., Nature 322: 541 (1986)), the stimulation of collagenase and production of prostaglandin E2 (Dayer, J. -M et al., J. Exp. med 162: 2163 (1985)); and immunomodulatory actions, including activation of T cells (Yokota, S. et al., J. Immunol.140: 531 (1988)), of B cells (Kehrl, JH et al., J. Exp. Med. 166: 186 (1987)), from monocytes (Philip, R. et al., Nature 323: 86 (1986)), from thymocytes (Ranges, GE et al., J. Exp. Med. 167 : 1412 (1988)), and the stimulation of the surface expression of the cells of the class I and class II molecules of the main complex of hi-tocomp t ibity (MHC, for its acronym in English) (Collins, T. et al., Proc. Nati. Acad, Sci. USA 83: 446 (1986), Pujol-Borrel, R. et al. Nature 326: 304 (1987)). It is noted that TNF has proinflammatory actions that result in tissue damage, such as the induction of procoagulant activity in vascular endothelial cells (Pober, JS et al., J. Immunol., 136: 1680 (1986). ), increased adherence of neutrophils and lymphocytes (Pober, JS et al., J. Immunol., 138: 3319 (1987)), and stimulation of the release of platelet activation factor by macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 166: 1390 (1987)). 'Recent evidence implicates TNF in the pathogenesis of many infections (Cerami, A. et al., Immunol. Today 9:28 (1988)), immunological disorders, neoplastic pathology, for example, in the cachexia that accompanies some malignancies (Oliff, A. et al., Cell 50: 555 (1987)), and in autoinological pathologies and in graft versus host pathology (Piguet, P.-F. et al., J. Exp. Med. 166: 1280 (1987)). The association of TNF with cancer and with infectious pathologies is often related to the catabolic state of the host. A major problem in patients with cancer is weight loss, usually associated with .anorexia. The resulting extensive wear is known as "cachexia" (Kern, K.A. et al., J. Parent, Enter, Nutr. 22: 286-298 (1988)). Cachexia includes progressive weight loss, anorexia, and persistent erosion of body mass in response to malignant growth. The cachectic state is thus associated with significant morbidity and is responsible for most of the cancer mortality. A number of studies have suggested that TNF is an important mediator of cachexia in cancer, in infectious disease, and in other catabolic states. It is believed that TNF plays a central role in the pathophysiological consequences of sepsis by Gram-negative bacteria and endotoxic shock (Michie, HR et al., Br. J. Surg. 76: 610-611 (1989); Debets JM et al., Second Vienna Shock Forum, pp. 463-466 (1989), Simpson, S. Q., et al., Crit. Care Clin. 5: 21-41 (1989)), including fever, malaise. , anorexia, and cachexia. Endotoxin is a potent activator of monocytes / macrophages, which stimulates the production and secretion of TNF (Kornbluth, S.K. et al., J. Immunol., 137: 2585-2591 (1986)) and other cytokines. Because TNF could simulate many biological effects of endotoxin, it was concluded that it was a central mediator responsible for the clinical manifestations of endotoxin-related disease. TNF and other monocyte-derived cytokines mediate metabolic responses and rheumatoid neuro- pathy to endotoxin (Michie, H.R. et al., N. Eng. J. Med. 328: 1481-1486 (1988)). Administration of endotoxin to human volunteers produces an acute illness with influenza-like symptoms which include fever, tachycardia, increased metabolic rate and release of hormones by stress (Revhaug, A. et al., Surg., 123: 162-110 (1988)). Elevated levels of circulating TNF have also been found in patients suffering from sepsis from Gram-negative bacteria (Waage, A, et al., Lancet 2: 355-357 (1987)).; Hammerle, A. F. et al. , Second Vienna Shock Forum p. 715-718 (1989); Debets, J. M. H. et al. , Crit. Care med. 27: 489-497 (1989); Calandra, T. et al. , J. Infect Dis. 161: 982-981 (1990)). Passive immunotherapy directed to neutralizing TNF can have a beneficial effect in sepsis by Gram negative bacteria and in endotoxemia, based on the increased production of TNF and the high levels of TNF in these pathological states, as discussed above. Antibodies to a "modulator" material that was characterized as cachectin (which was later found to be identical to TNF) were described by Cerami et al. (EPO Patent Publication 0,212,489, March 4, 1987). It was mentioned that these antibodies were useful in diagnostic immunoassays and in shock therapy in bacterial infections. Rubin et al (Patent Publication EPO 0,218,868, of 22 April 1987) describes monoclonal antibodies to human TNF, the hybridomas that secrete those antibodies, methods for the production of those antibodies, and the use of those antibodies in TNF immunoassay. Yone et al (Patent Publication EPO 0,288,088, October 26, 1988) discloses anti-TNF antibodies, including mAbs, and their usefulness in the diagnosis of pathologies by means of immunoassays, in particular the pathology of Kawasaki and bacterial infection . It was mentioned that the body fluids of patients in the pathology of Kawasaki (Febrile, infantile, acute, knot syndrome in the mucocutaneous lymph; Kawasaki, T., Allergy 16: 118 (1967); Kawasaki, T., Shonica (Pediatrics) 26: 935 (1985)) contained high levels of TNF that were related to the progress of the pathology (Yone et al., Supra). Other investigators have described specific mAbs for recombinant human TNF that had a neutralizing activity in vitro (Liang, CM, et al, Biochem, Biophys, Res. Comm. 137: 841-854 (1986); Meager, A; et al., Hybridoma ^: 305-311 (1987), Fendly et al., Hybridoma 6 ~: 359-369 (1987); Bring an, TS et al., Hybridoma 6: 489-501 (1987): Hirai , M. et al., J. Immunol., Meth. 96: 51-62 (1987); Moller, A et al.

(Citokine 2: 162-169 (1990)). Some of these mAbs were used to map epitopes of human TNF and develop enzyme immunoassays (Fendly et al., Supra; Hirai et al., Supra; Moller et al., Supra) and to aid in the purification of recombinant TNF (supra); Bringmank et al., Supra). However, these studies do not provide a basis for the production of neural anti-TNF antibodies that can be used for in vivo diagnosis or for therapeutic uses in humans, due to immunogenicity, lack of specificity and / or pharmaceutical convenience. It has been shown that neutralizing antisera or mAbs, for TNF, in mammals other than humans, abolish adverse physiological changes and prevent death after the lethal challenge in endotoxemia and experimental bacteremia. This effect has been demonstrated, for example, in lethality tests in rodents and in pathological model systems in primates (Mathison, JC et al., J. Clin. In est. 82: 1925-1937 (1988); Beutler, B et al., Science 229: 869-811 (1985); Tracey, KJ et al., Nature 330: 662-664 (1987); Shimamoto, Y. et al., Immunol. Lett. 17: 311-318 ( 1988), Silva, AT et al., Infect. Dis. 162: 421-427 (1990), Opal, SM et al., J. Infecí., Dis. 252: 1148-1152 (1990); Hinshaw, LB et al. ., Circ.Shock 30: 279-292 (1990)). To date, experience with anti-TNF mAb therapy in humans has been limited but shows beneficial therapeutic results, for example, in arthritis and sepsis. See, for example, Elliott, M.J. et al. , Bailliers Clin. Rheumatol 9: 633-52 (1995); Feldmann M, et al. , Ann. N. Y. Acad. Sci. USA 766: 212-8 (1995); van der Poli, T. et al. , Shock 3: 1-12 (1995); Wherry et al. , Crit. Care Med. 22: S436-40 (1993); Tracey K. J., et al. , Crit Care. Med. 22: S415-22 (1993). The development of mammals is dependent on both the proliferation and differentiation of cells and the programmed cell death which occurs through apoptosis (Walker, et al., Methods Achiev., Exp. Pathol. 13: 18 (1988) Apoptosis plays a critical role in the destruction of immune thymocytes that recognize their own antigens.The failure of this normal process of elimination may play a role in autoimmune diseases (Gammon et al., Immunology Today 22: 193 (1991)) Itoh et al (Cell 66: 233 (1991)) describes a cell surface antigen, Fas / CD23, that mediates apoptosis and that is involved in the clonal deletion of T cells. Fas is expressed in activated T cells, in B cells, in neutrophils and in the thymus, liver, heart and lung and ovaries in adult mice (Wat anabe- Fukunaga et al., J. Immunol.; 1214 (1992)) in addition to activated T cells, B cells, and neutrophils. In experiments where a monoclonal Ab is cross-linked to Fas, apoptosis is induced (Yonehara et al., J. Exp. Med. 169: 1741 (1989); Trauth et al., Science 245: 301 (1989)). In addition, there is an example in which the binding of a monoclonal Ab to the Fas is T cell imaging, under certain conditions (Alderson et al., J. Exp. Med. 178: 2231 (1993)). The Fas antigen is a protein on the surface of the cell of a relative molecular weight of 45 Kd. Both human and murine genes, for the Fas, have been cloned by Watanabe-Fukunaga et al. , (J. Immunol., 148: 1214 (1992)) and by Itoh et al. (Cell 66: 2 3 3 (1991)). The proteins encoded by these genes are both transmembrane proteins with structural homology to the receptor superfamily of the Nerve Growth Factor / Tumor Necrosis Factor, which includes two TNF receptors, the receptor for the Nerve Growth Factor. of low affinity and CD40, CD27, CD30, and OX40. Recently the Fas ligand has been described (Suda et al., Cel l 75: 1169)). The amino acid sequence indicates that the Fas ligand is a type II transmembrane protein belonging to the TNF family. In this manner the Fas ligand polypeptide comprises three major domains: a short intracellular domain at the amino terminal end and a longer extracellular domain at the carboxy terminal end, connected by a hydrophobic tr ansmembric domain. The Fas ligand is expressed in splenocytes and thymocytes, consistent with T-cell mediated cytotoxicity. The purified Fas ligand has a molecular weight of 40 kD. It has recently been shown that interactions between Fas / Fas ligands are required for apoptosis, followed by the activation of T cells (Ju et al, Na t ure 3 73 -.? 4 4 (1995); Brunner et al., Na ture 373: 441 (1995)). Activation of the T cells induces both proteins on the surface of the cell. The subsequent interaction between the ligand and the receptor results in apoptosis of the cells. This supports the possibly regulatory role of apoptosis induced by the interaction between Fas / Fas ligands during normal immunological responses. Accordingly, there is a need to provide cytokines similar to TNF that are involved in pathological conditions. These novel cytokines could be used to produce novel antibodies or other antagonists that bind to these TNF-like cytokines for the therapy of disorders related to TNF-like cytokines.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides isolated nucleic acid molecules comprising a polynucleotide that encodes a cytokine that is structurally similar to TNF and related cytokines and is believed to have similar biological effects and activities. This cytokine is called Neutrocin oc and the invention includes Neutrocin oc polypeptides having at least a portion of the amino acid sequence of Figure 1 (SEQ ID NO: 2) or of the amino acid sequences encoded by the cDNA clone. deposited in a bacterial host such as the ATCC Deposit of October 22, 1996. The nucleotide sequence determined by sequencing the deposited Neutrocin c or c clone, which is shown in Figure 1 (SEQ ID NO: 1), contains a open reading frame encoding a complete polypeptide of 285 amino acid residues including an N-terminal methionine, a predicted intracellular domain of about 46 amino acid residues, a predicted amino acid domain of approximately 26 amino acids, an approximately predicted extracellular domain 213 amino acids, and a deduced molecular weight, for the entire protein, of approximately 31 kDa. As for other transmembrane proteins of type II, the soluble forms of Neutrocin or c include all or a portion of the extracellular domain separated from the transmembrane t-domain and a polypeptide containing the entire Neutrocin c polypeptide which lacks the transmembrane t domain. say, the extracellular domain linked to the intracellular domain. Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a full-length Neutrocin oc polypeptide having the complete sequence of amino acids shown in Figure 1 (SEQ ID NO: 2) or as encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996; (b) a nucleotide sequence encoding the predicted extracellular domain of the Neutrocin oc polypeptide having the amino acid sequence at positions 73 through 285 as shown in Figure 1 (SEQ ID NO: 2) or such as encoded by the cDNA clone in the ATCC Deposit of October 22, 1996; (c) a nucleotide sequence encoding a polypeptide comprising the intracellular domain of Neutrocin oc (amino acid residues from about 1 to about 46 in Figure 1 (SEQ ID NO: 2)) or as encoded by the clone of cDNA contained in the ATCC Deposit of October 22, 1996; (d) a nucleotide sequence encoding a polypeptide comprising the transmembrane t-domain of Neutrocin ce (amino acid residues from about 47 to about 72 in Figure 1 (SEQ ID NO: 2) or as encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996; (e) a nucleotide sequence encoding a soluble Neutrocin, a polypeptide having the extracellular and intracellular domains but lacking the transmembrane domain, and (f) ) a complementary nucleotide sequence for any of the nucleotide sequences of subsections (a), (b), (c), (d) or (e) above Additional embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical, to any of the nucleotide sequences that they are found in subsections (a), (b), (c), (d), (e) or (f) above, or a polynucleotide that hybridizes under severe hybridization conditions, to a polynucleotide such as those found in subsections (a), (b), (c), (d), (e) or (f) above. These hybridizing polynucleotides do not hybridize under severe hybridization conditions, in a polynucleotide having a nucleotide sequence consisting solely of residues A or only of residues T. An additional embodiment of nucleic acid of the invention, refers to an isolated nucleic acid molecule comprising a polynucleotide that encodes the amino acid sequence of a portion that contains an epitope of a Neutrocin 8 polypeptide having an amino acid sequence such as those found in items (a), (b), (c), (d) or (e-) above. The present invention also relates to recombinant vectors that include the isolated nucleic acid molecules of the present invention, and to host cells that contain the recombinant vectors, as well as to the methods for the manufacture of those vectors and host cells and to use them for the production of polypeptides or peptides from Neutrocin or c through recombinant techniques. The invention further provides an isolated Neutrocin oc polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the full length Neutrocipa oc polypeptide, having the complete amino acid sequence that is shown in Figure 1 (SEQ ID No. 2) or as encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996; (b) the amino acid sequence of the predicted extracellular domain of the Neutrocin oc polypeptide having the amino acid sequence at positions 73 to 285 found in Figure 1 (SEQ ID NO: 2) or as is encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996; (c) the amino acid sequence of the intracellular domain of Neutrocin oc (amino acid residues from about 1 to about 46 in Figure 1 (No: SEC ID: 2)) or as encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996; (d) the transmembrane sequence of Neutrocin oc (amino acid residues - from about 47 to about 72 in Figure 1 (SEQ ID No.) 2) or as encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996; and (e) the amino acid sequence of the soluble Neutrocin oc polypeptide having the extracellular and intracellular domains but lacking the transmembrane t domain, wherein each of these domains is as defined above. The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity to those described in subparagraphs (a), (b), ( c), (d) or (e) above, as well as polypeptides having at least 80% identical amino acid sequence, more preferably at least 90% identical, and even more preferably 95%, 96%, 97%, 98%, or 99% identical to the previous ones. A further embodiment of this aspect of the invention relates to a peptide or polypeptide having the amino acid sequence of a portion containing an epitope, of a Neutrocin polypeptide or, having an amino acid sequence described in the subparagraphs (a ), (b), (c), (d) or (e) above. Peptides or polypeptides having the amino acid sequence of an epitope-containing portion of a Neutrocin oc polypeptide of the invention include portions of those polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although polypeptides containing the epitope of any length are included in the invention, even until they include the entire amino acid sequence of a polypeptide of the invention described above. In another embodiment, the invention provides an isolated antibody that specifically binds to a polypeptide having an amino acid sequence described in subparagraphs (a), (b), (c), (d) or (e) above. The invention further provides methods for isolating antibodies that specifically bind to a Neutrocin oc polypeptide having an amino acid sequence such as that described herein. These antibodies are diagnostically or therapeutically useful, as described post-operatively. The invention also provides pharmaceutical compositions comprising Neutrocin or soluble polypeptides, particularly Neutrocin or human polypeptides, which can be used, for example, to treat tumors and tumor metastases, infections by bacteria, viruses and other parasites, immunodeficiencies , inflammatory diseases, lymphadenopathy, autoimmune diseases, graft versus host disease, and to stimulate peripheral tolerance, destroy some transformed cell lines, mediate cellular activation and proliferation, and are functionally associated as primary mediators of immune regulation and inflammatory responses. The invention further provides compositions containing a Neutrocin-c polynucleotide or a Neutrocin-C polypeptide for administration to invading cells, to ex vivo cells and to viral cells, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a Neutrocin oc polynucleotide for the expression of a Neutrocin oc polypeptide in a host organism, for the treatment of the disease. In this aspect, particularly preferred is expression in a human patient for the treatment of a dysfunction associated with the endogenous aberrant activity of a Neutrocin oc gene.

The present invention also provides a method of selective sorting for. identify compounds capable of enhancing or inhibiting a cellular response induced by Neutrocin oc, which involves contacting the cells expressing Neutrocin or the candidate compound, subjecting to analysis or assay a cellular response, and comparing the cellular response with a standard cellular response, and the standard is tested when contact is made in the absence of the candidate compound; whereby, an increased cellular response to the standard indicates that the compound is an agonist, and a decreased cellular response to the standard indicates that the compound is an antagonist. In another aspect a method is provided for identifying Neutrocin oc receptors, as well as for the selective classification assay of agonists and antagonists, using those. receivers. This assay involves determining the effect that a candidate compound has on the binding of Neutrocin or the Neutrocin oc receptor. In particular the method involves contacting a Neutrocin oc receptor with a Neutrocin oc polypeptide and a candidate compound, and determining whether the binding of Neutrocin oc polypeptide to Neutr.ocina oc receptor is increased or decreased due to the presence of the candidate compound. Antagonists can be used to prevent septic shock, inflammation, cerebral malaria, HIV virus activation, graft-host rejection, bone resorption, rheumatoid arthritis and cachexia (wasting or malnutrition). The inventors of the present have discovered that Neutrocin c is expressed not only in neutrophils but also in the kidneys, lung, peripheral leukocytes, bone marrow, T-cell lymphoma, B-cell lymphoma, activated T cells, cancer in the stomach, smooth muscle, macrophages, umbilical cord blood tissue. For a number of disorders of these tissues and cells, such as tumor and tumor metastasis, infection by bacteria, viruses and other parasites, immunode fi ciency, septic shock, inflammation, cerebral malaria, activation of the HIV virus, graft-host rejection , host resorption, bone resorption, rheumatoid arthritis and cachexia (wasting or poor nutrition), it is believed that significantly higher or lower levels of Neutrocin gene expression can be detected or, in certain tissues (for example, in the medulla bone) or in bodily fluids (for example, in serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual who has one of these disorders, in relation to a gene expression level of Neutrocin or "standard" , that is, the level of expression of Neutrocin or in the tissue or bodily fluids of an individual who does not have the disorder. In this way the invention provides a useful diagnostic method by diagnosing a disorder, which involves: (a) testing or analyzing the level of gene expression of Neutrocin c in cells or in a body fluid of an individual; (b) compare the level of gene expression of Neutrocin or c with a level of gene expression of Neutrocin or standard, thus an increase or decrease in the level of gene expression of Neutrocin or c tested, compared to the level of standard expression, is indicative of a disorder. A further aspect of the invention is related to a method of treating an individual in need of an increased level of Neutrocin oc activity in the body, which comprises administering to that individual a composition containing a therapeutically effective amount of a Neutrocin or iso isolated, of the invention, or an agonist thereof. A still further aspect of the invention relates to. a method for the treatment of an individual in need of a decreased level of activity of Neutrokine ac in the body, which comprises administering to that individual a composition containing a therapeutically effective amount of a Neutrocin ce antagonist. Preferred antagonists for use in the present invention are antibodies specific for Neutrocin oc.

Brief Description of the Figures Figure 1 shows the nucleotide sequences (SEQ ID NO: 1) and the deduced amino acid sequence, (SEQ ID NO: 2) of the protein Neutrocin oc. The amino acids from 1 to 46 represent the intracellular domain, the amino acids from 47 to 72 the transmembrane domain (the underlined sequence), and the amino acids from 73 to 285 the extracellular domain (the rest of the sequence). Figure 2 shows the regions of identity between the amino acid sequences of the protein Neutrocin oc and TNF-oc (SEQ ID NO: 3), of TNF-β (1 infotoxin) (SEQ ID NO: 4) and ligand FAS (No. SEC ID: 5), determined by the routine "Megalign" which is part of the computer program called "DNAStar". Figure 3 shows an analysis of the amino acid sequence of Neutrocin ac. The alpha, beta, return and winding regions are shown; hydrophilicity and hydrophobicity; the amphipathic regions; the flexible regions; the antigenic index and the superficial probability. In the graph "Antigenic Index - Jameson-Wol f", the indicated location of the highly antigenic regions of the neutrocyl or c protein can be obtained, ie the regions from which the peptides containing the epitope can be obtained, from the invention. Figure 4 shows the alignment of the nucleotide sequence of Neutrocin oc, determined from the cDNA deposited in the ATCC Deposit of October 22, 1996 with related human cDNA clones of the invention, which have been called HSOAD55R (No SEC ID: 7); HSLAH84R (SEQ ID NO: 8) and HLTBM08R (SEQ ID NO: 9).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides isolated nucleic acid molecules, comprising a polynucleotide encoding the Neutrocin oc polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID NO: 2), which was determined by sequencing a Neutrocin or of cloned cDNA. The nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) was obtained by sequencing the clone HNEDU15, which was deposited on October 22, 1996 at the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland. The deposited clone is contained in the pBluescript SK (-) plasmid (Stratagene, La Jolla, CA). The Neutrocin oc protein of the present invention shares the sequence homology with the translation product of the mRNAs for TNF-oc, TNF-β and Fas ligand (Figure 2). As noted above, it is believed that TNF-oc is an important cytokine that plays a role in cytotoxicity, necrosis, apoptosis, co-stimulation, proliferation, lymph node formation, change in immunoglobulin class, differentiation, antiviral activity, regulation of adhesion molecules and other cytokines and growth factors.

Nucleic acid molecules Unless indicated otherwise, all nucleotide sequences determined by the sequencing of a DNA molecule herein were determined. using an automated DNA sequencer (such as model 373 from Applied Biosystems, Inc., Foster City, CA), and all amino acid sequences of polypeptides encoded by DNA molecules, determined in the present, were predicted by the translation of a certain DNA sequence. as previously. Therefore, as is known in the art, for any DNA sequence determined through this automated approach, any nucleotide sequence of / terminated herein may contain some errors. The nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the current nucleotide sequence of the sequenced DNA molecule. The actual sequence can be determined more accurately through other approaches that include manual DNA sequencing methods well known in the art. As is also well known in the art, a single insertion or deletion in a given nucleotide sequence, compared to the actual sequence, will cause a displacement of the structure, in the translation of the nucleotide sequence, such that the predicted amino acid sequence, encoded by a given nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, starting at the point of that insertion or deletion. By "nucleotide sequence" of a nucleic acid molecule or polynucleotide, it is understood, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA or polynucleotide molecule, the corresponding sequence of ribonucleotides (A, G, C and U), in which each thymidine deoxyribody (T) in the deoxyribatinocyte sequence is replaced by the uridine (U) of the ribonucleotide. Using the information provided herein, such as the nucleotide sequence of Figure 1, a nucleic acid molecule of the present invention, which encodes a Neutrocin oc polypeptide, can be obtained using standard cloning and standard selective sorting procedures, such as those to clone the cDNAs using mRNA as the initial material. Illustrative of the invention, the nucleic acid molecule described in Figure 1 (SEQ ID NO: 1) was discovered in a cDNA library derived from neutrophils. In the neutrophils, fragments of the expressed sequence corresponding to a portion of the cDNA of Neutrocin oc were also found. The Neutrocin oc gene contains an open reading frame that encodes a protein of approximately 285 amino acid residues, an intracellular domain of about 46 amino acids (amino acid residues from about 1 to about 46 in Figure 1 (SEQ ID NO: 2) )), a transmembrane domain of approximately 26 amino acids (amino acid residues from about 47 to about 72 in Figure 1 (SEQ ID NO: 2)), an extracellular domain of approximately 213 amino acids (amino acid residues from about 73 to about 285 in Figure 1 (SEQ ID No. 2)); and a deduced molecular weight of approximately 31 kDa. The protein Neutrocin oc shown in Figure 1 (SEQ ID NO: 2) is approximately 20% similar and approximately 10% identical to the human TNF-oc which can be accessed at GenBank with Accession No. 339764. As one skilled in the art will appreciate, due to the possibilities of the sequencing errors discussed above, the actual, complete Neutrocin oc polypeptide, encoded by the deposited cDNA, comprising approximately 285 amino acids, it can be somewhat shorter. In particular, the determined Neutrocin c coding sequence, contains a second methionine codon that can serve as an alternative start codon for translation of the open reading frame, at nucleotide positions 210-213 in Figure 1 (No SEC ID: 1). More generally, the actual open reading structure can be anywhere in the range of ± 20 amino acids, and more likely in the range of ± 10 amino acids of the predicted from any methionyl codon, either the first or the second, of the term N shown in Figure 1 (SEQ ID No. 1). It will further be appreciated that, depending on the analytical criteria used for the identification of several functional domains, the exact "direction" of the extracellular, intracellular and transmembrane extern d domains of the Neutrocin oc polypeptide may differ slightly. For example, the exact location of the extracellular domain of Neutrocin or c in Figure 1 (SEQ ID No. 2) may vary slightly (for example, the address may be "displaced" by approximately 1 to approximately 20 residues, more likely from about 1 to about 5 residues) depending on the criteria used to define the domain. In this case, the ends of the transmembrane domain and the start of the extracellular domain were predicted based on the identification of the hydrophobic amino acid sequence in the positions indicated above, as shown in Figure 3. In any case, as will be discussed further below, the invention further provides polypeptides having various residues deleted from the N-terminus of the complete polypeptide, including polypeptides lacking one or more amino acids of the N-terminus of the extracellular domain described herein, which constitutes soluble forms of the domain extracellular of the pro tein Neutrocin oc. As indicated, the nucleic acid molecules of the present invention may be in the form of RNA such as mRNA, or in the form of DNA, including, for example, cDNA and genomic DNA obtained by cloning or synthetically produced. DNA can be double-stranded or single-stranded. The single-stranded DNA or RNA can be the coding strand, also known as the sense strand, or it can be > the non-coding strand which is also referred to as the antisense strand. By "isolated molecule" nucleic acid molecule (s) is meant a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Additional examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or DNA molecules purified (partially or substantially) in solution. The molecules of. Isolated RNAs include RNA transcripts, i n vi v or i n vi t ro, of the DNA molecules of the present invention. The nucleic acid molecules isolated in accordance with the present invention further include those synthetically produced molecules. The isolated nucleic acid molecules of the present invention include the DNA molecules which. comprise an open reading frame (ORF) with an initiation codon at positions 147-149 of the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1). In addition nucleic acid molecules isolated, of the invention, include DNA molecules comprising a sequence substantially different from those described above, but due to the degeneracy of the genetic code, they still encode the protein Neutrocin or. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above. In another aspect, the invention provides isolated nucleic acid molecules that encode the Neutrocin ce polypeptide having an amino acid sequence encoded by the cDNA contained in the plasmid deposited on October 22, 1996. Preferably this nucleic acid molecule will comprise a sequence encoding the extracellular domain of the polypeptide encoded by the deposited cDNA clone, described above. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 1 (SEQ ID No. 1) or the nucleotide sequence of the cDNA of Neutrocin or c contained in the deposited clone, described above, or a nucleic acid molecule having a sequence complementary to one of the above sequences. These isolated molecules, particularly the DNA molecules, are useful as probes for the mapping of genes, through in situ hybridization with chromosomes and to detect the expression of Neutrocin c genes in human tissue, for example, by means of the analysis of Stained or Northern Transfer (Northern Blot). The present invention is also focused on molecules of. nucleic acids encoding portions of the nucleotide sequences described herein as well as fragments of the isolated nucleic acid molecules, described herein. In particular the invention provides a polynucleotide having a nucleotide sequence which represents the portion of SEQ ID NO: 1 which consists of positions 1-1001 of SEQ ID NO: 1. In addition, the invention includes a polynucleotide comprising a sequence of at least 95% identical. to any portion of at least about 30 contiguous nucleotides, preferably at least about 50 nucleotides, of the sequence from nucleotide 1 to nucleotide 809 in Figure 1 (SEQ ID NO: 1). More generally, by a fragment of a molecule, of isolated nucleic acid having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) are understood to be fragments of at least about 15 nt , and more preferably at least about 20 nt, and even more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length, which are useful as diagnostic probes and primers , as discussed in the present. Of course larger fragments ranging from 50 to 300 nt in length are also useful in accordance with the present invention, as are the fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or the which is shown in figure 1 (No. SEC ID: 1). For a fragment of at least nt in length, for example, means fragments that include 20 or more contiguous bases of the nucleotide sequence of the deposited cDNA or of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1). Preferred nucleic acid fragments of the present invention include nucleic acid molecules that encode the epitope-containing portions of the Neutrocin oc polypeptide or such as defined in FIG. 1 and described in greater detail below. In another aspect the invention provides a molecule. of isolated nucleic acid comprising a polynucleotide that hybridizes under stringent hybridization conditions in a portion of the polynucleotide in a nucleic acid molecule of the invention, described above, for example, the cDNA clone contained in the ATCC Deposit of October 22 1996. By "stringent hybridization conditions" is meant a whole night of incubation at 40 ° C in a solution that. contains: 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate) 50 mM disodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and salmon sperm DNA, subjected to shear, followed by washing the filters in O.lx SSC at a temperature of approximately 65 ° C. For a polynucleotide that hybridizes to a "portion" of a polynucleotide, is meant a polynucleotide (either DNA or RNA) that hybridizes at least about 15 nucleotides (nt), and most preferably at least about 20 nucleotides , still more preferably at least about 30 nucleotides, and in shape, even more preferably about 30-70 (for example 50) nucleotides of the reference polynucleotide. These are useful as diagnostic probes and primers, such as, discussed above and in more detail later. For a portion of a polynucleotide of "at least 20 nucleotides in length", for example, is understood to be 20 or more contiguous nucleotides of the nucleotide sequence of the reference polynucleotide (for example, the deposited cDNA or the nucleotide sequence shown in Figure 1 (SEQ ID No.) : 1) ) . Of course, a polynucleotide that hybridizes only in a poly A sequence (such as the terminal poly (A) 3 'extension of the Neutrokine cDNA shown in Figure 1 (SEQ ID NO: 1)), or in a The complementary stretch of T (or U) residues would not be included in an in-built po 1 of the invention used to hybridize to a portion of the nucleic acid of the invention, since that polynucleotide would hybridize to any nucleic acid molecule containing a nucleic acid molecule. poly stretch (A) or its complement (for example, practically any clone of double-stranded cDNA). As indicated, the nucleic acid molecules of the present invention that encode a Neutrocin oc polypeptide can include, but are not limited to, those that encode the amino acid sequence of the extracellular domain of the polypeptide, per se; and the coding sequence for the extracellular domain of the polypeptide and additional sequences, such as those encoding the sequences of the transmembrane and intracellular domain, or a sequence of a preprotein, or proprotein or preprotein; the coding sequence of the extracellular domain of the polypeptide, with or without the additional coding sequences mentioned above. Also encoded by nucleic acids of the invention are the above protein sequences together with additional non-coding sequences including, for example, but not limited to, the introns and the 5 'and 3' non-coding sequences, such as the transcribed sequences, untranslated, which play a role in the transcription, in the processing of the mRNA, including the signals of bonding and binding and polyadenylation, for example the binding of ribosomes and the stability of the mRNA; an additional coding sequence that codes for additional amino acids such as those that provide additional functionalities. Thus, the sequence encoding the polypeptide can be fused to a marker sequence such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-hi s t idin peptide, such as the tag or tag sequence provided in the pQE vector.

(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz et al., Pro c. Na t i. Ac a d. S c i. USA 8: 821-824 (1989), for example, hexa-histidine provides convenient purification of the fusion protein. The tag or "HA" marker sequence is another peptide useful for purification that corresponds to an epitope derived from the influenza hemagglutinin protein which has been described by Wilson et al., Cel l 3 7: 1 6 1 (1984) . As discussed below, other such fusion proteins include Neutrocin or fc fused at the N or C terminus.

Polynucleo tidps Varian tes y Mu tan tes The present invention also relates to variants of the nucleic acid molecules of the present invention which encode portions, analogs or derivatives of the protein Neutrocin oc. Variants can occur naturally, such as a natural allelic variant. By an "allelic variant" is meant one of several alternative forms of a gene occupying a given site of a chromosome or of an organism. Gen e s II, Lewin, B., ed., John Wiley & Sons, New York (1985). Variants that do not occur naturally can be produced using mutagenesis techniques used in the field. These variants include those produced by substitutions, eliminations or nucleotide additions. Substitutions, deletions or additions may involve one or more nucleotides. The variants can be altered in coding regions, in non-coding regions or in both. Alterations in the coding regions can produce, deletions or additions, conservative or non-conservative amino acid substitutions. Especially preferred among these are the silent substitutions, additions and deletions that do not alter the properties and activities of the Neutrocin protein or portions of it. Also especially preferred in this respect are conservative substitutions. The most highly preferred are nucleic acid molecules that encode the extracellular domain of the protein having the amino acid sequence shown in Figure 1 (SEQ ID No. 2) or the extracellular domain of the amino acid sequence of Neutrocin c or coded by the deposited cDNA clone. Additional embodiments include an isolated nucleic acid molecule comprising a polynucleotide having at least 90% identical nucleotide sequence, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to a selected polynucleotide of the group consisting of: (a) a nucleotide sequence encoding the Neutrocin oc polypeptide having the complete amino acid sequence of Figure 1 (SEQ ID NO: 2); (b) a nucleotide sequence encoding the predicted extracellular domain of the Neutrocin oc polypeptide having the amino acid sequence at positions 73-285 in Figure 1 (SEQ ID NO: 2); (c) a nucleotide sequence encoding the Neutrocin oc polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996; (d) a nucleotide sequence encoding the extracellular domain of the Neutrocin oc polypeptide having the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996; and (e) a complementary nucleotide sequence for any of the nucleotide sequences of parts (a), (b), (c) or (d) above. For a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a Neutrocin oc polypeptide, it is understood that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence encoding the Neutrocin oc polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or a number of nucleotides Up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These reference sequence mutations can occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, either interspersed individually between the nucleotides found in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleotide sequence shown in the figure below. the nucleotide sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the program Bestfit (Wisconsin Sequence Analysis Package, Version 8 for Unix, University Research Park, 575 Science Drive, Madison, Wl 53711). The Bestfit uses the local homology algorithm of Smith and Waterman, Adva n c e s i n Appl i ed Ma th em a t i c s 2: 482-489 (1981), to find the best homology segment between the two sequences. When the Bestfit or any other program is used for the alignment of the sequence, to determine if a particular sequence is, for example, 95% identical to the reference sequence according to the present invention, the parameters are of course adjusted to such that the percentage of identity is calculated over the entire length of the reference nucleotide sequence and that clearances or cleavages in homology are allowed, up to 5% of the total number of nucleotides in the reference sequence. The present application is focused on nucleic acid molecules at least 90%91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figure 1 (SEQ ID No. 1) or the nucleic acid sequence of the deposited cDNA, regardless of whether they encode a polypeptide having an activity such as that of Neutrocin oc. This is because although a particular nucleic acid molecule does not encode a polypeptide having an activity of Neutrocin or c, a person skilled in the art will still know how to use the nucleic acid molecule, for example, as a hybridization probe or a primer for polymerase chain reaction (PCR). The uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having an activity of Neutrocin or c include, inter alia, (1) isolating the Neutrocin c gene or allelic variants thereof in a cDNA library.; (2) Insitu hybridization (eg "FISH") for metaphase chromosome spreads, to provide accurate chromosomal location of the Neutrocin oc genes, as described by Verma et al., Human Chromo somes: A Man ual of Ba si c Techni ques, Pergamon Press, New York (1988); and the analysis of Spotting or Northern Transfer to detect the expression of the mRNA of Neutrocin oc in specific tissues. However, nucleic acid molecules having at least 90%, 95%, 96%, 97%, 98% or 99% sequences identical to the nucleic acid sequence shown in Figure 1 are preferred (SEQ ID NO. : 1) or to the nucleic acid sequence of the deposited cDNA which in fact encodes a polypeptide having an activity of the protein Neutrocin oc. By "a polypeptide having Neutrocin oc activity" is meant polypeptides that exhibit activity similar, though not necessarily identical, to an activity of the extracellular domain of the full-length protein Neutrocin ce of the invention, as measured in a particular biological trial. For example, the Neutrocin oc protein of the present invention modulates cell proliferation, cytotoxicity and cell death. An assay for cell proliferation in vi tro, cytotoxicity and cell death, to measure the effect of a protein on certain cells, can be performed using well-known reagents commonly available in the art to detect cellular reduplication and / or cell death For example, numerous of these assays for protein activities related to TNF are described in the various references, in the background section of this description. Briefly, one of these assays involves collecting human or animal (eg mouse) cells and mixing them with (1) a supernatant from the transfected host cell containing the Neutrocin protein oc (or a candidate polypeptide) or (2) the control of the supernatant of the non-transfected host cells, and to measure the effect on the number of cells or on the viability after the incubation of a certain period of time. Those activities of modulating cell proliferation, as can be measured in this type of assay, are useful for treating tumors, tumor metastasis, infections, inflammation by autoimmune diseases and other related immunological diseases. Neutrocin oc modulates cell proliferation and differentiation in a dose-dependent manner in the assay described above. Thus, "a polypeptide having an activity of the protein Neutrocin oc" includes polypeptides that also exhibit any of the same cell modulation activities (particularly immunomodulatory) in the assays described above, in a dose-dependent manner. Although the dose dependent activity level need not be identical to that of the Neutrocin oc protein, preferably "a polypeptide having an activity of the Neutrocin oc protein" will exhibit substantially similar dependence on a given activity when compared to the protein Neutrocin oc ( that is, the candidate polypeptide will exhibit greater activity or no more than about 25 times less and, preferably, no more than about 10 times the activity relative to the reference Neutrocin protein or c). Like other members of the TNF family, Neutrocin oc exhibits activity in leukocytes including, for example, monocytes, lymphocytes and neutrophils. For this reason Neutrocin ce is active in directing the proliferation, differentiation and migration of these cell types. This activity is useful for the intensification or immunological suppression, for the mi pro loc tion, for the mobilization of the stem or stem cells, for the acute and chronic inflammatory control and for the treatment of the leukemia. Tests for the measurement of this activity are known in the art. For example, see Peters et al. Imm a. Today 1 7: 2 1 3 (1996); Young et al. , j.

Exp. Med. 182: 1111 (1995); Caux et al. , Nature 390: 258 (1992); and Sant iago-Schwar z et al. , Adv. Exp. Med. Bio. 378: 1 (1995). "] Of course, due to the degeneracy of the genetic code, a person of ordinary skill in the art will immediately recognize that a large number of nucleic acid molecules having a sequence of at least 90%, 95 %, 96%, 97%, 98% or 99% identical to the nucleic acid sequences of the cDNA deposited to the nucleic acid sequence shown in Figure 1 (SEQ ID NO: 1) will encode a polypeptide "having an activity of the protein Neutrocin oc. "Indeed, since the degenerate variants of these nucleotide sequences encode all the same polypeptide, this will be clear to the one skilled in the art even without performing the comparison test described above. in the art that, for those nucleic acid molecules that are not degenerate variants, a reasonable number also encodes a polypeptide having an activity of the Neutrokine protein This is because the one skilled in the art is fully aware of amino acid substitutions that are less likely or that are not likely to significantly effect protein function (for example, replacing an aliphatic amino acid with a second aliphatic amino acid), as will be described further below.

Vectors and Host Cells The present invention also relates to vectors that include the isolated DNA molecules of the present invention, host cells which have been genetically engineered with the recombinant vectors, and the production of Neutrocin oc polypeptides or fragments thereof , using recombinant techniques. The vector can be, for example, a phage, a plasmid, or a viral or retroviral vector. Retroviral vectors may be competent in reduplication or defective in reduplication. In the latter case, viral propagation will generally occur only in complementary host cells. The polynucleotides can be linked to a vector that contains a selectable marker for propagation in a host. Generally, a plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or into a complex with a charged lipid. If the vector is a virus, it can be packaged using an appropriate packaging cell line, and then transduced into the host cells. The DNA insert should be functionally linked to an appropriate promoter, such as the phage lambda PL promoter, the a c, t rp, ph or A of E. c ol i and the t a c promoters, the early and late SV40 promoters and the retroviral LTR promoters, to name a few. Other suitable promoters will be known to those skilled in the art. The expression constructs will additionally contain sites for the initiation of transcription, for the termination thereof and, for the region described, a site for the binding of the ribosome for translation. The coding portion of the extracellular domain of the transcripts expressed by the constructs will preferably include a translation that starts at the beginning and at a stop codon (UAA, UGA or UAG) appropriately located at the end of the polypeptide to be translated. As indicated, the expression vectors will preferably include at least one selectable marker. These markers include dihydro folate reductase, G418 or neomycin resistance for a culture of eukaryotic cells and genes resistant to tetracycline, kanamycin or ampicillin for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. c ol i, the Trep tomi ees and the Salmon cells the l a typh im uri um; fungal cells such as yeast cells; insect cells such as Drosophila S2 cells and Spodoptera Sf9; animal cells such as CHO, COS, 293 and Bowes melanoma cells; and the plant cells. The appropriate culture media as well as the appropriate conditions for the host cells described above are known in the art.

Preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc., supra; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNHldA, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, p-DR540, pRIT5 available from Pharmacia. Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and the pSVL available from Pharmacia. Other suitable vectors will be readily apparent to one skilled in the art. The introduction of the construct into the host cell can be effected by transfection with calcium phosphate, by transfection mediated with DEAE-dexthan, by transfection mediated with cationic lipid, by electroporation, transduction, infection or other methods. These methods are described in many standard laboratory manuals such as the Davis et al., Basic Methods in Molecular Biology (1986). The polypeptide can be expressed in a modified form such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N terminus of the polypeptide, to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, to facilitate purification, portions of peptides can be added to the polypeptide. These regions can be eliminated before the final preparation of the polypeptide. The addition of the peptide portions to the polypeptides, to engender the secretion or excretion, to improve the stability and to facilitate the purification, among others, are familiar and routine techniques in the field. A preferred fusion protein comprises a heterologous immunoglobulin region that is useful for stabilizing and purifying proteins. For example, EP-A-0 464 533 (Canadian Counterpart 2045869) discloses fusion proteins comprising several constant region portions of immunoglobulin molecules, together with another human protothein or part thereof. In many cases, the Fc part in a fusion protein is completely advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A-0232262). On the other hand, for some uses it would be desirable to be able to remove the Fc part after the fusion protein is expressed, detected and purified in the advantageous manner described. This is the case when the Fc portion proves to be an impediment to use in therapy and diagnosis, for example, when the fusion protein is to be used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high throughput selective screening assays, to identify antagonists of hIL-5. See, D. Bennett et al., J. Molecular Recognition 8: 52-58 (1995) and K. Johanson et al. , J. Biol. Chem. 270: 9459-9411 (nineteen ninety five) . The Neutrocin c protein can be recovered and purified from recombinant cell cultures, through well-known methods, including precipitation with ammonium sulfate or with ethanol, extraction with acid, anionic or cation exchange chromatography, chromatography with fos focelulose, chromatography by hydrophobic interaction, affinity chromatography, chromatography with hydroxy lapa tit and chromatography with lectin. Most preferably, high-performance liquid chromatography is used for purification ("HPLC"). The polypeptides of the present invention include the naturally purified products, the products of the synthetic chemical processes, and products produced through recombinant techniques from a prokaryotic or eukaryotic host, including, for example, the bacterial cells of yeasts, higher plants, insects and mammals. Depending on the host employed in a recombinant production process, the polypeptides of the present invention can be glycosylated or they can be non-glycosylated. In addition, the polypeptides of the invention may also include a modified, initial methionine residue, in some cases as a result of processes mediated by the host.

Neutrocin 8 and Fragment polypeptides cough The invention further provides an isolated Neutrocin oc polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence shown in Figure 1 (SEQ ID NO: 2), or a peptide or polypeptide comprising a portion of the above polypeptides.

Polypeptides Varian tes and Mutan tes To improve or alter the characteristics of Neutrocin 8 polypeptides, engineering manipulation of proteins can be employed. Recombinant DNA technology, known to those skilled in the art, can be used to create novel mutant proteins or "muteins" that include single or multiple amino acid substitutions, deletions thereof, additions thereof, or fusion proteins . These modified polypeptides can show, for example, enhanced activity or increased stability. In addition, they can be purified in high yields and show a better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.

So many Elimination N-terminal and C- terminals For example, for many proteins that include the extracellular domain or the mature form (s) of a secreted protein, it is known in the art that one or more amino acids can be deleted from the N term or the C term, without substantial loss of biological function. For example, Ron et al., J. Bi ol. Ch em 268: 2 98 4 - 2 98 8 (1993) reported modified KGF proteins that had a binding activity to heparin, even if 3, 8, or 27 amino-terminal amino acid residues were lost. In the present case, since the protein of the invention is a member of the TNF-polypeptide family, the deletions of the N-terminal amino acids up to the residue Gly (G) at position 191 of Figure 1 (No. SEC: 2) may retain some biological activity such as cytotoxicity towards appropriate target cells. Polypeptides having additional N-terminal deletions including the Gly (G) residue would not be expected to retain those biological activities because this residue in the TNF-related polypeptides is known to be at the start of the conserved domain, required for the biological activities. However, even if this removal of one or more amino acids from the N-terminus of a protein results in a modification of loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened protein to induce and / or bind to antibodies that recognize the complete or extracellular domain of the protein will generally be conserved when the N terminus is removed from most of the residues of the entire or extracellular domain. If a particular polypeptide that lacks N-terminal residues of a complete protein, retains those activities unological or not, it can be readily determined by routine methods described herein and otherwise known in the art. Accordingly, the present invention further provides polypeptides having one or more residues of the amino terminus of the amino acid sequence of Neutrocin ce shown in Figure 1 (SEQ ID NO: 2), up to residue Gly 191 of the amino terminus, and polynucleotides encoding those polypeptides. In particular, the present invention provides polypeptides having the amino acid sequence of residues n-190 of SEQ ID NO: 2, wherein n is an integer that is in the range of 2 to 190 and 191 is the position of the first residue of the N terminus of the complete Neutrocin oc polypeptide (shown in SEQ ID NO: 2) that is believed to be required for the activity of the Neutrocin oc protein. More particularly, the invention provides polynucleotides encoding polypeptides having the amino acid sequence of residues2-285, 3-285, 4-285, 5-285, 6-285, 7-285, 8-285, 9-285, 10-285, 11-285, 12-285, 13-285, 14-285, 15 -285, 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285 , 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285,. 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52- 285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 71-285, 72-285, 73-285, 74-285, 75-285, 76-285, 77- 285, 78-285, 79-285, 80-285, 81-285, 82-285, 83-285, 84-285, 85-285, 86-285, 87-285, 88-285, 89-285, 90-285, 91-285, 92-285, 93-285, 94-285, 95-285, 96-285, 97-285, 98-285, 99-285, 100-285, 101-285, 102- 285, 103-285, 104-285, 105-285, 106-285, 107-285, 108-285, 109-285, 110-285, 111-285, 112-285, 113-285, 114-285, 115-285, 116-285, 117-285, 118-285, 119-285, 120-285, 121-285, 122-285, 123-285, 124-285, 125-285, 126-285, 127- 285, 128-285, 129-285, 130-285, 131-285, 132-285, 133-285, 134-285, 135-285, 136-285, 137-285, 138-285, 139-285, 140-285, 141-285, 142-285, 143-285, 144-285, 145-285, 146-285, 147-285, 148-285, 149-285, 150-285, 151-285, 152- 285, 153-285, 154-285, 155-285, 156-285, 157-285 , 158-285, 159-285, 160-285, 161-285, 162-285, 163-285, 164-285, 165-285, 166-285, 167-285, 168-285, 169-285, 170 -285, 171-285, 172-285, 173-285, 174-285, 175-285, 176-285, 177-285, 178-285, 179-285, 180-285, 181-285, 182-285 , 183-285, 184-285, 185-285, 186-285, 187-285, 188-285, 189-285, 190-285, of SEQ ID NO: 2. Polynucleotides encoding these polypeptides are also provided. . Similarly, many examples of biologically functional C-terminal elimination muteins are known. For example, interferon gamma shows up to 10 times higher activity by eliminating 8 to 10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J. Biotech ch ol olgy 7: 199-216 (1988). that the protein of the present is a member of the TNF polypeptide family, the eliminations of the amino acids • C-terminal is that it reaches the Leu at position 284, it is expected to retain the majority, if not all the activity biological, such as binding to the receptor and modulation of duplication Polypeptides having deletions of up to about 10 additional C-terminal residues (ie, up to residue Gly at position 273) can also retain some activity such as binding to the receptor, although those, for example, would lack a portion of the conserved TNF domain, starting approximately at Leu 284. However, even if the elimination of one or more amino acids, of the C term of a protein, results in the modification of the loss of one or more biological functions of the protein, other biological activities could still be retained. Thus, the ability of the shortened protein to induce and / or bind to antibodies that recognize the complete or mature protein will generally be retained when less than the majority of the complete or mature protein residues are removed from the C terminus. If a particular polypeptide lacking the C-terminal residues of a complete protein, whether or not it retains those immunological activities, can be readily determined by routine methods described herein and otherwise known in the art. Accordingly, the present invention further provides polypeptides having one or more residues of the carboxy terminus of the amino acid sequence of Neutrocin oc shown in Figure 1 (SEQ ID NO: 2), up to the Gly274 residue of the carboxy terminus, and the polynucleotides encoding those polypeptides. In particular, the present invention provides polypeptides having the amino acid sequence of the 1-m residues of the amino acid sequence in SEQ ID NO: 2, where m is any integer that is in the range of 274 a 284. More particularly, the invention provides polynucleotides encoding polypeptides having the amino acid sequence of residues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1 -282, 1-283 and 1-284 of SEQ ID NO: 2. Nucleic acid polymers encoding these polypeptides are also provided. Also provided are polypeptides having one or more amino acids deleted from both the amino and carboxyl terms, which can be described, in general, as having n-m residues of SEQ ID NO: 2.; where n and m are integers as described above. Also included is a nucleotide sequence encoding a polypeptide consisting of a portion of the amino acid sequence of Neutrocin c, complete, encoded by the cDNA clone contained in the ATCC Deposit of October 22, 1996 where this portion excludes the 190 amino acids of the amino terminus or from one to 11 amino acids of the C-terminus of the complete amino acid sequence (or any combination of these N-terminal and C-terminal deletions) encoded by the cDNA clone in the deposited clone. Polynucleotides encoding all of the above elimination polypeptides are also provided.

Other Mutan tes In addition to the terminal deletion forms of the protein discussed above, a person skilled in the art will recognize that some of the amino acid sequences of the Neutrocin oc polypeptide can be varied without having any significant effect on the structure or function of the protein. . If those differences are contemplated in the sequence, it should be remembered that there will be critical areas in the protein that determine the activity. In this manner the invention further includes variations of the Neutrocin c polypeptide that show a substantial activity of the Neutrocin c polypeptide or that include regions of the Neutrocin c protein such as the protein portions discussed below. These mutants include deletions, insertions, inversions, repeats, and type substitutions, selected according to general rules known in the art to have a small effect on activity. For example, guidance is provided concerning how to produce substitutions of typically silent phenoid amino acids, in Bowie, JU et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Subs t t i ts," S ci en ce 247 : 1306-1310 (1990), where the authors indicate that there are two main approaches to study the tolerance of an amino acid sequence, to the change. The first method is based on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to induce amino acid changes at specific positions of a cloned gene, and selections or selective classifications, to identify sequences that maintain functionality. As the authors state, these studies have revealed which proteins are surprisingly tolerated by amino acid substitutions. The authors also indicate that changes in amino acids are likely to be permissive at a certain position of the protein. For example, the most hidden amino acid residues require non-polar side chains, while generally few features of the surface side chains are retained. Other typical and activated phenol subscripts are described in Bowie, J. U. et al. , upra, and the references cited therein. Typically seen as conservative substitutions are the replacements, one for the other, between the aliphatic amino acids Ala, Val, Leu and lie; the exchange of the hydroxyl residues, Ser and Thr, the exchange of the acid residues Asp and Glu, the substitution between the amide residues Asn and Gln, the exchange of the basic residues Lys and Arg and the replacements between the aromatic residues Phe, Tyr. Thus, the derivative or analogue fragment of the polypeptide of Figure 1 (SEQ ID NO: 2), or that encoded by the deposited cDNA, can be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and a substituted amino acid residue, such that it may or may not be encoded by the genetic code, or (ii) one in which one or more of the residues of amino acids include a substituent group, or (iii) one in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (eg, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the extracellular domain of the polypeptide, such as a peptide or guide or secretory sequence, of the IgG Fc fusion region, or a sequence that is used for the purification of the extracellular domain of the polypeptide or a protein sequence. These fragments, derivatives or analogs are considered to be within the scope of the teachings of those skilled in the art. Thus, Neutrocin oc of the present invention may include one or more substitutions, deletions or additions, of amino acids, either from natural mutations or by human manipulation. As indicated, changes of a minor nature, such as conservative amino acid substitutions, that do not significantly affect the folding or activity of the protein are preferable (see Table 1).

Table I: Amino Acid Preservative Subsitutions Table I (Continued) Amino Acid Conservative Substitutions The amino acids in the Neutrocin ce protein of the present invention, for function, can be identified by methods known in the art, such as site-directed mutagenesis or alanine tracking mutagenesis (Cunningham and Wells, Science 244). : 1081-1085 (1989)). The last procedure introduces unique alanine mutations, in each residue in the molecule. The resulting mutant molecules are then analyzed for biological activity such as binding to the receptor or proliferating activity in vitro or in vivo. Of particular interest are substitutions of charged amino acids with other charged or neutral amino acids that can produce highly desirable enhanced character proteins, such as reduced aggregation. Aggregation may not only produce the activity, but may also be problematic when pharmaceutical formulations are prepared, because the aggregates may be immunogenic (Pinckard et al., Clin Exp. Immunol., 2: 331-340 (1967).; Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993) .Amino acid replacement can also change the binding selectivity of a ligand to cell surface receptors For example, Ostade, et al., Nature 361: 266-268 (1993) describes certain mutations that result in the selective binding of TNF-oc to only one of the two Known types of TNF receptors Since Neutrocin oc is a member of the TNF family of polypeptides, it is likely that mutations similar to those occurring in TNF-oc have similar effects in Neutrocin oc. for linking 1 i gan Do-receptor can also be determined through structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity-labeled (Smith et al. , J. Mol. Biol. 224: 899-904 (1992) and de Vos et al. Science 255: 306-312 (1992)). Since Neutrocin oc is a member of the family of proteins related to TNF, to modulate rather than completely eliminate the biological activities of Neutrocin oc, the mutations are preferably performed on sequences encoding amino acids in the conserved domain of TNF, that is, in positions 191-284 of Figure 1 (SEQ ID NO: 2), more preferably in residues that are within this region and that are not conserved in all members of the TGF family. by performing a specific mutation in Neutrocin or in the position where that conserved amino acid is typically found in related TNFs, Neutrocin or oc will act as an antagonist, thus possessing angiogenic activity. Accordingly, the polypeptides of the present invention include the Neutrocin oc mutants. These Neutrocin oc mutants are comprised of the full length extracellular domain or preferably the extracellular domain of the amino acid sequence shown in Figure 1 (SEQ ID NO: 2). Also forming part of the present invention are the isolated polynucleotides comprising nucleic acid sequences encoding the Neutrocin c or earlier mutants. The polypeptides of the present invention are preferably provided in an isolated form, and are preferably substantially purified. A recombinantly produced version of the Neutrocin oc polypeptide can be substantially purified through the one step method described in Smith and Johnson, Gen e 57: 31-40 (1988). The polypeptides of the present invention include the entire polypeptide encoded by the deposited cDNA, which includes the intracellular, transdermal, and extracellular domains of the polypeptide encoded by the deposited cDNA., the extracellular domain minus the intracellular and transmembrane domains of the protein, the complete polypeptide of Figure 1 (SEQ ID NO: 2), the extracellular domain of Figure 1 (SEQ ID NO: 2) minus the intracellular domains and t ransmembrani co, as well as polypeptides having at least 90% similarity, more preferably at least 95% similarity, and even more preferably at least 96%, 97%, 98% or 99% similarity with those described above. Additional polypeptides of the present invention include polypeptides that are at least 80% identical, more preferably at least 90% or 95% identical, and even more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA or the polypeptide of Figure 1 (SEQ ID NO: 2), and also include portions of those polypeptides with at least 30 amino acids and more preferably at least 50 amino acids. By "% similarity" for two polypeptides, we mean a similarity score produced when comparing the amino acid sequences of the two po 1 -eptides, using the Bestift program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 53711) and the default settings, to determine similarity. The Bestfit program uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489, 1981) to find the best segment of similarity between two sequences. For a polypeptide having an amino acid sequence, for example, at least 95% "identical" to a reference amino acid sequence, of a Neutrocin oc polypeptide, it is understood that the amino acid sequence of the polypeptide is identical to the sequence of reference, except that the polypeptide sequence may include up to 5 alterations in the amino acid per 100 amino acids of the reference amino acid of the Neutrocin polypeptide or. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence can be removed or replaced with another amino acid, or A number of amino acids of up to 5% of the total amino acid residues in the reference sequence can be removed or inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal, intermixed positions, and either individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, it can be determined, for example, whether a particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical, for example, to the amino acid sequence shown in Figure 1 (SEQ ID No. 2) or the amino acid sequence encoded by the deposited cDNA clone, and this determination can be made conventionally using known computer programs, such as the Bestfit program (Wiscosin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 53711). When the Bestfit or any other program is used for sequence alignment, to determine whether a particular sequence is, for example, 95% identical to a reference sequence in accordance with the present invention, the parameters are, of course, adjusted in such a way that the percent identity is calculated across the entire length of the reference amino acid sequence and that spaces or differences in homology are allowed, up to 5% the total number of amino acid residues in the sequence reference. The polypeptide of the present invention could be used as a marker for molecular weights on SDS-PAGE gels or gel filtration columns with molecular sieves, using methods well known to those skilled in the art. As described in detail below, the polypeptides of the present invention can also be used to elevate or increase polyclonal and monoclonal antibodies, which are useful in assays for detecting the expression of the Neutrocin c protein as described below, or as agonists. and antagonists capable of enhancing or inhibiting the function of the protein Neutrocin oc. In addition, these polypeptides can be used in a yeast two-hybrid system to "capture" proteins that bind Neutrocin or protein that are also candidate agonists and candidate antagonists according to the present invention. The yeast two-hybrid system is described in Fields and Song, Nature 340: 245-246 (1989).

Portions Containing a Capped Cap In another aspect the invention provides a peptide or polypeptide comprising a portion containing an epitope, of a polypeptide of the invention. The epitope of this portion of the polypeptide is an epitope, immunogenic or antigenic, of a polypeptide of the invention. An "immunogenic epitope" is defined as a part of a protein that evokes an antibody response when the entire protein is the immunogen. On the other hand, a region of a protein molecule to which an antibody can be linked is defined as an "antigenic epitope". The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al. , Pro c. Na t i. Aca d. Sci. USA 82: 3998-4002 (1983). Regarding the selection of peptides or polypeptides that contain an antigenic epitope (ie, they contain a region of a protein molecule to which an antibody can be linked), it is well known in the art that relatively short, synthetic peptides, which resemble part of a protein sequence, are routinely capable of evoke an antiserum that reacts with the partially resembled protein. See, for example, Sutcliffe, J.G., Shinnick, T.M., Green, N. and Learner, R.A. (1983) "Antibodies that react with predetermined sites on proteins", Science, 219: 660-666. Peptides capable of producing sera reactive with the protein, 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 immunodominic regions or protein intact (i.e. , immunogenic epitopes) or amino or carboxyl terminals. The peptides and polypeptides containing antigenic epitopes of the invention are therefore useful for increasing antibodies, including monoclonal antibodies, which specifically bind to a polypeptide of the invention. See, for example, Wilson et al. , Cell 37: 1-118 (1984) in 777. The peptides and polypeptides containing antigenic epitopes of the invention preferably contain a sequence of at least seven, more preferably at least nine and more preferably between about 15 and about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. Non-limiting examples of antigenic polypeptides or peptides, which can be used to generate antibodies specific for Neutrocin, and include: a polypeptide comprising amino acid residues from about Phe 115 to about Leu 147 of Figure 1 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about 150 to about Tyr 163 of Figure 1 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser 171 to about Phe 194 of Figure 1 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Glu 223 to about Tyr 247 of Figure 1 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser 271 to about Phe 278 of Figure 1 (SEQ ID NO: 2). It has been determined that these polypeptide fragments contain antigenic epitopes of the protein Neutrocin oc, and this has been done by the analysis of the Jameson-Wolf antigenic index, as shown in Figure 3, above. The peptides and polypeptides comprising the epitope of the invention can be produced by any conventional method. See, for example, Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigenic-ant ibody interaction at the level of individua.l amino acids. Pro c. Na t i Aca d. Sci. USA 82: 5131-5135; this process of "Simultaneous Multiple Peptide Synthesis (SMPS)" is further described in U.S. Patent No. 4,631,211 issued to Houghten et al. (1986). Peptides and polypeptides containing an epitope, of the present invention, are used to induce antibodies, in accordance with methods well known in the art. See, for example, Sutcliffe et al., Supra; Wilson et al., Supra; Chow, M. et al., Proc. Nati, Acad.

Sci. USA 82: 910-914; and Bittle, F. J. et al., J. Gen. Virol. 66: 2347-2354 (1985). Epitope-containing, immunogenic peptides of the invention, ie, those portions of a protein that elicit an antibody response when all of the protein is the immunogen, are identified according to methods well known in the art. See, for example, Geysen et al., S upra. In addition, US Patent No. 5, 194,392 issued to Geysen (1990) describes a general method to detect or determine the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (for example a "mimotope") that is complementary to a particular paratope (site of binding to the antigen) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 issued to Geysen (1989) discloses a method for detecting or determining a sequence of monomers that is a topographic equivalent of a ligand that is complementary to the site of binding of the ligand, a particular recipient of interest. Similarly, U.S. Patent No. 5,480,971 issued to Houghten, RA et al (1996), which refers to Mixtures of 01 and Peralkylates, describes olipeptides for which alkyl with one to seven carbon atoms. , linear, and groups and libraries of t peptides, as well as to methods for using those groups and libraries of Igitope, to determine the sequence of a peralkylated oligopeptide that is preferentially linked to an accepting molecule of interest. In this manner, non-peptide analogs of the epitope-possessing peptides of the invention can also be produced routinely by t methods.

Fusion Theories As one skilled in the art will appreciate, the polypeptides of the present invention and epitope-possessing fragments thereof, described above, can be combined with portions of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. . T fusion proteins facilitate purification and show an increased half-life, in vivo. This has been demonstrated, for example, for chimeric proteins consisting of the first two domains of the human CD4 polypeptide and several domains of the constant regions of the heavy or light chains of mammalian immunoglobulin (EP A 394,827; Traunecker et al., Nature 331: 84-86 (1988)). Fusion proteins that have a disulfide linked dimeric structure, due to the IgG part, may also be more efficient in binding and neutralizing other molecules, than the Neutron or monomeric protein or protein fragment alone (Fountoul aki s et al., J. Bi or ch em. 2 70: 3958-3964 (1995)).

Diagnosis of Trasnos Related to the Immunological System The inventors of the present have discovered that Neutrocin oc is expressed in various tissues and particularly in neutrophils. For a number of disorders related to the immune system, substantially altered (increased or decreased) levels of Neutrocin c gene expression can be detected in tissues of the immune system or other cells or body fluids (eg, sera, plasma, urine, synovial fluid or spinal fluid) taken from an individual who has such a disorder, in relation to a level of gene expression of Neutrocin or "normal" that is, the level of expression of Neutrocin or in tissues of the immune system or fluids bodily of an individual who does not have the disorder in the immune system. In this way the invention provides a diagnostic method, useful during the diagnosis of a disorder of the system, which involves measuring the level of expression of the gene encoding the protein Neutrocin ce in the tissue of the immune system or other cells or body fluids. , of an individual, and compare the level of genetic expression measured, with a level of gene expression of Neutrocin oc, whereby a decrease or increase in the level of genetic expression, compared with normal, is indicative of a disorder of the immune system. In particular it is believed that certain tissues found in mammals with cancer of the immune system express significantly increased or reduced levels of the protein Neutrocin oc and the mRNA that encodes the protein Neutrocin oc, when compared to a corresponding "normal" level. In addition, it is believed that increased or decreased levels of the Neutrocin ce protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) of mammals with cancer, when compared to mammalian sera. of the same species that do not have cancer. In this way, the invention provides a diagnostic method useful during the diagnosis of an immune system disorder, including cancers of this system, which involves measuring the level of expression of the gene encoding the protein Neutrocin or c in tissues of the immune system or other cells or body fluid of an individual, and compare the level of gene expression measured with a level of gene expression of neutrocy ce, normal or pattern, whereby an increase or decrease in the level of gene expression compared to the pattern, it is indicative of a disorder of the immune system. When a diagnosis of a disorder in the immune system, including the diagnosis of a tumor, already been performed, according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting a genetic expression of Neutrokine or c, increased or decreased, will experience a worse clinical outcome in relation to patients who express the gene at a level closer to the normal level. By "assaying the level of expression of the gene encoding the protein Neutrocin oc" is meant measuring or estimating, qualitatively or quantitatively, the level of the protein Neutrocin or oe 1 level of the mRNA that encodes the protein Neutrocin or c in a first biological sample, either directly (for example, by determining or estimating the absolute level of proteins or the absolute level of mRNA) or relatively (for example, by comparing Neutrocin c protein level or mRNA level in a second sample biological). Preferably, the level of protein Neutrocin oc or the level of mRNA in the first biological sample is measured or estimated, and compared to a level of protein Neutrocin oc or mRNA level, normal, and the pattern is taken from a second biological sample obtained from an individual who does not have the disorder or is determined by averaging levels from a population of individuals who do not have an immune system disorder. As will be appreciated in the art, once the normal level of the neutrocylco protein or the normal level of mRNA is known, it can be repeatedly used as a standard or standard for comparison. By "biological sample" is meant any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source containing the protein Neutrocin or the mRNA. As indicated, biological samples include bodily fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) that contain the free extracellular domains of the Neutrocin ce protein, immune system tissue, and other tissue sources that have been found that they express the free or complete extracellular domain of Neutrocin sc or a Neutrocin oc receptor. Methods for obtaining tissue and body fluid biopsies from mammals are well known in the art. Where the biological sample will include mRNA, a tissue biopsy is the preferred source. The present invention is useful for the diagnosis or treatment of various disorders related to the immune system, in mammals, preferably in humans. These disorders include, but are not limited to, tumors and tumor metastases, infections by bacteria, viruses and other parasites, immunode fi ciencies, inflammatory diseases, 1 infadenopathy, autoimmune diseases, and graft-versus-host disease. Total cellular RNA can be isolated from a biological sample, using any suitable technique such as the one-step method using guanidini or-1-iocynate-phenol-chloroform, described in Chomczynski and Sacchi, Anal, Biochem. 162: 156-159 (1987). Then, the mRNA levels that encode the protein Neutrocin oc are analyzed, using some suitable method. These include the analysis of staining or Northern blot, the mapping of nuclease SI, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) , and reverse transcription in combination with the ligase chain reaction (RT-LCR). The assay or analysis of the levels of the protein Neutrocin oc, in a biological sample, can occur using techniques based on antibodies. For example, the expression of the protein Neutrocin ce in tissues can be studied with classical immunological methods (Jalkanen, M., et al., J. Cell, Biol. 202: 976-985 (1985).; Jalkanen, M., et al. , J. Cell. Biol. 105: 3081-3096 (1987)). Other antibody-based methods, useful for detecting the gene expression of the Neutrocin oc protein, include the immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Labels or labels suitable for assays with antibodies are known in the art and include enzyme labels, such as glucose oxidase, and radioisotopes, such as iodine (125 I, 121 I), carbon (14 C), sulfur (35 S) , tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin. In addition to analyzing the levels of the protein Neutrocin oc, in a biological sample obtained from an individual, the protein Neutrocin oc can also be detected in vivo through imaging. Antibody labels or markers, for in vivo imaging, of the Neutrocin c protein, include those that can be detected by X-ray, NMR or ESR. For X-ray, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not manifestly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which can be incorporated into the antibody by labeling or labeling nutrients for the relevant hybridoma. An antibody or antibody fragment, specific for the protein Neutrocin or c, which has been labeled with a detectable, appropriate portion for imaging, such as a radioisotope (eg, 131I, 112In, 99mTc), a radiopaque substance or a material detectable by nuclear magnetic resonance, is introduced (eg, parenterally, subcutaneously or int aperitoneally) to the mammal to be examined for the disorder to the immune system. It will be understood in the art that the size of the subject and the imaging system used will determine the amount of the portion for imaging necessary to produce diagnostic images. In the case of a portion of radioisotope, for a human subject, the amount of radioactivity injected will normally vary from about 5 to 20 millicuries of 99 Tc. The antibody or labeled antibody fragment will then accumulate preferentially in the location of the cells containing the protein Neutrocin oc. Tumor imaging, in vivo, is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radi ochemi cal Detection of Cancer, SW Burchiel and BA Rhodes , eds., Masson Publishing Inc. (1982)).

Antibodies Antibodies specific for the protein Neutrocin or c, for use in the present invention, can be produced against the intact Neutrocin protein or an antigenic polypeptide fragment thereof, which can be presented together with a carrier protein, such as a albumin, for an animal system (such as rabbit or mouse) or, if it is long enough (of at least approximately 25 amino acids), without a carrier. As used herein the term "antibody" (Ab) or "monoclonal antibody" (Mab) includes intact molecules as well as antibody fragments (such as for example Fab fragments and F (ab ') 2 fragments) which are capable of binding specifically to the protein Neutrocin ce. The Fab and F (ab ') 2 fragments lack the Fc fragment of the intact antibody, are more rapidly removed from the circulation, and may have less non-specific binding to the tissue, of an intact antibody (Wahl et al., J. Nu cit. I d .. 24: 316-325 (1983)). Thus, these fragments are preferred. The antibodies of the present invention can be prepared by any variety of methods. For example, cells expressing the Neutrocin protein oc or an antigenic fragment thereof, can be administered to an animal, to induce the production of sera containing monoclonal antibodies. In a preferred method, a Neutrocin ce protein formulation is prepared and purified to render it substantially free of natural contaminants. That preparation is then introduced to an animal, to produce polyclonal antisera of higher specific activity.

In the most preferred method the antibodies of the present invention are monoclonal antibodies (or binding fragments to the Neutrocin protein oc, thereof). These monoclonal antibodies can be prepared using hybridoma technology (Kdhler et al., Nature 256: 495 (1975); Kohler et al., Eur. J. Immunol., 5: 511 (1976); Kohler et al., Eur. J Immunol 6: 292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybrodomas, Elsevier, NY, (1981) pp. 563-681). In general, these methods involve the immunization of an animal (preferably a mouse) with an antigen of the protein Neutrocin or, more preferably, with a cell that expresses the protein Neutrocin or c. Suitable cells can be recognized by their ability to bind to the anti-Neutrocin oc protein antibody. Those cells can be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Eagle's modified Eagle's medium, supplemented with 10% fetal bovine serum (inactivated at a temperature of approximately 56 ° C, and supplemented with approximately 10 g / L of non-essential amino acids, approximately in 1000 U / ml penicillin, and approximately 100 μg / ml streptomycin Mouse splenocytes are extracted and fused with a suitable myeloma cell line Any suitable myeloma cell line can be used in accordance with the present invention; however, it is preferred to employ the related myeloma cell line (SP20) available from the American Type Culture Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells are selectively maintained in a HAT medium, and then cloned by limiting the dilution as described by Wands et al. (Gastroenterology 80: 225-232 (1981)). The hybridoma cells obtained through this selection are then subjected to an analysis to identify the clones that secrete the antibodies capable of binding to the Neutrocin oc protein antigen. Alternatively, additional antibodies capable of binding Neutrocin ce protein antigen can be produced in a two-step procedure, through the use of idiotypic antibodies. One such method makes use of the fact that the antibodies are themselves antigens, and that it is therefore possible to have an antibody that binds to a second antibody. In accordance with this method, antibodies specific for the protein Neutrocin oc are used to immunize an animal, preferably a mouse. The splenocytes of that animal are then used to produce hybridoma cells, and the hybridoma cells are selectively sorted to identify clones that produce an antibody whose ability to bind to the antibody specific for the Neutrocin protein or, can be blocked by the antigen of the protein Neutrocin oc. These antibodies comprise idiotypic antibodies to the antibody specific for the Neutrocin c protein and can be used to immunize an animal and induce the formation of antibodies specific for the neutrocyl or additional protein. It will be appreciated that Fab and F (ab ') 2 and other fragments of the antibodies of the present invention can be used, according to methods described herein. These fragments are typically produced through proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ') 2 fragments). Alternatively, fragments of binding to the protein Neutrocin ce can be produced through the application of recombinant DNA technology or through synthesis chemistry. Where in vivo imaging is used to detect increased levels of the Neutrocin c protein, for human diagnosis, it may be preferable to use "humanized" chimeric monoclonal antibodies. Those antibodies can be produced using genetic constructs derived from the hybridoma cells that produce the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229: 1202 (1985); Oi et al., Bi oTechniques 4: 214 (1986); Cabilly et al., U. S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312: 643 (1984); Neuberger et al., Nature 314: 268 (1985).

TREATMENT OF DISORDERS RELATED TO THE IMMUNE SYSTEM As noted above, Neutrocin a polynucleotides and polypeptides are useful for diagnosing conditions that involve abnormally high or low expression of Neutrocin a activities. Given the cells and tissues in which Neutrokine is expressed, as well as the activities modulated by Neutrokine a, it is readily apparent that a level, substantially altered (increased or decreased) of the expression of Neutrocin a in an individual, compared with the standard or "normal" level produces pathological conditions related to the body system (s) in which Neutrocid is expressed and / or is active. A person skilled in the art will also appreciate that, since the Neutrocin protein a of the invention is a member of the TNF family, the extracellular domain of the protein can be released in soluble form from the cells expressing the -Neutrocin to by proteolytic cleavage and therefore, when the protein Neutrocin a (particularly a soluble form of the extracellular domain) is added from an exogenous source of cells, tissues or the body of an individual, the protein will exert its modulating activities on any of its cells objective of that individual. Also, the cells that express this type II tr ansmembrany protein can be added to the cells, tissues or to the body of an individual, whereby the added cells will bind to the cells that express the receptor for Neutrocin a, so which cells expressing Neutrocin a can cause actions (eg, cytotoxicity) in the target cells that contain the receptor. Therefore, it will be appreciated that conditions caused by a decrease in the standard or normal level of Neutrokine activity, in an individual, particularly immune system disorder, can be treated through administration of the Neutrocin protein to (in the form of the soluble extracellular domain or of cells that express the complete protein). Thus, the invention also provides a method of treating an individual in need of an increased level of Neutrokine activity, which comprises administering to that individual a pharmaceutical composition containing an amount of a Neutrokine polypeptide to an isolated, the invention, effective to increase the activity level of Neutrocin in that individual. Since Neutrocin a belongs to the TNF superfamily it should also modulate angiogenesis. In addition, since Neutrocin a inhibits immunological cellular functions, it will have a wide range of anti-inflammatory activities. Neutrokine can be used as an anticoagulant agent to treat solid tumors by stimulating the invasion and activation of host defense cells, for example, cytotoxic T cells and macrophages and by the inhibition of tumor angiogenesis. Those skilled in the art will recognize other indications not against cancer where the proliferation of blood vessels is not desired. It can also be used to intensify host defenses against chronic and acute infections, resistant, for example, myobacterial infections, through the attraction and activation of microbicidal leukocytes. Neutrocin A can also be used to inhibit the proliferation of T cells, by inhibiting the biosynthesis of IL-2 for the treatment of autoimmune diseases mediated by T cells and by leukemias 1 inf ocí ticas. Neutrocin a can also be used to stimulate wound healing, both by cleaning impurities and inflammatory cells that promote connective tissue. In this same way, Neutrocin a can also be used to treat other fibrotic disorders, which include liver cirrhosis, osteoarthritis and pulmonary fibrosis. Neutrokine a also increases the presence of eosinophils that have the distinctive function of exterminating larvae of parasites that invade tissues, as in schistosomiasis, trichinosis and ascariasis. It can also be used to regulate hematopoiesis, regulating the activation and differentiation of several progenitor hematopoietic cells, for example, to release mature leukocytes from the bone marrow, followed by chemotherapy, that is, in the mobilization of stem cells. Neutrokine can also be used to treat s eps is.

Formulations The polypeptide composition of Neutrocin a (preferably containing a polypeptide that is a soluble form of the extracellular domain) will be formulated and dosed in a manner consistent with good medical practice, taking into account the clinical condition of the individual patient (especially side effects) of the treatment with the Neutrocin polypeptide only), with respect to the site of the Neutrocin a polypeptide composition delivery, the method of administration, the administration regimen, and other factors known to the doctors. The "effective amount" of Neutrocin a polypeptide, for purposes herein, is thus determined by those considerations. As a general proposition, the total pharmaceutically effective amount of Neutrocin a polypeptide, administered parenterally per dose, will range from about 1 μg / kg / day to 10 mg / kg / day per patient body weight, although, as mentioned above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg / kg / day, most preferably, for humans, between about 0.01 and 1 mg / kg / day for the hormone. If supplied continuously, the Neutrocin a polypeptide is typically administered at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour, either by an amount of 1 to 4 injections per day or by infusions. continuous subcutaneous, for example, using a mini pump. An intravenous bag solution can also be used. The duration of treatment necessary to observe changes, and the interval followed by treatment for responses to occur, seems to vary depending on the desired effect. The pharmaceutical compositions containing the Neutrocin a of the invention can be administered orally, rectally, parenterally, intrasistemally, intimavaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patches), buccally, or as an oral or nasal. By "pharmaceutically acceptable carrier" is meant a solid, semi-solid or liquid filler material, a diluent, encapsulating material or auxiliary formulation of any kind, non-toxic. The term "parenteral," as used herein, refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intimal injection and infusion. The Neutrocin a polypeptide is also conveniently administered through sustained release systems. Suitable examples of compositions for sustained release include semipermeable polymer matrices in the form of patterned articles, eg, films or microcapsules. Matrices for sustained release include polylactides (U.S. Patent No. 3,773,919, EP 58,881), L-glutamic acid copolymers, and gamma-ethyl L-glutamate (Sidman, U. et al., Biopolymers 22: 547-556 ( 1983)), poly (2-hydroxy methacrylate) (R. Langer et al., J. Biomed, Mater. Res. 15: 167-277 (1981), and R. Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id) or pol i -D- (-) - 3 -hydroxybutyl alcohol (EP 133,988). Neutrocin a polypeptide compositions, for sustained release, also include the Neutrocin polypeptide entrapped in liposomes. Liposomes containing the Neutrocin a polypeptide are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Nati Acad. Sci. (United States of America)) 82: 3688-3692 (1985); Hwang et al., Proc. Nati Acad. Sci (United States of America)) 77: 4030-4034 (1980): EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; The Japanese patent application. 83-118008; The North American Patents. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small unilamellar type (approximately 200 to 800 Angstroms) in which the lipid content is greater than about 30% in mol of cholesterol, and the selected portion is adjusted for optimal therapy with the Neutrocin polypeptide. . For parenteral administration, the Neutrocin a polypeptide is generally formulated by mixing it, to the desired degree of purity, in an injectable unit dosage form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is not toxic to the containers, to the dosages and concentrations used, and that is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be harmful to the polypeptides. Generally, the formulations are prepared by contacting the Neutrocin polypeptide to, uniformly and intimately with liquid carriers, or finely divided solid carriers, or both. Then, if necessary, the product is shaped to obtain the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood in the container. Examples of such carriers include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier conveniently contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. These materials are not toxic to containers at the concentrations and dosages used, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; Antioxidant is such as ascorbic acid; low molecular weight polypeptides (less than about ten residues), for example polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers, or polyethylene glycol (PEG). The Neutrocin a polypeptide is typically formulated in these vehicles, at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1 to 10 mg / ml, at a pH of about 3 to 8. It will be understood that the use of certain preceding carrier or stabilizer excipients will result in the formation of neutral Neutral polypeptide salts. The Neutrocin polypeptide to be used for therapeutic administration must be sterile. Sterility is easily achieved through filtration in membranes for sterile filtration (eg, 0.2 micron membranes). The therapeutic Neutrokine a polypeptide compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or a vial having a plug that can be punctured with the needle of a hypodermic injection. ca The Neutrocin polypeptide will ordinarily be stored in single-dose or multi-dose containers, eg, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10 ml vials are filled with 5 ml sterile filtered 1% (w / v) Neutrocin polypeptide aqueous solution, and the resulting mixture is lyophilized. The solution for infusion is prepared by reconstituting the freeze-dried Neutrocin or polypeptide, using water for bac terial injection.

The invention also provides a pharmaceutical package or kit containing one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated to that (those) recipient (s) can be found a note in the form predescribed by a governmental agency that regulates the manufacture, use or sale of pharmaceutical or biological products, note that reflects the approval by the agency, of the manufacture, use or sale for administration to humans. In addition, the polypeptides of the present invention can be used in conjunction with other therapeutic compounds.

AGONISTS AND ANTAGONISTS - TESTS AND MOLECULES The invention also provides a method for the selective classification of compounds, to identify those that enhance or block the action of Neutrocin a on cells, such as their interaction with molecules that bind to Neutrocin, such as receptor molecules.

An agonist is a compound that increases the natural biological functions of Neutrokine a, or that functions in a manner similar to that of neurotin, while antagonists decrease or eliminate those functions. In another aspect of this embodiment, the invention provides a method for identifying a receptor protein or other protein for binding to the ligand, which specifically binds to a Neutrocin polypeptide. For example, a cell compartment, such as a membrane or a preparation thereof, can be prepared from a cell that expresses a molecule that binds to Neutrocin a. The preparation is incubated with labeled Neutrocin and Neutrocin complexes bound to the receptor or other binding protein, isolated and characterized according to routine methods known in the art. Alternatively, the Neutrocin polypeptide can be bound to a solid support in such a way that the binding molecules solubilized from the cells are bound or bound to the column and then eluted and characterized according to routine methods. In the assay of the invention, for agonists or antagonists, a cellular compartment, such as a membrane or a preparation thereof, can be prepared from a cell that expresses a molecule that binds to Neutrocin a such as a molecule of a signaling or regulatory path, modulated by Neutrocin a. The preparation is incubated with Neutrocin-a-labeled, in the absence or presence of a candidate molecule that may be an agonist of Neutrocin a or an antagonist thereof. The ability of the candidate molecule to bind to the binding molecule is reflected in the decreased binding of the labeled ligand. Molecules that link freely, that is, without inducing the effects of Neutrocin a in the binding to the binding molecule of Neutrocin a, are the most likely to be good antagonists. Molecules that bind well and produce effects that are the same or that are closely related to Neutrocin are agonists.

Effects similar to those of Neutrocin a, of potential agonists and antagonists, can be measured, for example, by determining the activity of a second messenger system, followed by the interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of Neutrocin a or molecules that cause the same effects as Neutrocin a. Second messenger systems that may be useful in this regard include, but are not limited to, guani AMP the t oci c lasa, second messenger systems of the ion channel or the hydrolysis of fos fo inos i t uro. Another example of an assay for Neutrokine a antagonists is a competitive assay that combines Neutrokine a and a potential antagonist with receptor molecules that bind to the membrane or Neutrokine receptor molecules to recombinants, under appropriate conditions, for a competitive inhibition assay. Neutrokine can be labeled, such as by radioactivity, such that the number of Neutrokine molecules bound to a receptor molecule can be accurately determined to evaluate the effectiveness of the potential antagonist. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists can also be small organic molecules, a peptide, a polypeptide such as a tightly linked protein or an antibody that binds to the same sites in a binding molecule such as a receptor molecule, without inducing the activities induced by the Neutrokine a, thereby preventing the action of Neutrokine a through the exclusion of Neutrocin a from binding. Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA or through the formation of a triple helix. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); "Oligodeoxynucleot ides as Antisense Inhibitors of Gene Expression, CRC 'Press, Boca Raton, FL (1988) .The formation of the triple helix is discussed, for example, in Lee et al., Nucleic Acids Research 6: 3073 (1979); et al., Science 241: 456 (1988), and Dervan et al., Science 251: 1360 (1991) .The methods are based on the binding of a polypeptide to a complementary DNA or RNA. the 5 'coding portion of a polynucleotide encoding the extracellular domain of the polypeptide of the present invention can be used to design an oligonucleotide with antisense RNA, from about 10 to 40 base pairs in length.An oligonucleotide with DNA is designed To be complementary to a region of the gene involved in transcription, which prevents the transcription and production of Neutrocin A. The oligonucleotide with RNA that has antisense, hybrid to mRNA in vi and blocks the translation of the molecule of mRNA in Neutrocin polypeptide a. The oligonucleotides described above can also be delivered to cells, such that the RNA or DNA with antisense can be expressed in order to inhibit the production of Neutrocin a. The agonists and antagonists can be employed in a composition with a pharmaceutically acceptable carrier, as described for example above. The antagonists can be used for example to inhibit Neutrokine a, chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some subsets of T cells, eg, cytotoxic T cells activated and CD8 and natural killer cells, certain infectious diseases and in autoimmune and chronic inflammatory diseases. Examples of autoimmune diseases include multiple sclerosis, and insulin-dependent diabetes. Antagonists can also be used to treat infectious diseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosis, preventing the clustering and activation of mononuclear phagocytes. They can also be used to treat hyperostosis or idiopathic syndrome, preventing the production and migration of eosinophils. Endotoxic shock can also be treated through the antagonists, preventing the migration of macrophages and their production of the human chemokine polypeptides of the present invention. Antagonists can also be used for the treatment of atherosclerosis, preventing the infiltration of monocytes into the wall of the arteries. Antagonists can also be used to treat histamine-mediated allergic reactions and immune disorders including late-stage allergic reactions, chronic urticaria, and atopic dermatitis, by inhibiting mast cells induced by chemosin and dehydration. of basophils and the release of histamine. IgE-mediated allergic reactions such as allergic asthma, renitis, and eczema can also be treated. Antagonists can also be used to treat chronic and acute inflammation, preventing the attraction of monocytes to an area of a wound. They can also be used to regulate normal populations of pulmonary macrophages, since pulmonary, inflammatory, chronic and acute diseases are associated with the sequestration of mononuclear phagocytes in the lung. Antagonists can also be used to treat rheumatoid arthritis, preventing the attraction of monocytes in the synovial fluid in the joints of patients. The influx of monocytes and activation plays a significant role in the pathogenesis of both degenerative and inflammatory arthropathies. Antagonists can also be used to interfere with harmful cascades attributed mainly to IL-1 and TNF that prevent the biosynthesis of other inflammatory cytokines. In this way the. Antagonists can be used to prevent inflammation. Antagonists can also be used to inhibit prostaglandin-independent fever, induced by chemokines. Antagonists can also be used to treat cases of bone marrow deterioration, for example, aplastic anemia and myelodysplastic syndrome. Antagonists can also be used to treat asthma and allergy, preventing the accumulation of eosinophils in the lung. Antagonists can also be used to treat fibrosis in the subepithelial basement membrane, which is a prominent feature of the asthmatic lung. Antibodies against Neutrocin a can be used to bind Neutrocin to and inhibit activity, to treat ARDS, preventing the infiltration of neutrophils into the lung after damage. The antagonists can be employed in a composition with a pharmaceutically acceptable carrier, for example, as described below.

TESTS WITH CHROMOSOMES The nucleic acid molecules of the present invention are also valuable for the identification of chromosomes. The sequence is specifically arrived at and can hybridize to a particular location on an individual human chromosome. In addition, there is a current need to identify particular sites on the chromosome. Few reagents to mark chromosomes, based on current frequency data (repeat polymorphisms) are currently available to mark the location of chromosomes. The mapping of the DNAs in the chromosomes, in accordance with the present invention, is an important first step in the correlation of those sequences with genes associated with a disease. In certain preferred embodiments in this regard, the cDNA described herein is used to clone genomic DNA from a Neutrocin a protein gene. This can be done using a variety of well-known techniques as well as well-known libraries, which are generally commercially available. Genomic DNA is then used for the mapping of chromosomes i n si t u, using techniques well known for this purpose. In addition, in some cases, maps of the sequences in the chromosomes can be prepared by preparing PCR primers (preferably 15-25 bp) from the cDNA. The computed analysis of the 3 'untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used to selectively classify the PCR of somatic cell hybrids containing individual human chromosomes. Fluorescence hybridization i n s i t u ("FISH") of a cDNA clone in a metaphase chromosomal propagation can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA probes, as short as 50 or 60 bp. To review this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamos Press, New York (1988). Once the map of a sequence has been lifted, at a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with data from the genetic map. These data are found, for example, in V. McKusick, Mendelian Inheritance In Man, available online through Johns Hopkins University, Welch Medical Library. The relationship between the genes and the diseases to which the map has been raised, in the same chromosomal region, are then identified through linkage analysis (common inheritance of physically adjacent genes). Then, it is necessary to determine the differences in the cDNA or in the genomic sequence between affected and unaffected individuals. If a mutation is observed in some affected individuals or in all of these individuals, but not in any of the normal individuals, then it is likely that the mutation is the causative agent of the fermeda. Having described the invention in a general manner, it will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention.

EXAMPLES Example: Expression and Purification of Neutrocin to "Marked with His" in E. Coli The bacterial expression vector pQE9 (pD10) is used for bacterial expression in this example. (QIAGEN, Inc., supra). pQE9 codes for resistance to the antibiotic ampicillin ("Ampr") and contains a bacterial origin of duplication ("ori"), an IPTG-inducible promoter, a ribosome binding site ("RBS"), six codons that encode histidine residues that allow affinity purification, using nickel-nitrile-triacetic acid ("Ni-NTA) which are the affinity resins sold by QIAGEN, Inc., supra, and sites for the cleavage of the appropriate single restriction enzyme. These elements are arranged in such a way that a fragment of inserted DNA encodes a polypeptide that expresses the polypeptide with six His residues (ie, "6 X His tag") covalently linked to the amino terminus of a polypeptide.

The DNA sequence encoding the desired portion of the Neutrocin a protein comprising the extracellular domain sequence is amplified from the deposited cDNA clone, using primers of the PCR oligonucleotide that are reinforced to the amino terminal sequences of the desired portion of the Neutrocin protein a and the sequences found in the 3 'construct deposited, for the sequence encoding the cDNA. In the sequences of the 5 'and 3' primer, respectively, additional nucleotides containing the restriction sites are added, to facilitate cloning in the vector pQE9. For cloning the extracellular domain of the protein, the 5 'primer has the sequence 5' GTGGGATCCAGGGCAGAGCTG 3 '(SEQ ID NO: 10) containing the underlined BamH I restriction site, followed by 18 nucleotides of the amino terminal coding sequence of the extracellular domain of the Neutrocin sequence a of Figure 1. Of course, a person skilled in the art would appreciate that the point in the coding sequence of the protein, where the 5 'primer starts, can be varied to amplify a segment of DNA encoding any desired portion of the Neutrocin protein to complete, shorter or longer than the extracellular domain of the form. The 3 'primer has the sequence 5' GTGAAGCTT TTATTACAGCAGTTTCAA GCACC3 '(SEQ ID NO: 11) containing the underlined Hind III restriction site, followed by two stop codons and 18 complementary nucleotides up to the 3T end of the coding sequence of the DNA sequence of Neutrocin a, of Figure 1. The DNA fragment of Neutrocin a, amplified, and the vector pQE9, is digested with BamH I and Hind III and the digested DNAs are then joined to each other. The insertion of Neutrocin a DNA into the restricted pQE9 vector places the coding region of the Neutrocin protein a, downstream of the IPTG inducible promoter and in the structure, with a starter AUG and the six histidine codons. The binding mixture is transformed into E cells. c and i 'competent, using standard procedures such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). The strain of E is used. c ol i, M15 / rep4, which contains multiple copies of plasmid pREP4, which expresses the lac repressor and confers resistance to Kanamycin ("Kanr"), to carry out the illustrative example described herein. This strain, which is the only one of many that are suitable for expressing the protein Neutrocin a, is commercially available from QIAGEN, Inc., s upra. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. The plasmid DNA was isolated from resistant colonies and the identity of the cloned DNA was confirmed by restriction analysis, PCR and DNA sequencing. Clones containing the desired constructs are grown overnight ("O / N") in liquid culture in LB medium supplemented with both ampicillin (100 μg / ml) and kanamycin (25 μg / ml). The O / N culture is used to inoculate a large culture, at a dilution of about 1:25 to 1: 250. The cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6. Then Isopropyl-β-D-thiogalactopyranoside ("IPTG") is added to a final concentration of 1 mM to induce transcription from the lac repressor responsive promoter, inactivating the lacl repressor. The cells are subsequently incubated for an additional 3 to 4 hours. The cells are then harvested by centrifugation. The cells are then agitated for 3 to 4 hours at 4 ° C in 6M guanidine-HCl, pH 8. The cell debris is removed by centrifugation, and the supernatant containing Neutrocin a is loaded onto a column. which has nickel-nit ryl-acid-triacetic affinity resin ("NiNTA") (available from QIAGEN, Inc., s upra). The 6x His Tag proteins bind to Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIA expre ssi oni st, 1995, QIAGEN, Inc., s upra ). Briefly, the supernatant is loaded onto the column in guanidine-HCl, 6M, pH 8, first the column is washed with 10 volumes of guanidine-HCl, 6M, pH 8, then washed with 10 volumes of guanidine- HCl, 6M, pH 6, and finally Neutrocin a is eluted with 6M guanidine-HCl, pH 5. The purified protein is then naturalized again by dialysis against phosphate buffered saline (PBS) or Na acetate buffer, pH 6.50 M, plus 200 mM NaCl. Alternatively, the protein can multiply successfully again, while immobilized on the Ni-NTA column. The recommended conditions are as follows: re-naturalize using a linear gradient of 6M-1M urea, in 500mM NaCl, 20% glycerol, 20mM Tris / HCl, pH 7.4, containing the protease inhibitors. The renaturation should be done over a period of 1.5 hours or more. After renaturation the proteins can be eluted by the addition of 250 mM imidazole. The imidazole is removed through a final dialysis step against PBS or 50mM sodium acetate buffer, pH 6, plus 200mM NaC. The purified protein is stored at a temperature of 4 ° C or frozen at -80 ° C.

Example Ib: Expression and purification of Neutrocin a in E. coli The bacterial expression vector pQE60 is used for bacterial expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311). PQE60 encodes resistance to the antibiotic ampicillin ("Ampr") and contains a bacterial origin of duplication ("orí"), an IPTG-inducible promoter, a ribosome binding site ("RBS"), six codons that encode residues histidine that allows affinity purification, using 1-nyne-tri-acetic acid ("Ni-NTA") affinity resin sold by QIAGEN, Inc., upra, and enzyme break sites restriction, unique, adequate. These elements are arranged or arranged in such a way that a DNA fragment encoding a polypeptide can be inserted in such a manner to produce that polypeptide with all six His residues (ie, a "6 His tag") covalently linked. to the carboxyl terminus of that polypeptide. However, in this example, the coding sequence of the polypeptide is inserted such that translation of the six His codons is prevented and, therefore, the polypeptide is produced without the 6X His tag. The DNA sequence encoding the desired portion of the Neutrocin a protein comprising the extracellular domain sequence is amplified from the deposited cDNA clone, using PCR oligonucleotide primers that are boosted at the amino terminal frequencies of the desired portion of the Neutrocin protein. aya and the sequences found in the deposited 3 'construct, in the coding sequence of the cDNA. Additional nucleotides containing restriction sites to facilitate cloning in the pQE60 vector are added in the 5 'and 3' sequences, respectively. To clone the extracellular domain of the protein, primer 5"has the sequence 5 'GTGTCATGAGCCTCCGGGCAGAGCTG3' (SEQ ID NO: 12) containing the underlined BspH restriction site, followed by 17 nucleotides of the amino terminal coding sequence of the domain extracellular sequence of Neutrocin a of Figure 1. The person skilled in the art will of course appreciate that the point, in the coding sequence of the protein, where the 5 'primer starts, can be varied to amplify a desired portion of the protein. the complete protein, shorter or longer than the extracellular domain of the form The 3 'primer has the sequence 5' GTGAAGCTTTTATTACAGCAGTTTCAATGCACC3 '(SEQ ID NO: 13) containing the underlined Hind III restriction site, followed by two codons of stoppage and 18 complementary nucleotides up to the 3 'end of the coding sequence in the DNA sequence of Neutrocin a, from Figure 1. The Neutrocin DNA fragments a, amplified, and the vector pQE60, are digested with BspH I and Hind III, and the digested DNAs are then linked to each other. The insertion of the Neutrocin a DNA into the restricted pQE60 vector places the coding region of the Neutrocin protein a which includes its associated stop codon, downstream of the IPTG-inducible promoter and in the structure, with an initiating AUG. The associated stop codon prevents translation of the six histidine codons, downstream of the insertion point. The binding mixture is transformed into E cells. c ol i. competent, using standard procedures, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). The strain E is used. c ol i M15 / rep4, which contains multiple copies of plasmid pREP4, which expresses the lac repressor and confers resistance to kanamycin ("Kanr"), to carry out the illustrative example described herein. This strain, which is only one of many that are convenient for expressing the protein Neutrocin a, is commercially available from QIAGEN, Inc., s upra. Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. The plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA is confirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight ("O / N") in liquid culture in LB medium supplemented with both ampicillin (100 μg / ml) and kanamycin (25 μg / ml). The O / N culture is used to inoculate a large culture, at a dilution of about 1:25 to 1: 250. The cells are grown to an optical density at 600 nm ("OD600") of between 0.4 and 0.6. Then i sopropil-b-D-thiogalactopyranoside ("IPTG") is added to a final concentration of 1 mM to induce transcription of the lac repressor responsive promoter, inactivating the lacl repressor. The cells are subsequently incubated for an additional 3 to 4 hours. Then the cells are collected by centrifugation. The cells are then agitated for 3 to 4 hours at 4 ° C, in 6M guanidine-HCl, pH 8. The cell debris is removed by centrifugation, and the supernatant, which contains Neutrokine a, is dialyzed against 50 mM sodium acetate buffer, pH 6, supplemented with 200 mM NaCl. Alternatively, the protein can be successfully re-multiplied by dialyzing against 500 mM NaCl, 20% glycerol, 25 mM Tris / HCl, pH 7.4, containing protease inhibitors. After renaturation, the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, an affinity chromatography step, such as an antibody column, can be used to obtain the pure Neutrocin protein. The purified protein is stored at 4 ° C or frozen at -80 ° C.

Example 2: Cloning and Expression of the Neutrocin a Protein, in a Baculovirus Expression System In this illustrative example, the shuttle or transfer vector, of the pA2 GP plasmid, is used to insert the cloned DNA encoding the extracellular domain of the protein, which lacks its naturally associated intracellular and transmutaneous sequences, into a baculovirus, to express the extracellular domain of the Neutrocin a protein, using a baculovirus guide and standard methods such as those described in Summers et al., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No 1555 (1987). This expression vector contains the strong polyhedrin promoter of the nuclear polyhedrosis virus Au t ographa ca li forni ca (AcMNPV) followed by the secretory signal peptide (guide) of the baculovirus gp67 protein and convenient restriction sites such as the BamH I, Xba I and Asp718. The polyadenylation site of the simian virus 40 ("SV40") is used for an efficient polio. For easy selection of the recombinant virus, the plasmid contains the beta-galactosidase gene, from E. c ol i, under the control of a weak Drosophila promoter, in the same orientation, followed by the signal of 1 iadeni 1 ation of the polyhedrin gene. The inserted genes are flanked on both sides, by viral sequences for the recombination of cell-mediated homologs, a wild-type viral DNA, to generate viable viruses that express the cloned polynucleotide.

Many other baculovirus vectors could be used. instead of the previous vector, such as pAc373, pVL941 and pAcIMl, which could easily be appreciated by one skilled in the art, as long as the construct provides signals, appropriately located, for typing, translation, secretion and the like, including a signal peptide and an AUG in the structure, as required. These vectors are described, for example, in Luckow et al., Vi rol ogy 1 70: 3 1 - 3 9 (1989). The cDNA sequence encoding the extracellular domain of the Neutrocin protein a, in the deposited clone, lacking the AUG initiation codon and the sequences of the intracellular and transmembrane domain, naturally and associated, shown in Figure 1 (SEQ ID No.) : 2), are amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequence of the gene. The 5 'primer has the sequence 5' GTGGGATCCCCGGGCAGAGCTGCAGGGC3 ' (SEQ ID No. 14) which has the BamH I restriction enzyme site underlined, followed by 18 nucleotides of the sequence of the extracellular domain of the Neutrocin protein a shown in Figure 1, starting with the N-terminus indicated, extracellular domain of the protein. The 3 'primer has the sequence 5' GTGGGATCCTTATTACAGCAGTTTCAATGCACC3 '(SEQ ID NO: 15) containing the underlined Bam Hl restriction site, followed by two stop codons and 18 complementary nucleotides, up to the 3T coding sequence of Figure 1 The amplified fragment is isolated on a 1% agarose gel, using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca). The fragment is then digested with BamH I and again purified on a 1% agarose gel. This fragment is designated herein as Fl. The plasmid is digested with Bam Hl restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel, using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca). This vector DNA is referred to herein as "VI".

The fragment Fl and the plasmid dephosphorylated VI bind to each other with T4DNA ligase. The HB101 of E. col i or other suitable hosts of E. As the XL-1 Blue cells (Statagene Cloning Systems, La Jolla, CA) are transformed with the binding mixture and spread on culture plates. Bacteria containing the plasmid are identified, with the Neutrocinai to human gene, by digesting the DNA from individual colonies, using Bam Hl and then analyzing the digestion product through electric filters in gel. The sequence of the cloned fragment is confirmed through DNA sequencing. This plasmid is referred to in the present Neutrocin as pA2GP. Fec five μg of the neutrocytic plasmid was transferred to pA2GP, with 1.0 μg of commercially available linearized baculovirus DNA ("Baculo Gold (MR) baculovirus DNA", Pharmingen, San Diego, CA), using the lipofection method. described by Felgner et al., Proc. Nati Acad. Sci. USA 84: 7413-7417 (1987). 1 μg of Gold Bone Virus (MR) DNA and 5 μg of the Neutrocin plasmid are mixed to pA2GP in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg , MD). After that, 10 μl of Lipofectin plus 90 μl of Grace's medium are added, mixed and incubated for 15 minutes at room temperature, then the transfection mixture is added dropwise to Sf9 insect cells (ATCC CRL 1711) seeded in a culture dish with 35 mm tissue, with 1 ml of Grace's medium, without serum. The plate is then incubated for 5 hours at 27 ° C. Then the transfection solution is removed from the plate, and 1 ml of Grace's insect medium is added, supplemented with 10% fetal calf serum. Then the culture is continued at 27 ° C for four days. After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of clones that express gal, which produce the blue-stained plates. (A detailed description of such a "plate assay" can also be found in the user's guide for insect cell cultures and baculovirology, distributed by Life Technologies Inc., Gaithersburg, page 9-10). After the appropriate incubation, the blue-stained plates are collected with the tip of a micropipette apparatus (e.g., Eppendorf). The agar containing the recombinant viruses is then suspended in a microcentrifuge tube containing 200 μl of Grace's medium, and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then stored at 4 ° C. The recombinant virus is called V-Neutrocin a. To verify the expression of the Neutrocin gene at, Sf9 cells are grown in Grace's medium supplemented with thermally inactivated FBS, 10%. The cells are infected with the recombinant baculovirus V-Neutrocin a with a multiplicity of infection ("MOI") of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine. (available from Life Technologies Inc., Rockville MD). After 42 hours, 5 μCi of 35 S-methionine and 5 μCi 35 S-Cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then harvested by centrifugation. The proteins found in the sobriente, as well as the intracellular proteins, are analyzed by SDS-PAGE followed by aut oradiography (if radiolabeled). The micro sequence of the amino acid sequences of the amino terminus, of the purified protein, can be used to determine the aminotermin 1 sequence of the extracellular domain of the protein and thus the point of cleavage and the length of the signal peptide. secretory Example 3: Cloning and Expression of Neutrocin a in mammalian cells A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of the mRNA, the coding sequence of the protein, and the signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include in t ens i f i ctor, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA binding. Highly efficient transcription can be achieved with the early and late promoters of SV40, the long terminal repeats (L P.s) of Retrovirus, eg, RSV, HTLVI, HIVI and the cytomegalovirus early promoter (CMV). However, cellular elements can also be used (eg, the human actin promoter). Suitable expression vectors, for use in the practice of the present invention, include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146 ) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1 cells, Cos 7 and CV1, quail QC1-3, mouse L cells and ovarian cells of Chinese hamster (CHO). Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. Co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin, allow the identification and isolation of transfected cells. The transfected gene can also be amplified to express large amounts of encoded protein. The DHFR marker (dihydrofolate reductase) is useful for developing cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227: 277777-279 (1991), Bebbington et al., Bi o / Te chn ol ogy 1 0: 169-175 ( 1992)). Using these arcs, the mammalian cells are cultured in a selective medium and the cells with the highest resistance are selected. These cell lines contain the (the) amplified gene (s) integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for protein production. The pCI and pC4 expression vectors contain the strong promoter (LTR) of the Rous Sarcous Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV int ensi fi er ( Boshart et al., Cell 41: 521-530 (1985)). Multiple cloning sites, for example, with the cleavage sites of the restriction enzyme BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3 'intron and the polyadenylation signal and termination / of the rat prepoinsulin gene.

Example 3 (a): Cloning and Expression in COS cells The expression plasmid, pNeutrocin a HA, is manufactured by cloning a portion of the deposited cDNA encoding the extracellular domain of the Neutrocin a protein into the pcDNA / Amp or pcADMIII expression vector (available from Invitrogen, I c.) . To produce a soluble, secreted polypeptide form, the extracellular domain is fused to the secretory leader sequence of the IL-6 gene. The pcDNA / amp expression vector contains: (1) a duplication origin of E. c ol i cash for propagation in E. c or i and other prokaryotic cells (2) an ampicillin resistance gene for the selection of prokaryotic cells containing plasmid; (3) an SV40 duplication origin for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron; (5) several codons encoding a fragment of hemagglutinin (ie, an "HA" tag to facilitate purification) followed by a stop codon and a polyadenylation signal arranged such that a cDNA can be conveniently placed control or expression of the CMV promoter and functionally bind to the SV40 intron and the polyadenylation signal, through the restriction sites found in the polylinker. The HA marker corresponds to an epitope derived from the influenza hemagglutinin protein, described by Wilson et al., Cel l 3 7: 767 (1984). The fusion of the HA marker to the target proteins allows the easy detection and recovery of the recombinant protein with an antibody that recognizes the HA epitope. The pcAD-NIII contains, in addition, the selectable Neomycin marker. A DNA fragment encoding the ex tranexular domain of the Neutrocin a polypeptide is cloned into the polyenlazant region of the vector, such that the expression of the recombinant protein is driven by the CMV promoter. The construction strategy of the plasmid is as follows. The Neutrokine cDNA of the deposited clone is amplified using primers containing the convenient restriction sites, more as described above for the construction of ectodres for the expression of Neutrocin or in E. c ol i. Suitable primers include the following, which are used in this example. The 5 'primer, containing the underlined Bam Hl site, a Kozak sequence, or AUG start codon, a sequence encoding the secretory guiding peptide of the human IL-6 gene, and 18 nucleotides of the 5' coding region of the extracellular domain of the Neutrocin protein, has the following sequences: 5 'GCGGGATCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCGGTC.C? GGCG.TTC.TC.CCTGGGC.TGCC.CTGTTTGCC.T CTGCCTTCCCTGCCCCAGTTGTGAGACAAGGGGACCTGGCCAGC3' (SEQ ID NO: 16). The 3 'primer, which contains the restriction site Bam Hl, underlined and 18 of the complementary nucleotides for the 3' coding sequence immediately before the stop codon, has the following sequence: 5 'GTGGGATCCTTACAGCAGTTTCAATGCACC3' (No SEC ID: 17 ). The amplified DNA fragment, PCR, and the vector, pcDNA / Amp, are digested with Bam Hl and then linked or bound. The binding mixture is transformed into the E strain. c ol i SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037), and the transformed culture is applied to plates on plates with ampicillin media which are then incubated to allow growth of the ampicillin-resistant colonies. The plasmid DNA is isolated from resistant colonies and examined by restriction analysis or other means for the presence of the fragment that blocks the extracellular domain of Neutrocin a. For the expression of Neutrokine to recombinant, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for example, in Sambrook et al., Molecular Cloning: a Laboratory manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989). The cells are incubated under conditions for the expression of Neutrocin a by the vector. The expression of the HA fusion protein of Neutrocin a is detected by radiolabelling and immunoprecipitation, using the methods described for example in Harlow et al., Antibodies: A Laboratorv Manual, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Yor (1988). Up to this point, two days after transfection, the cells are labeled by incubation in a medium that has 35 S-cysteine for 8 hours. The cells and media are pooled, and the cells are washed and lysed with RIPA buffer containing the detergent: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5 DOC %, 50 mM TRIS, pH 7.5, as described by Wilson et al. I mentioned earlier. The proteins are precipitated from the cell lysate and from the culture media, using a monoclonal antibody specific for HA. The precipitated proteins are then analyzed by SDS-PAGE and author adiogr af í a. An expected expression product of the size is observed in the cell lysate, which is not observed in the negative controls.

Example 3 (b): Cloning and Expression in CHO cells The vector pC4 is used for the expression of the protein Neutrocin a. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (accession No. ATCC, 37146). To produce a soluble, secreted form of the Neutrocin a polypeptide, the portion of the deposited cDNA, which encodes the extracellular do-ness, is fused to the secretory leader sequence of the IL-6 gene. The plasmid vector contains the mouse DHFR under the control of the SV40 early promoter. Chinese hamster ovary cells, or other cells, which lack the activity of dihydro foliate, which are transfected with these plasmids, can be selected by culturing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of DHFR genes in methotrexate-resistant cells (MTX) has been well documented (see, for example, Alt, FW, Kellems, RM, Bertino, JR, and Schimke, RT, 1978, J. Biol. Chem. 253: 1357-1370, Hamlin, JL and Ma, C. 1990, Biochem., And Biophys., Acta, 1097: 107-143, Page, MJ and Sydenham, MA 1991, Biotechnology 9: 64-68). Cells grown in increasing concentrations of MTX develop drug resistance, excessively producing the target, DHFP. , as a result of the amplification of the DHFR gene. If a second gene binds to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the ampli fi ed gene (s). When the methotrexate is taken up, cell lines containing the amplified gene, integrated into one or more chromosomes of the host cell, are obtained. Plasmid pC4 contains to express the gene of interest, the long-terminal repeat (LTR) promoter of the Rouse Sarcoma virus (Cullen, et al., Mol e ul for Cell Bi ul or Bi, March 1985: 438-447) plus an isolated fragment enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41: 521-530 (1985)).

Downstream of the promoter are the unique restriction enzyme cleavage sites that allow the integration of the genes: Ba HI, Xba I, and Asp "718. Behind these cloning sites the plasmid contains the intron 3! the site of po i i adeni lation of the rat pr epro insulin gene, other high efficiency promoters can also be used for the expression, for example, of the Beta-actin promoter hum.ana, for the SV40 early or late promoters or the long terminal repeats of other retroviruses, for example, of HIV and HTLVI The Tet - Off * - 7 Tet - On gene expression systems of Clontech and similar systems can be used to express Neutrokine in a regulated form , in mammalian cells (Gossen, M., &Bujard, H. 1992, Proc. Nati, Acad. Sci. USA 89: 5547-5551) For the pol i adeni ation of mRNA, other such signals, for example, of human growth hormone or gn genes. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected during cotransfection with a selected marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selected marker at the beginning, for example, methotrexate G418 plus. Plasmid pC4 is digested with the restriction enzymes Bam Hl and then dephosphorylated using calf intestinal phosphates, by procedures known in the art. The vector is then isolated from a 1% agarose gene. The DNA sequence encoding the extracellular domain of the Neutrocin protein a. it is amplified using the PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. The 5 'primer, which contains the underlined Bam Hl site, a Kozak sequence, an AUG start codon, a sequence encoding the secretory guiding peptide of the human IL-6 gene, and 18 nucleotides of the 5' coding region of the extracellular domain of the protein Neutrocin, has the following sequences: 'GCGGGATCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCG G CC? GTTGCCT C CCC GGGGC GC CC GGTGTTGCC GC G CCTTCCCTGCCCCAGTTGTGAGACAAGGGGACCTGGCCAGC3' (SEQ ID NO: 16). The 3 'primer, containing the underlined Bam Hl and 18 of the complementary nucleotides for the 3' coding sequence immediately before the stop codon, has the following sequence: 5 'GTGGGATCCTTACAGCAGTTTCAATGCACC3"(SEQ ID NO: 17). The amplified fragment is digested with the Bam Hl endonucleases and then purified again on a 1% agarose gel. The isolated fragment and the phosphorylated vector are then ligated with the T4 DNA ligase. The HB101 or XL-1 Blue cells of E. c or l i are then transformed and those bacteria containing the fragment inserted in the plasmid pC4 are identified, using, for example, the analysis of restriction enzymes. For the treatment, the Chinese hamster ovary lacking an active DHFR gene were sanctified. Five μg of the expression plasmid pC4 was co transfected with 0.5 μg of the pSVneo plasmid using lipofectin (Felgner et al., Upra). Plasmid pSV2-neo contains a dominant selectable marker, the n eo gene of Tn5 encodes an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg / ml of G418. After 2 days, the cells are treated with trypsin and seeded in plates for the cloning of hybridomas (Greiner, Germany) in alpha minus MEM supplemented with 10, 25 or 50 ng / ml of methotrexate plus 1 g / ml of G418. After a period of about 10-14 days the single clones are treated with trypsin and then seeded in 6-well petri dishes or 10 ml bottles, using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM , 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 M, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. The expression of the desired genetic product is analyzed, for example, by SDS-PAGE and Spotting or Western Blot or by reverse phase HPLC analysis.

Example 4: Tissue distribution, expression of neutropenia mRNA The Stained or Transfer analysis Northern is carried out to examine the gene expression of Neutrocin a in human tissues, using the methods described for example, among others, by Sambrook et al., Cited above. A cDNA probe containing the entire nucleotide sequence of the Neutrokine protein (SEQ ID NO: 1) is labeled with 32 P using the re ipr ime (MR) DNA marking system (Amersham Life Science), in accordance with the instructions manufacturer. After labeling, the probe is purified using a CHROMA SPIN-100MR column (Clontech Laboratories, Inc.), in accordance with the manufacturer's protocol number PT1200-1. The labeled and purified probe is then used to screen several human tissues for the Neutrocin a mRNA. Multiple stained or Northern Tissue Transfers (MTN) are obtained which contain several human tissues (H) or human immune system (IM) tissues from Clontech and are examined with the labeled probe using the solution for Express hybridization sHybMR (Clonte.ch ) in accordance with the manufacturer's protocol number PT1190-1. Following hybridization and washing, the stains are mounted and exposed on film, at -70 ° C overnight, and the films are developed in accordance with standard procedures. It will be clear that the invention can be practiced in a manner different from that which was particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in view of the foregoing teachings and are therefore within the scope of the appended claims. The total description of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are incorporated herein by reference.

LIST OF SEQUENCES (1. GENERAL INFORMATION (i) APPLICANT: YU, GUO-LIANG EBNER, REINHARD NI, JIAN., ii) TITLE OF THE INVENTION: NEUTROCINA ALPHA (iii) NUMBER OF SEQUENCES: 17 (iv) ADDRESS FOR CORRESPONDENCE: '&) RECIPIENT: FUMAN GENOME SCIENCES, INC. (B ~) STREET: 9410 KEY WEXT AVENUE (C) CITY: ROCKVTLLE (D) STATE: MD (E) COUNTRY: United States of America (F) ZIP: 20850 (v) COMPUTER LEGIBLE FORM: (A) TYPE MEDIUM: Flexible disk (B) COMPUTER: Compatible with IBM (C) OPERATING SYSTEM: PC-DOS / MS-DOS ÍD) SOFTWARE: Parenüln Reeléase «1.0, Version # 1.30 (vi) COMMON DATA OF THE APPLICATION: (A) APPLICATION NUMBER: (ü) ttCHA JJ-. f tSn. iATION: (C) CLASIrICATION: (viii) INFORMATION FROM THE LAWYER / MANDATORY: (A) NAME: 3ENS0N, R03ERT H (B) REGISTRATION NUMBER: 30,446 ÍC) REFERENCE NUMBER OF FILE: PF343 (ix) INFORMATION FOR TELECOMMUNICATION: '( A) TELEPHONE: '301) 309-8504 (B) TELEEAX: (301) 309-8512 INFORMATION FOR SEC. ID SEC (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1100 base pairs (B) TYPE: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (LOCATION): 147..1UU1 (ix) CHARACTERISTICS : (A.) NAME / KEY: sis stight (LOCATION): b5..3dA (ix) FEATURE: (A) NAME / KEY: mat néntido (LOCATION): 147..IUUI "(xi) DESCRIPTION OF THE SEQUENCE: No. ID S? C: 1: AAATTCAGGA_ TAACTCTCCT GAGGGGTsAG CCAAGCCCTG CCATGTAGTG CACGCAGGAC SO ATCAACAAAC ACAGATAACA GGAAATGATC CATTCCCTGT GG CACTTAT TCTAAAGGCC t? 0 CCAACCTTCA AAGTTCAAGT AGTGAT ATG GAT GAC TCC ACA GAA AGG GAG CAG 173 Mee A = p Asp Ser Thr Glu Arg Glu Gln 1 5 TCA CGC CTT ACT TCT TGC CTT AAG AAA AGA GAA GAA ATG AAA CTG AAG 221 Ser Arg Leu Thr Ser Cys Leu Lys Lys Arg Glu Glu Mee Lys Leu Lys 10 15 20 25 GAG TGT GTT TCC ATC CTC CCA CGG AAG GAA AGC CCC TCT GTC CGA TCC 269 Glu Cys Val Ser lie Leu Pro Arg Lys Glu Ser Pro Val Arg Ser 30 35 40 TCC AAA GAC GGA AAG CTG CTG GCT GCA ACC TTG CTG CTG GCA CTG CTG 317 Ser Lys Asp Gly Lys Leu Leu Wing Wing Thr Leu Leu Leu Wing Leu Leu 43 50 55 TCT TGC TGC CTC ACG GTG GTG TCT TTC TAC CAG GTG GCC GCC CTG CAA 3S5 Ser Cys Cys Leu Thr Val Val Ser Phe Tyr Gln Val Wing Ala Leu Glp SO 55 70 GGG GAC CTG GCC AGC CTC CGG GCA GAG CTG CAG GGC CAC CAC GCG GAG. 413 Gly Asp Leu Ala Ser Leu Arg Ala Glu Lau Gln Gly His HIS 'Ala Glu 75 80 as AAG CTG CCA GCA GGA GCA GCC ICC CCC AAG GCC GGC CTG GAG GAA GCT SI Lys Leu Pro Wing Gly Wing Gly Wing Pro Lys Wing Gly Leu Glu Glu Wing 90 95 100 105 CCA GCT GTC ACC GCG GGA CTG AAA ATC TTT GAA CCA CCA GCT CCA GGA 509 Pro Wing Val Thr Wing Gly Leu Lys lie Phe Glu Pro Pro Wing Pro Gly 110 115 120 GAA GGC AAC TCC AGT CAG AAC AGC AGA AAT AAG CGT GCC GTT CAG GGT 557 Glu Gly Asn Ser Ser Gln Asn Ser Arg Asa Lys Arg Wing Val Glp Gly 125 130 135 CCA GAA GAA ACA GTC ACT CAA GAC TGC TTG CAA CTG ATT GCA GAC AGT SOS Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu lie Wing Asp Ser 140 145 150 GAA ACA CCA ACT ATA CAA AAA GGA TCT TAC ACA TTT GTT CCA TGG CTT S53 Glu Thr Pro Thr He Gla Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu 155 ISO 1S5 CTC AGC TTT AAA AGG GGA AGT GCC CTA GAA GAA AAA GAG AAT AAA ATA 701 Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu? Sn Lys lie 170 175 180 1 = 5 TTG GTC AAA GAA ACT GGT TAC TTT TTT ATA TAT GGT CAG GTT TTA TAT 749 Leu Val Lys Glu Thr Gly Tyr Phe Phe lie Tyr Gly Gla Val Leu Tyr 190 195 200 Ac GAT? AG AC = TAC GCC Ats GGA CAT CTA Att CAG AG3 AAG AAG GTC 797 Thr Asp Lys Thr Tyr ? the Met: Gly His Leu Zle Gla Arg Lys Lys Val 205 210 215 CAT GTC TTT GGG GAT GAA TTG AGT CTG GTG ACT TTG TTT CGA TGT ATT 345 His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys lie 220 225 230 CAA AAT ATG CCT GAA ACA CTA CCC AAT AAT TCC TGC TAT TCA GCT GGC 393 Gla Asr. Met Pro Glu Thr Leu Pro Handle Asa Ser Cys Tyr Ser Wing Gly 235 240 245 ATT GCA AAA CTG GAA GAA GGA GAT GAA CTC CAA CTT GCA ATA CCA AGA 941 lie Ala Lys Leu Glu Glu Gly Asp Glu Leu Gla Leu Ala "" Pro Arg 250 255 260 265 GAA AAT GCA CAA ATA TCA CTG GAT GGA GTC GTC ACA TTT TTG GGT GCA 989 Glu Asa Wing Gln He Ser Leu Asp Gly Asp Val Thr Phe Phe sGly Wing 270 275 280 TTG AAA CTG CTG TGACCTACTT ACACCATGT TTCCTCCCTT 1041 Leu Lys Leu Leu 235 TCTCTGTACC TCTAAGAAGA AAGAATCTAA CTGAAAATAC CAAAAAAAAA AAAAAAAAA 1100, (2) INFORMATION FOR NO. SEC ID: (i) SEQUENCE CHARACTERISTICS: (A.) LENGTH: 285 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: NO. ID SEC Met Asp Asp Ser Thr Glu Arg Glu Gla Ser Arg Leu Tar Ser Cys Leu 1 5 10 15 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser He Lau Pro 20 25 30 Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Lau Leu 35 40 45 Wing Wing Thr Leu Leu Wing Leu Leu Ser Cys Cys Leu Thr Val Val 50 55 SO Ser Phe Tyr Gin Val Wing Wing Lau Gla Gly Asp Leu Wing Ser Leu Arg 55 70 75 80 Ala Glu Leu Gla Gly HAS Kis Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95 Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105 110 Lys He Phe Glu Pro Pro Ala Pro Gly Glu Gly Asa Ser Ser Gla Asa 115 120 125 Ser Arg Asa Lys Arg Ala Val Gla Gly Pro Glu Glu Thr Val Thr Gla 130 135 140 As? Cys Leu Gla Leu He Wing A = p Ser Glu Thr Pro Thr -le Gla Lys 145 150 155 1S0 Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser 1S5 170 175 Ala Leu Glu Glu Lys Glu Asa Lys lie Leu Val Lys Glu Thr Gly Tyr 180 135 190 Í Phe Phe lie Tyr Gly Gla Val Leu Tyr Thr A = p Lys Thr Tyr Ala Mee 195 200 205 Gly Kis Leu He Gla Arg Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215 220 Ser Leu Val Thr Leu Phe Arg Cys He Gla Met Pro Pro Glu Thr Leu 225 230 235 240 Pro Asa Asa Ser Cys Tyr Ser Wing Gly He Wing Lys Leu Glu Glu Gly 245 250 2S5 Asp Giu Leu Gla Leu Ala Ua. Pro Arg Giu Asa Wing Gla He Ser Leu 260 265 270 sp Gly Asp Val Thr Phe Phe Gly A_a Leu Lys Leu Leu 275 280 285 (2) INFORMATION FOR NO. SEC ID: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 233 amincacidos (B) TYPE: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 3; Mee Ser Thr Glu Ser Mee He Arg Asp al Glu Lau Wing Glu Glu Wing 1 5 10 15 Leu Pro Lys Lys Thr "Gly Gly Pro Gia Gly Ser Arg Arg Cys Leu Phe 20 .25 30 Leu Ser Leu Phe Ser Phe Leu He Val Wing Ala Thr Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val He Gly Pro Gia Arg Glu Glu Ser Pro 50 55 SO Arg Asp Leu Ser Leu He Ser Pro Leu Ala Gla Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val Wing His Val Val Wing Asa Pro ace 90 95 Gla Glu Gla Gla Gla Gla Leu Gla Trp Leu Asa Arg Arg Ala Asa Ala Leu 100 105 110 Leu Ala Asa Giy Val Glu Leu Arg Asp Asa Gla Leu Val Val Pro Ser 115 - 120 125 Glu Gly Leu Tyr Leu lie Tyr Ser Gla Val Leu Phe Lys Gly Gla Gly 130- 135 140 Cys Pro Ser Thr His Val Leu Leu Thr Kis Thr He Ser Arg lie Wing 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asa Leu Leu Ser Wing He Lys Ser Pro 165 170 175 Cys Gla Arg Glu Thr Pro Glu Gly Wing Glu Wing Lys Pro Trp Tyr Glu 180 '185 190 Pro He Tyr Leu Gly Gly Val Phe Gla Leu Glu Lys Gly A = p Arg Leu 195 200 205 Ser Wing Glu He Asa Arg Pro Asp Tyr Leu Asp Phe Wing Glu Ser Gly 210 215 220 Gln Val Tyr Phe Giy He He Ala Ala 225 - 230 (2) INFORMATION FOR NO. SEC ID: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 205 amino acids (B) TYPE: amino acids (C) HEBRAS: unique •: D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: NO. ID S? C Mee Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr 1 5 10 15 His Leu Leu Leu Leu Leu Glu Leu Leu Val Leu Pro Gly Wing 20 25 30 Gla Gly Lau Pro Gly Val Gly Leu Thr Pro Ser Ala Wing Gla Thr Wing 35 40 45 Arg Gia His Pro Lys Mee His Leu Wing His Ser Thr Leu Lys Pro Wing 50 55 60 Wing His Leu He Gly Asp Pro Ser Lys Gla. Asa Ser Leu Leu Trp Arg 65 70 75 80 Ala Asa Thr Asp Arg Ala Phe Leu Gia Asp Gly Phe Ser Leu Ser Asa 35 90. i 95 Asa Ser Lau Leu Val Pro Thr Ser Gly He Tyr Phe Val Tyr Ser Gla 100 105 110 Val Val Phe Ser Gly Lys Wing Tyr Sez Pro Lys Wing Pro Ser Pro 115 120 125 Leu Tyr Leu Wing Kis Glu Val Gla Leu Phe Ser Ser Gla Tyr Pro Phe 130 135 140 His Val Pro Lau Leu Ser Ser Gla Lys Mee Val Tyr Pro Gly Leu Gla 145 150 155 ISO Glu Pro Trp Leu His Ser Met Tyr His Gly Wing Wing Phe Gla Leu Thr 1S5 170 175 Gla Gly Asp Gla Leu Ser Thr? Is Thr Asp Gly He Pro His Leu Val ISO 135 190 Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu 195 200 205 INFORMATION FOR NO. SEC ID: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 244 amino acids (B) TYPE: amino acids (C) HEBRAS: single (D) TOPOLOGY: linear (ii) TYPE OF MOL CULA: protein (xi) DESCRIPTION OF THE SEQUENCE .: THE NO. SEC ID: 5: Mee Giy Ala Leu Gly Leu Glu Gly Arg Gly Giy Arg Leu Gla Gly Arg 1 5 10 15 Gly Ser Leu Leu Leu Wing Val Wing Gly Wing Thr Ser Leu Val Thr Leu 20 25 30 Leu Lau Wing Val? Rc He Thr Val Leu Wing Val Leu Wing Leu Val Pro 35 40 45 Gla Asp Gla Gly Gly Leu Val Thr Glu Thr Wing Asp Pro Gly Wing Gla 55 55 60 Wing Gla Gla Gly Leu Gly Phe Gla Lys Leu Pro Glu Glu Glu Pro Glu 65 70 75 30 Thr Asp Leu Ser Pro Gly Leu Pro Wing Ala His Leu lie Gly Ala Pro 85 90 '95 Leu Lys Gly Gla Gly Leu Giy Trp Giu Thr Thr Lys Glu Gla Wing Phe 100 103 110 Leu Thr Ser Gly Thr Gia Phe Ser A = p Wing Glu Gly Leu Wing Leu Pro 115 120 125 Gla Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg 130 135 140 Wing Pro Pro Gly Gly Gly Asp Pro Gla Gly Arg Ser Val Thr Leu Arg 145 150, 155 ISO Be Ser Lau Tyr Arg Wing Gly Wing Gly Wing Pro Gly Thr Pro Glu 165 170 175 Leu Leu Glu Gly Wing Glu Thr Val Thr Pro Val Leu Asp Pro Wing 180 185 130 Arg Arg Gla Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly 195 200 205 Gly Leu Val Gia Leu Arg Arg Gly Glu Arg Val Tyr Val Asa Ha Ser 210 215 220 His Pro Asp Met Val As? Pha Wing Arg Gly Lys Thr Phe Pha Gly Wing 225 230 235 240 -. c Val Mae Val Gly "• > • - r-7 <" -.-? 3t, iri < - '< ? T 2 NO ID S? C "C" (i) CHARACTERISTICS OF THE SEQUENCE: 20 ÍA) LENGTH: 231 amino acids (3) TYPE: amino acids ÍC) HEBRAS: unique (D) TOPOLOGY: linear 25 (ii) TYPE OF MOLECULE : protein (xi) SEQUENCE DESCRIPTION: NO. SEC ID: 6: Mee Gla Gla Pro Phe Asa Tyr Pro Tyr Pro Gla lie Tyr Trp Val Asp 1 5 10 15 • ~ l Being Ser Wing Being Pro Pro Trp Wing Pro Pro Gly Thr Val Leu Pro Cys 20 2S 30 Pro Thr Ser Val Pro Arg Arg Pro Giy Gla Arg Arg Pro Pro Pro 4D 35 _ 40 45 > Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro 50 Pro Pro Pro Pro 55 Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Acte Pro Leu Cys Leu Leu Val Mee Phe Phe Mee Val Leu Val Ala Leu Val Gly 50 85 90 95 Leu Gly Leu Gly Mee Phe Gia Leu Phe Kis Leu Gla Lys Glu Leu Ala 100. IOS 110 55 Glu Leu Arg Glu Ser Thr Ser Gla Mee His Thr Ala Ser Ser Leu Glu 115 120 125 end Lys Gla He Gly? is Pro Pro Pro Pro Glu Lys Lys Glu Leu Arg 130 135 140 Lys Val Wing His Leu Thr Gly Lys Ser Asa Ser Arg Ser Mee Pro Leu 145 150 155 - ISO Glu Trp Glu Asp Thr Tyr Gly He Val Leu Leu Ser Gly Val Lys Tyr 1S5 170 175 Lys Lys Gly Gly Leu Val He Asa Giu Thr Gly Lau Tyr Phe Val Tyr 180 185 190 Ser Lys Val Tyr Phe Arg Gly Gia Ser Cys Asa Asa Lau Pro Lau Sar ..195. 200 205 His Lys Val Tyr Met Arg Asa Ser Lys Tyr Pro Gla Asp Leu Val Mee 210 21S 220 Mat Giu Gly Lys Mßt Mae Ser Tyr Cys Thr Thr Gly Gla Mee Trp Wing 225 230 235 240 Arg Ser Ser Tyr Lau Gly Ala Val Phe Asa Leu Thr Ser Wing Asp Kis 245 250 255 Leu Tyr Val Asa Val Ser Giu Leu Ser Leu Val Asa Pha Glu Glu Sei 2S0 263 270 Gla Thr Phe Phe Giy Leu Tyr L s Leu 275 280 (2) PAPA INFORMATION. HE DOES NOT. ID S? C: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 338 base pairs (tí) 'üj ^ O: nucleic acid (C) HEBRAS: unique ID) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (qenomic) (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 7: AGG TAACTC TCCTGAGGGG TGAGCCAAGC CCTGCCATGT AGTGCACGCA GGACATCANC SO AAACACANNN NNCAGGAAAT AATCCATTCC CTGTGGTCAC TTATTCTAAA GGCCCCAACC 120 TTCAAAGTTC AAGTAGTGAT ATGGATGACT CCACAGAAAG GGAGCAGTCA CGCCTTACTT 180 CTTGCCTTAA GAAAAGAGAA GAAATGAAAC TGNAAGGAGT GTGTTTCCAT CCTCCCACGG 240 AAGGAAAGCC CCTCTNTCCG ATCCTCCAAA GACGGAAAGC TGCTGGCTGC AACCTTGNTG 300 NtsGCATTGT GTTCTTGCTG NCTCAAGGTG GTGTTUTT (2) INFORMATION FOR NO. SEC ID: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 509 base pairs (B) TiPO: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA ( genomic) (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 8: AATTCGGCA AGNAAACTGG TTACTTTTTT ATATATGGTC AGGTTTTATA TACTGATAAG 60 ACCTACGCCA TGGGACATCT AGTTCAGAGG AAGAAGGTCC ATGTCTTTGG GGATGAATTG 120 AGTCTGGTGA CTTTGTTTCG ATGTATTCAA AATATGCCTG AAACACTACC CAATAATTCC 180 TGCTATTCAG CTGGCATTGC AAAACTGGNA GGAAGGAGAT GAACTCCAAC TTGCAATACC 240 AGGGGAAAAT GCACAATTAT CACTGGGATG GAGATGTTCA CATTTTTTGG GTGCCATTGA 300 AACTGCTGTG ACCT CTTAC ANCANGTGCT GTTNGCTATT TTJTCCTNCCT NTTCT-ITGGT 3 SO AACCTCTTAG GAAGGAAGGA TTCTTAACTG GGAAATAACC CAAAAAAANN TTAAANGGGT 420 ANGNGNNANA NGNGGGGNiíG TTNNCMNGNN GNNTTTTNGG N ATNTT T TNGGGNNN 430 NGTAAAAATG GGGCC ANGG GGGNTTTTT 509 (2) INFORMATION PAPA EL NO. SEC ID: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 497 base pairs (B) _I? 0: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE .: THE NO. SEC ID: 9: AATTCGGCAC GAGCAAGGCC GGCCTGGAGG AAGCTCCAGC TGTCACCGCG GGACTGAAAA SO TCTTTGAACC ACCAGCTCCA GGAGAAGGCA ACTCCAGTCA GAACAGCAGA AATAAGCGTG 120 CCGTTCAGGG TCCAGAAGAA ACAGTCACTC AAGACTGCTT sCAACTGNTT GCAGACAGTG 180 AAACACCAAC TATACAAAAA GGCTCC TTC TGNTGCCACA TTTGGGCCAA GGAATGGAGA 240 GATTTCTTCG TCTGGAAACA TTTTGCCAAA C CTTCAGAT ACTCrrTNCT CTCTGGGAAT 300 CAAAGGAAAA TCTCTACTTA GATTN? CACA TTTGTTCCCA TsGGTNTCrT AAGTTTTAAA 3SO AGGG AGTGC CCTTAGGAGG AAAAGGGGAT AAATATTGGC CAAGG1ÍACTG GTGANTTTNT 420 AAATATGGTC AGGTTTOTAT ANCTGGTAGG CCTCGCCATG GGCATTNATT CANGGNGAGG 480 497 NCNNTCTTTT GGGUTGA (2) INFORMATION FOR NO. SEC ID: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (a) Tlt: nucleic acid (C) HEBRAS: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA ( genomic) (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 10: (2) INFORMATION FOR NO. SEC ID: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TiPC: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY .: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: THE MO. SEC ID: 11: GTGAAGCTTT TATTACAGCA GTTTCAATGC ACC (2) INFORMATION FOR NO. SEC ID: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) THE GITUDE: 26 base pairs (B) TYPE: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF SEQUENCE: NO. SEC ID: 12: GTGTCATGAG CCTCCGGGCA GAGCTG (2) INFORMATION FOR NO. SEC ID: 13: ii) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (OR HEBRAS: unique ID) TOPOLOGY: linear fii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 13: GTGAAGCTTT TATTACAGCA GTTTCAATGC ACC (2) INFORMATION FOR NO. SEC ID: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A.) LENGTH: 28 base pairs (tí) TxfO: nucleic acid ÍC! HEBRAS: io ca * "(D) TOPOLOGY .: linear (li) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 14: jvju vx CCC CGGGCAGAGC TGCAGC (2) INFORMATION FOR NO. SEC ID: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA ( genomic) (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 15: GTGGGATCCT TATTACAGCA GTTTCAATGC ACC (2) INFORMATION FOR NO. SEC ID: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A.) LENGTH: 129 base pairs (B) TYPE: nucleic acid (C) H? 3RAS: mica (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID: 16: GCGGGATCCG CCACCATGAA CTCCTTCTCC ACAAGCGCCT TCGGTCCAGT TGCCTTCTCC SO CTGGGGCTGC TCCTGGTGTT GCCTGCTGCC TTCCCTGCCC CAGTTGTGAG ACAAGGGGAC 120 CTGGCCAGC 129 (2) INFORMATION FOR NO. SEC ID: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) HEBRAS: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA ( genomic) (xi) DESCRIPTION OF THE SEQUENCE: NO. SEC ID 17: GTGGGATCCT TACAGCAGTT TCAATGCACC It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned intion is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property: i "". 0

Claims (21)

1. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding the Neutrocin a polypeptide having the sequence of complete amino acids shown in Figure 1 (SEQ ID NO: 2); (b) a nucleotide sequence encoding the Neutrocin a polypeptide, which has the complete amino acid sequence encoded by the cDNA clone contained in the ATCC deposit of October 22, 1996; (c) a nucleotide sequence encoding the extracellular domain of the Neutrocin a polypeptide; (d) a nucleotide sequence encoding the transmembrane t-domain of the Neutrocin a polypeptide; (e) a nucleotide sequence encoding the intracellular domain of Neutrocin polypeptide; (f) a nucleotide sequence encoding a soluble Neutrokine a polypeptide, comprising the tracelullar and intracellular domains but lacking the tr ansmembi an domain; and (g) a nucleotide sequence complementary to any of the nucleotide sequences of subsections (a), (b), (c), (d), (e) or (f) above.

2. The nucleic acid molecule according to claim 1, characterized in that the polynucleotide has the complete nucleotide sequence shown in Figure 1 (SEQ ID NO: 1).

3. The nucleic acid molecule according to the rei indication 1, characterized in that the polynucleotide has the nucleotide sequence shown in FIG. Figure. 1 (SEQ ID NO: 1) encoding the Neutrocin polypeptide having the complete amino acid sequence shown in Figure 1 (SEQ ID NO: 2).

4. The nucleic acid molecule according to claim 1, characterized in that the polynucleotide has the nucleotide sequence encoding a soluble Neutrokine polypeptide, comprising the extracellular domain shown in Figure 1 (SEQ ID NO: 2) .

5. An isolated nucleic acid molecule characterized in that it comprises a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence that consists of residues n-285 of SEQ ID NO: 2, where n is an integer that is in the range of 2-190; (b) a nucleotide sequence encoding a polypeptide having the amino acid sequence consisting of residues 1-m of SEQ ID NO: 2, wherein m is an integer that is in the range of 274-284; (c) a nucleotide sequence encoding a polypeptide having the amino acid sequence consisting of the nm residues of SEQ ID NO: 2, where n and m are integers as defined respectively in subparagraphs (a) and (b) ) previous; and (d) a nucleotide sequence encoding a polypeptide consisting of a portion of the amino acid sequence of Neutrocin a, complete, encoded by the cDNA clone contained in the ATCC deposit of October 22, 1996, wherein the portion excludes from I to 190 amino acids from the amino terminus and from 1 to II amino acids of the C term of the complete amino acid sequence.

6. The nucleic acid molecule according to claim 1, characterized in that the polynucleotide has the complete nucleotide sequence of the cDNA clone contained in the ATCC deposit of October 22, 1996.

7. The nucleic acid molecule according to claim 1, characterized in that the polynucleotide has the nucleotide sequence encoding the Neutrocin a polypeptide, which has the complete amino acid sequence encoded by the cDNA clone contained in the ATCC deposit of October 22 of 1996.

8. The nucleic acid molecule according to claim 1, characterized in that the polynucleotide has the nucleotide sequence encoding a soluble Neutrokine a polypeptide, comprising the extracellular domain encoded by the cDNA clone contained in the ATCC deposit of 22 October 1996

9. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide that hybridizes under stringent hybridization conditions in a polynucleotide having a nucleotide sequence identical to a nucleotide sequence of those in (a), (b), (c), (d), (e) 0 (f) of claim 1, wherein the hybridizing polynucleotide does not hybridize under stringent hybridization conditions in a polynucleotide having a nucleotide sequence consisting only of residues A or only of the waste T.

10. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide encoding the amino acid sequence of a portion containing an epitope, of a Neutrocin a polypeptide, having an amino acid sequence such as those found in clauses ( a), (b), (c), (d), (e) or (f), in accordance with the claim 1.

11. The nucleic acid molecule, isolated in accordance with the rei indication 10, characterized in that it encodes a portion containing an epitope, of a Neutrocin a polypeptide, selected from the group consisting of: a polypeptide comprising amino acid residues from about Phe 115 to about Leu 147 (No. ID S? C: 2); a polypeptide comprising amino acid residues from about 150 to about Tyr 163 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser 171 to about Phe 194 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Glu 223 to about Tyr 247 (SEQ ID NO: 2); and a polypeptide comprising amino acid residues from about Ser 271 to about Phe 278 (SEQ ID NO: 2).

12. A method for manufacturing a recombinant vector, characterized in that it comprises inserting an isolated nucleic acid molecule, in accordance with rei indication 1, into a vector.

13. A recombinant vector, characterized in that it is produced through the rei indication 12 method.

14. A method for manufacturing a recombinant host cell, characterized in that it comprises introducing the recombinant vector of claim 13 into a host cell.

15. A recombinant host cell characterized in that it is produced by the method of claim 14.

16. A recombinant method for producing a Neutrocin a polypeptide, characterized in that it comprises culturing the recombinant host cell of claim 15 under conditions such that the polypeptide is expressed and the polypeptide is recovered.

17. An isolated Neutrocin polypeptide, characterized in that it comprises at least a 95% amino acid sequence identical to a sequence selected from the group consisting of: (a) the amino acid sequence of the Neutrocin a polypeptide having the complete amino acid sequence of the Figure 1 (No. SEC ID: 2); (b) the amino acid sequence of the Neutrocin a polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the ATCC deposit of October 22, 1996; (c) the amino acid sequence of the extracellular domain of the polypeptide of

Neutrocin a; (d) the amino acid sequence of the transmembrane domain of the Neutrocin a polypeptide; 10 (e) the amino acid sequence of the intracellular domain of the Neutrocin a polypeptide; (f) the amino acid sequence of a soluble Neutrocin polypeptide, which 15 comprises the domain; and (g) the amino acid sequence of a portion containing an epitope, of any one of the polypeptides of part (a),. { b), (c), (d), (e) or (f). ? 18. A polypeptide isolated in accordance with rei indication 17, characterized in that it comprises a portion containing an epitope, of the protein Neutrocin a, wherein the portion is selected from the group consisting: a polypeptide comprising amino acid residues from about Phe 115 to approximately Leu 147 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about 150 to about Tyr 163 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser 171 to about Phe 194 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Giu 223 to about Tyr 247 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser 271 to about Phe 278 (SEQ ID NO: 2).

19. An isolated antibody that binds specifically to a polypeptide of Neutrocin according to claim 17.

20. A pharmaceutical composition characterized in that it comprises a polypeptide according to claim 17 and a pharmaceutically acceptable carrier.

21. An isolated nucleic acid molecule, characterized in that it comprises a polynucleotide having at least one sequence 95% identical to a sequence selected from the group consisting of: (a) the nucleotide sequence of clone HSOAD55R (SEQ ID NO: 7); (b) the nucleotide sequence of clone HSLAH84R (SEQ ID NO: 8); (c) the nucleotide sequence of clone HLTBM08R (SEQ ID NO: 9); (d) the nucleotide sequence of a portion of the sequence shown in Figure 1 (SEQ ID No. 1) wherein the portion comprises at least 30 contiguous nucleotides from nucleotide 1 to nucleotide 809; and (e) a complementary nucleotide sequence for any of the nucleotide sequences shown in items (a), (b), (c) or (d) above.