HK1023600A - NEUTROKINEα - Google Patents

Description

Neutrophil leucocyte factor alpha

no marking

The technical field to which the invention belongs

The present invention relates to a novel cytokine which is expressed by neutrophils and thus is called neutrophil factor (Neutrokine) alpha protein ("neutrophil factor alpha"). In particular, the invention provides isolated nucleic acid molecules encoding neutrophil factor alpha protein. The invention also provides neutrophil factor alpha polypeptide, carrier for producing the polypeptide, host cell and recombination method. Related prior art

Human tumor necrosis factors (TNF-. alpha.) and (TNF-. beta., or lymphotoxins) are relevant members of a broad class of polypeptide modulators, including interferons, interleukins, and growth factors, collectively referred to as cytokines (Beutler, B. and Cerami, A., Annu. ret.,. Immunol.,7: 625-. Sequence analysis of cytokine receptors has identified several subfamilies of membrane proteins: (1) ig superfamily, (2) hematopoietins (cytokine receptor superfamily), (3) Tumor Necrosis Factor (TNF)/Nerve Growth Factor (NGF) receptor superfamily (for a review of TNF superfamily, see Gruss and Down, blood 85(12): 3378-. The TNF/NGF receptor superfamily contains at least 10 different proteins. Gruss and Dower, supra. Ligands for these receptors have been identified, and they belong to at least two cytokine superfamilies. Gruss and Dower, supra.

Tumor necrosis factor (a mixture of TNF- α and TNF- β) was originally discovered for its anti-tumor activity, however, now it is considered to be a pleiotropic cytokine with many biological activities, including causing some transformed cells to appear, regulating cell activation and proliferation, and playing an important role in immune regulation and inflammation as well.

Members of the TNF-ligand superfamily known to date include TNF- α, TNF- β (lymphotoxin), LT- β, OX40L, Fas ligand, CD30L, CD27L, CD40L and 4-IBBL. The ligands of the TNF ligand superfamily are acidic, TNF-like molecules with about 20% (range, 12% -36%) sequence homology in the extracellular domain and exist only as membrane-bound forms with biologically active forms as trimeric multimeric complexes. To date, only TNF, LTP, and Fas ligands have been identified in soluble forms of the TNF ligand superfamily (for a general review, see Gruss, H. and Dower, S.K., blood, 85(12): 3378-. These proteins are involved in the regulation of cell proliferation, activation, and differentiation, including the control of cell survival or death by apoptosis or cytotoxicity (Armitage, r.j., curr. opin. immunol.6:407 (1994)) and Smith, CA., cell 75:959 (1994)).

Tumor necrosis factor-alpha (TNF α; also known as cachectin; hereinafter "TNF"), a soluble homologous triplet of 17kD protein subunits, is secreted primarily by monocytes and macrophages in response to endotoxin or other stimuli (Smith, R.A et al, J. Biochem. 262:6951-6954 (1987)). The 26kD membrane-bound precursor form of TNF has also been described (Kriegler, M., et al, cell 53:45-53 (1988)).

Accumulating evidence suggests that TNF is a regulatory cytokine with pleiotropic biological activity. These activities include: inhibition of lipoprotein lipase synthesis ("cachectin" activity) (Beutler, B. et al, Nature 316:552(1985)), activation of polymorphonuclear leukocytes (Klebanoff S.J. et al, J. Immunol 136:4220 (1986); Perussia, B., et al, J. Immunol 138:765(1987)), inhibition of cell growth or stimulation of cell growth (Vilcek, J. et al, J. Immunol 163:632 (1986); Sugarman, B.J. et al, science 230:943 (1985); Lachman, L.B. et al, J. 138: 1987), cytotoxic effects on certain transformed cell types (Sachman, L.B. et al, supra; Darzyneywicz, Z. et al, Cane.Res.44: 83: 1984), antiviral activity (Sachman, L.B. et al, Surpra, J. 1986: Nature W: 547 et al, J. Oakohol. 1986, J. Ehrlich.11, J. Equ et al, Nature W.11: 1986, Nature W: 547 et al, (1986), stimulation of Nature W322, J. Ehrlich. Eq. Stimulation of collagenase and prostaglandin E2 production (Dayer, J. -M. et al., J.Experimental J.162: 2163 (1985)); and immunomodulating effects, including T cells (Kehrl, J.H., et al, J.H., 166:786(1987)), monocytes (Philip, R. et al, Nature 323:86(1986)), thymocytes (Ranges, G.E., 167:1472(1988)), and stimulation of cell-surface expression of Major Histocompatibility Complex (MHC) class I and II molecules (Collins, T. et al, Proc. Natl. Acad. Sci. USA 83:446 (1986); Pujol-Borrel, R et al, Nature 326:304 (1987)).

TNF has been noted for its pro-inflammatory effects on tissue damage, such as inducing procoagulant activity on vascular endothelial cells (Pober, J.S et al, J.Immunol 136:1680(1986)), increasing neutrophil and lymphocyte adhesion (Pober, J.S. et al, J.Immunol 138:3319(1987)), and stimulating platelet activating factor release from macrophages, neutrophils, and vascular endothelial cells (Camussi, G et al, J.Immunol 166.1390 (1987)).

Recent evidence suggests that TNF is involved in the pathology of many infections (Cerami, A. et al, modern immunology 9:28(1988)), immune diseases, neoplasias, for example, in cachexia associated with some malignancies (Cliff, A et al, cell 50:555(1987)) and in autoimmune pathology and graft-to-host pathology (Piguet, P. -F. et al, J.EXPERIMENT MEDICAL 166:1280 (1987)). The association of TNF with cancer and infectious pathologies is often associated with the catabolic state of the host. One major problem in cancer patients is weight loss, often associated with anorexia. The strong wasting it causes is called "cachexia" (Kern, K.A. et al J.Parent.enter.Nutl:12: 286-. Cachexia includes progressive weight loss, anorexia, and permanent loss of body weight in response to malignant growth. Thus, cachexia is associated with a major morbidity and contributes to most cancer deaths. Several studies have suggested that TNF is an important mediator of cachexia, infectious disease, and other catabolic states in cancer.

TNF is thought to play a central role in the pathophysiological consequences of gram-negative sepsis and endotoxic shock, including fever, malaise, anorexia, and cachexia (Michie, H.R. et al, Br.J.Surg.76:670-671 (1989); Debets, J.M.H. et al, Second Yiennnashock Forum, p.463-466 (1989); Simpson, S.Q. et al, Crit.Care Clin.5:27-47 (1989)). Endotoxin is a potent monocyte/macrophage activator that stimulates the production and secretion of TNF and other cytokines (Kornbruth, SK. et al, J. Immunol 137:2585-2591 (1986)). Since TNF mimics the biological effects of many endotoxins, it was concluded to be a central mediator of the emergence of clinical toxoid-related diseases. TNF and other monocyte-derived cytokines modulate the metabolism of endotoxins and neurohormonal responses (Michie, H.R. et al, N.Eng.J.Med.318:1481-1486 (1988)). Administration of endotoxin to human volunteers produces acute diseases with influenza-like symptoms, including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al, Arch. Surg.123:162-170 (1988)). Elevated circulating TNF levels have also been detected in patients with gram-negative sepsis (Waage, A. et al, Lancet1:355-357 (1987); Hammerle, A.F. et al, Second Yieena Shock Forum p.715-718 (1989); Debets, J.M.H. et al, Crit. Care Med.17:489-497 (1989); Calandra, T. et al, J.Oenothers 161:982-987 (1990)).

Based on the increased TNF production and elevated TNF levels in these pathological conditions discussed above, passive immunotherapy against neutralizing TNF may have beneficial effects in gram-negative sepsis and endotoxemia. Cerami et al (EPO patent publication No. 0,212,489,1987, 3/4) disclose antibodies directed against "modulator" substances that are characterized as cachexins (later found to be identical to TNF. such antibodies are believed to be useful in diagnostic immunoassays and in the treatment of shock from bacterial infections. Rubin et al (EPO patent publication No. 0,218,868,1987, 4/22) disclose monoclonal antibodies to human TNF, hybridomas that secrete such antibodies, methods of producing such antibodies, and the use of these antibodies in TNF immunoassays.Yone et al (EPO patent publication No. 0,288,088,1998, 10/26) disclose anti-TNF antibodies, including mAbs, and their utility in pathological immunoassay diagnostics, particularly in Kawasaki disease and bacterial infections. body fluids from patients with Kawasaki disease (infant acute febrile mucocutaneous lymph node syndrome; Kawasaki, T., allergy 16:178 (1967); Kawasaki, t. Shonica (pediatrics) 26:935(1985) is believed to contain elevated TNF levels, which are associated with disease progression (Yone et al, supra).

Other investigators have described mAbs specific for recombinant human TNF having in vitro neutralizing activity (Liang, C-M., et al Biochemical Biophysical research Commission 137:847-854 (1986); meager, A. et al, hybridoma 6:305-31l (1987); fendly et al, hybridoma 6:359-369 (1987); bringman, TS et al, hybridoma 6:489-507 (1987); hirai, M.et al, J.Immunol.96: 57-62 (1987); moiler, A. et al (cytokine 2:162-169 (1990)). some of these mAbs were used to map epitopes to human TNF, and development of enzyme immunoassays (Fendly et al, supra; hirai et al, supra; moiler et al, supra), and aids in recombinant TNF purification (Bringman et al, supra), however, due to immunogenicity, lack of specificity and/or drug adaptability, these studies do not provide the basis for the generation of TNF neutralizing antibodies that can be used for diagnostic and therapeutic uses in humans.

Neutralizing antisera or mAbs against TNF have been shown to abrogate adverse physiological changes in non-human mammals and prevent death following lethal challenge in experimental endotoxemia and bacteremia. This effect has been demonstrated, for example, in rodent lethality assays and in primate pathology model systems (Mathison, J.C. et al, J.C. J.Res.81: 1925-.

To date, experience with anti-tnmab therapy in humans has been limited, but has shown beneficial therapeutic results, for example, in rheumatoid arthritis and sepsis. See, e.g., Elliott, M.J., et al, Baillieres Clin. Rharmatol.9:633-52 (1995); feldmann M, et al, Ann.N.Y.Acad.Sci.USA 766:272-8 (1995); van der Poll, T.et al, shock 3:1-12 (1995); wherry et al, grid.Care.Med.21: S436-40 (1993); tracey K.J., et al, grit. Care Med.21: S415-22 (1993).

Mammalian development is dependent on cellular proliferation and differentiation, as well as apoptosis by apoptosis (Walker, et al, Methods achiev. exp. Pathol.13:18 (1988.) apoptosis plays a role in the destruction of immune thymocytes that recognize self-antigens.

Itoh et al (cell 66:233(1991)) describe the cell surface antigen Fas CD23, which regulates apoptosis and is involved in clonal deletion of T-cells. Fas is expressed in activated T-cells, B-cells, neutrophils and, in addition to activated T-cells, B-cells, neutrophils, and in the thymus, liver, heart and lung, ovaries of adult mice (except for activated T-cells, B-cells, neutrophils) (Watanabe-Fukunaga et al, J. Immunol 148:1274 (1992)). Apoptosis is induced in monoclonal antibody cross-linking experiments with Fas (Yonehara et al, J.Imai Med.169: 1747 (1989); Trauth et al, science 245:301 (1989)). Furthermore, there are instances where the binding of monoclonal antibodies to Fas is stimulatory to T-cells (Alderson et al, J. Im. Med. 178:2231 (1993)).

The Fas antigen is a cell surface protein with a relative molecular weight of 45 Kd. Both the human and murine genes of Fas have been cloned by Watanabe-Fukunaga et al (J. Immunol 148:1274(1992)) and Itoh et al (cell 66:233 (1991)). Both proteins from the surface of these genes are transmembrane proteins with structural homology to the nerve growth factor/tumor necrosis factor receptor superfamily, which includes two TNF receptors, the low affinity nerve growth factor receptor and CD40, CD27, CD30, and OX 40.

Shortly before, Fas ligand has been described (Suda et al, cell 75:1169 (1993)). The amino acid sequence shows that Fas ligand is a II-type transmembrane protein, belonging to TNF family. Thus, Fas ligand polypeptides comprise three major domains: a short intracellular domain at the amino terminus and a long extracellular domain at the carboxy terminus are linked by a hydrophobic transmembrane domain. Fas ligand is expressed in splenocytes and thymocytes, consistent with T-cell regulated cytotoxicity. The purified Fas ligand has a molecular weight of 40 kD.

Shortly before, it was indicated that Fas ligand interaction is required for apoptosis following T-cell activation (Ju et al, Nature 373:444 (1995); Brunner et al, Nature 373:441 (1995)). Activation of T-cells induces two proteins on the cell surface. The subsequent interaction between the ligand and the receptor results in apoptosis of the cell. This fact supports this conclusion: during a normal immune response, a possible regulatory role for apoptosis is induced by Fas ligand interactions.

Therefore, there is a need to provide cytokines similar to TNF that are involved in pathological conditions. Such novel cytokines can be used to prepare novel antibodies or other antagonists that bind these TNF-like cytokines for the treatment of TNF-cytokine related disorders.

Summary of The Invention

The present invention provides isolated nucleic acid molecules comprising polynucleotides encoding cytokines that are structurally similar to TNF and related cytokines and are believed to have similar biological effects and activities. This cytokine is designated neutrophil factor alpha, and the invention includes neutrophil factor alpha polypeptides having at least a portion of the amino acid sequence of FIG. 1(SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone contained in the ATCC deposit deposited at 22.10.1996. The nucleotide sequence shown in FIG. l (SEQ ID NO:2) determined by sequencing of the deposited neutrophil factor alpha clone contains an open reading frame encoding the entire polypeptide of 285 amino acid residues, including an N-terminal methionine, a predicted intracellular domain of about 46 amino acid residues, a predicted transmembrane domain of about 26 amino acids, a predicted extracellular domain of about 213 amino acids, and a predicted complete protein molecular weight of about 31 kDa. For other class ii transmembrane proteins, all or part of the extracellular domain from which soluble forms of neutrophil factor α are cleaved from the transmembrane domain comprises a polypeptide in which the entire neutrophil factor α polypeptide lacks the transmembrane domain (i.e., 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 neutrophil factor alpha polypeptide having the complete amino acid sequence of figure 1(SEQ ID NO:2) or encoded by a cDNA clone contained in the ATCC deposit deposited at 10/22 1996; (b) a nucleotide sequence encoding the expected extracellular domain of a neutrophil factor α polypeptide having the amino acid sequence 73-285 of figure 1(SEQ ID NO:2) or encoded by a cDNA clone contained in the ATCC deposit deposited at 22.10.1996; (c) a nucleotide sequence encoding a polypeptide comprising the intracellular domain of a neutrophil factor alpha polypeptide (from about amino acids 1 to about 46 in FIG. 1(SEQ ID NO: 2)) or encoded by a cDNA clone contained in the ATCC deposit deposited at 10/22 1996; (d) a nucleotide sequence encoding a polypeptide comprising the neutrophil factor alpha polypeptide transmembrane domain (from about amino acids 47 to about 72 in FIG. 1(SEQ ID NO: 2)) or encoded by a cDNA clone contained in the ATCC deposit deposited at 10/22 1996; (e) a nucleotide sequence encoding a soluble neutrophil factor α polypeptide comprising extracellular and intracellular domains but lacking a transmembrane domain; and (f) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c), (d) or (e) above.

Other embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90%, preferably at least 95%, 96%, 97%, 98% or 99% identical to the sequence of any of (a), (b), (c), (d), (e) or (f) above, or which hybridizes under stringent hybridization conditions to a polynucleotide of (a), (b), (c), (d), (e) or (f) above, which hybridizes under stringent hybridization conditions without hybridizing to a polynucleotide having a nucleotide sequence consisting of only a residues or only T residues. A further nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide encoding the amino acid sequence of an epitope-bearing portion of a neutrophil factor alpha polypeptide having the amino acid sequence in (a), (b), (c), (d) or (e) above.

The invention also relates to recombinant vectors comprising the isolated nucleic acid molecules of the invention, host cells comprising the recombinant vectors, and methods of making such vectors and host cells and using them to produce neutrophil factor alpha polypeptides or peptides via recombinant techniques.

The present invention further provides an isolated neutrophil factor α polypeptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence of a full length neutrophil factor alpha polypeptide having the complete amino acid sequence of figure 1(SEQ ID NO:2) or encoded by a cDNA clone contained in the ATCC deposit deposited at 22/10/1996; (b) an amino acid sequence of the expected extracellular domain of a neutrophil factor α polypeptide having the amino acid sequence from position 73 to 285 of figure 1(SEQ ID NO:2) or encoded by a cDNA clone contained in the ATCC deposit deposited at 22.10.1996; (c) an amino acid sequence of a polypeptide comprising the intracellular domain of a neutrophil factor α polypeptide (from about amino acids 1 to about 46 in FIG. 1(SEQ ID NO: 2)) or encoded by an eDNA clone contained in the ATCC deposit deposited at 22.10.1996; (d) an amino acid sequence of a polypeptide comprising the transmembrane domain of a neutrophil factor alpha polypeptide (from about amino acids 47 to about 72 in FIG. 1(SEQ ID NO: 2)) or encoded by a cDNA clone contained in the ATCC deposit deposited at 22.10.1996; (e) an amino acid sequence of a soluble neutrophil factor alpha polypeptide comprising extracellular and intracellular domains but lacking a transmembrane domain, wherein each of these domains is as defined above.

The polypeptides of the invention also include polypeptides having at least 90% similarity, more preferably at least 95% similarity to those described in (a), (b), (c), (d) or (e) above, as well as polypeptides having an amino acid sequence which is at least 80% identical, more preferably at least 90% identical, more preferably 95%, 96%, 97%, 98% or 99% identical to those described above.

Another embodiment of this aspect of the invention relates to a peptide or polypeptide having the amino acid sequence of an epitope-bearing portion of a neutrophil factor alpha polypeptide, said polypeptide having the amino acid sequence described in (a), (b), (c), (d) or (e) above. The peptides or polypeptides of the invention having an epitope of a neutrophil factor alpha polypeptide-bearing the amino acid sequence of this portion include portions of polypeptides having at least six or seven, preferably at least nine, more preferably at least about 30 to about 50 amino acids, although epitope-bearing polypeptides of any length (up to and including the entire amino acid sequence of the polypeptides of the invention described above) are also included in the invention. In another embodiment, the invention provides an isolated antibody that specifically binds to a polypeptide having an amino acid sequence described in (a), (b), (c), (d), or (e) above.

The invention further provides methods of isolating antibodies that specifically bind to a neutrophil factor α polypeptide having an amino acid sequence described herein. These antibodies are useful diagnostically and therapeutically, as described below.

The invention further provides pharmaceutical compositions comprising soluble neutrophil factor α or polypeptides, particularly human neutrophil factor α polypeptides, which may be used in the treatment of, for example, tumors and tumor metastases, bacterial, viral, and other parasitic infections, immunodeficiency disorders, inflammatory diseases, lymphadenopathy, autoimmune diseases, graft rejection host disease, for stimulating peripheral tolerance, eliminating some transformed cell lines, modulating cell activation and proliferation, and are functionally associated as primary regulators of immunoregulation and inflammatory responses.

The invention further provides compositions comprising a neutrophil factor α polynucleotide or neutrophil factor α polypeptide for administration to cells in vitro, cells from vivo and cells in vivo or to multicellular organisms. In particularly preferred embodiments of this aspect of the invention, the composition comprises a neutrophil factor α polynucleotide for expressing neutrophil factor α for treating a disease in a host organism. Particularly preferred in this regard is expression in human patients to treat dysfunction associated with aberrant endogenous activity of the neutrophil factor alpha gene.

The invention also provides a screening method for identifying a compound capable of increasing or inhibiting a cellular response induced by neutrophil factor alpha, the method comprising contacting a cell expressing neutrophil factor alpha with a candidate compound, measuring the cellular response, comparing the cellular response to a standard cellular response (the standard being measured by contact in the absence of the candidate compound). Thus, an increased cellular response above the standard indicates that the compound is an agonist, and a decreased cellular response below the standard indicates that the compound is an antagonist.

In another aspect, the invention provides methods for identifying neutrophil factor alpha receptors and screening assays for agonists and antagonists utilizing such receptors. This assay involves determining the effect of a candidate compound on binding to neutrophil factor alpha receptor. More specifically, the method comprises contacting a neutrophil factor α receptor with a neutrophil factor α polypeptide and a candidate compound, and determining whether binding of the neutrophil factor α receptor to the neutrophil factor α polypeptide is increased or decreased due to the presence of the candidate compound. Said antagonists can be used to prevent septic shock, inflammation, cerebral malaria, HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis and cachexia (wasting or malnutrition).

The present inventors have found that neutrophil factor α is expressed not only in neutrophils but also in kidney, lung, peripheral leukocytes, bone marrow, T-cell lymphoma, B-cell lymphoma, activated T-cells, gastric cancer, smooth muscle, macrophages, chordae blood tissue. For certain diseases of these tissues and cells, such as tumor and tumor metastasis, bacterial, viral and other parasitic infections, immunodeficiency diseases, septic shock, inflammation, cerebral malaria, HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis, and cachexia (wasting or malnutrition), it is believed that significantly higher or lower levels of neutrophil factor α gene expression may be detected in some tissues (e.g., bone marrow) or body fluids (e.g., serum, plasma, urine, synovial fluid) taken from individuals having such diseases, relative to "standard" neutrophil factor α gene expression levels, i.e., the expression levels of neutrophil factor α in tissues and body fluids taken from individuals not having the disease. Thus, the present invention provides a diagnostic method useful during the diagnosis of a disease, the method comprising (a) determining the expression level of the neutrophil factor alpha gene in cells or body fluid of an individual; (2) comparing the expression level of the neutrophil factor alpha gene with a standard expression level of the neutrophil factor alpha gene, whereby an increase or decrease in the measured expression level of the neutrophil factor alpha gene as compared to the standard expression level is indicative of a disease.

Another aspect of the invention relates to a method of treating an individual in need of increased neutrophil factor α activity in vivo, comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated neutrophil factor α polypeptide of the invention or an agonist thereof.

Yet another aspect of the invention relates to a method of treating an individual in need of reduced neutrophil factor α activity in vivo, comprising administering to such an individual a composition comprising a therapeutically effective amount of a neutrophil factor α antagonist. Preferred antagonists for use in the present invention are neutrophil factor alpha-specific antibodies.

Brief description of the drawings

FIG. 1 shows the nucleotide (SEQ ID NO:1) and putative amino acid (SEQ ID NO:2) sequences of the neutrophil factor alpha protein. Amino acids 1 to 46 represent the intracellular domain, amino acids 47 to 72 represent the transmembrane domain (underlined sequence), and amino acids 73 to 285 represent the extracellular domain (remaining sequence).

FIG. 2 shows the regions of identity between the neutrophil factor alpha protein and the amino acid sequences TNF-alpha (SEQ ID NO:3), TNF-beta (lymphotoxin) (SEQ ID NO:4) and FAS ligand (SEQ ID NO:5) as determined by the "Megalign" program, which is part of a computer program called "DNAStar". The solid squares in the figure are residues that are perfectly paired with the consensus sequence.

FIG. 3 shows the amino acid sequence analysis of neutrophil factor α. Showing the α, β, turn and helical regions; hydrophilic and hydrophobic; an amphiphilic region; a flexible region; antigen index and surface probability. In the antigen index-Jameson-Wolf diagram, the position of the high antigen region of the neutrophil factor α protein (i.e., the region from which the epitope-carrying peptide of the present invention can be obtained) is indicated.

FIG. 4 shows a sequence alignment of the neutrophil factor α nucleotide sequence determined from the human cDNA contained in the ATCC deposit deposited at 22.10.1996 with the relevant human cDNA clones of the invention which have been designated HSOAD55R (SEQ ID NO:7), HSLAH84R (SEQ ID NO:8) and HLTBM08R (SEQ ID NO: 9).

Detailed Description

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a neutrophil factor alpha polypeptide having the amino acid sequence shown in figure 1(SEQ ID NO:2), said sequence being determined by sequencing cloned cDNA neutrophil factor alpha. The nucleotide sequence shown in FIG. 1(SEQ ID NO:1) was obtained by sequencing the HNEDU15 clone deposited at the American type culture Collection (12301 Park Lawn Drive, Rockville, Maryland) at 10.22.1996. The deposited clone was contained in pBluescript SK (-) plasmid (Stratagene, La Jolla, Calif.).

The neutrophil factor alpha protein of the present invention has sequence homology with the translation products of human mRNAs for TNF-alpha, TNF-beta and Fas ligand (FIG. 2). As mentioned above, TNF- α is considered to be a cytokine that plays a role in cytotoxicity, necrosis, apoptosis, costimulation, proliferation, lymph node formation, immunoglobulin class switching, differentiation, antiviral activity, adhesion molecules and regulation of other cytokines and growth factors. Nucleic acid molecules

Unless otherwise indicated, all nucleotide sequences determined by sequencing DNA molecules of the invention are determined using an automated DNA sequencer (e.g., model 373 from Applied Biosystems, Inc., Foster City, Calif.). And all amino acid sequences of polypeptides encoded by the DNA molecules determined by the present invention are predicted by translation of the DNA sequences identified above. Thus, as is known in the art for any DNA sequence determined by this automated method, any nucleotide sequence determined by the present invention may contain some errors. The nucleotide sequence determined by automation is typically at least about 90% identical, more typically at least about 95% to about at least 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more accurately determined by other methods, including manual DNA sequencing methods known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence, as compared to the actual sequence, results in a reading frame shift in translation of the nucleotide sequence such that at the beginning of the point of such insertion or deletion, the predicted amino acid sequence encoded by the determined nucleotide sequence is completely different from the amino acid sequence actually encoded by the sequenced DNA molecule.

"nucleotide sequence" of a nucleic acid molecule or polynucleotide means, for a DNA molecule or polynucleotide, a deoxyribonucleotide sequence, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U), wherein each thymidine deoxyribonucleotide (T) in a particular deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequence in fig. 1, the nucleic acid molecules of the invention encoding a neutrophil factor α polypeptide can be obtained using standard cloning and screening methods, such as those that clone cDNA using mRNA as a starting material. The nucleic acid molecule depicted in FIG. 1(SEQ ID NO:1), which is illustrative of the present invention, was detected from a neutrophil-derived cDNA library. Expressed sequence tags corresponding to the neutrophil factor alpha cDNA portion were also detected in neutrophils.

The neutrophil factor alpha gene contains an open reading frame encoding a protein of 285 amino acid residues, an intracellular domain of about 46 amino acid residues (amino acid residues about 1 to about 46 of FIG. 1(SEQ ID NO: 2)), a transmembrane domain of about 26 amino acids (amino acid residues about 47 to about 72 of FIG. 1(SEQ ID NO: 2)), an extracellular domain of about 213 amino acids (amino acid residues about 73 to about 285 of FIG. 1(SEQ ID NO: 2)), a putative molecular weight of about 31 kDa. FIG. 1(SEQ ID NO:2) shows that the neutrophil factor α protein is about 20% similar and about 10% identical to human TNF- α, which can be found in GenBank under accession number 339764.

As will be clear to one skilled in the art, because of the possibility of sequencing errors discussed above, the actual whole neutrophil factor α polypeptide encoded by the deposited cDNA (which comprises about 285 amino acids) may be shorter. Specifically, the neutrophil factor α coding sequence was determined to contain a second methionine codon which serves as an alternative initiation codon for open reading frame translation (at nucleotide 210-213 in FIG. 1(SEQ ID NO: 1)). More generally, the actual open reading frame may be within. + -.20 amino acids, more likely anywhere within. + -.10 amino acids, predicted from the first or second methionine codon from the N-terminus shown in FIG. 1(SEQ ID NO: 1). It is also clear that the exact "address" of the extracellular, intracellular and transmembrane domains of the neutrophil factor alpha polypeptide may vary slightly depending on the assay criteria used to identify the various functional domains. For example, depending on the criteria used to determine the domain, the exact position of the extracellular domain of neutrophil factor c in FIG. 1(SEQ ID NO:2) may vary slightly (e.g., the address may be "shifted" by about 1 to about 20 residues, more likely by about 1 to about 5 residues. in this case, the end of the transmembrane domain and the beginning of the extracellular domain predicted based on the hydrophobic amino acid sequence identifying the positions indicated above are shown in FIG. 3. in any case, as discussed further below, the invention also provides polypeptides having various residues deleted from the entire polypeptide, including polypeptides of one or more amino acids N-terminal to the extracellular domain described herein, which constitute the extracellular domain of the soluble form of the neutrophil factor α protein.

As indicated, the nucleic acid molecules of the invention may be in the form of RNA, such as mRNA, and in the form of DNA, including, for example, cDNA and genomic DNA (obtained from a clone or produced synthetically). The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also referred to as the sense strand) or the non-coding strand (also referred to as the antisense strand).

An "isolated" nucleic acid molecule is intended to mean a nucleic acid molecule, DNA or RNA, which has been isolated from its natural environment. For example, a recombinant DNA molecule contained in a vector is considered isolated for the purposes of the present invention. Other examples of isolated DNA molecules include recombinant DNA molecules or purified (partial or substantial) DNA molecules maintained in a heterologous host cell. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the invention. Isolated nucleic acid molecules according to the invention also include synthetically produced such molecules.

The isolated nucleic acid molecules of the present invention include DNA molecules comprising an Open Reading Frame (ORF) having the start codon at position 147-149 of the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1). Furthermore, the isolated nucleic acid molecules of the present invention include DNA molecules comprising sequences that are substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode the neutrophil factor α protein. Of course, the genetic code is known in the art. Thus, it is routine for one skilled in the art to produce degenerate variants as described above. In another aspect, the invention provides an isolated nucleic acid molecule encoding a neutrophil factor α polypeptide having an amino acid sequence encoded by a cDNA clone contained in a plasmid deposited at 22/10/1996. Preferably, this nucleic acid molecule comprises a sequence encoding the extracellular domain of the polypeptide encoded by the deposited cDNA clone described above.

The invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown in FIG. 1(SEQ ID NO:1) or a nucleotide sequence comprising the deposited cloned neutrophil factor alpha cDNA as described above, or a nucleic acid molecule having a sequence complementary to any of the above sequences. Such isolated molecules, particularly DNA molecules, are useful as probes for gene mapping (using in situ hybridization of chromosomes) and in detecting expression of the neutrophil factor alpha gene in human tissue (e.g., via Northern blot analysis).

The invention also relates to nucleic acid molecules encoding portions of the nucleotide sequences described above as well as fragments of the isolated nucleic acid molecules described herein. Specifically, the present invention provides a polynucleotide having a nucleotide sequence representing a part of SEQ ID NO:1 (which consists of 1-1001 of SEQ ID NO: 1).

Furthermore, the present invention includes polynucleotides comprising a sequence that is at least 95% identical to a portion of at least about 30 contiguous nucleotides, preferably at least about 50 nucleotides, of the sequence from nucleotide 1 to nucleotide 809 of FIG. 1(SEQ ID NO: 1).

More generally, a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in FIG. 1(SEQ ID NO:1) means a fragment of at least about 15nt, preferably at least about 20nt, more preferably at least about 30nt, and most preferably at least about 40nt in length, which is useful as a diagnostic probe and primer, as discussed herein. Of course, larger fragments of 50-300nt in length, which correspond to most, if not all, of the deposited cDNA or the nucleotide sequence shown in FIG. 1(SEQ ID NO:1), are also useful according to the invention. For example, a fragment of at least 20nt in length means a fragment comprising 20 or more contiguous bases of the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1). Preferred nucleic acid fragments of the invention include nucleic acid molecules encoding the epitope-bearing portion of the neutrophil factor alpha polypeptide (as defined in figure 1 and described in detail below).

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide that hybridizes under stringent hybridization conditions to a portion of the polynucleotide of the nucleic acid molecule of the invention described above, e.g., a cDNA clone contained in the ATCC deposit deposited at 10/22 1996. "stringent hybridization conditions" means an overnight incubation at 42 ℃ in a solution comprising 50% formamide, 5XSSC (150mM NaCl,15mM trisodium citrate), 50mM sodium phosphate pH7.6),5 XDenhardt's solution, 10% dextran sulfate and 20. mu.g/ml denatured sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at about 65 ℃.

A polynucleotide that hybridizes to a "portion" of a polynucleotide means a polynucleotide (DNA or RNA) that hybridizes to at least about 15 nucleotides (nt), preferably at least about 20nt, more preferably at least about 30nt, and most preferably about 30-70 (e.g., 50) nt of a reference sequence. These are useful as diagnostic probes and primers, as described above and in detail below.

A portion of a polynucleotide "at least 20nt in length" means, for example, 20 or more contiguous nucleotides of the nucleotide sequence of the reference polynucleotide (deposited cDNA or the nucleotide sequence shown in FIG. 1(SEQ ID NO: 1)). Of course, polynucleotides that hybridize only to poly A (e.g., the 3' terminal poly (A) fragment of the neutrophil factor α cDNA shown in FIG. 1(SEQ ID NO: 1)) sequences or to the T (or U) residue complementary backbone would not be included in the polynucleotides used to hybridize to portions of the nucleic acids of the present invention, as such polynucleotides would hybridize to any nucleic acid molecule containing the poly (A) backbone or its complement (e.g., virtually any double stranded cDNA clone).

As indicated, the nucleic acid molecules of the invention encoding a neutrophil factor α polypeptide may include, but are not limited to, those that themselves encode the amino acid sequence of the intracellular domain of the polypeptide; coding sequences for the intracellular domain and additional sequences of said polypeptide, such as those encoding extracellular and transmembrane domain sequences or pre-, pro-or prepro-protein sequences; a coding sequence for an intracellular domain of said polypeptide with or without the previously mentioned additional coding sequences.

Also encoded by the nucleic acids of the invention are the above protein sequences as well as additional non-coding sequences, including but not limited to, for example, introns and non-coding 5 'and 3' sequences, such as transcribed non-translated sequences that play a role in transcription and mRNA processing, including splicing and polyadenylation signals, e.g., ribosome binding and stability mRNA; additional coding sequences encoding additional amino acids, such as those providing additional functional groups.

Thus, a sequence encoding a polypeptide may be fused to a tag sequence, for example a sequence encoding a polypeptide which facilitates purification of the fused polypeptide. In a preferred embodiment of this aspect of the invention, the tag amino acid sequence is a hexahistidine polypeptide, such as the tag provided by the pQE vector (QIAGEN Inc., 9259Eton Avenue, Chatsworth, CA,91311), many of which are commercially available. As described by Gentz et al, Proc. Natl.Acad.Sci.USA 86:821-824(1989), for example, hexa-histidine provides convenience for protein purification. "HA" is another polypeptide useful for purification, which corresponds to an epitope from the influenza hemagglutinin protein, which is described by Wilson et al, cell 37:767 (1984). Other such fusion proteins include neutrophil factor α fused to Fc at the N-or C-terminus, as discussed below. Variant and mutant polynucleotides

The invention also relates to variants of the nucleic acid molecules of the invention, which encode a part, an analogue or a derivative of the neutrophil factor alpha protein. The variant may be naturally occurring, such as a natural allelic variant. "allelic variant" means one of several alternative forms of a gene having a given locus on a chromosome of an organism. Gene II, Lewis, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants can be generated using mutagenesis techniques known in the art.

Such variants include those resulting from nucleotide substitutions, deletions or additions. Substitutions, deletions or additions may include one or more nucleotides. Variants may vary in coding regions, non-coding regions, or both. Changes in the coding regions may result in conservative or non-conservative amino acid substitutions, deletions or additions. Among these, particularly preferred are silent substitutions, additions and deletions, which do not alter the properties and activity of the neutrophil factor α protein or a part thereof. In this connection, conservative substitutions are particularly preferred.

Most preferred are nucleic acid molecules that encode the intracellular domain of a protein having the amino acid sequence shown in FIG. 1(SEQ ID NO:2), or the extracellular domain of the amino acid sequence of neutrophil factor α encoded by the deposited cDNA clone. Other embodiments include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90%, preferably at least 95%, 96%, 97%, 98% or 99% identical to a polynucleotide selected from the group consisting of: (a) a nucleotide sequence encoding a neutrophil factor alpha polypeptide having the complete amino acid sequence of figure 1(SEQ ID NO: 2); (b) a nucleotide sequence encoding the predicted extracellular domain of a neutrophil factor α polypeptide having the amino acid sequence from position 73 to 285 of figure 1(SEQ ID NO: 2); (c) a nucleotide sequence encoding a neutrophil factor α polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the ATCC deposit deposited at 22/10/1996; (d) a nucleotide sequence encoding the expected extracellular domain of a neutrophil factor α polypeptide having the amino acid sequence encoded by the cDNA clone contained in the ATCC deposit deposited at 22/10/1996; and (e) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c), (d) or (e) above.

A polynucleotide having a nucleotide sequence that is at least, e.g., 95% "identical" to a reference nucleotide sequence encoding a neutrophil factor α polypeptide means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence encoding a neutrophil factor α polypeptide. In other words, in order 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 may be deleted or substituted with other nucleotides, or nucleotides in an amount of 5% of the total nucleotides of the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5 'or 3' terminal position of the reference nucleotide sequence, or anywhere in between, interspersed either individually within the nucleotide sequence of the reference sequence or in one or more contiguous groups within the reference sequence.

Indeed, 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 FIG. 1 or the nucleotide sequence of a deposited cDNA clone, it can be routinely determined using known computer programs, such as the Bestfit program (Wisconsin sequence analysis software package, Unix version 8, genetic computer group, university institute, Science 575 Drive, Madison, Wis.53711). Bestfit uses Smith and Waterman to apply mathematical developments: 482-489(1981) found the best segment homology between the two sequences. When using Bestfit or any other sequence comparison program to identify whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, the parameters should of course be set so that the percentage of identity is calculated from the complete reference nucleotide sequence, allowing gaps of homology of up to 5% of the total number of nucleotides in the reference sequence.

The present application relates to nucleic acid molecules which are at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIG. 1(SEQ ID NO:1) or the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having neutrophil factor alpha activity. This is because even if a particular nucleic acid molecule does not encode a polypeptide having neutrophil factor α activity, the skilled artisan will still know how to use said nucleic acid molecule, for example, as a hybridization probe or Polymerase Chain Reaction (PCR) primer. Use of a nucleic acid molecule of the invention which does not encode a polypeptide having neutrophil factor α activity in particular (1) isolating the neutrophil factor α gene or an allelic variant thereof in a cDNA library; (2) metaphase chromosome in situ hybridization (e.g., "FISH") to provide the precise chromosomal location possessed by neutrophil factor α, as described by Verma et al chromosome: basic technical Manual, pergamon Press, New York (1988); and northern blot analysis to detect expression of neutrophil factor alpha mRNA in specific tissues.

However, preferred are nucleic acid molecules which have a sequence which is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in FIG. 1(SEQ ID NO:1) or the sequence of the deposited cDNA and which do encode a polypeptide having neutrophil factor alpha activity. By "polypeptide having neutrophil factor α activity" is meant a polypeptide that exhibits activity similar to (but not necessarily identical to) the extracellular domain or full length neutrophil factor α protein of the present invention, when assayed in a particular biological assay. For example, the neutrophil factor α proteins of the present invention regulate cell proliferation, cytotoxicity and cell death. Cell proliferation, cytotoxicity and cell death assays for determining the effects of proteins on certain cells can be performed by employing reagents for detecting cell replication and/or death that are well known in the art and commercially available. For example, many such assays for TNF-related protein activity are described in the references in the background section above this specification. Briefly, such assays involve collecting human or animal (e.g., mouse) cells and mixing with the following (1) transfected host cell-supernatant (or candidate polypeptide) containing neutrophil factor α or 2) non-transfected host cell-supernatant control, and after a certain period of culture, determining the effect on cell number and viability. Such cell proliferation modulating activity that can be measured in this type of assay is useful for treating tumors, tumor metastases, infections, autoimmune diseases, inflammation and other immune-related diseases.

In the assays described above, neutrophil factor α modulates cell proliferation and differentiation in a dose-dependent manner. Thus, "a polypeptide having neutrophil factor α protein activity" includes polypeptides that also exhibit any of the same cell modulating (particularly immunomodulatory) activity in a dose-dependent manner in the assays described above. Although the degree of dose-dependent activity need not be equivalent to the neutrophil factor α protein, preferably, the "polypeptide having neutrophil factor α protein activity" exhibits substantially similar dose-dependence at a given activity as compared to the neutrophil factor α protein (i.e., the candidate polypeptide exhibits greater activity or no more than about 25-fold, preferably, no more than about 10-fold, activity relative to the reference neutrophil factor α protein).

Like other members of the TNF family, neutrophil factor α exhibits activity on leukocytes, including, for example, monocytes, lymphocytes and neutrophils. Thus, neutrophil factor α is active in directing the proliferation, differentiation, and migration of these cell types. Such activity is useful for immune enhancement or suppression, spinal cord protection, stem cell fixation, control of acute and chronic inflammation, and treatment of leukemia. Assays for measuring such activity are known in the art. See, e.g., Peters et al, modern immunology, 17:273 (1996); yong et al, J.Ex.Med. 182:1111 (1995); caux et al, Nature 390:258 (1992); and Santiago-Schwarz et al, Experimental medical biology progress 378:7 (1995).

Of course, because of the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that: nucleic acid molecules having a sequence which is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in FIG. 1(SEQ ID NO:1) each encode a polypeptide having "neutrophil factor alpha protein activity". In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, it will be clear to the skilled worker even without comparative testing as described above. It will also be recognized in the art that for such nucleic acid molecules that are not degenerate variants, a reasonable number of the molecules will also encode a polypeptide having neutrophil factor α protein activity. This is because amino acid substitutions that are unlikely or unlikely to significantly affect protein function (e.g., replacement of a first aliphatic amino acid with a second aliphatic amino acid) are well known to the skilled artisan, as described further below. Vectors and host cells

The invention also relates to vectors comprising the isolated DNA molecules of the invention, genetically engineered host cells using said recombinant vectors, and methods for producing neutrophil factor alpha polypeptides or fragments thereof by recombinant techniques. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors can be replication competent or replication defective. In the latter case, propagation of the virus generally occurs only in the complementing host cell. The polynucleotide may be ligated into a vector comprising a selectable marker for propagation in a host. Typically, the plasmid vector is introduced into a precipitate (e.g., a calcium phosphate precipitate) or a lipid-loaded complex. If the vector is a virus, it may be extracorporeally packaged with a suitable packaging cell line and then transduced into a host cell.

The DNA insert should be operably linked to suitable promoters such as the phage lambda PL promoter, the E.coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and the retroviral LTRs promoter, among others as are known to those skilled in the art. The expression construct further contains transcription initiation, termination sites, and a translational ribosome binding site in the transcribed region. The coding portion of the extracellular domain of the transcript expressed by the construct preferably includes a translation initiation site at the start and stop codons (UAA, UGA or UAG), which are suitably located at the ends of the polypeptide being translated.

As illustrated, the expression vector preferably includes at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for culturing eukaryotic cells, and tetracycline, kanamycin or ampicillin resistance genes for culturing E.coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as E.coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells if Musca S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,293 and Bowes melanoma cells; and plant cells. Suitable media and conditions for the above-described host cells are known in the art.

Among the vectors, preferred for use in bacteria include pQE70, pQE60, and pQE9, provided by QIAGEN inc; pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A supplied by Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 supplied by Pharmacia. Preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG supplied by Stratagene; and pSVK3, pBPV, pMSG, and pSVL supplied by Pharmacia. Other suitable vectors will be apparent to the skilled person.

The construct may be introduced into the host cell by: calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection and other methods. Such methods are described in many standard laboratory manuals, e.g., Davis et al, basic methods of molecular biology (1986).

The polypeptides may be expressed in modified form (e.g., as fusion proteins) and may include not only secretion signals, but additional heterologous functional regions. For example, additional amino acid regions, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and constancy in the host cell during purification, or during subsequent processing and storage. In addition, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides (to cause secretion or excretion, to improve stability, and to facilitate purification, etc.) is well known and routine in the art. Preferred fusion proteins comprise heterologous regions of an immunoglobulin useful for stabilizing and purifying the protein. For example, EP-A-O464533 (Canadian family 2045869) discloses fusion proteins comprising an immunoglobulin molecule and various parts of the constant region of another human protein or part thereof. In many cases, the Fc part in the fusion protein is entirely advantageous for therapeutic and diagnostic purposes and thus leads, for example, to improved pharmacokinetic properties (EP-A0232262). On the other hand, for certain applications, it may be desirable to delete the Fc portion after the fusion protein is expressed in the manner described above, detected and purified. This is the case when the Fc portion demonstrates that the disorder is useful in therapy and diagnosis, for example, when the fusion protein is used as an immunogen. In drug discovery, for example, human proteins such as hIL-5 are fused to an Fc portion for the purpose of high-throughput screening assays to identify hIL-5 antagonists, see, D.Bennett et al, J.M.ID.8: 52-58(1995) and K.Johanson et al, J.Biol.Chem.270: 9459-9471 (1995).

The neutrophil factor alpha protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, High Performance Liquid Chromatography (HPLC) is used in the purification. Polypeptides of the invention include naturally purified products, products of chemical synthetic methods, and products produced by recombinant techniques in prokaryotic or eukaryotic hosts, including, for example, bacteria, yeast, higher plant, insect, and mammalian cells. Depending on the host used in the recombinant production method, the polypeptides of the invention may be glycosylated or may be non-glycosylated. In addition, the polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Neutrophil factor alpha polypeptides and fragments

The invention further provides an isolated neutrophil factor alpha polypeptide having the amino acid sequence encoded by the deposited cDNA or the amino acid sequence in FIG. 1(SEQ ID NO:2), or a peptide or polypeptide comprising a portion of the foregoing polypeptides. Variant and mutant polypeptides

In order to improve or alter the characteristics of the neutrophil factor alpha polypeptide, protein engineering may be used. Recombinant DNA techniques known to those skilled in the art can be used to create new muteins or fusion proteins comprising single or several amino acid substitutions, deletions, additions. Such modified polypeptides may exhibit, for example, increased activity or increased stability. Furthermore, they can be purified in higher yields and show better solubility than the corresponding native polypeptide, at least under certain purification and storage conditions. N-terminal and C-terminal deletion mutants

For example, for many proteins, including extracellular domains or mature forms of secreted proteins, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function. For example, Ron et al, J. Biochem., 265:2984-2988(1993) report that modified proteins having heparin-binding activity even if 3,8, or 27 amino-terminal amino acid residues are deleted.

In this case, because the proteins of the invention are members of the TNF polypeptide family, deletion of the N-terminal amino acid to the Gly (G) residue at position 191 in FIG. 1(SEQ ID NO:2) may still retain some biological activity, such as cytotoxicity against the appropriate target cell. Polypeptides with further N-terminal deletions (including Gly (G) residues) are not expected to retain such biological activity because this residue in TNF-related peptides is known to be in the start site of the conserved domain required for biological activity. However, even if one or more amino acids are initiated from the N-terminus of the protein resulting in a modification that results in loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, but removing no more than the majority of the residues of the full or extracellular domain of the protein from the N-terminus, the ability of the shortened protein to induce and/or bind antibodies that recognize the full or extracellular domain of the protein is generally maintained. Whether a particular polypeptide lacking the N-terminal residue of the intact protein retains such immunological activity can be readily determined by the routine methods described herein and by other methods known in the art.

Accordingly, the present invention further provides a polypeptide having one or more residues from the amino terminus to Gly191 residues counted from the amino terminus of the amino acid sequence of neutrophilin α shown in FIG. 1(SEQ ID NO:2) and a polynucleotide encoding such a polypeptide. In particular, the invention provides polypeptides having the amino acid sequence of residues N-190 of SEQ ID NO:2, wherein N is an integer in the range of 2-190 and 191 is the position of the first N-terminal residue of the complete neutrophil factor alpha polypeptide (shown in SEQ ID NO:2) which is believed to be required for neutrophil factor alpha protein activity. More specifically, the present invention provides polynucleotides encoding a polypeptide having the amino acid sequence of SEQ ID NO 2: 2-285,3-285,4-285,5-285,6-285,7-285,8-285,9-285,10-285,11-285,12-285,13-285,14-285,15-285,16-285,17-285,18-285,19-285,20-285,21-285,22-285,23-285,24-28285,27-285,28-285,29-285,30-285,31-285,32-285,33-285,34-285,35-285,36-285,37-285,38-285,39-285,40-285,4285,45-285,46-285,47-285,48-285,49-285,50-285, 51-285,52-285,53-285,54-285,55-285,56-285,57-285,58-285,59-285,66-285,61-285,62-285,63-285,64-285,65-285,66-285,67-285,68-285,69-285,70-285,71-285,72-285,73-285,74-285,75-285,76-285,77-285,78-285,79-285,80-285,81-285,82-285,83-285,84-285,85-285,86-285,87-285,88-285,89-285,90-285,91-285,92-285,93-285, 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. The invention also provides polynucleotides encoding these polypeptides.

Likewise, many examples of biologically functional C-terminal deletion muteins are known. For example, interferon γ exhibits up to ten-fold greater activity by deleting 8-10 amino acid residues from the carboxy-terminus of the protein (Dabeli et al, J. Biotechnology 7:199-216 (1988.) because the proteins of the present invention are members of the TNF polypeptide family, C-terminal amino acid deletions to Leu at position 284 are expected to retain most, if not all, of the biological activities, such as receptor binding and regulation of cell replication.Polypeptides having deletions to reach about 10 additional C-terminal residues also retain some activity (i.e., reach the Gly residue at position 273), such as receptor binding, although such polypeptides would lack the conserved TNF domain start at about Leu 284. however, other biological activities can be retained even if the deletion of one or more amino acids at the C-terminus of the protein results in a change in one or more biological functions of the protein, the ability of the shortened protein to induce and/or bind antibodies that recognize the intact or mature protein will generally be maintained when no more than most of the residues of the intact or mature protein are removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of the intact protein retains such immunological activity can be readily determined by the routine methods described herein and by other methods known in the art.

Accordingly, the present invention further provides a polypeptide having one or more residues from the carboxy terminus to Gly274 residue counted from the carboxy terminus of the amino acid sequence of neutrophilin α shown in FIG. 1(SEQ ID NO:2) and a polynucleotide encoding such a polypeptide. In particular, the present invention provides polypeptides having the amino acid sequence of residues 1-m of SEQ ID NO. 2, wherein m is an integer in the range of 274-284, and more particularly polynucleotides encoding polypeptides having the amino acid sequence of residues SEQ ID NO. 2: 1-274,1-275,1-276,1-277,1-278,1-279,1-280,1-281,1-282,1-283 and 1-284. The invention also provides polynucleotides encoding these polypeptides.

The invention also provides polypeptides having one or more amino acids deleted from the amino and carboxy termini, which can be described as having the n-m residues of SEQ ID NO 2, where n and m are integers as described above. Also included in the present invention is a nucleotide sequence encoding a polypeptide consisting of a portion of the complete neutrophil factor alpha amino acid sequence encoded by the cDNA clone contained in the ATCC deposit deposited at 22.10.1996, which is excluded from amino acids 1-190 from the amino terminal of the complete amino acid sequence encoded by the cDNA clone in the deposited clone (or any combination of these N-and C-termini) or amino acids 1-11 from the C-terminus. The present invention also provides polynucleotides encoding the above deletion polypeptides. Other mutants

In addition to the terminal deletion forms of the proteins discussed above, one of ordinary skill in the art will recognize that the amino acid sequence of some neutrophil factor alpha polypeptides may be varied without having a significant effect on the structure or function of the protein. If such differences in sequence are taken into account, it should be borne in mind that there will be regions on the protein that are critical for activity.

Thus, the invention also includes variants of neutrophil factor α polypeptides that substantially exhibit neutrophil factor α polypeptide activity or include regions of the neutrophil factor α protein, such as portions of the proteins discussed below. Such mutants include deletions, insertions, inversions, repeats and substitutions of the type selected according to the general rules known in the art (so as to have a minor effect on activity). For example, Bowie J.U., et al, "deciphering information in protein sequences: tolerance to amino acid substitutions," guidance on how to perform phenotypically silent amino acid substitutions is provided in science 247: 1306-. The authors indicate that there are two main approaches to studying amino acid sequence changes, the first of which relies on evolutionary processes in which mutations are accepted or not by natural selection. The second approach utilizes genetic engineering to introduce amino acid changes at specific positions of cloned genes and selects or screens to identify sequences that retain function.

As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors also indicate that an amino acid change is likely to be allowed at a certain position in the protein. For example, most hidden amino acid residues require nonpolar side chains, while few side chain surface properties are generally conserved. Such phenotypically silent substitutions are described in Bowie j.u. et al, supra, and are incorporated herein by reference. Conservative substitutions are typically seen as one substitution between the aliphatic amino acids Ala, Val, Leu and Ile; the interchange of the hydroxyl residues Ser and Thr, the interchange of the acidic residue Asp with Glu, the substitution between the amide residues Asn and Gln, the interchange of the basic residues Lys and Arg and the substitution between the aromatic residues Phe, Tyr.

Thus, a fragment, derivative or analogue of the polypeptide of FIG. 1(SEQ ID NO:2) or encoded by the deposited cDNA may be (i) one in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), which may or may not be encoded by the genetic code; or (ii) one in which one or more amino acid residues comprise a substituent; or (iii) one in which the extracellular domain of the polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide (e.g., polyethylene glycol); or (iv) one in which additional amino acids are fused to the extracellular domain of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence, and the sequence of the extracellular domain used for purification of the polypeptide or proprotein sequence. Such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art from the teachings herein.

Thus, the neutrophil factor α of the present invention may comprise one or more amino acid substitutions, deletions or additions, whether from natural mutations or artificial manipulations. As noted, the change is preferably a minor property change, e.g., a conservative amino acid substitution that does not significantly affect the folding or activity of the protein (see table 1).

TABLE 1 conservative amino acid substitutions

Aromatic phenylalanine

Tryptophan

Tyrosine

Hydrophobic leucine

Isoleucine

Valine

Polar glutamine

Asparagine

Basic arginine

Lysine

Histidine

Acidic aspartic acid

Glutamic acid

Small alanine

Serine

Threonine

Methionine

Glycine

The amino acids of the neutrophil factor alpha protein of the present invention, which are functionally important, can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Well, science 244:1081-1085 (1989)). The latter method introduces a single alanine mutation at all residues in the molecule. The resulting mutant molecules are then tested for biological activity, such as receptor binding or in vitro proliferative activity.

Of particular interest is the substitution of charged amino acids with other charged amino acids or neutral amino acids, which can result in compositions with highly desirable improved characteristics, such as less aggregation. Aggregation can not only reduce activity but can be problematic in the preparation of pharmaceutical formulations because the polymer can be immunogenic (Pinckard et al, clinical Experimental immunology 2:331-340 (1967); Robbins et al, diabetes 36: 838-845; Cleland et al, Crit. Rev. therapeutic drug Carrier Systems 10:307-377 (1993).

Amino acid substitutions may also alter the selectivity of ligand binding to cell surface receptors. For example, Ostade et al, Nature 361:266-268(1993) describe certain mutations that result in TNF- α binding selectively to only one of two known types of TNF receptors. Because neutrophil factor α is a member of the TNF polypeptide family, mutations similar to those in TNF- α are likely to have similar effects in neutrophil factor α.

The critical sites for ligand-receptor binding can be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. mol. biol. 224:899-904(1992) and Vos et al, science 255:306-312 (1992)). Since neutrophil factor α is a member of the TNF-related family of proteins, it is preferred to make mutations in the sequence encoding amino acids in the conserved domain of TNF (i.e., position 191-284 of FIG. 1(SEQ ID NO: 2)), and more preferably in residues in regions that are not conserved among all members of the TGF family, in order to modulate, but not completely eliminate, the biological activity of neutrophil factor α. Neutrophil factor α acts as an antagonist by making specific mutations in neutrophil factor α at the positions where such conserved amino acids are commonly found in the relevant TNFs, and thus has antagonistic activity. Accordingly, the polypeptides of the invention include neutrophil factor alpha mutants. Such a neutrophil factor alpha mutant consists of the full-length or extracellular domain (preferably) of the neutrophil factor alpha amino acid sequence shown in FIG. 1(SEQ ID NO: 2). Also forming part of the invention are isolated polynucleotides comprising a nucleic acid sequence encoding the above-described neutrophil factor alpha mutants.

The polypeptides of the invention are preferably provided in isolated form, preferably in substantially purified form. Recombinantly produced forms of the neutrophil factor alpha polypeptide may be substantially purified by the one-step method described in Smith and Johnson genes 67:31-40 (1988).

The polypeptides of the invention include the complete polypeptides encoded by the deposited cDNAs, including the intracellular, transmembrane and extracellular domains of the polypeptides encoded by the deposited cDNAs, the extracellular domain of the protein minus the intracellular and transmembrane domains, the complete polypeptide of FIG. 1(SEQ ID NO:2), the extracellular domain of FIG. 1(SEQ ID NO:2) minus the intracellular and transmembrane domains, and polypeptides having at least 90%, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% similarity to those described above.

The invention also encompasses a polypeptide which is at least 80% identical, preferably at least 90% or 95% more preferably at least 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNA or the polypeptide of FIG. 1(SEQ ID NO:2), as well as a portion of such a polypeptide having at least 30 amino acids, more preferably at least 50 amino acids.

The percent similarity of the two polypeptides is meant by using the Bestfit program (wisconsin sequence analysis software package, Unix version 8, genetic computer group, university institute, 575 scientific drive, Madison, WI 53711). Comparing the sequences of the two polypeptides the similarity scores generated by comparing the amino acid sequences of the two polypeptides and determining the value scale (default setting) for similarity. Bestfit uses Smith and Waterman to apply mathematical developments: 482-489(1981) found the best segment homology between the two sequences.

A polypeptide having an amino acid sequence that is at least, e.g., 95% "identical" to the reference amino acid sequence of a neutrophil factor α polypeptide means that the amino acid sequence of said polypeptide is identical to the reference sequence except that said polypeptide may include up to 5 amino acid changes per 100 amino acids of the reference amino acid sequence of a neutrophil factor α polypeptide. In other words, in order to obtain a polypeptide having an amino acid sequence at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with other amino acids, or amino acids in an amount of 5% of the total residues of the reference sequence may be inserted into the reference sequence. These changes to the reference sequence can occur at the amino or carboxy terminal position of the reference amino acid sequence, or anywhere between these two terminal positions, interspersed either individually within the amino acid sequence of the reference sequence or in one or more contiguous groups within the reference sequence.

Indeed, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the amino acid sequence shown in FIG. 1(SEQ ID NO:2) or the amino acid sequence encoded by the deposited cDNA clone, it can be routinely determined using known computer programs, such as the Bestfit program (Wisconsin sequence analysis software package, Unix version 8, genetic computer group, university institute, 575 scientific drive, Madison, Wis.53711). When using Bestfit or any other sequence comparison program to identify whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, the parameters should of course be set so that the percentage of identity is calculated from the complete reference amino acid sequence, allowing gaps of homology of up to 5% of the total number of amino acids in the reference sequence.

The polypeptides of the invention can be used as molecular weight markers on SDS-PAGE gels or molecular sieve gel filtration columns using methods intended by the person skilled in the art.

As described in detail below, the polypeptides of the invention may also be used to generate polyclonal and monoclonal antibodies that are useful in assays for detecting expression of neutrophil factor α protein as described below or as agonists and antagonists capable of enhancing or inhibiting neutrophil factor α protein function. In addition, such polypeptides may be used in yeast two-hybrid systems to "capture" neutrophil factor alpha protein binding proteins, which are also candidate agonists and antagonists in accordance with the present invention. The yeast two-hybrid system is described in Fields and Song, Nature 340:245-246 (1989). Epitope-bearing moieties

In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of the polypeptide of the invention. "immunogenic epitope" is defined as a portion of a protein that elicits an antibody response when the entire protein is an immunogen. In another aspect, the region of the protein molecule to which an antibody can bind 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, Proc. Natl. Acad. Sci. USA 81: 3998-.

For the selection of peptides or polypeptides that carry an epitope (i.e., a region containing a protein molecule to which an antibody can bind), it is known in the art that relatively short synthetic peptides that mimic portions of a protein sequence can often elicit antisera that react with portions of the mimicking protein. See, e.g., Sutcliffe, j.g., Shinnick, t.m., Green, n, and Learner, R.A (1983) "antibodies that react with predetermined sites on proteins", science 219: 660-. Peptides capable of eliciting protein-reactive sera are generally represented by the primary sequence of a protein and can be characterized by a set of simple chemical properties that are neither restricted to the immunodominant regions of the intact protein (i.e., immunogenic epitopes) nor to the amino or carboxyl termini. Thus, the antigenic epitope-bearing peptides and polypeptides of the invention are useful for the production of antibodies, including monoclonal antibodies, that specifically bind to the polypeptides of the invention. See Wilson et al, cell 37:767-778(1984), p 777.

Preferably the antigenic epitope-bearing peptides and polypeptides of the invention preferably contain at least seven, preferably at least nine, more preferably between about 15 and about 30 amino acids contained within the amino acid sequence of the polypeptide of the invention. Non-limiting examples of antigenic polypeptides or peptides that can be used to generate antibodies specific for neutrophil factor α include: a polypeptide comprising amino acid residues from about Phe115 to about Leu147 of figure 1(SEQ ID NO: 2); a polypeptide comprising the amino acid residues from about Ile150 to about Tyr163 of FIG. 1(SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser171 to about Phe194 of FIG. 1(SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Glu223 to about Tyr247 of FIG. 1(SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser271 to about Phe278 of FIG. 1(SEQ ID NO: 2); these polypeptide fragments have been determined to carry epitopes of the neutrophil factor alpha protein by Jameson-Wolf antigen index analysis, as shown in FIG. 3.

The epitope-bearing peptides and polypeptides of the invention can be produced by any conventional method. See, e.g., Houghten, R.A, (1985), solid phase methods for rapid mass synthesis of peptides: specificity of antigen-antibody interaction at the single amino acid level, proceedings of the national academy of sciences USA 82: 51315135; this "Simultaneous Multiple Peptide Synthesis (SMPS)" approach is further described in U.S. Pat. No. 4,631,211 to Houghten et al (1986).

The epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods known in the art. See, e.g., Sutcliffe et al, supra; wilson et al, supra; chow, M, et al, Proc. Natl. Acad. Sci USA 82: 910914; and Bittle, F.J., et al, J.Gen.Virol.66: 2347-. The immunogenic epitope-bearing peptides of the invention, i.e., when the entire protein is an immunogen, those portions of the protein that elicit an antibody response are identified according to methods known in the art. See, e.g., Geysen et al, supra. In addition, U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general method for detecting or determining the sequence of a monomer (amino acid or other compound) which is a topologically equivalent epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes methods for detecting or determining the sequence of monomers which are topologically equivalent ligands which are complementary to the ligand binding site of a particular receptor of interest. Similarly, Houghten, R.A. et al (1996) U.S. Pat. No. 5,480,971 discloses linear C1-C7-alkyl overalkylated oligopeptides in overalkylated (Peralkylated) oligopeptide mixtures and sets and libraries of such peptides, and methods for determining overalkylated oligopeptides that preferentially bind to receptor molecules of interest using such sets and libraries of oligopeptides. Thus, non-peptide analogs of the epitope-bearing peptides of the invention can also be prepared routinely by these methods. Fusion proteins

It will be clear to the skilled person that the above described neutrophil factor alpha polypeptides of the present invention, epitope-bearing fragments thereof, may be combined with portions of the constant binding domain of an immunoglobulin (IgG) to form a chimeric polypeptide. These fusion proteins are easy to purify and exhibit an increased half-life in vivo as has been shown for chimeric proteins consisting of the first two domains of the human CD 4-polypeptide and various domains of the heavy or light chain constant region of a mammalian immunoglobulin (EP394,827; Traunker et al, Nature 331:84-86 (1988)). Fusion proteins with disulfide-linked dimeric structures may also bind or neutralize other molecules more efficiently than monomeric neutrophil factor alpha protein or protein fragments alone, due to the IgG moiety (Foutoulakis et al, J. Biochem. 270: 3958-. Diagnosis of immune system-related diseases

The present inventors have examined the expression of neutrophil cytokine α in various tissues, particularly neutrophils. For some immune system-related diseases, substantially altered (increased or decreased) levels of neutrophil factor α gene expression may be detected in immune system tissue or other cells or body fluids (e.g., serum, plasma, urine, synovial fluid, and spinal fluid) taken from an individual with such a disease relative to "standard" neutrophil factor α gene expression levels, i.e., neutrophil factor α expression levels in immune system tissue or body fluids from individuals not suffering from the immune system disease. Thus, the present invention provides a diagnostic method useful during diagnosis of a systemic disease, the method comprising detecting the expression level of a gene encoding neutrophil factor alpha protein in immune system tissue or other cells or body fluids obtained from an individual and comparing the measured gene expression level with a standard neutrophil factor alpha gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune system disease.

In particular, it is believed that increased or decreased levels of neutrophil factor α protein and mRNA encoding neutrophil factor α protein are significantly expressed in mammals with cancer when compared to the corresponding "standard" levels. Furthermore, it is believed that increased or decreased levels of neutrophil factor α protein may be detected in certain bodily fluids (e.g., serum, plasma, urine, and spinal fluid) of a mammal having such cancer when compared to serum from a mammal of the same species that does not have the cancer.

Thus, during the diagnosis of immune system diseases, including cancers of this system, the present invention provides a useful diagnostic method comprising detecting the expression level of a gene encoding neutrophil factor alpha protein in the immune system tissue or other cells or body fluids obtained from an individual and comparing the measured gene expression level to a standard neutrophil factor alpha gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune system disease.

The present invention is useful as a prognostic indicator when there has been a diagnosis of immune system diseases (including tumor diagnosis) according to conventional methods, whereby patients showing increased or decreased expression of the neutrophil factor alpha gene will reach a worst clinical conclusion compared to patients expressing the gene at a level close to the standard level.

By "determining the expression level of a gene encoding a neutrophil factor α protein" is meant qualitatively or quantitatively determining or estimating the level of the neutrophil factor α protein or the level of the mRNA encoding the neutrophil factor α protein in a first biological sample, either directly (e.g., by determining or estimating the absolute protein level or the mRNA level) or relatively (e.g., by comparing to the neutrophil factor α protein level or the mRNA level in a second biological sample). Preferably, the level of neutrophil factor α protein or mRNA in a first biological sample is determined or estimated and compared to the level of neutrophil factor α protein or mRNA of a standard taken from a second biological sample obtained from an individual not suffering from the disease and determined from the average level of a population of individuals not suffering from the immune system disease. It is clear in the art that once the standard neutrophil factor alpha protein level or mRNA level is known, it can be reused as a comparative standard.

By "biological sample" is meant any biological sample, bodily fluid, cell line, tissue culture, or other source containing neutrophil factor alpha protein or mRNA obtained from an individual. As noted, biological samples include body fluids (e.g., serum, plasma, urine, synovial fluid, and spinal fluid) containing the free extracellular domain of the neutrophil factor α protein, immune system tissues, and other sources of tissue found to express neutrophil factor α or the intact or free extracellular domain of the neutrophil factor α receptor. Methods for obtaining tissue biopsy samples and body fluids from mammals are known in the art. Where the biological sample comprises mRNA, a tissue biopsy sample is a preferred source.

The present invention is useful for diagnosing or treating various immune system-related diseases in mammals, preferably humans. Such diseases include, but are not limited to, tumors and tumor metastases, infections, viruses and other parasitic bacteria, immunodeficiency disorders, inflammatory diseases, lymphadenopathy, autoimmune diseases, and diseases of the graft rejection host.

Total cellular RNA can be isolated from a biological sample using any suitable technique, using the one-step guanidinium thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, analytical biochemistry 162: 156-. The level of mRNA encoding the neutrophil factor alpha protein is then determined by any suitable method. These include northern blot analysis, S1 nuclease mapping, Polymerase Chain Reaction (PCR), reverse transcription coupled to polymerase chain reaction (RT-PCR), and reverse transcription coupled to ligase chain reaction (RT-LCR).

The level of neutrophil factor alpha protein can be determined in a biological sample using antibody-based techniques. For example, expression of neutrophil factor alpha protein in tissues can be studied using classical immunohistological methods (Jalkanen, M., et alJournal of cell biology 101:976-985 (1985); jalkanen, M., et al, J.Cell.Biol.105: 3087-3096 (1987)). Other antibody-based methods useful for detecting neutrophil factor alpha protein gene expression include immunoassays, such as enzyme linked immunosorbent assays (ELISA) and Radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and include radioisotopes such as iodine (I), (II), and (III)¹²⁵I,¹²¹I) Carbon (C)¹⁴C) Sulfur (S) (S)³⁵S), tritium (³H) Indium (I)¹¹²In), technetium (^99mTc), and fluorescent labels such as fluorescein and rhodamine, and biotin.

In addition to measuring neutrophil factor α protein in a biological sample obtained from an individual, neutrophil factor α protein can be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of neutrophil factor alpha protein include those detectable by X-ray radiography, NMR or ESR. For X-ray radiography, suitable labels include radioisotopes, such as barium or cesium, which emit detectable radiation but are not harmful to the subject. Suitable labels for NMR and ESR include those with a detectable characteristic rotation, such as deuterium, which can be incorporated into the antibody by labeling the relevant hybridoma nutrients.

A neutrophil factor alpha protein-specific antibody or antibody fragment, which has been labeled with a suitable detectable contrast component, such as a radioisotope (e.g.,¹³¹I,¹¹²I,^99mtc), radiopaque substances, or materials detectable by nuclear magnetic resonance. It will be appreciated in the art that the size of the subject and the contrast system utilized will determine the amount of contrast component required to produce a diagnostic audio image. In the case of radioisotope compositions for use in human subjects, the amount of radioactivity injected is typically between about 5-20 milliCuries^99mTc is in the range. The labeled antibody or antibody fragment then preferentially accumulates at the site of the cells containing the neutrophil factor alpha protein.In vivo tumor imaging is described in S.W.Burchiel et al, "immunopharmacology of radiolabeled antibodies and fragments thereof" (Chapter 13 in tumor imaging: radiochemical detection of cancer, S.W.Burchiel and B.A.Rhodes, eds., Masson publishing Co. (1982)). Antibodies

The neutrophil factor α protein-specific antibody used in the present invention may be produced from an anti-whole neutrophil factor α protein or an antigenic polypeptide fragment thereof, which may be present together with a carrier protein such as albumin of an animal system (e.g., rabbit or mouse); if it is of sufficient length (at least about 25 amino acids), then there is no carrier.

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (e.g., Fab and F (Ab') 2 fragments) capable of specifically binding to the neutrophil factor alpha protein. Fab and F (ab') 2 fragments lack the Fc fragment of intact antibodies, cycle significantly more rapidly, and can have less non-specific tissue binding of intact antibodies (Wahl et al, J. Nuclear medicine, 24:316-325 (1983)). Thus, these fragments are preferred.

The antibodies of the invention can be prepared by any of a variety of methods. For example, cells expressing the neutrophil factor α protein or antigenic fragments thereof can be administered to an animal in order to induce the production of serum comprising polyclonal antibodies. In a preferred method, the neutrophil factor alpha protein is prepared so as to be substantially free of natural contaminants. This preparation is then introduced into animals to produce more specific polyclonal antisera.

In most preferred methods, the antibody of the invention is a monoclonal antibody (or a neutrophil factor α protein-binding fragment thereof). Such monoclonal antibodies can be prepared using hybridoma technology (Kijhler et al, Nature 256:495 (1975); Kiihler et al, J.Eur. Immunol.6: 511 (1976); K  hlei et al, J.Eur. Immunol.6: 292 (1976); Hammerling et al, monoclonal antibodies and T-cell hybridomas, Elsevier, N.Y., (1981) pp.563-681). In general, such methods involve immunizing an animal (preferably a mouse) with a neutrophil factor α protein antigen or, more preferably, with a cell expressing a neutrophil factor α protein. Suitable cells can be identified by their ability to bind anti-neutrophil factor alpha protein antibodies. Such cells may be cultured in any suitable tissue culture machine; however, it is preferred to culture the cells in Eagle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56 ℃) and with about 10g/l non-essential amino acids, about 1,000U/ml penicillin, and about 100. mu.g/ml streptomycin. Splenocytes from such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be used in accordance with the present invention; more preferably, however, a parent myeloma cell line (SP20) is used, and may be obtained from the american type culture collection (Rockville, maryland). After fusion, the hybridoma cells formed are selectively maintained in HAT medium and then cloned by limiting dilution as described by Wands et al (gastroenterology 80:225-232 (1981)). The hybridoma cells obtained by this selection are then examined to identify clones that secrete antibodies capable of binding to the neutrophil factor alpha protein antigen.

Furthermore, additional antibodies capable of binding to the neutrophil factor alpha protein may be generated by a two-step process using anti-idiotype antibodies. Such a method utilizes the fact that an antibody is an autoantigen, and, therefore, it is possible to obtain an antibody that binds to a second antibody. According to this method, antibodies specific for the neutrophil factor alpha protein are used to immunize animals, preferably mice. Splenocytes from such animals are then used to generate hybridoma cells, which are screened to identify clones that produce antibodies whose ability to bind antibodies specific for the neutrophil factor α protein can be blocked by the neutrophil factor α protein antigen. Such antibodies include anti-idiotypic antibodies directed against antibodies specific for the neutrophil factor alpha protein and can be used to immunize animals to induce the formation of other antibodies specific for the neutrophil factor alpha protein.

It will be appreciated that Fab and F (ab') 2 and other fragments of the antibodies of the invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce (Fab') 2 fragments). In addition, neutrophil factor α protein-binding fragments can be produced by applying recombinant DNA techniques or synthetic chemistry.

Where in vivo imaging is used in human diagnostics to detect elevated levels of neutrophil factor α protein, it may be more preferred to use a "humanized" chimeric monoclonal antibody. Such antibodies can be produced by using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. For a review see, Morrison, science 229:1202 (1985); oi et al, Biotechnology 4:214 (1986); cabilly et al, U.S. Pat. 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 immune system-related disorders

As described above, neutrophil factor α polynucleotides and polypeptides are useful for the diagnosis of diseases involving abnormally high or low expression of neutrophil factor α activity. In the case of cells and tissues expressing neutrophil factor α and having activity modulated by neutrophil factor α, it is readily apparent that a substantial change (increase or decrease) in the level of expression of neutrophil factor α in an individual as compared to a standard or "normal" level results in a pathological condition associated with the systemic system in which neutrophil factor α is expressed and/or active.

It is also clear to those skilled in the art that since the neutrophil factor α proteins of the present invention are members of the TNF family, the extracellular domain of the protein can be released in soluble form from cells that express neutrophil factor α by proteolytic cleavage, and thus, when added from an exogenous source to the cells, tissues, and body of an individual, the neutrophil factor α protein (particularly the soluble form of the extracellular domain) will exert its regulatory activity on any target cell of the individual. In addition, cells expressing type ii transmembrane proteins can be added to cells, tissues or the body of an individual, whereby the added cells will bind to cells expressing the receptor for neutrophil factor α, such that the cells expressing neutrophil factor α can act (e.g., be cytotoxic) against the target cells bearing the receptor.

Thus, it will be appreciated that diseases caused by a reduction in the normal or normal level of neutrophil factor α activity in an individual, particularly immune system diseases, may be treated by administration of neutrophil factor α protein (either in the form of a soluble extracellular domain or cells expressing the intact protein). Thus, the present invention also provides a method of treating an individual in need of increased levels of neutrophil factor α activity, comprising administering to such an individual a pharmaceutical composition comprising an isolated neutrophil factor α polypeptide of the present invention in an amount effective to increase the levels of neutrophil factor α activity in such an individual.

Since neutrophil factor α belongs to the TNF superfamily, it should also regulate angiogenesis. In addition, since neutrophil factor α inhibits immune cell function, it will have various anti-inflammatory activities. Neutrophil factor α can be used as an anti-neovascularization agent to treat solid tumors by stimulating invasion and activation of host defense cells (e.g., cytotoxic T cells, as well as macrophages) and by inhibiting angiogenesis. Those skilled in the art will recognize other non-cancerous indications where vascular proliferation is not desired. They may also be used to improve host defense against chronic and acute infections, for example against myobacterial infections via the attraction and activation of microbicidal leukocytes. Neutrophil factor alpha can also be used to inhibit T-cell proliferation by inhibiting IL-2 biosynthesis for the treatment of T-cell mediated autoimmune diseases and lymphocytic leukemia. Neutrophil factor α can also be used to stimulate wound healing, both of which promote inflammatory cells via debris clearance and connective tissue. In this same manner, neutrophil factor α can also be used to treat other fibrotic diseases, including cirrhosis of the liver, osteoarthritis and pulmonary fibrosis. Neutrophil factor α also increases the presence of eosinophils, which have the obvious function of killing tissue-infesting parasite larvae as in schistosomiasis, trichinosis and ascariasis. It can also be used to regulate hematopoiesis by regulating the activation and differentiation of various hematopoietic progenitor cells, e.g., the release of mature leukocytes from the bone marrow following chemotherapy, i.e., on stem cell activation. Neutrophil factor α may also be used to treat sepsis. Preparation

The neutrophil factor alpha polypeptide composition (preferably comprising a polypeptide which is an extracellular domain in soluble form) is formulated and administered in a manner consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with a neutrophil factor alpha polypeptide alone), the site of delivery of the neutrophil factor alpha polypeptide composition, the method of administration, the time course of administration, and other factors known to the physician. An "effective amount" of a neutrophil factor α polypeptide for the purposes described herein is determined by such considerations.

As a general rule, a pharmaceutically effective amount of the neutrophil factor α polypeptide administered parenterally per dose is from about 1 μ g/kg/day to 10 μ g/kg/day by patient weight, although as noted above, this will be judged by treatment. More preferably, this dose is at least 0.01 μ g/kg/day, most preferably between about 0.01 and 1 μ g/kg/day of hormone for human patients. If administration is continued, the neutrophil factor alpha polypeptide is typically administered at a dosage rate of from about 1 μ g/kg/hour to about 50 μ g/kg/hour for 1-4 injections per day or for continuous subcutaneous infusion, e.g., using a mini-pump. Intravenous bag solutions may also be used. The length of treatment required to observe changes and the interval after treatment at which responses appear to vary with the effect desired.

Pharmaceutical compositions comprising the inventive neutrophil factor α can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (typically as a powder, ointment, droplet, or eye patch), orally, or as an oral or nasal spray. "pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of auxiliary agent. The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The neutrophil factor alpha polypeptide is suitable for administration via a sustained release system. Suitable examples of sustained release compositions include semipermeable polymer matrices in the form of shaped particles, e.g., films, or microcapsules. Sustained release matrices include polylactide (U.S. Pat. No. 3,773,919, EP58,481), a heterogeneous polymer of L-glutamic acid and ethyl gamma-L-glutamate (Sidman, U.S. et al, biopolymer 22:547-556(1983)), poly (2-hydroxyethylmethacrylate) (R.Langer et al, J.biomed.Mater.Res.15:167-277(1981), and R.Langer, chemical engineering 12:98-105(1982)), vinyl acetate (R.Langer et al, supra) or poly-D- (-) -3-hydroxybutyric acid (EP133,988). The sustained release neutrophil factor α polypeptide composition also includes a lipid entrapped neutrophil factor α polypeptide. Liposomes comprising the neutrophil factor alpha polypeptide are prepared by previously known methods: DE3,218,121; epstein et al, proceedings of the national academy of sciences USA 82: 3688-; hwang et al, Proc. Natl. Acad. Sci. USA 77:4030:4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; japanese patent application 83-118008; U.S. patent nos. 4,485,045 and 4,544,545; and EP102,324. Typically, liposomes are small (about 200-800 angstroms) lamellar in which the lipid content is greater than about 30mol percent cholesterol, the proportions selected being adjusted for optimal neutrophil factor α polypeptide therapy.

In one embodiment, for parenteral administration, the neutrophil factor α polypeptide is generally formulated by mixing it (in a unit dose injectable form (solution, suspension or emulsion) at the desired degree of purity) with a pharmaceutically acceptable carrier that is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other components of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are harmful to the polypeptide.

In general, the formulations are prepared by uniformly, consistently and intimately admixing the neutrophil factor alpha polypeptide with liquid carriers or finely divided solid phase carriers or both. Then, if necessary, the product is prepared into a desired formulation form. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carriers include water, saline, Ringer's solution, and dextrose solution. Non-aqueous carriers (e.g., fixed oils and ethyl oleate) and liposomes are also useful herein.

The carrier suitably contains minor amounts of additives such as substances which enhance isotonicity and chemical stability. Such species are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetate, and other organic acids or their salts; antioxidants, such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrin; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions, such as sodium; and/or a non-ionic surfactant, such as polysorbate, poloxamer, or PEG.

The neutrophil factor alpha polypeptide is typically formulated into such a carrier at a concentration of about 0.1mg/ml to 100mg/ml, preferably at a concentration of 1 to 10mg/ml, at a pH of about 3 to 8. It will be appreciated that the use of certain of the aforementioned excipients, carriers, or stabilizers will result in the formation of a neutrophil factor α polypeptide salt.

The neutrophil factor alpha polypeptide for therapeutic administration must be sterile. Sterility is readily achieved by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). The therapeutic neutrophil factor α polypeptide composition is typically placed into a container having a sterile access port, for example, an intravenous fluid bag or a vial having a stopper pierceable by a hypodermic injection needle.

The neutrophil factor alpha polypeptide is typically stored in a single-dose or multi-dose container, e.g., an ampoule or vial sealed with an aqueous solution or a lyophilized preparation for reconstitution. As an example of a lyophilized preparation, a 10ml vial was filled with 5ml of a sterile-filtered 1% (W/V) aqueous neutrophil factor alpha polypeptide solution, and the resulting mixture was lyophilized. Infusion solutions were prepared by reconstituting lyophilized neutrophil factor alpha polypeptide with water for injection.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the invention. Some of the matters associated with such containers may be noted in the form prescribed by governmental agencies governing the manufacture, use or sale of samples and biological products, such comments reflecting approval by human pharmaceutical manufacturing, use or sale authorities. In addition, the polypeptides of the invention may be combined with other therapeutic compounds. Agonist and antagonist-assays and molecules

The invention also provides methods of screening compounds to identify those that enhance or block the effect of neutrophil factor alpha on cells, such as their interaction with neutrophil factor alpha binding molecules (e.g., receptor molecules). Agonists are compounds that increase the natural biological function of neutrophil factor α or act in a manner similar to neutrophil factor α, while antagonists reduce or eliminate such function.

Another aspect of this embodiment of the invention provides a method for identifying receptor proteins or other ligand-binding proteins that specifically bind to a neutrophil factor α polypeptide, e.g., a cellular compartment such as a membrane or preparation thereof can be prepared from cells expressing a molecule that binds to neutrophil factor α. The preparation is incubated with labeled neutrophil factor alpha, complexes of neutrophil factor alpha binding receptors or other binding proteins are isolated, and characteristics are determined according to conventional methods known in the art. In addition, the neutrophil factor alpha polypeptide can be bound to a solid support so that soluble binding molecules from the cells are bound to the column, which is then eluted and characterized according to conventional methods.

In an agonist or antagonist assay of the invention, a cellular compartment (e.g., a membrane or preparation thereof) can be prepared from cells expressing a molecule that binds to neutrophil factor α, e.g., a molecule of a signaling or regulatory pathway regulated by neutrophil factor α. The preparation is incubated with labeled neutrophil factor alpha in the presence or absence of a candidate molecule that may be an agonist or antagonist of neutrophil factor alpha. The ability of the candidate molecule to bind to the binding molecule is reflected by a decrease in binding of the labeled ligand. Molecules that bind without reason, i.e., do not induce the effect of neutrophil factor α on binding to neutrophil factor α binding molecules, are likely to be good antagonists. Molecules that bind well and elicit effects similar to or closely related to neutrophil factor alpha are agonists.

The neutrophil-like factor α effects of potential agonists and antagonists can be determined, for example, by the following methods: after interaction of the candidate molecule with the cell or an appropriate cell preparation, the activity of the second messenger system is determined and the effect is compared with that of neutrophil factor alpha or a molecule that elicits the same effect as neutrophil factor alpha. Second messenger systems that may be useful in this regard include AMP guanylate cyclase, ion channels or phosphoinositide hydrolysis second messenger systems.

Another example of an assay for a neutrophil factor α antagonist is a competitive assay, which combines a neutrophil factor α and a potential antagonist with a membrane-bound receptor molecule or a recombinant neutrophil factor α receptor molecule under suitable conditions of a competitive inhibition assay. The neutrophil factor α can be labeled (e.g., radiolabeled) so that the number of neutrophil factor α molecules bound to the receptor molecule can be accurately measured, assessing the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to and thereby inhibit or eliminate the activity of the polypeptides of the invention. Potential antagonists may also be small organic molecules, peptides, polypeptides, such as closely related proteins or antibodies, that bind to the same site on a binding molecule (e.g., a receptor molecule) without inducing neutrophil factor α -induced activity, thereby inhibiting the effects of neutrophil factor α by excluding neutrophil factor α from binding.

Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression by antisense DNA or RNA or triple-helix formation, as described, for example, in Okano, J. Neurochem.56: 560 (1991); "oligodeoxynucleotides as inhibitors of gene expression, as discussed in CRC Press, Boca Raton, FL (1988). Triple-helix formation is described, for example, in Lee et al, nucleic acids Res 6:3073 (1979); gooney et al, science 241:456 (1988); and Dervan et al, science 251:1360 (1991). The method is based on binding of polynucleotides to complementary DNA or RNA. For example, the 5' coding portion of a polynucleotide encoding an RNA for the extracellular domain of a polypeptide of the invention can be used to design an antisense RNA from an oligonucleotide of about 10-40 base pairs in length. The DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription in order to prevent transcription and neutrophil factor alpha production. Antisense RNA oligonucleotides hybridize in vivo to mRNA and inhibit translation of mRNA molecules into neutrophil factor alpha polypeptides. The oligonucleotides described above may also be delivered to cells to allow in vivo expression of antisense RNA or DNA to inhibit neutrophil factor alpha production.

Agonists and antagonists may be used in the compositions with a pharmaceutically acceptable carrier, e.g., as described previously.

In certain auto-immune and chronic inflammatory and infectious diseases, antagonists may be used, for example, to inhibit chemotaxis and activation of neutrophil factor α, macrophages and their precursors, as well as chemotaxis and activation of neutrophils, basophils, B lymphocytes and some subset of T-cells, such as activated and CD8 cytotoxic T cells and natural killer cells. Examples of auto-immune diseases include multiple sclerosis, and insulin-dependent diabetes. Antagonists may also be used to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis by preventing eosinophil and monocyte activation. They may also be used to treat idiopathic hypereosinophil syndrome by inhibiting the production and migration of eosinophils. Endotoxin shock can also be treated by antagonists by inhibiting macrophage migration and their production of the human chemokine polypeptides of the invention. Antagonists may also be used to treat atherosclerosis by inhibiting monocyte infiltration in the arterial wall. Antagonists may also be used to treat histamine-mediated allergic reactions and immunological diseases (including late phase allergic reactions), chronic urticaria, and atopic dermatitis, by chemokine-induced degranulation and release of histamine from mast cells and basophils. IgE-mediated allergic reactions, such as allergic asthma, rhinitis, and eczema, can also be treated. Antagonists may also be used to treat chronic and acute inflammation by inhibiting attraction of monocytes to the wound area. They can also be used to regulate the normal macrophage population of the lung because chronic and acute inflammatory lung diseases are associated with isolated mononuclear phagocytes in the lung. Antagonists may also be used to treat rheumatoid arthritis by inhibiting synovial fluid that attracts monocytes into the patient's joints. Monocyte influx and activation play an important role in the pathology of degenerative and inflammatory joint diseases. Antagonists can be used to interfere with deleterious cascades primarily attributed to IL-1 and TNF, which inhibit the biosynthesis of other inflammatory cytokines. In this way, antagonists may be used to inhibit inflammation. Antagonists may also be used to inhibit prostaglandin-independent fever induced by chemical promoters (chemokins). Antagonists may also be used to treat cases of bone marrow failure, e.g., aplastic anemia and myelodysplastic syndrome. Antagonists may also be used to treat asthma and allergy by inhibiting eosinophil accumulation in the lung. Antagonists may also be used to treat subepithelial basement membrane fibrosis, a prominent feature of asthmatic lungs.

Antibodies directed against neutrophil factor α can be used to bind to and inhibit neutrophil factor α activity to treat ARDS by inhibiting neutrophil invasion of the lung following injury. The antagonist can be used in the composition with a pharmaceutically acceptable carrier, as described below. Chromosome assay

The nucleic acid molecules of the invention are also valuable for chromosome identification. Specifically targeting sequences that can hybridize to specific locations on the individual's human chromosome. In addition, there is a need to identify specific sites on chromosomes. Few chromosome identification reagents based on actual sequence data (repeat polymorphisms) are currently available to identify chromosomal locations. The DNA according to the invention is an important first step in the mapping of chromosomes in connection with those sequences and genes associated with diseases.

In certain preferred embodiments of this regard, the cDNA disclosed herein is used to clone the genomic DNA of the neutrophil factor alpha protein gene. This can be accomplished using a variety of known techniques and libraries, which are generally commercially available. The genomic DNA is then used for in situ chromosome mapping for this purpose using known methods.

Furthermore, in some cases, the sequence can be mapped to the chromosome by preparing PCR primers (preferably 15-25bp) from the cDNA. In silico analysis of the 3' untranslated region of a 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 for PCR screening of somatic hybrids containing individual human chromosomes. In one step, fluorescence in situ hybridization ("FISH") of cDNA clones to mid-stage chromosomal bands can be used to provide precise chromosomal locations. This technique can be used with probes from cDNA as short as 50 or 60 bp. For a review of this technology, see Verma et al, human chromosome: basic technical Manual, Pergamon Press, New York (1988).

Once the sequence is mapped precisely to the chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data can be found in v.mckusick, human mendelian law genetics (available online through Welch medical library, university of Johns Hopkins). Then, the correlation between the gene and the disease that has been mapped to the same chromosomal region is identified by linkage analysis (co-inheritance of physically adjacent genes).

Second, it is necessary to determine differences in cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals, but not in any normal individuals, then the mutation is likely to be the cause of the disease.

Having generally described this invention, the same will be more readily understood through reference to the following examples, which are provided for purposes of illustration and are not intended to be limiting of the present invention. EXAMPLES example 1a expression and purification of "His-tagged" neutrophilic cytokine alpha in E.coli

The bacterial expression vector pQE9(pD10) was used for bacterial expression in this example (QIAGEN, supra). pQE9 encodes ampicillin antibiotic resistance ("Ampr") and contains the bacterial origin of replication ("ori"), the IPTG inducible promoter, the ribosome binding site ("RBS"), six codons encoding histidine residues which allow affinity purification using nickel-nitrilo-tri-acetic acid ("nickel-NTA") affinity resin (sold by QIAGEN, supra) and a suitable single restriction enzyme cleavage site. These elements are arranged so that an inserted DNA fragment encoding a polypeptide having 6 histidine residues covalently attached to its amino terminus (i.e., "6 × His tag") is expressed.

The DNA sequence encoding the desired portion of the neutrophil factor α protein (including the extracellular domain sequence) is amplified from the deposited cDNA clone using PCR oligonucleotide primers that anneal to the amino terminal sequence of the desired portion of the neutrophil factor α protein and to the 3' sequence of the deposited construct cDNA coding sequence. Additional nucleotides containing restriction sites facilitating cloning in the pQE9 vector were added to the 5 'and 3' primer sequences, respectively.

For the extracellular domain of the cloned protein, the 5 ' primer has the sequence 5 ' GTGGGATCCAGCCTCCGGGCAGAGCTG3 ' (SEQ ID NO:10) which contains the underlined BamHI restriction site followed by 18 nucleotides of the amino-terminal coding sequence of the extracellular domain of the neutrophil factor alpha sequence in FIG. 1. Of course, it will be clear to one of ordinary skill in the art that the starting point of the 5' primer in the protein coding sequence may be varied to amplify a DNA segment encoding any desired portion of the complete neutrophil factor alpha protein that is shorter or longer than the extracellular domain of the form in question. Primer 3 'has the sequence 5' GTGAAGCTTTTATTACAGCAGTTTCAATGCACC3 '(SEQ ID NO:11) which contains the underlined Hind III restriction enzyme site followed by two stop codons and 18 nucleotides complementary to the 3' end of the coding sequence of the neutrophil factor alpha DNA sequence in FIG. 1.

The amplified neutrophil factor alpha DNA fragment and vector pQE9 were digested with BamHI and HindIII, and the digested DNAs were ligated. Insertion of neutrophil factor α DNA into the restriction digested pQE9 vector places the neutrophil factor α protein coding region downstream of the IPTG-inducible promoter and in frame with the initiating AUG and 6 histidine codons.

The ligation mixture is transformed into competent E.coli using standard methods, for example, in Sambrook et al, molecular cloning: A laboratory Manual, second edition; cold spring harbor laboratory Press, cold spring harbor, NY (1989). Coli strain M15/rep4 contains multiple copies of plasmid pREP4 (which expresses the lac repressor and confers kanamycin resistance ("Kan"), which is used to carry out the illustrative examples described herein. this strain is only one of many strains suitable for expressing the neutrophil factor alpha protein, available from QIAGEN, Inc. (supra.) transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. At a dilution of about 1:25 to 1: 250. Cells were grown to an optical density of between 0.4 and 0.6 at 600nm ("OD 600"). isopropyl-beta-D-thiogalactopyranoside ("IPTG") was then added to a final concentration of 1mM to induce transcription from the lac repressor sensitive promoter by inactivation of the lacI repressor. The cells were then further cultured for 3-4 hours. Cells were then harvested by centrifugation.

The cells were then stirred in 6M guanidine hydrochloride (pH8) at 4 ℃ for 3-4 hours. Cell debris was removed by centrifugation and the supernatant containing neutrophil factor α was loaded onto a nickel-nitrilo-tri-acetic acid ("nickel-NTA") (by QIAGEN, provided supra) affinity resin column. Proteins with 6XHis tags bind with high affinity to nickel-NTA resin and can be purified in the simplest single-step purification procedure (see: QIAexpressinst, 1995, QIAGEN, supra). Briefly, the supernatant was loaded onto the column with 6M guanidine hydrochloride (pH8), first washed with 10 volumes of 6M guanidine hydrochloride (pH6), and finally eluted with 6M guanidine hydrochloride (pH5) for neutrophil factor α.

The purified protein was then renatured by dialysis against phosphate-buffered saline (PBS) or 50mM sodium acetate (pH6) buffer plus 200mM NaCl. Furthermore, proteins can be successfully folded when immobilized on a nickel-NTA column. The recommended conditions are as follows: renaturation was carried out using a linear 6M-1M urea gradient (containing protease inhibitors in 500mM NaCl, 20% glycerol, 20mM Tris/HCl pH 7.4). Renaturation should be carried out for 1.5 hours or more. After renaturation, the protein can be eluted by adding 250mM immidazole. Immidazole was removed by a final dialysis step against 200mM NaCl in PBS or 50mM sodium acetate (pH6) buffer. The purified protein is stored at 4 ℃ or frozen at-80 ℃. Example 1 b: expression and purification of neutrophil factor alpha in colibacillus

The bacterial expression vector pQE60 was used for bacterial expression in this example (QIAGEN, 9259Eton Avenue, Chatsworth, CA, 91311). pQE60 encodes ampicillin antibiotic resistance ("Ampr") and contains the bacterial origin of replication ("ori"), the IPTG inducible promoter, the ribosome binding site ("RBS"), six codons encoding histidine residues which allow affinity purification using nickel-nitrilo-tri-acetic acid ("nickel-NTA") affinity resin (sold by QIAGEN, supra) and a suitable single restriction enzyme cleavage site. These elements are arranged so that a DNA fragment encoding a polypeptide can be inserted in a manner that results in a polypeptide having 6 histidine residues covalently attached to its carboxy terminus (i.e., a "6 × His tag"). However, in this example, the polypeptide coding sequence was inserted in such a way that translation of the six histidine codons was inhibited, and thus, the resulting polypeptide did not have a 6 × His tag.

The DNA sequence encoding the desired portion of the neutrophil factor α protein (including the extracellular domain sequence) is amplified from the deposited cDNA clone using PCR oligonucleotide primers that anneal to the amino terminal sequence of the desired portion of the neutrophil factor α protein and to the 3' sequence of the deposited construct cDNA coding sequence. Additional nucleotides containing restriction sites facilitating cloning in the pQE60 vector were added to the 5 'and 3' primer sequences, respectively.

For the extracellular domain of the cloned protein, the 5 ' primer has the sequence 5 ' GTGTCATGAGCCTCCGGGCAGAGCTG3 ' (SEQ ID NO:12) which contains the underlined BspH restriction site followed by 17 nucleotides of the amino-terminal coding sequence of the extracellular domain of the neutrophil factor α sequence in FIG. 1. Of course, it will be clear to one of ordinary skill in the art that the starting point of the 5' primer can be varied in the protein coding sequence to amplify a desired portion of the entire protein that is shorter or longer than the extracellular domain of the form in question. Primer 3 'has the sequence 5' GTGAAGCTTTTATTACAGCAGTTTCAATGCACC3 '(SEQ ID NO:13) which contains the underlined Hind III restriction enzyme site followed by two stop codons and 18 nucleotides complementary to the 3' end of the coding sequence of the neutrophil factor alpha DNA sequence in FIG. 1.

The amplified neutrophil factor alpha DNA fragment and vector pQE60 were digested with BspH I and HindIII, and then the digested DNAs were ligated. Insertion of neutrophil factor α DNA into the restriction digested pQE60 vector places the neutrophil factor α protein coding region, including its associated stop codon, downstream of the IPTG-inducible promoter and in frame with the initiating AUG.

The ligation mixture is transformed into competent E.coli using standard methods, such as those described in Sambrook et al, molecular cloning: a laboratory manual, second edition; cold spring harbor laboratory Press, cold spring harbor, NY (1989). Coli strain M15/rep4 contains multiple copies of plasmid pREP4 (which expresses the lac repressor and confers kanamycin resistance ("Kan"), which is used to perform the illustrative examples described herein.

Clones containing the desired construct were grown overnight ("O/N") in liquid culture on LB medium supplemented with ampicillin (100. mu.g/ml) and kanamycin (25. mu.g/ml). O/N cultures were used to inoculate large cultures at dilutions between about 1:25 and 1: 250. Cells were grown to an optical density of between 0.4 and 0.6 at 600nm ("OD 600"). isopropyl-b-D-thiogalactopyranoside ("IPTG") was then added to a final concentration of 1mM to induce transcription from the lac repressor sensitive promoter by inactivation of the lacI repressor. The cells were then further cultured for 3-4 hours. Cells were then harvested by centrifugation.

The cells were then stirred in 6M guanidine hydrochloride (pH8) at 4 ℃ for 3-4 hours. Cell debris was removed by centrifugation and the supernatant containing neutrophil factor α was dialyzed against 50mM sodium acetate (pH6) buffer (supplemented with 200mM NaCl). Furthermore, the protein was successfully folded by dialysis against 500mM NaCl, 20% glycerol, 25mM Tris/HCl (pH7.4) containing protease inhibitors. After renaturation, the protein can be purified by ion exchange, hydrophobic interaction and sieve chromatography. In addition, affinity chromatography steps (e.g., antibody columns) may also be used to obtain purified neutrophil factor α protein. The purified protein was stored at 4C or frozen at-80 ℃. Example 2: cloning and expression of neutrophil factor alpha protein in baculovirus expression system

In this illustrative example, plasmid shuttle vector pA2GP was used to insert cloned DNA encoding the extracellular domain of a protein, lacking its native intracellular and transmembrane sequences, into baculovirus using baculovirus and standard methods as described in Summers et al, handbook of baculovirus vectors and methods for insect cell culture, Texas agricultural laboratory Handbuch 1555 (1987) to express the extracellular domain of neutrophil factor alpha. This expression vector contains the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV), followed by the secretory signal peptide (leader sequence) of baculovirus gp67 protein and convenient restriction sites such as BamH I, Xba I and Asp 718. The polyadenylation site of monkey virus ("SV 40") is used for efficient polyadenylation. For easy selection of recombinant viruses, the plasmid contains the E.coli beta-galactosidase gene under the control of a weak Drosophila promoter in the same orientation, followed by a polyadenylation signal for the polyhedrin gene. Two measures of the inserted gene are cell-mediated viral sequences that recombine homologously with the wild-type viral DNA (producing viable viruses that express the cloned polynucleotide).

Many other baculovirus vectors may be substituted for the above vectors, such as pAc373, pVL941 and pAcIM1, as will be readily understood by those skilled in the art, so long as the construct provides the appropriate targeting signals for transcription, translation, secretion, etc., including, if desired, a signal peptide and AUG in reading frame. Such vectors are described, for example, in Ldckow et al, virology 170:31-39 (1989).

The cDNA sequence encoding the extracellular domain of the neutrophil factor alpha protein in the deposited clones, lacking the AUG initiation codon and the naturally associated intracellular and transmembrane domain sequences shown in FIG. 1(SEQ ID NO:2), was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. The 5 ' primer has the sequence 5 ' GTGGGATCCCCGGGCAGAGCTGCAGGGC3 ' (SEQ ID NO:14) which contains the underlined BamHI restriction enzyme site followed by 18 nucleotides of the sequence of the extracellular domain of the neutrophil factor alpha protein shown in FIG. 1, starting at the N-terminus of the extracellular domain of the protein shown. Primer 3 'has the sequence 5' GTGGGATCCTTATTACAGCAGTTTCAATGCACC3 '(SEQ ID NO:15) which contains the underlined BamHI restriction enzyme site followed by two stop codons and 18 nucleotides complementary to the 3' coding sequence in FIG. 1.

The amplified fragments were separated from 1% agarose gel using a commercially available kit ("Geneclean," BIO101 inc., LaJolla, Ca.). The fragment was then digested with BamHI and purified again on a 1% agarose gel. This fragment is herein designated F1.

The plasmid was digested with the restriction enzyme BamHI and optionally dephosphorylated with calf intestinal phosphatase by conventional methods known in the art. Then, a commercially available kit ("Geneclean," BIO101 Inc., La Jolla, Ca.) separates DNA from 1% agarose gel. This vector DNA is designated herein as "V1".

The fragment Fl and the dephosphorylated plasmid V1 were ligated with T4DNA ligase. Coli HB101 or other suitable E.coli hosts, e.g., XL-1Blue (Statagene cloning System, La Jolla, CA) cells, were transformed with the ligation mixture and propagated on culture plates. Bacteria containing plasmids of the human neutrophil factor alpha gene were identified by digesting DNA from individual colonies with BamH I, followed by analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragment was confirmed by DNA sequencing. This plasmid is designated herein as pA2GP neutrophil factor alpha.

1.0. mu.g of commercially available linearized baculovirus DNA ("Bacuiogold") was used for lipofection as described by Felgner et al, Proc. Natl. Acad. Sci. USA 84:7413-^TMBaculovirus DNA ", Pharmingen, San Diego, CA) was co-transfected with 5 μ g of plasmid pA2GP neutrophil alpha. 1 microgram of Bacuiogold^TMViral DNA and 5 micrograms of plasmid pA2GP neutrophil factor α were mixed in wells of a microtiter plate containing 50 microliters of serum-free Grace's medium (life technologies inc., Gaithersburg, MD). Thereafter, 10. mu.l Lipofectin (Lipofectin) and 90. mu.l Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then, 2 drops of the transfection mixture were added to Sf9 insect cells (ATCC CRL1711) seeded in 35mM tissue culture plates with 1ml of serum-free Grace's medium. The plates were then incubated at 27 ℃ for 5 hours. The transfection solution was then removed from the plates and 1ml of Grace's insect medium supplemented with 10% fetal bovine serum was added. The culture was then continued at 27 ℃ for four days.

Four days later, supernatants were collected and plaque assays were performed as described by Summers and Smith, supra. Agarose gels with "Blue Gal" (Life Technologies Inc., Gaithersburg) were used to facilitate the identification and isolation of Blue-plaque producing Gal-expressing clones (a detailed description of this type of "plaque assay" can also be found in the insect cell culture and baculovirus biology user guide provided by Life Technologies Inc., Gaithersburg, p 9-10). After an appropriate incubation period, the blue plaques are picked with the tip of a micropipette (e.g., Eppendorf). Agar containing the recombinant virus was then suspended in a microcentrifuge tube containing 200 microliters of Grace's medium and the suspension containing the recombinant baculovirus used to infect Sf9 cells seeded in 35mM dishes. Four days later, the supernatants of these dishes were collected and then stored at 4 ℃. This recombinant virus is called V-neutrophil factor alpha.

To confirm the expression of the neutrophil factor α gene, Sf9 cells were cultured in Grace's medium supplemented with 10% heat-inactivated FBS. Cells were infected with recombinant baculovirus V-neutrophil factor alpha with an infection multiplicity ("MOI") of about 2. If a radiolabeled protein is desired, after 6 hours, the medium is removed and replaced with SF900 II medium (reduced methionine and cysteine) (supplied by Technologies Inc., Rockville, Md.). After 42 hours, 5. mu. Ci were added³⁵S-methionine and 5. mu. Ci³⁵S-cysteine (supplied by Amersham). The cells were further cultured for l6 hours and then harvested by centrifugation. The proteins in the supernatant as well as intracellular proteins are analyzed by SDS-PAGE and subsequently by autoradiography (if radiolabeled).

Minisequencing of the amino acid sequence of the amino terminus of the purified protein can be used to determine the amino-terminal sequence of the extracellular domain of the protein, thereby determining the cleavage site and length of the secretory signal peptide. Example 3: cloning and expression of neutrophil cytokine alpha in mammalian cells

Typical mammalian expression vectors contain promoter elements that regulate the initiation of transcription of mRNA, protein coding sequences, and certain signals (required for transcription termination and polyadenylation of the transcript). Additional elements include enhancers, Kozak sequences and insertions (flanking donor and acceptor sites for RNA splicing). High transcription efficiency can be achieved by the SV40 early and late promoters, the Long Terminal Repeats (LTRs) of retroviruses (e.g., RSV, HTLVI, HIVI), and the Cytomegalovirus (CMV) early promoter. However, cellular elements (e.g., the human actin promoter) may also be used. 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 (ATCC37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that may be utilized include human Hela,293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos1, Cos7 and CV1, qualil QC1-3 cells, mouse L cells and Chinese Hamster Ovary (CHO) cells.

Furthermore, the gene can be expressed in stable cell lines containing the gene integrated into the chromosome. Co-transfection with selectable markers (e.g., dhfr, gpt, neomycin, hygromycin) allows for the identification and isolation of transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful for developing cell lines carrying hundreds or even thousands of copies of the gene of interest. Another useful selectable marker is the enzyme Glutamine Synthase (GS) (Murphy et al, J. Biochem.227: 277-279 (1991); Bebbington et al, Biotechnology 10: 169-175). Using these markers, mammalian cells are cultured in selective media and the cells with the highest resistance are selected. These cell lines contain the amplified gene integrated into the chromosome. Chinese Hamster Ovary (CHO) and NSO cells are often used to produce proteins.

Expression vectors pC1 and pC4 contain the strong promoter (LTR) of Rous sarcoma virus (Cullen et al, molecular and cellular biology, 438-447 (3 months 1985)) plus a CMV-enhancer fragment (Boshart et al, cell 41:521-530 (1985)). Multiple cloning sites, for example, restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate cloning of the gene of interest. In addition, the vector contained 3 introns, polyadenylation of the rat preproinsulin gene and termination signals. Example 3 (a): cloning and expression in COS cells

Expression plasmid pNeutrokine alpha HA was prepared by cloning part of the deposited cDNA encoding the extracellular domain of the neutrophil factor alpha protein into the expression vector pcDNA I/Amp or pcDNA III (available from Invitrogen). To produce a soluble secreted form of the polypeptide, the extracellular domain is fused into the secretory leader sequence of the IL-6 gene.

The expression vector pcDNA I/amp contains: (1) an effective replication origin for propagation of E.coli and other prokaryotic cells; (2) ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) CMV promoter, polylinker, SV40 intron; (5) encodes a hemagglutinin fragment (i.e., an "HA" tag that facilitates purification) followed by a stop codon and a polyadenylation signal, arranged such that the cDNA can be conveniently placed under the expression control of the CMV promoter and operably linked to the SV40 intron and polyadenylation signal by means of restriction enzyme sites in a polylinker. The HA tag corresponds to the epitope from which the influenza hemagglutinin protein is derived as described by Wilson et al, cell 37:767 (1984). The fusion of the HA tag to the target protein allows for easy detection and recovery of the recombinant protein with antibodies that recognize the HA epitope. In addition, pcDNA III contains a selectable neomycin marker.

A DNA fragment encoding the extracellular domain of the neutrophil factor alpha polypeptide is cloned into the polylinker region of the vector so that expression of the recombinant protein is directed by the CMV promoter. The plasmid construction strategy is as follows. Amplification of the deposited cloned neutrophil factor alpha cDNA using primers containing convenient restriction sites is very similar to that described above in connection with the construction of vectors for expression of neutrophil factor alpha in E.coli. Suitable primers for use in this embodiment include the following, 5 'primers, a BamHI site containing underlining, a Kozak sequence, an AUG start codon, a sequence encoding a secretory leader peptide from the human IL-6 gene, a 5' coding region for the extracellular domain of the neutrophil factor alpha protein, having the following sequence: 5 'GCGGGATCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTTGTGAGACAAGGGGACCTGGCCAG 3' (SEQ ID NO: 16). The 3 'primer, containing the underlined BamHI restriction site and 18 nucleotides complementary to the 3' coding sequence immediately preceding the stop codon, has the following sequence 5 'GTGGGATCCTTACAGCAGTTTCAATGCACC 3' (SEQ ID NO: 17).

The PCR-amplified DNA fragment and the vector pcDNA I/Amp were digested with BamH I and ligated. The ligation mixture was integrated into the E.coli strain SURE (available from Stratagene cloning Systems,11099 North Torrey pipes Road, La Jolla, CA 92037), and the transformed cultures were plated onto ampicillin media plates, which were then cultured to grow ampicillin resistant colonies. Plasmid DNA was isolated from resistant colonies and the presence of a fragment encoding the extracellular domain of neutrophil factor α was detected by restriction analysis and other methods.

For the expression of recombinant neutrophil factor α, molecular cloning is used, for example, in Sambrook et al: a laboratory manual, second edition; DEAE-DEXTRAN described in Cold spring harbor laboratory Press, Cold spring harbor, NY (1989), COS cells were transfected with the expression vector described above. The cells are cultured under conditions in which the neutrophil factor alpha is expressed from the vector.

Using, for example, Harlow et al, antibodies: a laboratory manual, second edition; cold spring harbor laboratory Press, (1988) Cold spring harbor, N.Y., the expression of the neutrophil factor alpha HA fusion protein was detected by radiolabelling and immunoprecipitation. By the end of two days after transfection, cells were labeled by culturing in medium containing 35S-cysteine for 8 hours. The cells and medium were collected, washed, lysed with detergent-containing RIPA buffer as described by Wilson et al, cited above: 150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOG,50mM TRIS, pH 7.5. Proteins were precipitated from cell lysates and culture media using HA-specific monoclonal antibodies. The precipitated proteins were then analyzed by SDS-PAGE and autoradiography. Expression products of the expected size were visible in cell lysates, but not in the negative control. Example 3 (b): cloning and expression in CHO cells

The vector pC4 was used to express the neutrophil factor alpha protein. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC accession No. 37146). To produce a soluble, secreted form of the neutrophil factor α polypeptide, a portion of the deposited cDNA encoding the extracellular domain is fused into the secretory leader sequence of the IL-6 gene. The vector plasmid contained the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells lacking dihydrofolate activity (which have been transfected with these plasmids) can be selected by growth on selection media (α minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR gene in cells resistant to Methotrexate (MTX) is well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T.,1978, J.Biochem.253: 1357-. Due to the amplification of the DHFR gene, cells cultured in increasing concentrations of MTX developed resistance to the drug by overproducing the target enzyme DHFR. If a second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed, and this method can be used to develop cell lines carrying over 1000 copies of the amplified gene, as is known in the art. Thereafter, when methotrexate is withdrawn, a cell line is obtained that contains the amplifiable gene integrated into the chromosome of the host cell or cells.

Plasmid pC4 for expression of the gene of interest contains the strong promoter of the Rouse sarcoma virus Long Terminal Repeat (LTR) (Cullen et al, molecular and cellular biology, March 1985:438-447) and a fragment isolated from the human Cytomegalovirus (CMV) immediate early gene enhancer (Boshart et al, cell 41:521-530 (1985)). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow integration of the gene: BamHI, Xba I, and Asp 718. Following these cloning sites, the plasmid contains the 3' intron and the polyadenylation site of the rat preproinsulin gene. Other high efficiency promoters may also be used for expression, for example, the human β -actin promoter, the SV40 early or late promoter or other retroviral long terminal repeats, for example, HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express neutrophil factor alpha in a regulated manner in mammals (Gossen, M., & Bujard, H.1992, Proc. Natl. Acad. Sci. USA 89: 5547-. For polyadenylation of other signals of the mRNA, for example, the human growth hormone or globin genes may also be used. Suitable cell lines carrying the gene of interest integrated into the chromosome may also be selected on the basis of co-transfection with a selectable marker such as gpt, G418 or hygromycin. It may be advantageous to initially use more than one selectable marker, e.g., G418 plus methotrexate.

Plasmid pC4 was restriction digested with the restriction enzyme BamHI, followed by dephosphorylation with bovine intestinal phosphate and subsequent separation of the vector from a 1% agarose gel, as known in the art.

The DNA sequence encoding the extracellular domain of the neutrophil factor alpha protein was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. The 5 'primer, which contains the underlined BamH I site, Kozak sequence, AUG start codon, sequence encoding the secretory leader peptide of the human IL-6 gene, the 5' coding region of the extracellular domain of the neutrophil factor alpha protein, has the following sequence 5 'GCGGGATCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTTGTGAGACAAGGGGACCTGGCCAG 3' (SEQ ID NO: 16). A 3 'primer comprising the underlined bamhi restriction site and the 18 nucleotides immediately preceding the stop codon complementary to the 3' coding sequence, having the following sequence: 5 'GTGGGATCCTTACAGCAGTTTCAATGCACC 3' (SEQ ID NO: 17).

The amplified fragment was digested with the endonuclease BamH I and then purified again on a 1% agarose gel. The isolated fragment and dephosphorylated vector were ligated with T4DNA ligase. Coli or XL-1Blue cells were subsequently transformed. Bacteria containing the fragment inserted into plasmid pC4 are identified using, for example, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene were used for transfection. 5. mu.g of expression plasmid pC4 was co-transfected with 0.5. mu.g of plasmid pSVneo using a lipofectamine (Felgner et al, supra). Plasmid pSV2-neo contains the dominant selection marker, the neo gene of Tn5 (which encodes an enzyme that confers resistance to an antibiotic group, including G418). Cells were seeded in α minus MEM supplemented with 1mg/ml G418. After 2 days, cells were trypsinized and plated on hybridoma clone plates (Greiner, Germany) with α minus MEM supplemented with 10,25 or 50ng/ml metotrexate plus 1mg/ml G418. After about 10-14 days, single clones were trypsinized and then seeded in 6-well dishes or 10-ml flasks with varying concentrations of methotrexate (50nM,100nM,200nM,400nM,800 nM). Then, the clones grown in the highest concentration of methotrexate were transferred to new plates containing higher concentrations of methotrexate (1. mu.M, 2. mu.M, 5. mu.M, 10mM,20 mM). The same procedure was repeated until clones grown at a concentration of 100-200. mu.M were obtained. Expression of the desired gene product was analyzed by SDS-PAGE and Western blotting or by reverse phase HPLC analysis. Example 4: tissue distribution of neutrophil factor alpha mRNA expression

Northern blot analysis was performed to detect expression of the neutrophil factor alpha gene in human tissues using, for example, ambrook et al, described in the references cited above. According to the manufacturer's instructions, rediprime is used^TMThe DNA labeling system (Amersham Life science) labeled a probe containing cDNA of the entire nucleotide sequence (SEQ ID NO:1) of the neutrophil factor α protein with 32P. After labeling, the probes were purified using a CHROMA SPIN-100TM column (Clontech Laboratories, Inc.) according to the manufacturer's protocol PT 1200-1. The purified labeled probe was then used to examine various human tissues for neutrophil factor α mRNA.

Multi-tissue northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) were obtained from Clontech and used according to the manufacturer's protocol PT1190-1, ExpressHyb^TMThe hybridization solution (Clontech) was checked with a labeled probe. After hybridization and washing, the blot was mounted and the film was exposed overnight at-70 ℃ and developed according to standard methods.

It will be clear that the invention can be implemented in a different way than described in the above description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and thus such variations and modifications are to be considered within the purview of the appended claims.

The entire disclosure of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) is cited herein and incorporated by reference. Sequence listing (1) general information: the applicant (i): YU, GUO-LIANG

EBNER,REINHARD

NI, JIAN (ii) invention name: number of sequences of neutrophil factor α (iii): 17 (iv) communication address:

(A) the addressee: HUMAN GENOME sciences. inc.

(B) Street: 9410KEY WEST AVENUE

(C) City: ROCKVILLE

(D) State: MD

(E) The state is as follows: united states of America

(F) ZIP 20850 (v) computer-readable form:

(A) type of medium: flexible disk

(B) A computer: IBMPC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patent in Release #1.0, version #1.30 (vi) current application data:

(A) application No.:

(B) application date:

(C) classification number: (viii) attorney/proxy information:

(A) name: BENSON, ROBERTH

(B) Registration number: 30,446

(C) Certificate number: PF343 (ix) telecommunications information:

(A) telephone: (301)309-8504

(B) Faxing: (301)309-8512(2) information of SEQ ID NO:1: sequence characteristics:

(A) length: 1100 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: DNA (genome) (ix) feature:

(A) name/keyword: cds (b) position: 1001 (ix) feature:

(A) name/keyword: sig _ peptide

(B) Position: an 381 (ix) feature:

(A) name/keyword: mat _ peptide

(B) Position: the sequence of 1001 (xi): SEQ ID NO 1: AAATTCAGGA TAACTCTCCT GAGGGGTGAG CCAAGCCCTG CCATGTAGTG CACGCAGGAC 60ATCAACAAAC ACAGATAACA GGAAATGATC CATTCCCTGT GGTCACTTAT TCTAAAGGCC 120CCAACCTTCA AAGTTCAAGT AGTGAT ATG GAT GAC TCC ACA GAA AGG GAG CAG 173

Met Asp Asp Ser Thr Glu Arg Glu Gln

1 5TCA CGC CTT ACT TCT TGC CTT AAG AAA AGA GAA GAA ATG AAA CTG AAG 221Ser Arg Leu Thr Ser Cys Leu Lys Lys Arg Glu Glu Met Lys Leu Lys10 15 20 25GAG TGT GTT TCC ATC CTC CCA CGG AAG GAA AGC CCC TCT GTC CGA TCC 269Glu Cys Val Ser Ile Leu Pro Arg Lys Glu Ser Pro Ser Val Arg Ser

30 35 40TCC AAA GAC GGA AAG CTG CTG GCT GCA ACC TTG CTG CTG GCA CTG CTG 317Ser Lys Asp Gly Lys Leu Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu

45 50 55TCT TGC TGC CTC ACG GTG GTG TCT TTC TAC CAG GTG GCC GCC CTG CAA 365Ser Cys Cys Leu Thr Val Val Ser Phe Tyr Gln Val Ala Ala Leu Gln

60 65 70GGG GAC CTG GCC AGC CTC CGG GCA GAG CTG CAG GGC CAC CAC GCG GAG 413Gly Asp Leu Ala Ser Leu Arg Ala Glu Leu Gln Gly His His Ala Glu

75 80 85AAG CTG CCA GCA GGA GCA GGA GCC CCC AAG GCC GGC CTG GAG GAA GCT 461Lys Leu Pro Ala Gly Ala Gly Ala Pro Lys Ala GlY Leu Glu Glu Ala90 95 100 105CCA GCT GTC ACC GCG GGA CTG AAA ATC TTT GAA CCA CCA GCT CCA GGA 509Pro Ala Val Thr Ala Gly Leu Lys Ile Phe Glu Pro Pro Ala Pro Gly

110 115 120GAA GGC AAC TCC AGT CAG AAC AGC AGA AAT AAG CGT GCC GTT CAG GGT 557Glu Gly Asn Ser Ser Gln Asn Ser Arg Asn Lys Arg Ala Val Gln Gly

125 130 135CCA GAA GAA ACA GTC ACT CAA GAC TGC TTG CAA CTG ATT GCA GAC AGT 605Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser

140 145 150GAA ACA CCA ACT ATA CAA AAA GGA TCT TAC ACA TTT GTT CCA TGG CTT 653Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu

155 160 165CTC AGC TTT AAA AGG GGA AGT GCC CTA GAA GAA AAA GAG AAT AAA ATA 701Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile170 175 180 185TTG GTC AAA GAA ACT GGT TAC TTT TTT ATA TAT GGT CAG GTT TTA TAT 749Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr

190 195 200ACT GAT AAG ACC TAC GCC ATG GGA CAT CTA ATT CAG AGG AAG AAG GTC 797Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val

205 210 215CAT GTC TTT GGG GAT GAA TTG AGT CTG GTG ACT TTG TTT CGA TGT ATT 845His Val Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile

220 225 230CAA AAT ATG CCT GAA ACA CTA CCC AAT AAT TCC TGC TAT TCA GCT GGC 893Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly

235 240 245ATT GCA AAA CTG GAA GAA GGA GAT GAA CTC CAA CTT GCA ATA CCA AGA 941Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg250 255 260 265GAA AAT GCA CAA ATA TCA CTG GAT GGA GAT GTC ACA TTT TTT GGT GCA 989Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala

270 275 280TTG AAA CTG CTG TGACCTACTT ACACCATGTC TGTAGCTATT TTCCTCCCTT 1041Leu Lys Leu Leu

285TCTCTGTACC TCTAAGAAGA AAGAATCTAA CTGAAAATAC CAAAAAAAAA AAAAAAAAA 1100(2) information of SEQ ID NO:2: sequence characteristics:

(A) length: 285 amino acids

(B) Type (2): amino acids

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: 2 SEQ ID NO Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu 151015 Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro

20 25 30Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu

35 40 45Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val Val

50 55 60Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu Arg65 70 75 80Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly

85 90 95Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu

100 105 110Lys Ile Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn

115 120 125Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln

130 135 140Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys145 150 155 160Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser

165 170 175Ala Leu Glu Glu Lys Glu Ash Lys Ile Leu Val Lys Glu Thr Gly Tyr

180 185 190Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met

195 200 205Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu

210 215 220Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu225 230 235 240Pro Asn Asn Ser Cys Tyr Sar Ala Gly Ile Ala Lys Leu Glu Glu Gly

245 250 255Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu

260 265 270Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

275280285 (2) information of SEQ ID NO:3:

sequence characteristics:

(A) length: 233 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: 3: Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 151015 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe SEQ ID NO

20 25 30Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

35 40 45Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Ser Pro

50 55 60Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser65 70 75 80Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro

85 90 95Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu

100 105 110Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser

115 120 125Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly

130 135 140Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala145 150 155 160Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro

165 170 175Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu

180 185 190Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu

195 200 205Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly

210215220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225230 (2) information of SEQ ID NO:4:

sequence characteristics:

(A) length: 205 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: protein

(xi) sequence description: SEQ ID NO 4: Met Thr Pro Pro Glu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr 151015 Leu His Leu Leu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala

20 25 30Gln Gly Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala

35 40 45Arg Gln His Pro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala

50 55 60Ala His Leu Ile Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg65 70 75 80Ala Asn Thr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn

85 90 95Asn Ser Leu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln

100 105 110Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Pro Ser Ser Pro

115 120 125Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe

130 135 140His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln145 150 155 160Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln Leu Thr

165 170 175Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro His Leu Val

180 185 190Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu

195200205 (2) information of SEQ ID NO:5:

sequence characteristics:

(A) length: 244 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: protein

(xi) sequence description: SEQ ID NO 5: Met Gly Ala Leu Gly Leu Glu Gly Arg Gly Gly Arg Leu Gln Gly Arg 151015 Gly Ser Leu Leu Leu Ala Val Ala Gly Ala Thr Ser Leu Val Thr Leu

20 25 30Leu Leu Ala Val Pro Ile Thr Val Leu Ala Val Leu Ala Leu Val Pro

35 40 45Gln Asp Gln Gly Gly Leu Val Thr Glu Thr Ala Asp Pro Gly Ala Gln

50 55 60Ala Gln Gln Gly Leu Gly Phe Gln Lys Leu Pro Glu Glu Glu Pro Glu65 70 75 80Thr Asp Leu Ser Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro

85 90 95Leu Lys Gly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe

100 105 110Leu Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro

115 120 125Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg

130 135 140Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg145 150 155 160Ser Ser Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu

165 170 175Leu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro Ala

180 185 190Arg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly

195 200 205Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser

210215220 His Pro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly Ala 225230235240 Val Met Val Gly (2) information of SEQ ID NO:6:

sequence characteristics:

(A) length: 281 amino acids

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: protein

(xi) sequence description: 6: Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp 151015 Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys SEQ ID NO

20 25 30Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro

35 40 45Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro

50 55 60Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly65 70 75 80Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly

85 90 95Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala

100 105 110Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu

115 120 125Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg

130 135 140Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu145 150 155 160Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr

165 170 175Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr

180 185 190Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser

195 200 205His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met

210 215 220Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala225 230 235 240Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His

245 250 255Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser

260 265 270

Gln Thr Phe Phe Gly Leu Tyr Lys Leu

275280 (2) information of SEQ ID NO:7:

sequence characteristics:

(A) length: 338 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: SEQ ID NO 7: AGGNTAACTC TCCTGAGGGG TGAGCCAAGC CCTGCCATGT AGTGCACGCA GGACATCANC 60AAACACANNN NNCAGGAAAT AATCCATTCC CTGTGGTCAC TTATTCTAAA GGCCCCAACC 120TTCAAAGTTC AAGTAGTGAT ATGGATGACT CCACAGAAAG GGAGCAGTCA CGCCTTACTT 180CTTGCCTTAA GAAAAGAGAA GAAATGAAAC TGNAAGGAGT GTGTTTCCAT CCTCCCACGG 240AAGGAAAGCC CCTCTNTCCG ATCCTCCAAA GACGGAAAGC TGCTGGCTGC AACCTTGNTG 300NTGGCATTGT GTTCTTGCTG NCTCAAGGTG GTGTTNTT 338(2) information of SEQ ID NO 8:

sequence characteristics:

(A) length: 509 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: SEQ ID NO 8: AATTCGGCAN AGNAAACTGG TTACTTTTTT ATATATGGTC AGGTTTTATA TACTGATAAG 60ACCTACGCCA TGGGACATCT AGTTCAGAGG AAGAAGGTCC ATGTCTTTGG GGATGAATTG 120AGTCTGGTGA CTTTGTTTCG ATGTATTCAA AATATGCCTG AAACACTACC CAATAATTCC 180TGCTATTCAG CTGGCATTGC AAAACTGGNA GGAAGGAGAT GAACTCCAAC TTGCAATACC 240AGGGGAAAAT GCACAATTAT CACTGGGATG GAGATGTTCA CATTTTTTGG GTGCCATTGA 300AACTGCTGTG ACCTNCTTAC ANCANGTGCT GTTNGCTATT TTNCCTNCCT NTTCTNTGGT 360AACCTCTTAG GAAGGAAGGA TTCTTAACTG GGAAATAACC CAAAAAAANN TTAAANGGGT 420ANGNGNNANA NGNGGGGNNG TTNNCNNGNN GNNTTTTNGG NNTATNTTNT NNTNGGGNNN 480NGTAAAAATG GGGCCNANGG GGGNTTTTT 509(2) information of SEQ ID NO 9:

sequence characteristics:

(A) length: 497 base pair

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: DNA (genome) (xi) sequence description: SEQ ID NO 9: AATTCGGCAC GAGCAAGGCC GGCCTGGAGG AAGCTCCAGC TGTCACCGCG GGACTGAAAA 60TCTTTGAACC ACCAGCTCCA GGAGAAGGCA ACTCCAGTCA GAACAGCAGA AATAAGCGTG 120CCGTTCAGGG TCCAGAAGAA ACAGTCACTC AAGACTGCTT GCAACTGNTT GCAGACAGTG 180AAACACCAAC TATACAAAAA GGCTCCCTTC TGNTGCCACA TTTGGGCCAA GGAATGGAGA 240GATTTCTTCG TCTGGAAACA TTTTGCCAAA CTCTTCAGAT ACTCTTTNCT CTCTGGGAAT 300CAAAGGAAAA TCTCTACTTA GATTNACACA TTTGTTCCCA TGGGTNTCTT AAGTTTTAAA 360AGGGGAGTGC CCTTAGGAGG AAAAGGGGAT AAATATTGGC CAAGGNACTG GTTANTTTNT 420AAATATGGTC AGGTTTNTAT ANCTGGTAGG CCTCGCCATG GGCATTNATT CANGGNGAGG 480NCNNTCTTTT GGGNTGA 497(2) information of SEQ ID NO 10:

sequence characteristics:

(A) length: 27 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: SEQ TD NO:10: GTGGGATCCA GCCTCCGGGC AGAGCTG 27(2) information of SEQ ID NO:11:

sequence characteristics:

(A) length: 33 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: SEQ ID NO:11: GTGAAGCTTT TATTACAGCA GTTTCAATGC ACC 33(2) information of SEQ ID NO:12:

sequence characteristics:

(A) length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: SEQ ID NO:12: GTGTCATGAG CCTCCGGGCA GAGCTG 26(2) information of SEQ ID NO:13:

sequence characteristics:

(A) length: 33 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: SEQ ID NO 13: GTGAAGCTIT TATTACAGCA GTTTCAATGC ACC 33(2) information of SEQ ID NO 14:

sequence characteristics:

(A) length: 28 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: SEQ ID NO:14: GTGGGATCCC CGGGCAGAGC TGCAGGGC 28(2) information of SEQ ID NO:15:

sequence characteristics:

(A) length: 33 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: SEQ ID NO:15: GTGGGATCCT TATTACAGCA GTTTCAATGC ACC 33(2) information of SEQ ID NO:16:

sequence characteristics:

(A) length: 129 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: DNA (genome) (xi) sequence description: SEQ ID NO 16: GCGGGATCCG CCACCATGAA CTCCTTCTCC ACAAGCGCCT TCGGTCCAGT TGCCTTCTCC 60CTGGGGCTGC TCCTGGTGTT GCCTGCTGCC TTCCCTGCCC CAGTTGTGAG ACAAGGGGAC 120CTGGCCAGC 129(2) information of SEQ ID NO 17:

sequence characteristics:

(A) length: 30 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linearity

(ii) type of molecule: DNA (genome)

(xi) sequence description: 17: GTGGGATCCT TACAGCAGTT TCAATGCACC 30 SEQ ID NO

Claims (21)

1. An isolated nucleic acid molecule comprising 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 neutrophil factor alpha polypeptide having the complete amino acid sequence of figure 1(SEQ ID NO: 2);

(b) a nucleotide sequence encoding a neutrophil factor α polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the ATCC deposit deposited at 22/10/1996;

(c) a nucleotide sequence encoding the extracellular domain of a neutrophil factor α polypeptide;

(d) a nucleotide sequence encoding a neutrophil factor alpha polypeptide transmembrane domain;

(e) a nucleotide sequence encoding the intracellular domain of a neutrophil factor α polypeptide;

(f) a nucleotide sequence encoding a soluble neutrophil factor α polypeptide comprising extracellular and intracellular domains but lacking a transmembrane domain; and

(g) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above.

2. The nucleic acid molecule of claim 1, wherein said polynucleotide has the complete nucleotide sequence set forth in FIG. 1(SEQ ID NO: 1).

3. The nucleic acid molecule of claim 1, wherein said polynucleotide has the complete nucleotide sequence shown in figure 1(SEQ ID NO:1) encoding the neutrophil factor α polypeptide having the complete amino acid sequence shown in figure 1(SEQ ID NO: 2).

4. The nucleic acid molecule of claim 1, wherein said polynucleotide has a nucleotide sequence encoding a soluble neutrophil factor α polypeptide comprising the extracellular domain shown in figure 1(SEQ ID NO: 2).

5. An isolated nucleic acid molecule comprising 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 an amino acid sequence consisting of residues n-285 of SEQ ID No. 2, wherein n is an integer in the range of 2-190.

(b) A nucleotide sequence encoding a polypeptide having an amino acid sequence consisting of residues 1-m of SEQ ID NO 2, wherein m is an integer in the range 274-284.

(c) A nucleotide sequence encoding a polypeptide having an amino acid sequence consisting of residues n-m of SEQ ID NO 2, wherein n and m are integers as defined in (a) and (b) above, respectively.

(d) A nucleotide sequence encoding a polypeptide consisting of a portion of the complete neutrophil factor alpha amino acid sequence encoded by the cDNA clone contained in the ATCC deposit deposited at 22/10/1996, wherein said portion excludes amino acids 1 to 190 from the amino terminus and amino acids 1 to 11 from the C-terminus of said complete amino acid sequence.

6. The nucleic acid molecule of claim 1 wherein said polynucleotide has the complete nucleotide sequence of a cDNA clone contained in the ATCC deposit deposited at 22/10 th 1996.

7. The nucleic acid molecule of claim 1 wherein said polynucleotide has a nucleotide sequence encoding a neutrophil factor α polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the ATCC deposit deposited at 22/10 th 1996.

8. The nucleic acid molecule of claim 1, wherein said polynucleotide has a nucleotide sequence encoding a soluble neutrophil factor α polypeptide comprising the extracellular domain encoded by a cDNA clone contained in the ATCC deposit deposited at 22/10 th 1996.

9. An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a sequence identical to the sequence in claim 1(a), (b), (c), (d), (e) or (f), wherein said hybridized polynucleotide does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only a residues or only T residues.

10. An isolated nucleic acid molecule comprising a polynucleotide encoding the amino acid sequence of an epitope-bearing portion of a neutrophil factor α polypeptide having the amino acid sequence of claim 1(a), (b), (c), (d), (e) or (f).

11. The isolated nucleic acid molecule of claim 10, which molecule encodes a neutrophil factor α polypeptide epitope-bearing moiety selected from the group consisting of: a polypeptide comprising amino acid residues from about Phe115 to about Leu147 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ile150 to about Tyr163 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser171 to about Phe194 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Glu223 to about Tyr247 (SEQ ID NO: 2); and polypeptides comprising amino acid residues from about Ser271 to about Phe278 (SEQ ID NO: 2).

12. A method of making a recombinant vector comprising inserting the isolated nucleic acid molecule of claim 1 into a vector.

13. A recombinant vector produced by the method of claim 12.

14. A method of making a recombinant host cell, the method comprising introducing the recombinant vector of claim 13 into a host cell.

15. A recombinant host cell produced by the method of claim 14.

16. A recombinant method for producing a neutrophil factor α polypeptide, comprising culturing the recombinant host cell of claim 15 under conditions such that said polypeptide is expressed, and recovering said polypeptide.

17. An isolated neutrophil factor α polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of seq id no:

(a) an amino acid sequence of a neutrophil factor alpha polypeptide having the complete amino acid sequence of figure 1(SEQ ID NO: 2);

(b) an amino acid sequence of a neutrophil factor α polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the ATCC deposit deposited at 22/10/1996;

(c) an amino acid sequence of an extracellular domain of a neutrophil factor α polypeptide;

(d) an amino acid sequence of a neutrophil factor alpha polypeptide transmembrane domain;

(e) an amino acid sequence of an intracellular domain of a neutrophil factor α polypeptide; (f) an amino acid sequence of a soluble neutrophil factor alpha polypeptide comprising said domain; and

(g) an amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), (d), (e) or (f) above.

18. The isolated polypeptide of claim 17, comprising an epitope-bearing portion of a neutrophil factor α protein, wherein said portion is selected from the group consisting of: a polypeptide comprising amino acid residues from about Phe115 to about Leu147 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ile150 to about Tyr163 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser171 to about Phe194 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Glu223 to about Tyr247 (SEQ ID NO: 2); a polypeptide comprising amino acid residues from about Ser271 to about Phe278 (SEQ ID NO: 2).

19. An isolated antibody that specifically binds the neutrophil factor α polypeptide of claim 17.

20. A pharmaceutical composition comprising the polypeptide of claim 17 and a pharmaceutically acceptable carrier.

21. An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a sequence selected from the group consisting of seq id no:

(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 FIG. 1(SEQ ID NO:1), wherein said portion comprises at least 30 contiguous nucleotides from nucleotide 1 to nucleotide 809; and

(e) a nucleotide sequence complementary to any one of the nucleotide sequences in (a), (b), (c) or (d) above.